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Authors

Georg Büldt (FZJ) Dmitrij Frishman (HMGU) Udo Heinemann (MDC) Dirk Heinz (HZI) Harald Herrmann-Lerdon (DKFZ) Werner Mewes (HMGU) Uwe Müller (HZB) Michael Sattler (HMGU) Anne Ulrich (FZK) Edgar Weckert (DESY) Dieter Willbold (FZJ) Regine Willumeit (GKSS)

Coordination

Dirk Heinz (HZI) Michael Sattler (HMGU)

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Contents Glossary 4 Executiv e Summary 5 A. Introduction 6 B. Current status of structural biology in the Helmholtz Association 7 B1. Research activities 7 B2. Integration into Helmholtz research programmes 8 B3. Provision of infrastructure 9 B4. Synchrotron research for structural biology 9 B5. Biomolecular NMR spectroscopy at Helmholtz 11 B6. Helmholtz Protein Sample Production Facility (PSFP) 14 C. Helmholtz structural biology in the national and European conte xt 16 D. Perspectives of structural biology in the Helmholtz Associatio n 19 D1. Centre for Structural Systems Biology (CSSB) at DESY 20 D2. NMR centres in the Helmholtz Association 22 D3. Cryo-electron microscopy 23 D4. Helmholtz Protein Sample Production Facility (PSPF) 24 D5. Neutron scattering in biology 25 D6. Structural bioinformatics and systems biology 26 D7. Structural biology and chemical biology 27 D8. Training and visibility 28 E. Annex 30 Profiles of Helmholtz centres with activities in structural biology 30 F. Contact 44

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Glossary List of abbreviations used in this document: ANKA Synchrotron facility at Karlsruhe Research Centre BESSY Berliner Elektronen-Speicherring / Gesellschaft für Synchrotronstrahlung BNI Bernhard-Nocht-Institut für Tropenmedizin BNMRZ Bayerisches NMR Zentrum CD Circular dicroism DESY Deutsches Elektronen-Synchrotron DKFZ Deutsches Krebsforschungszentrum / German Cancer Research Centre EM Electron microscopy EMBL European Molecular Biology Laboratory EPR Electron paramagnetic resonance ESRF European Synchrotron Research Facility ESFRI European Strategy Forum on Research Infrastructures FESP Forum for European Structural Proteomics FLASH "F"reie-Elektronen-"LAS"er in "H"amburg / VUV free-electron laser at DESY FLI Friedrich-Loeffler-Institut FMP Leibniz-Institut für Molekulare Pharmakologie (Berlin) FRM-II Forschungsneutronenquelle Heinz Maier-Leibnitz FTIR Fourier-transformation IR spectroscopy FZB Forschungszentrum Borstel FZK Forschungszentrum Karlsruhe FZJ Forschungszentrum Jülich HPI Heinrich-Pette-Institut für Experimente Virologie und Immunologie HMGU Helmholtz Zentrum München / German Research Centre for Environmental Health HZB Helmholtz-Zentrum Berlin für Materialien und Energie HZI Helmholtz-Zentrum für Infektionsforschung INSTRUCT Integrated Structural Biology Infrastructure for Europe KIT Karlsruher Institut für Technologie MAD Multiwavelength anomalous dispersion OCD Oriented circular dicroism PETRA-III High-brilliance synchrotron radiation source at DESY PSPF Protein Sample Production Facility NMR Nuclear magnetic resonance SANS Small-angle neutron scattering SAR Structure activity relationship SAXS Small-angle X-ray scattering UKE Universitätsklinikum Hamburg-Eppendorf XFEL X-ray free-electron laser

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Executive Summary Modern biology and biomedicine increasingly rely on the knowledge of the three-dimensional structures of mac-

romolecules and their interactions within the cellular context. Structural biology comprising the well-established

experimental techniques X-ray crystallography, NMR spectroscopy and electron microscopy (EM) is capable of

providing precise information on the geometry and dynamics of large molecules and their assemblies. Recent

advances, like the use of brilliant synchrotron radiation, ultra-high magnetic fields as well as hybrid approaches

involving single particle cryo-EM, electron tomography, small-angle X-ray and neutron scattering will allow the

investigation of physiologically relevant biomacromolecular interactions of ever increasing complexity and dy-

namics.

Structural biology in the Helmholtz Association is highly interdisciplinary and an integral part of the research field

Health. Research of Helmholtz structural biologists has been internationally recognized and judged as excellent

to outstanding in recent programme and centre evaluations. Helmholtz centres provide large and expensive

infrastructure, in particular the synchrotron facilities at DESY, HZB and FZK, the NMR centres at HMGU, FZJ,

and FZK and the Protein Sample Production Facility at HZI and MDC. The storage rings at HZB and, in particu-

lar, the newly established synchrotron source PETRA III at DESY provide ultrabright X-rays enabling high reso-

lution structure determinations of the most challenging proteins and biomacromolecular assemblies. At the NMR

centres biological macromolecules and their molecular interactions are studied in a native-like environment and

their dynamic and transient properties are investigated in solution and the solid state. Together with chemical

biology, which is well developed at several Helmholtz centres, the mode of action of small molecules targeting

pathological processes can be investigated at high resolution paving the way towards novel lead compounds for

therapeutic intervention. The tight integration into specific health research programmes as well as the provision

of large-scale research infrastructure are the unique features of Helmholtz structural biology within the Helm-

holtz Association.

To meet the future challenges and to guarantee international competitiveness in structural biology additional

infrastructures and research activities are required. A planned new centre for structural systems biology (CSSB)

at DESY in Hamburg, should make use of the unique research opportunities provided by PETRA III, where the

Helmholtz Association and the Max-Planck Society will share a beamline for biomacromolecular crystallography

which will require long-term support and continued upgrading in the medium-term future.. Likewise the Euro-

pean free electron X-ray laser X-FEL, to become operational in 2014, may offer unprecedented but still poorly

understood for studying the structure and dynamics of single biomacromolecules. The imminent availability of

ultra-high magnetic fields (�

1 GHz) for NMR opens up the possibility of studying biomacromolecular assemblies

of highest complexity and biomedical significance by solution NMR techniques, as well as in heterogeneous and

solid phase. It will thus be important to install ultra-high field NMR instrumentation at the NMR centres of

HMGU, FZJ and FZK.

At present, the structural biology portfolio of the Helmholtz Association is strongly biased in favour of X-ray crys-

tallography and NMR spectroscopy with only modest representation of high-resolution electron microscopy or

tomography. Since these emerging techniques cover a gap between the molecular and (sub-)cellular levels of

analysis, it will remain a strategic decision of the Helmholtz Association to invest in these methods either on an

institutional basis or in cooperation with local partner institutions.

Thus, structural biology in the Helmholtz Association is well prepared and - with additional support as put for-

ward in this document - will provide an excellent environment and world-class infrastructure to support interdis-

ciplinary research in the newly emerging field of structural systems biology for understanding human disease

mechanisms and developing novel strategies for translational research.

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"Infection and Immunity" research programme, the HZI crystallographic and NMR spectroscopic groups concen-

trate on host-pathogen interactions and the molecular basis of infection processes. The MDC crystallographic

group focuses on the genome-wide structural analysis of human proteins and on protein-nucleic acid interac-

tions within the "Cancer Research" programme. FZJ participates with X-ray crystallography and NMR spectros-

copy in the programmes “Function and Dysfunction of the Nervous System” and “BioSoft: Macromolecular Sys-

tems and Biological Information Processing” of the research fields "Health" and “Key Technologies”, respec-

tively, with X-ray crystallographic and NMR spectroscopic studies on membrane-bound receptor signalling

pathways from membrane-bound receptors to the creation of electric signals by ion channels as well as the

investigation of the molecular basis of diseases such as AIDS, SARS and neurodegenerative disorders. The

NMR spectroscopic and synchrotron radiation (IR, CD) groups at the FZK characterize interactions taking place

at the membrane interface. The aim is to develop molecular tools to manipulate the membrane and thus design

a new generation of cellular transporters within the programme “Biointerfaces” of the research field “Key Tech-

nologies”.

B3. Provision of i nfrastructure

Both DESY and HZB provide excellent synchrotron sources (i.e. PETRA III and BESSY II) for high resolution X-

ray crystallography and small angle X-ray scattering (SAXS). HMGU, FZJ and FZK host three NMR centres that

provide infrastructure to study the structure and dynamics of biological macromolecules in solution and in the

solid state. GKSS, HZB and FZJ offer neutron scattering facilities for biological applications. The FZJ and GKSS

research groups on small angle neutron scattering (SANS) are based at the Forschungsreaktor II (FRM II) of the

Technical University of München, where HMGU contributes expertise in isotope labelling and sample production

and multidisciplinary approaches combining NMR spectroscopy with SANS. A further important activity of Helm-

holtz structural biology is the newly established protein sample production facility (PSPF), which is shared

equally by HZI and MDC, which aims to alleviate one the major bottlenecks in modern structural biology.

Several universities, Max-Planck and Leibniz groups benefit substantially from access to state-of-the-art facili-

ties of the Helmholtz Association. The unique research infrastructures offered by the Helmholtz centres have

attracted several non-Helmholtz institutes such as EMBL Hamburg, the Universities of Hamburg and Lübeck

and the Max-Planck Groups to the DESY campus. Likewise, the Leibniz Institute for Molecular Pharmacology

(FMP) Berlin is closely associated with MDC, linking chemical biology with structural biology. The University of

Karlsruhe will soon be merged with the FZK to form the Karlsruhe Institute of Technology (KIT).

The following sections will briefly review the expertise, large instrumentation and facilities currently available for

structural biology within the Helmholtz Association.

B4. Synchrotron research for structural biology

Macromolecular X-ray crystallography continues to be the leading method for the determination of 3D structures

of biological macromolecules at high, i.e atomic, resolution. Using this method, molecules and supramolecular

assemblies can be studied virtually without size limitation as long as they can be purified to homogeneity and

brought into a crystalline form. Crystallographic techniques can also be used to study short-lived structural in-

termediates, some of them even in the sub-nanosecond regime. In addition, macromolecular crystallography

plays a central role in pharmacological research where target validation, lead identification and optimisation are

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Figure 3: Vessel of the cryo cooled high heatload X-ray monochromator

usually guided by crystal structure analysis of receptor proteins and their complexes with lead or drug mole-

cules. Without doubt, the use of highly brilliant synchrotron radiation with tuneable wavelengths has made the

greatest impact on modern structural biology. Currently the three existing German synchrotrons are located in

Helmholtz institutions.

DESY Hamburg

The use of synchrotron radiation for structural biology has a long standing tradition at DESY, Hamburg mainly

carried out through the activities of the EMBL and MPG-ASMB outstations. First experiments in small angle

scattering from muscle tissue date back as early as 1970. At present EMBL operates five beamlines for macro-

molecular crystallography at the DORIS III storage ring, two of which are capable of multi-wavelength anoma-

lous dispersion (MAD) techniques. Another MAD-beamline for structural biology at DORIS III is operated by the

MPG-ASMB outstation at DESY. As a complementary analysis tool EMBL operates one beamline for Small

Angle X-ray Scattering (SAXS). Over the past 5 years approximately 600 user visits per year have been regis-

tered by the EMBL Hamburg.

At present DESY is commissioning the new third generation

synchrotron radiation source PETRA III (Figure 2 ) for high

brilliance applications which in future should also take over

all DORIS III activities. EMBL at present is setting up two

undulator beamlines for macromolecular crystallography and

an undulator SAXS beamline (BioSAXS) capable of time

resolved studies (Figure 3 ). The GKSS research centre will

contribute to the BioSAXS beamline at PETRA III. With fi-

nancial contribution from the Max-Planck society and the

Helmholtz research area “Health” DESY is currently establishing a

third beamline for macromolecular crystallography, which will also

provide a station for biological imaging using X-rays in the 2.4 – 10

keV photon energy range. In addition the universities of Lübeck and

Hamburg will contribute to the construction and operational costs of

the structural biology beamlines at PETRA III. In a future extension

of PETRA III a further curved magnet beamline for macromolecular

crystallography is planned. All PETRA III macromolecular crystallog-

raphy beamlines will provide capabilities for phasing using MAD.

Besides crystallography other techniques such as small angle scattering are having a pronounced impact on

structural biology. The combination of time resolved measurements with sophisticated data analytical tools open

up unmatched possibilities for studying the kinetics of biomolecules and macromolecular complexes. GKSS will

provide, in a common proposal procedure, the possibility for the application of complementary beamtime at the

small angle neutron scattering SANS-1 instrument of the FRM-II research reactor in Garching.

Free electron lasers are of crucial importance for the investigation of dynamic phenomena at time scales below

the bunch length of synchrotron storage rings (about 100 ps). DESY currently operates the world's first free

electron laser for soft X-rays FLASH to study dynamic properties of biomolecules using Raman techniques. The

future European X-ray free electron laser (XFEL), which is currently under construction, will enable studies in

the fs – regime and at atomic length scales, simultaneously, thus providing extraordinary possibilities.

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Figure 5: BESSY X-ray station

Figure 4: BESSY control room

BESSY Berlin

The new Helmholtz centre HZB currently operates three experimental

stations for macromolecular crystallography at the BESSY electron stor-

age ring in Berlin-Adlershof (Figure 4 ). Two of the stations are energy-

tuneable beamlines, and the third is a fixed-energy side station. The tune-

able beamlines are in regular user operation providing approximately 200

beam days and 600 user shifts annually to close to 50 European research

groups. Currently the beamlines are being upgraded in terms of automa-

tion and user access (Figu re 5). The currently very limited wet-laboratory

facilities will be expanded to provide an improved user service and sup-

port protein crystallographic research by BESSY staff. There are several

options regarding the future use of the fixed-energy beamline which in-

clude its use as a dedicated screening beamline. In addition to the three

stations for macromolecular crystallography, HZB/BESSY II operates 4

beamlines which are completely or partially dedicated to biological re-

search. These facilities include beamlines for circular dichroism spectros-

copy (in cooperation with Potsdam University), infrared spectroscopy and

nanometer-resolution X-ray fluorescence microscopy. The fourth beam-

line, dedicated to X-ray microscopy, offers perspectives for 3D imaging of

biological structures up to entire cells or organelles.

ANKA Karlsruhe

The synchrotron light source ANKA is operated as a user facility and for in-house research, offering beamlines

for X-ray spectroscopy, diffraction and imaging methods. Structural biology projects have been pursued mainly

by infra-red (IR) spectroscopy to date, but a circular dichroism (CD) beamline will soon be available. This beam-

line will also include a worldwide-unique oriented CD approach for measuring secondary structure and align-

ment of membrane proteins in oriented lipid bilayers. IR spectroscopy is used for the elucidation of protein struc-

ture and dynamics, as well as molecular reaction mechanisms by studying conformational changes, molecular

recognition and pathological misfolding. The future direction of strucltural biology at ANKA lies within the pro-

gramme “BioInterfaces” of the Helmholtz "Key Technologies" field. The CD and IR beamlines at ANKA, together

with the biomolecular NMR expertise at FZK constitutes a coordinated multi-technique structural biology plat-

form as a key tool of the BioInterfaces programme. As customary for all beamlines at ANKA, at least 50% of the

beamtime will go to external users via an independent peer review procedure.

B5. Biomolecular NMR spectroscopy a t Helmholtz

NMR spectroscopy is a unique tool for studying the three-dimensional structures, dynamics and molecular inter-

actions and thus functions of biomacromolecules in solution, solids and in heterogeneous phases. More re-

cently, in cell NMR spectroscopy is being developed, enabling NMR studies of biological macromolecules, their

posttranslational modifications and drug interactions in living cells. NMR spectroscopy is nowadays the standard

technique for structure determination of biomolecules in a native-like solution state. While standard protocols

are available for structure determination of proteins <30 kDa (Figure 6 ), strategies are being developed for the

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structural analysis of higher molecular weight complexes. NMR spectroscopy is

currently the only technique available that can monitor dynamic properties of

biomolecules over a wide range of timescales (10-12 – 104 s) at atomic resolu-

tion. Recent advances allow focussed studies of dynamics or molecular inter-

actions of very high molecular weight protein complexes such as the GroEL/ES

co-chaperone complex, the ribosome or the proteasome.

For membrane proteins and their complexes, as well as for micro-crystalline

protein precipitates and amyloid fibrils, solid-state NMR spectroscopy has devel-

oped into a powerful tool for elucidation of structure, dynamics, and conformatio-

nal distributions under native-like conditions. Structure determination of proteins in the solid state (i.e. in mem-

branes, fibrils, precipitates) by MAS (magic angle spinning) NMR spectroscopy or in oriented samples is now

possible, and dynamic properties can be studied.

NMR spectroscopy is a powerful method for the rapid characterization of biomolecular interactions and for map-

ping binding sites in protein-protein, protein-nucleic acid and protein-small molecule interactions. As cellular

functions are mediated by molecular interactions, the underlying mechanisms that relate to biological activity

can be directly and efficiently studied. The unique potential of NMR spectroscopy for characterizing protein-

small molecule interactions has provided novel tools (structure-activity relationship (SAR) by NMR), which were

crucial for the development of novel drugs, such as inhibitors of the apoptosis regulator Bcl-2.

Within the Helmholtz Association biomolecular NMR spectroscopy of proteins, nucleic acids and their com-

plexes is currently performed at NMR centres at HMGU together with the TU München, FZJ together with the

University of Düsseldorf, as well as FZK together with the University of Karlsruhe. In addition to these centres

NMR spectroscopy of biomolecules is performed at the HZI. The three Helmholtz NMR centres have comple-

mentary research foci (see Annex) and represent decentralized NMR infrastructure that provide access and

expertise for local and regional users (Table 1 ). The distributed provision of NMR measurement time (rather

than by a single central facility) is well suited and even required for biomolecular NMR studies, as they typically

involve substantial measurement time using various (isotope-labeled) samples. Fast local access is also re-

quired to exploit the unique potential of NMR spectroscopy for studying dynamic and transient properties of

biological macromolecules in a native-like environment.

NMR spectrum and structure ensemble of a protein-RNA complex

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Table 1. NMR centres of the Helmholtz Association

HMGU

Bayerisches NMR Zen trum (BNMRZ)

FZJ

Centre for Biomolecular NMR

FZK

BioNMR Centre at KIT

Infrastructure Liquid state: 900 MHz, 800 MHz, 750 MHz, 2x600 MHz, 500 MHz

Solid state: 500 wide bore;

Others: ca. 6 spectrometers (250-500 MHz) used by chem-ists and for teaching

Liquid state: 900 MHz, 800 MHz (also for solid state), 2x 600 MHz

Solid state: 2x 600 MHz wide bore

Solid state: 600 MHz, 2x 500 MHz, 300 MHz (all wide bore)

Liquid state: 600 MHz at University of Karlsruhe, 600 MHz at FZK (planned).

Users Ca. 10 research groups:

Groups at BNMRZ and groups from the south of Germany: Universities in Munich (TUM, LMU), Regensburg, Bayreuth and MPI Tübingen Additional users in collaborations.

Access to the high-field 900 MHz NMR spectrometer is pro-vided in the context of a DFG grant.

Ca. 12 research groups:

Groups at FZJ and University of Düsseldorf.

Further access for additional users from North-Rhine-Westphalia (MPI Dortmund, Universities of Duisburg-Essen and Bochum) and IBS Greno-ble. Irregular use by research-ers from FLI Jena.

Ca. 3 research groups

Groups from the KIT and from the south-west of Ger-many: Universities of Kars-ruhe, Heidelberg and Tübin-gen

International users from Spain, Portugal, Ukraine, Italy, Hungary, USA

Industrial cooperation with Merck

Unique features Advanced NMR Technology Platform: Protein and RNA iso-tope labelling, specific 2H label-ling schemes

Multidisciplinary approaches, combining NMR, X-ray, SAXS, SANS, single-particle cryo-EM

Preparation of isotope labelled samples of soluble, membrane and fibrillar proteins

Multidisciplinary approaches: NMR, X-ray, neutrons and single molecule spectroscopy

Preparation of non-natural 19F-labelled synthetic pep-tides and recombinant pro-teins for 19F NMR spectros-copy

Dedicated NMR hardware for 19F experiments

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Bayerisches NMR Zentrum (HMGU and TU München )

The Bayerisches NMR Zentrum (BNMRZ) (Figure 7 ) is a joint facil-

ity between the HMGU and the TU München and hosts several

research groups with expertise in the development of methodology

and the application of NMR spectroscopy for studying the structure,

dynamics and molecular interactions of biological macromolecules.

Research is focused on understanding the structural basis of bio-

logical pathways and disease mechanisms linked to the regulation

of gene expression (protein-RNA interactions) and signalling. NMR

contributes to chemical biology research at HMGU to monitor

ligand binding (peptides, RNA, small molecules). Specific expertise

is available on NMR pulse sequence design and NMR-based quan-

tum computing.

Centre for Biomolecular NMR (FZJ and University of Düsse ldorf)

The Centre of Biomolecular NMR is jointly run by the University of Düsseldorf and the Forschungszentrum

Jülich (FZJ). Fields of expertise of the structural biology research groups at FZJ include the development and

application of solid and liquid state NMR spectroscopy methods to investigate three-dimensional structures,

dynamics and molecular mechanisms of biologically and medically relevant molecules.

BioNMR Centre at KIT (FZK and Unive rsit y of Karlsruhe)

The NMR facility of the Institute of Biological Interfaces at FZK and the Institute of Organic Chemistry at the

University of Karlsruhe is part of the newly founded Karlsruhe Institute of Technology (KIT). Research is focused

on the structure and dynamic behaviour of membrane-bound proteins in their native bilayer environment, using

primarily solid state NMR spectroscopy applied to oriented membrane samples. These studies are comple-

mented with high-resolution NMR analysis in detergent micelles and method development. Specialized, home-

built NMR hardware is available to perform 19F NMR experiments as a highly sensitive and background-free

approach to biological systems in vitro as well as in-cell. Complementary biophysical analyses are carried out by

circular dichroism (CD), oriented circular dichroism (OCD) and Fourier-transform infrared (FTIR) spectroscopy

at the ANKA synchrotron.

B6. Helmholtz Protein Sample Production Facility ( PSPF)

The production of adequate quantities of pure and homogeneous macromolecular samples for structure deter-

mination still represents a major bottleneck in structural biology. Typically, proteins are produced recombinantly

using Escherichia coli as the expression system of choice. In addition to bacterial expression systems, eu-

karyotic expression systems are particularly suitable for the production of the usually more complex eukaryotic

proteins, which often require post-translational modifications for their stability and biological activity. In addition,

in vitro transcription/translation systems are becoming a possible alternative to cell cultivation. A survey of tech-

niques currently employed in the production of protein samples for structural biology and the major challenges

identified by a broad research community was recently published by the Forum for European Structural Pro-

teomics (FESP).

Figure 7: 900-MHz NMR spectrometer at HMGU

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Following an international evaluation of the protein production facilities for structural biology within the Helmholtz

Association, as recommended by the senate, the Protein Sample Production Facility (PSPF) was established in

2005. The PSPF is currently equally shared between MDC and HZI and focuses on the production of technically

challenging proteins, such as eukaryotic proteins, protein complexes and membrane proteins. While the MDC is

offering support in the high-throughput production of labelled and unlabelled proteins in E. coli, the HZI is provid-

ing infrastructure and expertise for sample production using eukaryotic expression systems, such as yeast, in-

sect and mammalian cells. The PSPF is open to structural biologist from academic institutions throughout Ger-

many. Project proposals, which can be submitted online (www.pspf.de), are judged for their feasibility and priori-

tized by a panel of scientists. As of now, 66 projects have been initiated.

In addition the Advanced NMR Technology Platform of the BNMRZ at HMGU provides special expertise in the

sample preparation and isotope labelling of RNA molecules as well as 2H labelling protocols of proteins for

structural studies by NMR and SANS experiments. The Biomolecular NMR Centre at FZJ provides special ex-

pertise in sample preparation and isotope labelling of soluble, transmembrane and fibrillar proteins.

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C. Helmholtz structural biology in the national and European context

The research activities of Helmholtz structural biologists are tightly integrated in the health research of the

Helmholtz Association. Besides their research activities they provide important infrastructure for the structural

biology community within and beyond the Helmholtz institutes. The integration into specific health research pro-

grammes as well as the provision of large-scale research infrastructure (see above) represents unique features

of structural biology within the Helmholtz Association. Considering these responsibilities for the wider research

community and compared to other research organizations the number of structural biologists in Helmholtz insti-

tutes is currently relatively small.

At present, structural biology is an important part of the research portfolio of every major university in Germany

(Figure 8). Within the Max-Planck Society there are large and focussed activities in structural biology. In addi-

tion, structural biology is also present at institutes of the Leibniz Association (e.g. FMP Berlin-Buch and FLI

Jena), the EMBL (Hamburg and Heidelberg) and the pharmaceutical industry (Figure 8). Taken together, the

number of principal investigators in the main disciplines of structural biology at German research institutions

probably exceeds 100, there are at least 60 independent protein crystallography groups and >30 NMR groups.

In the European context structural biology in Germany is thus well represented.

Figure 8: Structural biology in Germany

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Currently the three leading techniques in structural biology are macromolecular crystallography, NMR spectros-

copy and cryo-EM:

Macromolecular crystallography critically depends on brilliant and tuneable synchrotron radiation beams, both

for high resolution X-ray data collection and structure determination using MAD phasing. Complementary to

macromolecular crystallography X-ray small angle scattering (SAXS) beamlines enable the study of the static

and dynamic structure of proteins in solution at low resolution. In Germany the synchrotron radiation sources at

HZB (BESSY II) and at DESY (DORIS III and PETRA III) provide excellent facilities for biocrystallography, which

are comparable or will even surpass other European synchrotron facilities available for structural biology, such

as ESRF (France), Diamond (England), Swiss Light Source (Switzerland), Soleil (France), Elettra (Italy) and

MAX-Lab (Sweden) after commissioning of the PETRA III structural biology beamlines. The tunable energy MX-

beamlines at BESSY II (HZB) provide an excellent infrastructure for MAD and especially long wavelength phas-

ing. Additionally, the recently upgraded beamline BL14.1 offers a high degree of automation for crystal screen-

ing to the user community. Without doubt the Helmholtz Association is one of the leading providers of synchro-

tron radiation in Europe.

Research in NMR spectroscopy has been traditionally strong in Germany and the development of new methods

as well as the application of biomolecular NMR spectroscopy is internationally competitive and well-recognized.

The high-field NMR centres in Munich (BNMRZ), at the FMP in Berlin (focus on solid state NMR of membrane

proteins and aggregates), at the MPI in Göttingen (focus on methods development for liquid and solid state

NMR) and at the University in Frankfurt (NMR and electron paramagnetic resonance (EPR), currently hosting a

European Large Scale Facility) have pioneered research at the highest available magnetic field strengths in the

past. These NMR centres together with the Helmholtz NMR centres at FZJ and FZK are geographically well

distributed across Germany and provide access, support and complementary expertise for the development and

application of NMR methods to users all over Germany. Provision of such decentralized access for high-field

NMR is very appropriate and optimal for the application of NMR to biological macromolecules. This situation is

comparable to other European countries with substantial research in biomolecular NMR spectroscopy, such as

Switzerland, The Netherlands and the UK.

Over the past decade, cryo-EM has increasingly replaced the traditional methods of sample preparation (plastic

embedding, negative staining) which were notoriously prone to artifacts. Cryo-electron microscopy is a generic

term that refers to various electronmicroscopic imaging techniques when applied to samples embedded in

amorphous ice. Samples are vitrified by freezing and examined without chemical fixation or staining. Three ma-

jor branches of cryo-electron microscopy are relevant in the context of molecular structural biology: Electron

crystallography, single-particle analysis and electron tomography. The largest potential currently lies in the sin-

gle-particle analysis of macromolecular complexes and assemblies at resolutions around 10 Å or lower, which

can be complemented by higher resolution techniques, i.e. crystallography or NMR spectroscopy, in so-called

hybrid approaches. Currently there are a number of EM centres in Germany using high-end biomolecular in-

strumentation for structural biology, mainly located in the Max-Planck-Society i.e. MPI for Biochemistry, Martins-

ried; MPI for Biophysics, Frankfurt; MPI for Biophysical Chemistry, Göttingen, and a few universities, i.e. LMU

München and Humboldt University Berlin. Currently cryo-EM for structural biology is not available within the

Helmholtz Association.

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Helmholtz s tructural biology in the European Roadmap for Research Infr astructures (ESFRI)

The need to strengthen and integrate structural biology is reflected by a num-

ber of recent initiatives for large scale infrastructures in the UK ("Diamond

Light Source"), France ("The Partnership for Structural Biology" (PSB) initiative

by the EMBL, ESRF, IBS and ILL), but also the United States ("NIH road-

map").

At the European level, the European Strategy Forum on Research Infrastruc-

tures (ESFRI) has published a European Roadmap for Research Infrastruc-

tures. The Integrated Structural Biology Infrastructure (INSTRUCT,

www.instruct-fp7.eu, Figure 9 ) is one of these and aims to set up a framework consisting of distributed centres,

each of which will maintain a set of core technologies such as protein production, NMR spectroscopy, crystal-

lography and different forms of microscopy, including EM and combine this with a specific biological focus that

will drive the development of technological and methodological expertise, notably for the analysis of functional

complexes. Structural biology in the next two decades will face the integration of structural knowledge through-

out all available spatial and temporal levels into specific cellular questions of biomedical relevance. INSTRUCT

will link the information obtained by the major structural biology methods with state-of-the-art cell biology tech-

niques to provide a dynamic picture of key cellular processes. Major technological advances, ranging from high

throughput methods in protein production to hybrid methods in structural biology require continuous investments

in large and expensive infrastructure to maintain European competitiveness. The current preparatory phase of

INSTRUCT is forging a pan-European structure comprising a number of seven Core Centres. Two core centres

have been defined in Germany: the Max-Planck-Institutes for Biochemistry (Martinsried) and Biopyhsics (Frank-

furt). The EMBL with its locations in Hamburg (with its DESY beamlines) and Heidelberg is a third core centre.

In the current preparatory phase, a number of further associate centres, that should contribute additional com-

plementary expertise are being identified by the INSTRUCT consortium.

Unfortunately, at present the large scale structural biology infrastructures of Helmholtz are not well represented

in the INSTRUCT project. There are on-going activities to better link the synchrotron and NMR facilities in Ger-

many to INSTRUCT, which will be important for the future integration of structural biology infrastructures in

Germany (including those in the Helmholtz Association) into the European context.

The German NMR centres at HMGU (Helmholtz), in Frankfurt (DFG), Göttingen (MPI) and Berlin (Leibniz), as

well as the FZJ together with North-Rhine-Westphalian biomolecular NMR groups are seeking support for the

installation of the next generation 1.2-1.3 GHz spectrometers (expected in 2015). These ultra-high-field NMR

spectrometers will ensure that the German NMR community will remain at the forefront of biomolecular NMR

and should be part of a European network of structural biology infrastructures. Currently it is being discussed to

affiliate German and other European NMR centres within the ESFRI initiative INSTRUCT. These centres should

become part of a grid of European NMR centres that are associated with the core centre in Florence. The

BNMRZ (HMGU) is a member of the INSTRUCT Working Group O on solution state NMR spectroscopy and

actively involved in promoting this initiative.

Sample production is another important infrastructure for structural biology. A future participation of the Helm-

holtz PSPF in INSTRUCT will be essential to allow a Europe-wide contribution to cutting-edge structural biology

research. A case for an "associated centre" including the Helmholtz PSPF and the Institute of Biophysical

Chemistry at the University of Frankfurt to be included into the INSTRUCT Workpackage H (Sample Prepara-

tion) has recently been submitted and is currently under review.

Figure 9: INSTRUCT logo

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D. Perspectives of structural biology in the Helmholtz Association

At the molecular and cellular level, modern biology and biomedicine is squarely based on structural knowledge

of proteins, nucleic acids, membranes and their assemblies from which crucial functional insight is derived. Re-

cent technical developments in the main analytical methods of X-ray crystallography, NMR spectroscopy and

electron microscopy permit the study of the structure and dynamics of biological entities at various resolutions,

over a wide size scale and on many different time scales. By combining macromolecular structures with bioin-

formatics and biophysical approaches into hybrid models of large and sometimes transient subcellular assem-

blies structural biology is now evolving into structural systems biology.

Structural biologists active within the research field "Health" of the Helmholtz Association are part of this exciting

development that promises to yield unprecedented insight into the inner workings of cells under physiological

and pathological conditions. Their research is tightly linked to the work of cell and molecular biologists, pharma-

cologists and medical doctors in all research programmes of the Health field. Through close collaboration with

chemical biologists, protein crystallographers and NMR spectroscopists can play a central role in the identifica-

tion and characterization of small molecules modifying macromolecular and cellular activity and thus in transla-

tional research linked to early stages of drug development.

The various disciplines of structural biology strongly depend on large-scale facilities, many of which are beyond

the scope of universities, small research centres or companies. Nothing exemplifies these facilities better than

the electron storage rings which provide the brilliant X-ray beams needed to analyze crystal structures of large

proteins. The macromolecular crystallography beamlines at the electron synchrotrons at DESY and

HZB/BESSY serve large international user communities, and their continued support and upgrading is of central

importance for structural biology within and beyond the Helmholtz Association. In the following, additional new

developments and requirements are described that will allow Helmholtz structural biologists to maintain their

position at the forefront of their science, meet the exciting challenges of structural systems biology and contrib-

ute to translational research in molecular medicine.

� Centre for Structural Systems Biology (CSSB) at DESY

Establishment of an interdisciplinary centre on the DESY campus with a focus on structural systems biology in a

unique alliance with other research organisations

� NMR centres in the Helmholtz Association

Acquisition of the next generation of NMR magnets with greatly improved potential to yield molecular insight into

cellular pathways and the molecular basis of disease mechanisms not only through three-dimensional structures

of protein and nucleic acid complexes but also their temporal variations

� Cryo -electron microscopy

High priority acquisition of instrumentation for this important technique that is currently unavailable in the Helm-

holtz life sciences that bridges the size gap between molecular and sub-cellular structures and thus provides the

basis of structural systems biology

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� Helmholtz Protein Sample Production Facility (PSPF)

Continued support of the platform technology for the production of protein samples for structural studies in bac-

terial and eukaryotic cells and in cell-free systems, alleviating an important bottleneck in structural biology, for

partners from Helmholtz centres and other academic institutions

� Neutron scattering in biology

Further support of the neutron scattering activities that provide valuable structural and dynamic information on

proteins and other macromolecules in solution and crystals

� Structural bioinformatics and systems biology

Coordination of multidisciplinary and multi-scale approaches within the area of Health by combining comple-

mentary techniques, such as X-ray crystallography, NMR and EPR spectroscopy, electron microscopy and to-

mography, small angle scattering, CD, IR, and light microscopy, with bioinformatics, to provide complete struc-

tural descriptions of biological systems

� Structural biology and chemical biology

Promotion of strong collaborative initiatives of existing groups for the synthesis and use of small molecules that

modify the cellular activity of biological macromolecules either as research tools in cell biology or as leads for

drugs in pharmacology, by providing a rational basis from the knowledge of the three-dimensional structure of

the target macromolecule � Training and visibility

Further development of the strong position of Helmholtz structural biologists in providing research training at

world-class large-scale facilities and organising workshops and conferences, yielding international visibility and

ensuring representation in national and international research cooperations.

D1. Centre for Structural Systems Biology (CSSB) at DESY

In the near future DESY in Hamburg will be able to offer world-class synchrotron radiation (PETRA III) and un-

precedented free-electron lasers (FLASH, X-FEL) to address the most challenging projects in structural biology.

Currently structural biology on the DESY campus is performed by the EMBL Hamburg and the MPG-ASMB

groups as well as university groups from Hamburg and Lübeck. There is currently no Helmholtz structural biol-

ogy undertaking on the DESY campus. To match the developing unique physical infrastructures at DESY with

world-class biomedical research, the establishment of a new research centre (Centre for Structural Systems

Biology, CSSB) on the DESY campus is envisioned, in which research departments of research institutions and

regional universities together with DESY will examine the structure and dynamics of complex and disease-

related cellular processes at the highest spatial and temporal resolution. The CSSB will use advanced tech-

niques in structural biology spanning the entire resolution range relevant for biological and biomedical research.

Establishment of the CSSB includes a 70 Mio EUR investment for a new research building in the immediate

proximity to PETRA-III (Figure 10 ). The planned CSSB is comparable to recently established European institu-

tions such as the CISB (Centre for Integrated Structural Biology) on the ESRF campus (Grenoble, France) or

the Research Complex at the DIAMOND synchrotron light source (Oxfordshire, Great Britain). In contrast to the

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Research Complex which will mainly focus on high-throughput methods for structural biology, CISB and CSSB

will concentrate on questions related to human health. A scientific concept for the CSSB, including infection

research as a potential health related topic, is currently under review with the local and federal governments.

This concept includes contributions from partner institutions that include the Universites of Hamburg, Lübeck

and Kiel, UKE, MHH, the local Leibniz centres HPI, BNI

and FZB, the Helmholtz centres DESY, FZJ and HZI,

and the EMBL Hamburg. A decision on the CSSB is to

be expected by the end of 2009. The Helmholtz Asso-

ciation plans to install a new department of structural

biology (at W3-level) and a junior research group (W1)

at the CSSB, in an initiative currently coordinated by the

HZI. The junior research group will start in August 2009,

while negotiations with the future department head are

currently underway. In addition FZJ has decided to in-

stall a research group at the W2-level starting in 2010.

Exploitation of free electron laser technology for structural biology Dynamic studies at synchrotron radiation sources are limited to about 100 ps resolution determined by the

bunch length of these sources. This temporal resolution is

already sufficient for a large number of processes taking

place in biology. The experimental techniques applied are

time resolved SAXS and macromolecular crystallography. A

significant better temporal resolution for fast chemical and

biological reactions, by at least three orders of magnitude,

can be achieved by newly developed free electron lasers

(FELs). The first laser in the soft X-ray laser regime (FLASH,

Figure 11 ) is already in user operation at DESY in Hamburg,

where pioneering experiments are already being carried out

to study dynamics of matter by spectroscopic methods. An X-

ray free electron laser (XFEL) providing X-rays at fs time

scales will enable the study of reactions at atomic resolution on these time scales. The extremely short and

intense photon pulses of an XFEL are expected to open completely new possibilities for time-resolved bio-

molecular imaging which is barely understood at present. The first experiments at the soft X-ray laser FLASH

indicate that it is indeed possible to also image biological objects in a single shot at extremely high flux densities

since the photon pulses at a FEL are about 10 times shorter than it takes radiation damage effects to be come

visible. At an XFEL this might open the possibility of imaging structurally reproducible objects at very high reso-

lution without the need for crystallization. The limits of this method in terms of the achievable resolution and the

smallest possible particle size are still under active research.

Figure 10: Model of planned modular CSSB complex on the DESY campus

Figure 11: FLASH hall at DESY

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ronment and in a cellular context, ii) resolve dynamic features of macromolecular complexes, which are associ-

ated with field-dependent line-broadening, iii) improve resolution for studies of natively disordered proteins, fi-

brils and other molecules that are intermediary between the solution and solid phase, and iv) enable discoveries

of novel effects and experiments (in analogy to the invention of TROSY, which was stimulated by the availability

of 800 MHz NMR spectrometers).

In order to remain competitive and allow NMR investigations of protein complexes and systems of critical bio-

medical relevance with increasing molecular weight and complexity, it will be important to install NMR spec-

trometers at GHz field strengths at the BNMRZ in Munich and at the NMR centre at FZJ, as well as (sub) GHz

wide-bore instrumentation at FZK in Karlsruhe in the near future. The three NMR centres are geographically

well distributed and have a distinct research focus (see Annex).

In a midterm timeframe of 5-6 years the BNMRZ (Helmholtz), together with the NMR centres at the University of

Frankfurt, the FMP in Berlin and the MPI in Göttingen is seeking support for the installation of next generation

1.2-1.3 GHz spectrometers (expected in 2015). The installation at these sites will benefit from the demonstrated

track-record and know-how in NMR theory and methodology available at these centres. An ultra-high-field 1.2-

1.3 GHz spectrometer at BNMRZ would serve as a central facility in the Helmholtz Association and provide pre-

ferred NMR access for Helmholtz groups from the research area “Health”. The North-Rhine-Westphalian bio-

molecular NMR groups are likewise seeking support for the installation of a 1.2-1.3 GHz instrument at FZJ to

provide access to such instrumentation for groups from the research areas "Health" and “Key Technologies”.

D3. Cryo -electron m icroscopy

Novel approaches of high-resolution analysis of macromolecules, multi-molecular complexes and larger cellular

components have become possible by recent developments in the area of cryo-electron microscopy. New vitrifi-

cation protocols allow a detailed description of such complex

structures with an unprecedented spatial resolution (< 10 Å)

(Figure 14 ). Cryo-EM is a technique that is well suited for the

investigation of structures which are dynamic by nature (e.g.

the conformation of chromatin), or which are too complex to

organize in regular crystals. Cryo-EM, thus, perfectly meets the

current shift of scientific interest towards the structure of larger

macromolecules, molecular assemblies and complexes, which

is also taking place throughout the Helmholtz Association.

Cryo-EM is a technique that is highly complementary to X-ray

crystallography and multidimensional NMR spectroscopy, thus

providing the basis for the high-resolution description of larger

cellular components such as organelles. Cryo-techniques in

electron microscopy of biological samples are employed at two

levels: i. Vitrification and direct observation of the vitrified

specimen (via cryo-EM) allows the investigation the ultrastructure of single particles in their native, hydrated

state. Compared to X-ray diffraction, spatial resolution is in the 10 Å range. ii. Samples in the vitrified state can-

not be stained or labeled by immuno-histochemical procedures. However, freeze-substitution following cryo-

fixation provides samples of excellent structural preservation and antigenicity which can be post-treated and

observed at room temperature. Thus, this technology provides the means for modern cell biology to investigate

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the influence of sub-cellular topology on the proper function of macromolecular entities of interest. As a key-

requirement, cryo-fixation must achieve vitrification of entire cells. A method which has been shown to meet

these requirements is high-pressure freezing.

Single particle cryo-electron microscopy (Cryo-EM) will be an important branching point to link activities within

the Helmholtz research field “Health”, both within and between programmes. More specifically, within the pro-

gramme “Cancer Research” there are two topics “Signaling pathways, cell biology and cancer” and “Structural

and functional genomics”, which combine strong research activities within the DKFZ, the MDC and others in cell

biology and functional genomics. Cryo-EM of cellular organelles and larger macromolecular complexes will

greatly complement current activities in a number of cell biology projects (e.g. chromatin structure) and link

these to the multidimensional structure analysis of genomic information on the protein level. The experience on

the analysis of macromolecules could also be exploited within the other major Helmholtz health research pro-

grammes. It can be envisaged, that this methodology is also of interest for other Helmholtz Programmes such

as “Structure of Matter”. Thus, it provides an ideal tool for networking within the Helmholtz-Association.

Cryo-EM of vitrified specimen requires personnel experienced in operating the instrumentation as well as in the

elaborate analysis of the image data. Although cryo-EM is not currently available at a Helmholtz institution, there

is personnel with expertise at the DKFZ and the HZI. Thus, establishment of a cryo-EM facility, for instance at

the DKFZ would only require the investment of the respective instrumentation. This facility would be closely

linked with the HZI, where a long-standing tradition in electron microscopy exists. Scientifically, this facility will

perform further protocol development to optimize the method for the analysis of cellular components and organ-

elles such as the structures of the cell nucleus. However, this facility would be a complementary activity to other

programs within the Helmholtz Health Programme, which includes in particular the structure analysis of macro-

molecules using X-ray crystallography and NMR spectrscopy.

The cost of instruments for cutting edge research in biomolecular electron microscopy are high, and are increas-

ing steadily. Today, a 300-400 keV field emission instrument costs approx. 3 Mio EUR, and equipping it with

large area detectors (e.g. 8k x 8k), energy filters, autoloaders etc., adds substantially to the price. An investment

of approx. 10 Mio EUR is likely needed to establish a cryo-EM centre at a Helmholtz institute.

D4. Helmholtz Protein Sample Production Facility (PSPF)

The Helmholtz PSPF is currently establishing procedures to significantly

increase the throughput of structural biology projects. Scale-down by in-

creasing the productivity of the expression systems, reducing the devel-

opment time scale from clone to stable expression and automation are

major challenges to be addressed. The HZI branch of the PSPF has re-

cently established stable and highly productive mammalian master cell

lines. The performance and stability of these mammalian high-producer

cell lines have been tested during batch and continuous fermentation

(Figure 13 ). This approach is based on the rapid exchange of gene cas-

settes by specific recombination and is a major breakthrough for increas-

ing the throughput of difficult target proteins in the mammalian Chinese ������ � �� ����� ���� ���������� ����� �� ��� ���

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hamster ovary cell expression system. The expression of multi-protein complexes is extremely difficult and time

consuming. The new recombination mediated cassette exchange system is currently under further development

for the expression of multi-protein complexes. The strength of intermolecular interactions within the protein-

complex and the identification of stable protein domains essential for the assembly of the complex are major

bottlenecks for the efficient production and purification of protein assemblies. This new and rapid strategy will be

a unique tool for the production of complex mammalian protein assemblies for structural analysis. The revival of

the large scale production of biomaterials for the purification of native multi-protein complexes from cell cultures

(up to 300 L) will be another important source for the structural analysis of large assemblies by cryo-

electronmicroscopy and X-ray crystallography.

The Helmholtz PSPF is currently preparing the facility for the next decade. Latest developments for the expres-

sion and production of challenging protein, protein complexes and integral membrane proteins are being estab-

lished. The PSPF has recently initiated a partnership with the Institute of Biophysical Chemistry at the University

of Frankfurt, focusing on the emerging technology of cell-free expression. This institute has established tech-

nologies and facilities for the preparative scale production of currently > 200 membrane proteins covering large

transporters, G-protein coupled receptors, membrane integrated protease complexes and other pharmaceuti-

cally important targets. Besides establishing this national facility, additional efforts will be made to be part of a

pan-European centre of excellence for protein production for the structural biology community

A future participation in the ESFRI infrastructure initiative INSTRUCT will be essential to allow a European-wide

contribution to cutting-edge structural biology research. A case for an "associated center" including the Helm-

holtz PSPF and the Institute of Biophysical Chemistry at the University of Frankfurt to be included into the IN-

STRUCT Workpackage H (Sample Preparation) has recently been submitted and is currently under review.

D5. Neutron scattering in biology

Neutrons are scattered with different strengths by hydrogen (1H)

and deuterium (2H). Therefore, they are suitable probes for in-

vestigating the structure of biomolecules by variation of contrast

due to isotopic substitution which does not disturb their function.

The main applications in biology are Small Angle Neutron Scat-

tering (SANS) on macromolecules in solution, reflectometry/

diffraction on layered structures such as membranes and in-

eleastic measurements. At present GKSS is running its research

neutron reactor FRG-1 (Figure 16 ). In addition a neutron crystal-

lography beamline is being constructed for the FRM-II (operated

by FRM-II and FZJ). In order to investigate dynamic properties such as thermal equilibrium fluctuations of bio-

molecules, the unique feature of neutrons showing a large incoherent scattering cross-section for hydrogen is

used. Here variations in these fluctuations within a protein can be obtained from a protonated domain in a

deuterated environment. The situation of having low neutron fluxes from research reactors is greatly improved

by spallation sources with a suitable time structure now under construction in the US and Japan. The improve-

ment will yield a factor of 20 in flux for crystallography and of up to 1000 for inelastic experiments. The supply of

neutrons and the operation of instruments for the biological user community, amounts to about 20% of the total

beam time allocated to biological problemsand therefore is an important task of the Helmholtz centres involved.

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In the near future the main neutron sources and instrumentation for structural biology will be operated by the

Institut Laue-Langevin (Grenoble), HZB, FRM-II, FZJ and GKSS. Especially the possibilities offered by the more

pronounced engagement of the Helmholtz Association at the FRM-II will be of great benefit for biological struc-

ture research. However, the local support at the sources and instruments in terms of personnel as well as

equipment should be strengthened. HMGU and TUM are part of the neutron FP7 I3 activities and provide some

support for sample preparation at FRM-II. However the operation of a deuteration facility comparable to the

Deuteration Laboratory in Grenoble at one of the sources will help significantly to improve the scientific possibili-

ties as well as a possible commitment in the construction of a European Spallation Source, to be built most

likely in Lund (Sweden).

D6. Structural bioinformatics and systems biology

Systems biology is focussing on the modelling and exploitation of the relationships between individual molecular

components of the cell. The overall behaviour of biological systems at the cellular and sub-cellular level is de-

termined by interactions between bio(macro)molecules that have to be understood on the molecular and on the

systems level (e.g. the function of entire signalling networks or functional pathways). In recent years, bioinfor-

matics research, taking place for example at HMGU and DKFZ, has contributed substantially to the integration

of heterogeneous high-throughput data for investigating molecular networks. In particular, widely used genome

annotation and protein interaction databases are maintained at HMGU. Better understanding of how the topol-

ogy of various biological networks defines function and ultimately determines the phenotype will help uncover

the genetic architecture of complex organismal traits and diseases. So far, however, three-dimensional struc-

tural information on protein complexes has not been part of such studies. It is revealing that the term "protein

structure" is rarely found in systems biology publications. Recently, the need for reconstructing metabolic and

signaling pathways in structural detail has been underscored. The availability of high-resolution complex struc-

tures will greatly complement information about global (topology) and local (modules) network properties.

Structural biology research can be efficiently combined with already available high-throughput analysis pipelines

to help in understanding the mechanistic basis of cellular signalling. One example of such high-throughput tech-

nologies is quantitative phosphoproteomics which has become a powerful tool for characterizing signaling path-

ways, transcription networks and their downstream effects. Phosphorylation is a key event in eukaryotic signal

transduction, which is ultimately involved in the regulation of nearly every aspect of cell function, including dis-

ease. Large-scale data on expression states of whole proteomes, as well as quantitative, site specific and time

resolved information on phosphorylation have become available.

In order to gain molecular insight into the cellular functions it is crucial to describe not only the three-dimensional

structures of protein complexes but also their temporal variations. Understanding the spatial and temporal dy-

namics of proteins is therefore a key requirement for a systems description of biology. Crystallography can be

employed to efficiently determine structures of more "rigid" protein complexes. On the other hand, biomolecular

NMR spectroscopy is a powerful and reliable tool to validate and quantitate protein-protein interactions pre-

dicted by high-throughput methods. Taken together, crystallography and NMR spectroscopy combined with

systems biology approaches will have important roles to play in providing molecular and structural details in the

newly emerging field of structural systems biology.

Structural biology is also critically needed for deciphering the sequence and structure attributes of disease-

associated mutations and ultimately for understanding the molecular basis of disease and exploiting this knowl-

edge for designing novel therapeutic approaches. Disease-associated mutations have been shown to preferen-

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tially occur at functionally important and evolutionary conserved sites and to introduce more radical changes into

protein structures than benign mutations. Computational approaches have been developed to predict potentially

deleterious non-synonymous polymorphisms in globular proteins based on sequence conservation and struc-

tural features. Disease proteins are in general less designable, meaning that their structural folds allow for less

sequence diversity. It is therefore an emerging area of biomedical research to study and predict structural and

functional consequences of disease-related mutations based on a combination of experimental and theoretical

approaches. For example, the rich body of evidence on genetic variation associated with a number of human

disorders discovered at HMGU can be investigated by combining protein sequence analysis and molecular

modeling to identify particularly promising targets for subsequent structure determination. Mapping mutations on

protein architectures using experimental structural biology approaches will thus provide unique structural and

mechanistic insight into molecular pathophysiology of important syndromes.

In the future structural biology and systems biology, which are both well established in the health centres of the

Helmholtz Association, should develop or strengthen synergies to address questions of high biomedical rele

vance.

D7. Structural biology and chemical bio logy

The strong interdisciplinary research in molecular biology, biochemistry, cell biology and animal models at

Helmholtz Health centres has led to significant advances in our understanding of the molecular determinants of

human diseases and the interaction of cells with each other and the enviroment. Structural biology bridges the

gap between the cellular and the molecular (atomic) level by contributing high-resolution structural information

of these highly complex biological systems, to better understand molecular and mechanistic aspects of cellular

pathways and disease mechanisms. This knowledge also forms the basis for the development of new therapies.

In the various health programmes of the Helmholtz Association numerous proteins that are key players in the

regulation of protein networks and that are linked to human disease are being studied and characterized using a

wide range of methods, from in vitro studies to animal models. Many of these proteins are potential targets for

pharmacological interference. The identification of small chemical compounds that bind and inhibit target pro-

teins is a first step towards drug design. Small molecules are also efficient tools to alter the functional activity of

biological molecules. Thus, they provide novel tools for studying systemic effects upon the targeted inactivation

of key regulatory factors, for example proteins in signaling networks. For identified target proteins structural

biology allows rational and knowledge-driven approaches for the design of small molecule inhibitors.

Both NMR spectroscopy and X-ray crystallography allow

screening of low molecular weight compounds to identify

potential leads for pharmacological interference and/or to

probe molecular pathways. The information provided by

these methods is complementary to high-throughput assays

and is ideally suited for rational drug design. Specifically,

NMR spectroscopy is ideally suited to validate hits from cell

based screening efforts. The HZI has strong activities in the

field of chemical biology (Figure 15 ) and has already estab-

lished strong interactions with researchers at the FMP Ber-

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lin. Screening facilities have been established at the EMBL jointly with the DKFZ and at FMP. The HMGU is

currently establishing such a facility enabling chemical biology research in Munich.

The structural biology groups at these centres are already or will be in the future integrated into this chemical

biology research. It is anticipated and desired that these activities will be further coordinated across the centres

to exploit complementary expertise and synergies. The Helmholtz NMR centres and the crystallography groups

are already involved in local projects. In the future, these structural biology activities should be integrated into

larger cross-centre efforts in chemical biology, which are also being integrated at a European level.

D8. Training and visibility

Within the Helmholtz Association structural biologists are organized as a

special interest group since 2002. This group represents the ongoing struc-

tural biology activities in the Helmholtz Association and fosters collabora-

tion and technological exchange between Helmholtz centres. The group

actively participates in the strategic development of Helmholtz programmes

and cross-programme activities. To discuss general develop-

ments in the field, the group meets regularly and is involved in the organi-

zation of national or international workshops and conferences. As an ex-

ample the biannual international Murnau conference series on structural

biology of medically relevant topics (www.murnauconference.de, Figure

17), established in 2005 by D. Heinz (HZI), has become the leading forum

for structural biologist throughout Europe. In addition Helmholtz structural

biologists are involved in the organization of EMBO courses and workshops

such as the EMBO Practical Course on biomolecular NMR in Munich Jul/Aug 2009 or workshops on sample

production for structural biology as part of the EU FP6 Coordination Action NMR-Life. The BNMRZ has organ-

ized international conferences on advanced biomolecular NMR (for example, 2008 in Munich and in Murnau)

Helmholtz structural biologists are actively involved in numerous research networks, including DFG Clusters of

Excellence, Collaborative Research Centres (SFBs), Research Groups and Graduate Schools as well as inter-

national (e.g. EU) programs.

In addition they are routinely involved in the teaching curriculae (Bachelor, Masters, PhD) of the local universi-

ties. Education of PhD students in respect of a broad technological repertoire, excellent scientific qualification,

and transferable skills, is already realised in two international graduate schools at FZJ: The International Helm-

holtz Research School "BioSoft" and the North-Rhine-Westphalian Research School "BioStruct". In addition,

annual meetings of all biomolecular NMR research groups within North-Rhine-Westphalia at FZJ further

strengthens the know-how exchange with a focus on NMR hardware, software, sample production and network-

ing.The GKSS is involved in the coordination of the Marie-Curie Training Network BIOCONTROL which focuses

on interations at and with cell membranes as well as the EMBO Practical Course on Solution Scattering from

Biological Macromolecules organized by EMBL.

A new important activity of the group to be launched relates to regular, focused workshops to be held at the

different Helmholtz centres on a yearly basis. These meetings will serve to further enhance the awareness of

researchers working at Helmholtz centres for the strong expertise available within the different units. In addition,

group leaders working on various levels of the “life sciences” and medical sciences will have the opportunity to

discuss their results and team up with the leaders of the structural groups. This is thought to initiate collabora-

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tions within the centres that at present do not take place because of a lack of information about activities at the

various centres. To enhance the visibility of the individual structural groups and to specifically highlight their

expertise, it is suggested that every Helmholtz centre places on its internet start site a link to a new website

“Structural biology in the Helmholtz Association”, to be implemented within the next months. This should guide

interested researchers directly to a summary page plus links of the initiative. In addition, it should advertise ac-

tivities such as workshops and training programs for graduate students, postdocs and staff scientists (see be-

low).

In a further step following the implementation of the above activities, an optional rotation program will be set up

to train, within the framework of a Helmholtz graduate training in structural biology, students that need to acquire

deeper insight into methods of structural biology in order to progress in their research.

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E. Annex

Profiles of Helmholtz research centres with activities in Structural Biology

Health research centres

German Cancer Research Centre (DKFZ)

Current research activites Structural biology at the DKFZ is currently centered on two research topics. One focus of the division B060 'Mo-lecular Genetics' is ultrastructural research on the impact of nuclear architecture on nuclear function with special interest on the formation and maintenance of nuclear micro-compartments such as chromosome territories or the nuclear periphery. Topological parameters of nuclear organization are investigated by light microscopy, in particular fluorescence light microscopy to reveal the distribution-pattern of fluorescence-tagged or immuno-labeled proteins and of FISH-tagged nucleic-acid sequences, and electron microscopy to disclose their integra-tion within microenvironments at ultrastructural resolution. For this purpose, we have established a protocol for the correlative observation of identical structural entities by consecutive light and electron microscopy. This method enables us to perform a targeted preparation and relocation of cellular sites for electron microscopy, which had been selected and documented in advance by fluorescence microscopy

The group “Functional Architecture of the Cell”, B065, performs research on one of the three major filament systems of eukaryotic cells, i.e. intermediate filaments. These proteins constitute distinct systems both in the cytoplasm and in the nucleus. They are the primary determinants of cell plasticity. In particular, the lamin net-work, made from nuclear intermediate filament proteins, exhibits in addition distinct roles in organizing chroma-tin architecture and in DNA repair. This group explores both the structure of the assembly complexes at atomic resolution as well as the assembly mechanism of intermediate filaments from such basic complexes. The aim is to eventually reveal the molecular structure of an intermediate filament.

Equipment and expertise Leica TCSP2: Laser scanning microscope DeltaVision Core: Wide-field microscope using restorative image-deconvolution. Philips 400 conventional electron microscope Zeiss LEO912 electron microscope with imaging energy filter Zeiss Sesam 922 electron microscope with imaging energy filter and 200kV acceleration voltage DeltaVision Core: Wide-field microscope using restorative image-deconvolution Philips 400 conventional electron microscope Unique feature Possibility to perform ESI (Electron Spectroscopic Imaging) at 200 kV DeltaVision Core: Wide-field microscope using restorative image-deconvolution Philips 400 conventional electron microscope.

Collaboration with Helmholtz Centres none

Research groups and group leaders Prof. Peter Lichter: Molecular Genetics Dr. Harald Herrmann-Lerdon: Functional Architecture of the Cell Training and networking �Future Perspectives For cancer research, the imaging of distinct biomolecules within their cellular context at ultrastructural resolution is of paramount interest. In addition to our correlative approaches, we currently develop protocols for the evalua-

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tion of ESI-data from biological samples, acquired with the Sesam 922 energy-filtering electron microscope, in order to add element-contrast to the image information. Freeze-substitution of vitrified samples should further-more enable us to obtain sample preparations with improved structural preservation and immunogeneity as well. Therefore, our equipment needs to be supplemented with a high-pressure freezing machine.

For cancer research, the role of the cytoskeleton in coordinating signalling activities as well as cell migration through the extracellular matrix as well as through endothelia are not entirely understood, both in normal tissues and during metastasis. Hence, this group focuses its research on the basic structural properties of desmin (the muscle-specific intermediate filament protein), keratins (the hallmark structural proteins of epithelia) and lamins, in particular lamin A. For the latter protein, 330 human disease-causing mutations have been discovered, lead-ing to about 14 different disease entities. The most severe of these, impacting developmental programmes on various levels, is the Hutchinson-Gilford Progeria Syndrome (HGPS), or premature ageing. The latter mutations occur spontaneously in germ cell development, but have been recently also discovered to occur frequently in cells of elderly patients. Hence, it is very likely that lamin mutations may as well affect the progression of cells into tumorigenesis.

Selected publications

Richter, K., Reichenzeller, M., Görisch, S.M., Schmidt, U., Scheuermann, M.O., Herrmann, H. & Lichter, P. (2005). Characterization of a nuclear compartment shared by nuclear bodies applying ectopic protein expression and correlative light and electron microscopy. Exp. Cell. Res. 303, 128-137. Richter, K., Nessling, M. & Lichter, P. (2007). Experimental evidence for the influence of molecular crowding on nuclear architecture. J Cell Sci.120, 1673-1680. Kreplak, L., Richter, K., Aebi, U. & Herrmann, H. (2008). Electron microscopy of intermediate filaments: Teaming up with atomic force and confocal laser scanning microscopy. Methods Cell Biol. 88, 273-297. Richter, K. Nessling, M. & Lichter, P. (2008). Macromolecular crowding and its potential impact on nuclear function. Biochim. Biophys. Acta 1783, 2100-2107. Strelkov, S., Herrmann, H., Geisler, N., Wedig, T., Zimbelmann, R., Aebi, U. and Burkhard, P. (2002). Conserved segments 1A and 2B of the intermediate filament dimer: their atomic structures and role in filament assembly. EMBO J. 21,1255-1266. Strelkov, S.V., Schumacher, J., Burkhard, P., Aebi, U. & Herrmann, H. (2004). Crystal structure of the human lamin A coil 2B dimer: Implica-tions for the head-to-tail association of nucler lamins. J. Mol. Biol. 343, 1067-1080. Bär, H., Mücke, N., Sjöberg, G., Aebi, U. & Herrmann, H. (2005) Severe disease-causing desmin mutations interfere with in vitro filament assembly at distinct stages. Proc. Natl. Acad. Sci. USA,102, 15099-15104. Meier, M., Padilla, G.P., Herrmann, H., Wedig, T., Hergt, M., Patel, T.R., Stetefeld, J., Aebi, U. & Burkhard, P. (2009). Vimentin Coil 1A – a molecular switch involved in the initiation of filament elongation. J. Mol. Biol., in press.

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Helmholtz Centre for Infection Research (HZI)

Current research activities The Division of Structural Biology at the HZI is focussing on the structural elucidation of host-pathogen interac-tions involved in bacterial, fungal and viral infection processes. The study of these interactions at the atomic level will provide mechanistic insights for the development of new approaches to specifically interfere with infec-tion processes. The 3D-structures of microbial virulence factors and, if available, their complexes with host cell interaction partners and receptors is determined at high resolution using the well-established techniques of X-ray crystallography and NMR spectroscopy. The main emphasis is placed on virulence factors that contribute to microbial adherence and invasion, remodelling of the host cell cytoskeleton, components and effectors of type III secretion systems, key gene regulators of microbial virulence as well as heme and iron-uptake systems. An-other focus is on the structural investigation of physiological and pathological amyloid fibrils using NMR spec-troscopy. Finally together with the Department of Chemical Biology natural compounds are structurally eluci-dated bound to their molecular targets.

Equipment and expertise Protein crystallography: Rotating anode X-ray generator with image plate and CCD-detector NMR: 4 spectrometers: 300 MHz, 400 MHz, 2 x 600 MHz Protein production: State-of-the-art facilities for prokaryotic and eukaryotic (incl. mammalian cells) expression Unique features Helmholtz Protein Sample Production Facility (PSPF) Anaerobic preparation and crystallization of proteins High-end mass spectrometry and proteomics Protein sequencing (Edman) Collaborations with Helmholtz Centres With MDC: PSPF With DESY: New Helmholtz Department in Structural Biology at DESY (CSSB) Research groups and group leaders Prof. Dirk Heinz, Department of Structural Biology Dr. Wolf-Dieter Schubert, RG Molecular Host-Pathogen Interactions Dr. Victor Wray, RG Biophysical Analytics Dr. Joop van den Heuvel, RG Recombinant Protein Expression Prof. Christiane Ritter, JRG Macromolecular Interactions Dr. Thorsten Lührs, JRG Structure-based Infection Biology Training and networking HZI structural biologists are actively involved in teaching curriculae with TU Braunschweig, international training courses (InWEnt) and workshops. D. Heinz is founding head of the study group "Structural Biology" of the Ger-man Society for Biochemistry and Molecular Biology (GBM) and principal organizer of the Murnau Conference Series in Structural Biology. Future perspectives Structural biology at the HZI will continue to focus on the structural elucidation of proteins and protein com-plexes involved in host-pathogen interactions. These include interactions between pathogen's virulence factors with host cell receptors as well as host cell innate immune receptors with pathogen-specific molecules. Fur-thermore structure and assembly of physiological and pathological amyloid fibrils will be studied using NMR spectroscopy and other biophysical techniques.The PSPF will be further developed to provide excellent infra-structures for the production of high quality eukaroytic proteins for structural analysis. HZI is also actively in-volved in the process of establishing CSSB at DESY. Selected publications Niemann, H. N., Jäger, V., van den Heuvel, J., Schmidt, S., Ferraris, D., Gherardi, E. & Heinz, D. W. (2007). Structure of the receptor tyro-sine kinase Met in complex with the Listeria invasion protein InlB. Cell 130, 235-246. Wollert, T., Pasche, B., Rochon, M., Deppenmeier, S., van den Heuvel, J., Gruber, A. D., Heinz, D. W., Lengeling, A. & Schubert, W.-D. (2007). Extending the host range of Listeria monocytogenes by rational pathogen design. Cell 129, 891-902. Hagelüken, G., Adams, T. M., Wiehlmann, L., Widow, U., Kolmar, H., Tümmler, B., Heinz, D. W. & Schubert, W.-D. (2006). The crystal structure of SdsA1, an alkylsulfatase from Pseudomonas aeruginosa, defines an independent, third mechanistic class of sulfatases. Proc. Natl. Acad. Sci. U.S.A. 103, 7631-7636.

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Layer, G., Moser, J., Heinz, D. W., Jahn, D. & Schubert, W.-D. (2003). Unusual cofactor geometry of Radical SAM enzymes revealed by the crystal structure of coproporphyrinogen III oxidase HemN. EMBO J. 22, 6214-6224. Schubert, W.-D., Urbanke, C., Ziehm, T., Beier, V., Machner, M. P., Domann, E., Wehland, J., Chakraborty, T. & Heinz, D. W. (2002). Struc-ture of internalin, a major invasion protein of Listeria monocytogenes, in complex with its human receptor E-cadherin. Cell 111, 825-836.

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Helmholtz Zent rum München (HMGU) Current research activities The Institute of Structural Biology at the Helmholtz Zentrum München employs NMR spectroscopy and/or X-ray crystallography for studying the structure, molecular recognition and dynamics of proteins and complexes that play important roles in the regulation of gene expression and cellular signaling. Fundamental mechanisms of RNA-based regulation of gene expression, such as RNA splicing, localization and regulation by microRNAs are studied. We also investigate proteins in cellular signal transduction that are linked to genetic diseases. Our stu-dies provide a structural basis for molecular mechanisms of important biological pathways and their misregula-tion in human diseases. We are especially studying pathways that are linked to environmental health, e.g. re-flecting environmental changes and challenges in multifactorial diseases. Our structural and mechanistic studies are a starting point for the development of small molecule inhibitors using chemical biology. We use additional biophysical methods and complementary techniques (such as Small Angle X-ray and/or Neutron Scattering, and single particle cryo-electron microscopy) for the structural analysis of macromolecular complexes.

Equipment and expertise The Institute of Structural Biology is associated with the Chair for Biomolecular NMR Spectroscopy and the Ba-varian NMR Centre (BNMRZ; http://www.bnmrz.org) at the Technische Universität München. The BNMRZ hosts excellent state-of-the-art infrastructure for biomolecular NMR studies and has associated faculty that are experts in NMR methods development and biological chemistry. NMR spectrometer: BNMRZ: 900, 800, 750, 2*600 MHz, 500 wide bore, 500 MHz spectrometer; Advanced NMR Technology Platform: Protein and RNA isotope labeling for NMR and neutron scattering, protein interaction studies X-ray crystallography: rotating-anode generator, high-throughput screening robots and automated imaging facili-ties (access at the LMU Gene Centre and at the Max-Planck Institute for Biochemistry) Biophysical techniques: Isothermal Titration Calorimetry, Surface Plasmon Resonance Protein production: State-of-the-art facilities for prokaryotic and eukaryotic expression Unique features Bayerisches NMR Zentrum (BNMRZ) Advanced NMR Technology Platform (isotope labeling and biomolecular interaction) Multidisciplinary approaches: NMR, X-ray, SAXS, SANS, single-particle cryo-EM Collaborations with Helmholtz Centres With FZJ (SANS @ FMRII, Garching) Research groups and group leaders Prof. Michael Sattler, Biomolecular NMR, RNA-based regulation of gene expression, signaling Dr. Dierk Niessing, Helmholtz/Universiity Young Investigator Group, X-ray crystallography, RNA localization, intracellular vesicle transport Associated with the BNMRZ: Prof. Horst Kessler, Structural biology and biological chemistry Prof. Steffen Glaser, NMR methods and design, quantum computing PD Burkhard Luy, NMR methods Prof. Michael Nilges, (TUM/Inst Pasteur), molecular dynamics, structure calculation Training and networking EMBO Practical Course in biomolecular NMR (Jul 27-Aug 3, 2009), Workshops and conferences related to the application and development of biomolecular NMR techniques are organized (e.g. Murnau, 2008) BNMRZ is workpackage leader in the EU FP6 Coordination Action NMR-Life. Michael Sattler is member of the working group solution state NMR in the ESFRI structural biology initiative INSTRUCT. The research groups at the BNMRZ are involved in numerous research networks, including the DFG Cluster of Excellence: Centre for Integrated Protein Science Munich (CiPS-M), Sonderforschungsbereiche: including SFB594 - Molecular Machines; SFB646 Networks in genome expression and maintenance. The BNMRZ participates in various Graduate Schools especially in the Munich area. This include within the Elite Netzwerk Bayern (ENB): the International Research Schools “Protein Dynamics in Health and Disease” and “Quantum Computing, Control and Communication”. Members also participate in the Max-Planck Research School for Molecular and Cellular Life Sciences.

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Future perspectives We will continue structural biology research in the regulation of gene expression and signaling, with a focus on studying protein complexes, including those which involve weak/transient interactions and are thus often linked to regulatory processes. The structural studies combined with quantitative biophysical data provide information about molecular/mechanistic systems biology on the biological processes investigated. We are establishing a research group in solid state NMR spectroscopy to reinforce research on neurodegenera-tive diseases and diabetes. We are initiating the use of small molecules as tools to modulate cellular pathways towards the development of novel drug lead compounds using chemical biology approaches. For this we will also employ target-driven ap-proaches exploiting structural biology information that we generate in our studies. For these activities we will set-up some core expertise at HMGU and plan to interact and collaborate with other centres such as HZI, DKFZ/EMBL and the screening facility at the FMP/MDC in Berlin. Selected publications Corsini, L., Bonnal S., Basquin, J., Hothorn, M., Scheffzek, K., Valcárcel, J.& Sattler M. (2007). U2AF Homology Motif interactions are re-quired for alternative splicing regulation by SPF45. Nat. Struct. Mol. Biol. 14, 620-9. Heuck, A., Du, T.-G., Jellbauer, S., Richter, K., Kruse, C., Jaklin, S., Müller, M., Buchner, J., Jansen, R.-P.& Niessing, D. (2007). Monomeric myosin V uses two binding regions for the assembly of stable translocation complexes. Proc. Natl. Acad. Sci. USA 105, 19778-19783. Lingel, A., Simon, B., Izaurralde, E. & Sattler, M. (2003). Structure and nucleic acid binding of the Drosophila Argonaute2 PAZ domain. Nature 426, 465-9. Selenko, P., Gregorovic, G., Sprangers, R., Stier, G., Rhani, Z., Kramer, A. & Sattler, M. (2003) Structural Basis for the Molecular Recogni-tion between Human Splicing Factors U2AF(65) and SF1/mBBP. Mol. Cell 11, 965-76. Liu, Z., Luyten, I., Bottomley, M.J., Messias, A.C., Houngninou-Molango, S., Sprangers, R., Zanier, K., Krämer, A. & Sattler M. (2001). Struc-tural basis for recognition of the intron branch site RNA by splicing factor 1. Science 294, 1098-102.

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Max-Delbrück -Centre (MDC) Current research activities The structural biology groups at the MDC combine X-ray crystallography with various biochemical and biophysi-cal techniques to study the structures and interactions of human and other proteins involved in cancer and other diseases. NMR-supported research in structural biology is possible in collaboration with the Leibniz Institute for Molecular Pharmacology located next to the MDC on the Berlin-Buch research campus. Current research em-phasis is placed on the investigation of protein modules and protein-nucleic-acid interactions involved in cellular signaling, protein quality control, protein trafficking, and regulation of gene expression. Within the bench-to-bedside research environment provided by the MDC and cooperating clinics, structural biology contributes to molecular medicine and pharmacology by providing atomic-resolution insight into the molecular basis of cellular pathology. Equipment and expertise Protein production: Modern facilities for gene expression in prokaryotic and insect cells at elevated throughput Crystallogenesis: Nanoliter pipetting robotics and large-scale plate-storing and crystal growth control Protein crystallography: 2 rotating-anode X-ray generators with focussing optics, image plate and CCD-detector Unique features Helmholtz Protein Sample Production Facility (PSPF) MDC/FMP Screening Facility Collaborations with Helmholtz Centres With HZI: PSPF / With HZB: Protein Crystallography Beamlines at HZB/BESSY Research groups and group leaders Prof. Udo Heinemann: RG Macromolecular Structure and Interaction Dr. Oliver Daumke: JRG Structure and Membrane Interaction of G-Proteins Training and networking Participation in the Forum of European Structural Proteomics (FESP), an initiative of structural biologists, sup-ported by the European Commission (EC) to assess the state of structural proteomics in Europe and provide recommendation for funding; 2006-2008. Regular training activities in protein sample preparation and characterization within the Berlin branch of the Helmholtz PSPF; ongoing. Participation and workpackage coordination in the EC Integrated Project SPINE II – Complexes; ongoing. Practical course on the Biophysical Characterization of Macromolecular Complexes, supported by SPINE II – Complexes and TeachSG; Nov. 2008.. Future perspectives Structural biology at the MDC is embedded into a research environment that offers clinical research, animal models of cardiovascular and neurological disease and cancer, cell biology focussed on these diseases and pharmacology. Crystallographic, biochemical and biophysical studies of disease-related proteins and their inter-actions will be intensified to inform research in these areas. In this setting which includes a growing facility for compound screening, MDC structural biologists have a realistic chance to contribute both to unraveling the mo-lecular basis of important diseases and to developing tools for biological research, diagnostics and therapeutics. In close mutual collaboration with HZI, MDC will further develop the PSPF to provide excellent infrastructures for the production of high-quality eukaroytic protein samples for structural analysis. Selected publications Müller, J.J., Barbirz, S., Heinle, K., Freiberg, A., Seckler, R. & Heinemann, U. (2008). An intersubunit active site between supercoiled paral-lel � helices in the trimeric tailspike endorhamnosidase of Shigella flexneri phage Sf6. Structure 16, 766-775. Kümmel, D., Heinemann, U. & Veit, M. (2006). Unique self-palmitoylation activity of the transport protein particle component Bet3: A novel mechanism required for protein stability. Proc. Natl. Acad. Sci. USA 103, 12701-12706. Turnbull, A.P., Kümmel, D., Prinz, B., Holz, C., Schultchen, J., Lang, C., Niesen, F.H., Hofmann, K.-P., Delbrück, H., Behlke, J., Müller, E.-C., Jarosch, E., Sommer, T. & Heinemann, U. (2005). Structure of palmitoylated human BET3: Insights into TRAPP complex assembly and membrane localization. EMBO J. 24, 875-884. Khare, D., Ziegelin, G., Lanka, E. & Heinemann, U. (2004). Sequence-specific DNA binding determined by contacts outside the helix-turn-helix motif of the ParB homolog KorB. Nature Struct. Mol. Biol. 11, 656-663. Heinemann, U., Büssow, K., Mueller, U. & Umbach, P. (2003). Facilities and methods for the high-throughput crystal structure analysis of human proteins. Acc. Chem. Res. 36, 157-163.

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Other centres with health research Research Centre Geesthacht (GKSS) Current research activities GKSS contributes to the programme Regenerative Medicine in the Research field Health dealing with the re-generation of non-functional cells, tissues, and organs by biological replacement by e.g. in vitro grown tissues as well as by the stimulation of regeneration and repair processes in the own body. The Institute of Polymer Research, Research Campus Teltow, delivers competence for the development of degradable and stabile bio-materials that are processed into fibres, films, membranes and porous structures from solution or melt. The resulting products are evaluated concerning their toxicity and biocompatibility. Investigated material systems in the research area Biomaterials are biostabile, as well as synthetic, biodegradable materials, stimuli-sensitive polymer materials and biomimetic material modifications. A close link to the scattering activities of the Institute of Materials Research exists where the structural characterisation of the new materials is performed. The Departments “Structural Research on Macromolecules” and “Structural Research on New Materials”, Insti-tute of Materials Research at GKSS contribute to the development, construction and operation of neutron and synchrotron instrumentation as well as to biological structure research in the programme “Research with Pho-tons, Neutrons and Ions” in the Research Field “Structure of Matter” and to metallic biomaterial development (programme “Advanced Engineering Materials” in the Research Field “Key Technologies”). We are specialized on biomembrane related problems such as the understanding of biomembrane active peptides, peptide antibiot-ics or peptidomimetics interactions with cell membranes, the elucidation of structural properties of biological materials such as silk or wood and the elucidation of the function of lipids for biomaterials interactions. In the first case the mode of action, the influence on the biophysical behaviour of the lipid molecules and the mem-branes and the lipid specificity of small natural or synthetic molecules is studied by X-ray and neutron scattering or diffraction. The aim is to develop potent antimicrobial substances which act independent of protein receptors to prevent resistance development and to transfer this knowledge towards a new class of anti cancerous mole-cules. In the second case understanding of the structure-function relation of biological materials will help to de-sign biologically inspired new materials. In the third case lipids are used as implant coatings which improve the matrix formation by 30%. Here we aim at the understanding of protein and cell adhesion on a lipid coated per-manent metallic implant surface and the influence on cellular processes such as differentiation and matrix pro-duction. A similar approach is taken for the development of biodegradable metallic magnesium based implant materials. Equipment and expertise Operation of the small angle neutron scattering instrument SANS-1 and the reflectometer NeRO at FRG-1 (until summer of 2010) Operation of REFSANS and contributions to SANS-1 at the FRM-II Contribution to the small angle X-ray beamline BioSAXS operated by EMBL at PETRA III (building in 2009) and the microfocus beamline MINAXS (building in 2009). Construction and operation of the micro- and nanotomography beamline IBL at PETRA III (2009). Unique features Biophysical characterization of cell membrane. Materials development for implant design (especially biodegradable magnesium based materials) Cell culture (stem cells, primary osteoblasts and chondrocytes, glioblastoma cells). Collaborations with Helmholtz Centres With DESY: construction and operation of beamlines, an Engineering Materials Science Centre of GKSS will be build on the DESY site. With HZB: peptide antibiotics Planned/possible: FZJ – BioSoft and FZK - Biointerfaces Research groups and group leaders Prof. Regine Willumeit: Department of Structural Research on Macromolecules Prof. Martin Müller: Department of Structural Research on New Materials Future perspectives The study of the influence of lipid membrane properties on diseases – especially phase separated regions - and the possibilities of the tunability of these properties will become a more important field of research in the future. The larger focus however will be on the characterisation of the interaction of cells with surface modified metallic implant materials (permanent and biodegradable) or other medically relevant materials (e.g. magnetic nanoparticles).

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Training and networking Coordination of the Marie-Curie Training Network BIOCONTROL which focuses on interations at and with cell membranes. 13 PhD students are trained on various subjects relevant for structural biology: scattering, NMR, single molecule spectroscopy, modeling to name some topics. Participation in the EMBO Practical Course on Solution Scattering from Biological Macromolecules organized by EMBL. Lectures at the University of Hamburg contributing to bachelor and master education in “Molecular Life Science”. Selected publications Angelova, A., Angelov, B., Lesieur, S., Mutafchieva, R., Ollivon, M., Bourgaux, C., Willumeit, R., & Couvreur, P. (2008). Dynamic control of nanofluidic channels in protein drug delivery vehicles. J. Drug. Del. Sci. Tech .18 (1), 41-45. Pasquier, N., Keul, H., Heine, E., Moeller, M., Angelov, B., Linser, S. & Willumeit, R. (2008). Amphiphilic branched polymers as antimicrobial agents. Macromol. Biosci.10 (8), 903-915. DOI: 10.1002/mabi.200800121. Willumeit, R., Schuster, A., Iliev, P., Linser, S. & Feyerabend, F. (2007). Phospholipids as implant coatings. J. Mater. Sci. Mater. Med. 18, 367–380. Willumeit, R., Schossig, M., Clemens, H.& F. Feyerabend. (2007). In-vitro Interactions of Human Chondrocytes and Mesenchymal Stem Cells, and of Mouse Macrophages with Phospholipid-Covered Metallic Implant Materials. European Cells & Materials Journal 13, 11-25. Arnt, L., Rennie, J., Linser, S., Willumeit, R., Tew, G.N. (2006). Membrane Activity of Biomimetic Facially Amphiphilic Antibiotics. J. Phys. Chem. B. 110 3527-3532.

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Research Centre Jülich (FZJ) Current research activities Structural Biology carried out at FZJ focuses on the development and application of methods to precisely inves-tigate three-dimensional structures and molecular mechanisms of biologically and medically relevant macro-molecules involved in cellular processes like signalling pathways, protein synthesis, folding, misfolding and ag-gregation as well as protein-protein and protein-ligand interactions. These phenomena are studied by a variety of biophysical methods. For high spatial resolution studies X-ray crystallography, liquid and solid state NMR are employed, whereas spectroscopic methods like time-resolved absorption, infrared and fluorescence spectros-copy contribute to temporal resolution. We use and run instruments at various neutron sources (e.g. Oak Ridge and Munic) and run FRMII together with the Technische Universität München). Groups in the field of computa-tional structural biology complement the broad experimental techniques and make use of Europe's largest su-percomputing centre. Future efforts will increasingly be invested into structure and dynamics of fibril forming and membrane proteins, which are not readily amenable to standard structural investigation. The aim of these re-search projects is the characterization of basic processes in living cells and the investigation of the molecular basis of diseases like AIDS, SARS and neurodegenerative disorders. The relevant Helmholtz Programme (Bio-Soft) and the topic "Structural Biology" have been evaluated to be excellent.

Equipment and expertise X-ray crystallography: Robotic system for crystallization and a rotating anode X-ray generator equipped with Image plate detector. NMR spectrometers: 2x 600 MHz for liquid state NMR, 2x 600 MHz for solid state NMR, 800 MHz for liquid and solid state NMR, 900 MHz for liquid state NMR. Investment for a 1 GHz NMR has just obtained a clear positive recommendation of the POF review panel. Neutron instruments for small angle and ultra-small angle scattering (KWS-2, KWS-3) and for incoherent and coherent inelastic scattering (Neutron spin-echo, neutron back scattering) as well as a diffractometer with pola-rization option were build up by FZJ at the Munic outstation (FRM-II). Biophysical techniques: fluorescence correlation spectroscopy, fluorescence polarisation, isothermal titration calorimetry, surface plasmon resonance, static and dynamic light scattering, Protein production: State of the art facilities for prokaryotic and eukaryotic expression of soluble, transmem-brane and fibrillar proteins. Unique features High field NMR spectrometers for solid and/or liquid state NMR spectroscopy (800 MHz and 900 MHz). Isotope labeling of soluble, transmembrane and fibrillar proteins. Supercomputing. Strategic alliance with CEA including access to ESRF beamlines and PBS in Grenoble. Collaborations with Helmholtz Centres With DESY: New Helmholtz Department in Structural Biology at DESY Research groups and group leaders Prof. Dr. Georg Büldt: Molecular Biophysics PD Dr. Jörg Labahn: Structure Analysis of Membrane and Apoptosis Proteins PD Dr Jörg Granzin: Analysis of Water-Soluble Proteins PD Dr. Jörg Fitter: Molecular Dynamics in Proteins and Biological Membranes Prof. Dr Valentin Gordelii:Biophysics of Membrane Proteins and Lipid Systems Prof. Dr. Dieter Willbold: Structural Biochemistry Dr. Matthias Stoldt: Model Systems PD Dr. Bernd W. König: Protein Protein Interaction Analysis Prof. Dr. Henrike Heise: Solid State NMR PD Dr. Michael Bachmann: Soft Matter Systems Research Group Dr. Birgit Strodel: Multiscale Modelling of Protein-Protein Interactions Dr. Gunnar Schröder: Computational Structural Biology Training and networking The International Helmholtz-Research School on Biophysics on Soft Matter “BioSoft” offers an interdisciplinary graduate education at the interface between biology, chemistry, and physics for about 20 fellows and a compa-rable number of associate students. It is run by 12 research groups affiliated with the universities of Düsseldorf and Köln, the Research Centre caesar (Bonn) and the Forschungszentrum Jülich.

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The International Graduate School BioStruct offers 20 scholarships for PhD students in the field of structural biology in its application to molecular medicine and biotechnology (spokespersons: D. Willbold and L. Schmitt, University of Düsseldorf). The Virtual Institute for Structural Biology (VIBS) combines the efforts of ten structural biology groups located in the Rhine-Ruhr region. VIBS is headed by G. Büldt and supported by the “Helmholtz Impuls und Vernetzungs-fond” (VH-VI-013). The bio-N3MR network NRW combines biomolecular NMR activities of FZJ, the Max-Planck-Institute for Molecu-lar Physiology in Dortmund and the Universities of Duisburg/Essen, Düsseldorf and Bochum (spokesperson: D. Willbold). The recently founded cooperation between the Institute de Biologie Structurale Jean-Pierre Ebel (IBS), Com-missariat à l’Energie Atomique (CEA) in France and the ISB, allows access to synchrotron beam lines at Euro-pean Synchrotron Radiation Facility (ESRF) and to several technology platforms (electron microscopy, protein production and labelling, etc.). Further network activities include for example participation in European Networks of Excellence (NoE) and the DFG.Sonderforschungsbereich 575. Future perspectives Structural Biology at FZJ will continue to focus on the determination of precise geometric and dynamic informa-tion of biologically and medically highly relevant macromolecules as a basis for understanding their functions. A special focus will be on fibrillar and membrane proteins that are not easily amenable for structure determination. This will require further efforts towards methods development and sample production. Jülich will contribute to the planned Centre for Structural Systems Biology (CSSB) at DESY in Hamburg with a research group at the W2 level. This will allow access to one of the most brilliant synchrotron radiation sources (PETRA III) and the planned X-ray free electron laser (X-FEL). In parallel to ongoing experimental efforts the activities in the field of computational structural biology are concentrated within a new Institute of Advanced Simulation Sciences (IAS) and the newly founded German Research School for Simulation Sciences (GRS).

Selected publications Schuenke, S., Stoldt, M., Novak, K., Kaupp, U.B. & Willbold, D. (2009). Solution structure of the M.loti K1 channel cyclic nucleotide binding domain in complex with cAMP. EMBO Rep., in press. Schmidt, H., Hoffmann, S., Tran, T., Stoldt, M., Stangler, T., Wiesehan, K. & Willbold, D.. (2007). Solution structure of a Hck SH3 domain ligand complex reveals novel interaction modes. J. Mol. Biol. 365, 1517-1532

Moukhametzianov, R., Klare, J.P., Efremov, R., Baeken, C., Göppner, A., Labahn, J., Engelhard, M., Büldt, G. & Gordeliy, V.G. (2006). Development of the signal in sensory rhodopsin and its transfer to the cognate transducer. Nature 440, 115-119

Gordeliy, V.I., Labahn, J., Efremov, R., Moukhametzianov, R., Granzin, J., Schlesinger, R., Büldt, G., Savopol, T., Scheidig, A.J., Klare, J. P. & J. M. Engelhard. (2002). Molecular basis of transmembrane signalling by sensory rhodopsin II-transducer complex. Nature 419, 484 – 487

Sass, H.J., Büldt, G., Gessenich, R., Hehn, D., Neff, D., Schlesinger, R., Berendzen, J.& Ormos, P. (2000). Structural alterations for proton translocation in the M state of wild-type bacteriorhodopsin. Nature 406, 649-653

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Research Centre Karlsruhe (FZ K) Current research activities Expertise at FZK is focused on the 3D structure analysis of membrane-bound proteins, using NMR in the solid state and in solution, as well as circular dichroism (CD) and Fourier-transform infrared (FTIR) spectroscopy, supported by computer modelling and MD simulations. Some membrane proteins can be analyzed in detergent micelles, but most of them depend on a lipid bilayer to acquire their native conformation and be active. Solid state NMR is the most powerful method to obtain such structures with quasi-atomic resolution as well as dy-namic information on proteins that are embedded in a lipid environment. The NMR facility at FZK thus explores the conformation, alignment, oligomerization behaviour and mobility of membrane-active peptides (with antim-icrobial, cell penetrating, fusogenic, or cytotoxic functions) and of transmembrane proteins (forming ion chan-nels, transporters, or pores), to explore how these molecules interact with one another and with the lipid envi-ronment. A unique aspect of the solid state NMR activities is the use of highly sensitive 19F-NMR labels, having imple-mented a novel chemical labelling strategy and in-house hardware developments. This approach allows obser-vation of labeled peptides for the first time in natural membranes, such as erythrocyte ghosts or bacterial proto-plasts, in a background-free manner. Medium-throughput protocols are being established for 19F-, 15N- and 2H-labelling, sample preparation, NMR-measurements, and data analysis. Whenever possible, these studies will be complemented by liquid-state NMR in detergent micelles to measure residual anisotropic parameters to refine the structures or to reveal relative molecular orientations. A new Heisenberg-Professor with this expertise is currently being recruited. The analysis of anisotropic parameters in oriented samples (i.e. in biomembranes, stretched fibers, and weakly aligned gels) thus constitutes the special expertise of several complementary groups, using solid state NMR, oriented CD, and liquid-state NMR. Optimized protocols for oriented sample preparation and anisotropic data analysis are thus being jointly developed. The detailed but highly elaborate and costly NMR structure analysis of proteins is routinely complemented at FZK by rapid, sensitive, and inexpensive CD and FTIR analysis. Protein conformation is rapidly screened to monitor sample conditions, protein aggregation, or functionally relevant conformational changes, and simple difference-spectroscopy can reveal binding events. Both CD and FTIR are especially well suited for membrane proteins, i.e. using oriented CD (OCD) on oriented samples to determine the molecular alignment in a lipid bi-layer, and ATR-IR to measure conformational anisotropy. Structure refinement from the NMR and optical spectroscopy data is achieved by molecular modelling and MD simulations using a specialized force-field. Since for many proteins (soluble and membrane-bound) the 3D structures have not yet been determined, but there may be crystal structures available from related systems, we have also developed an all-atom approach for protein folding and structure prediction. In those cases the analy-sis and prediction of, e.g. suitable ligands, can be significantly sped up using computer aided homology model-ling. Likewise, very rapid methods have been developed for in silico high-throughput screening of small-molecule libraries to structurally characterize protein receptors. Equipment and expertise NMR: 5 spectrometers: 600 MHz, 500 MHz, 300 MHz wide-bore at FZK, 500 wide-bore at the University of Karlsruhe, 600 MHz standard-bore at the University of Karlsruhe Protein production: Molecular biology facilities for prokaryotic protein expression, purification, and functional tests Peptide synthesis: Large-scale synthesis and HPLC purification, multiple parallel synthesis at medium-throughput, SPOT synthesis Optical spectroscopy: 2 CD spectropolarimeters with home-built OCD cells for oriented samples, several FTIR spectrophotometers, dynamic light scattering, differential scanning calorimetry, etc. Computing: in-house all-atom MD simulation program COSMOS; POEM@HOME distributed computing initia-tive; FlexScreen for ligand docking Unique features NMR hardware for 19F-observation in the solid state, using static experiments on oriented membrane samples as well as MAS methods Peptide synthesis facility with organic chemistry expertise, especially to prepare 19F-labelled amino acids and incorporate them into synthetic peptides A new FTIR beamline has been recently installed at the synchrotron ANKA to exploit the high brilliance of this light source A new CD/OCD beamline will be installed in 2009 at the synchrotron ANKA to exploit the extended VUV wavelength-range and the orders of magnitude flux of this light source

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Collaborations with Helmholtz Centres Close networking within the new POF programme “BioInterfaces” at FZK with groups from IBG, ISS, INT, and IFG. Research groups and group leaders Institute of Biological Interfaces (Prof. Anne Ulrich, Chair of Biochemistry at University of Karlsruhe) RG Solution-state NMR (new Heisenberg-Prof. Burkhard Luy, IBG-2) RG Solid State NMR (Dr. Stephan Grage, IBG-2) RG Circular Dichroism (Dr. Jochen Bürck, IBG-2) RG Peptide Synthesis (Dr. Parvesh Wadhwani, IBG-2) RG Infrared Spectroscopy (Dr. David Moss, ISS) RG Molecular Modelling (Dr. Wolfgang Wenzel, INT) RG Receptor Ligand Interactions (Dr. Katja Schmitz, IFG) Training and networking The BioNMR Centre at KIT is part of the DFG-Centre for Functional Nanostructures (CFN) at Karlsruhe, offering infrastructure and expertise in the characterization of nanomaterials by solid state NMR. Biannual CFN summer schools on “Nano-Biology” are organized with prominent participation of graduate students since 2005. A Helmholtz Graduate School on “BioInterfaces” has been launched at FZK in 2009, with a practical and theoretical component in biomolecular structure analysis. Within the faculty of Chemistry and Bioscience, the Institute of Organic Chemistry and the IBG-2 have been responsible for establishing the new Bachelor and Master study course in “Chemical Biology” to commence in 2009, with a syllabus in structural biology and bioanalytics. The next international conference in the series on “Lipid-Protein Interactions” will be hosted at Karlsruhe by IBG-2. Future perspectives The IBG-2 will extend its studies of peptides and proteins in model membranes to include natural membrane systems prepared from bacterial or mammalian sources. Membrane-bound peptides and proteins will be further characterized especially with a view to intermolecular interactions (oligomerization, ligand-receptor binding, target recognition), to better understand their functional mechanisms and to use this information to construct new molecular tools for controlling cell behaviour. Novel types of cell-penetrating peptides and peptido-mimetics will be characterized in lipid membranes, and their bilayer perturbations will be examined. Knowledge of the specific peptide-lipid interactions and translocation mechanisms will help to extract the salient features that are required for a rational design of improved sequences. The solution-state NMR equipment at FZK has to be extended to cater for the new Heisenberg-Professor. The new CD beamline at ANKA is being implemented in 2009, and an international user facility will be estab-lished with access to OCD, combined CD and FTIR, as well as rapid-mixing and stopped-flow devices. The computational group will apply POEM@HOME for high-throughput protein structure prediction to elucidate protein function and protein-mediated cell-signalling processes. To regulate protein function we will perform small-molecule screens in silico with our in-house in silico high-throughput drug discovery approach FlexScreen. Selected publications Wadhwani, P., Buerck, J., Strandberg, E., Mink, C., Afonin, S., Ieronimo, M.& Ulrich, A.S. (2008). Using a sterically restrictive amino acid as a 19F-NMR label to monitor and to control peptide aggregation in membranes. J. Am. Chem. Soc. 130, 16515-16517. Afonin, S.E., Grage, S.L., Ieronimo, M., Wadhwani, P.& Ulrich, A.S. (2008). Temperature-dependent transmembrane insertion of the amphiphilic peptide PGLa in lipid bilayers observed by solid state 19F-NMR. J. Am. Chem. Soc. 130(49), 16512-16514. Mykhailiuk, P., Afonin, S., Gvozdovska, N.P., Shishkin, O.V., Ulrich, A.S. & Komarov, I.V. (2008). Synthesis of trifluoromethyl-substituted proline analogues as 19F-NMR labels for peptides in the polyproline II conformation. Angewandte Chem. 120, 5849-5851. Angewandte Chem. Intl. Ed. 47, 5765-5767. Bürck, J., Roth, S., Wadhwani, P., Afonin, S., Strandberg, E.& Ulrich, A.S. (2008). Conformation and membrane orientation of amphiphilic helical peptides by oriented circular dichroism. Biophys. J. 95, 3872-3881. Strandberg, E., Esteban-Martin, S., Salgado, J.& Ulrich, A.S. (2009). Orientation and dynamics of peptides in membranes calculated from 2H-NMR data. Biophys. J. 96, 3223-3232.

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Centres providing important infrastructure German El ectron Synchrotron (DESY) Synchrotron radiation provided by DORIS III and PETRA III (from 2010). Helmholtz protein crystallography beamline shared with Max-Planck at PETRA III. Free-electron lasers FLASH and X-FEL (from 2014). Planned Centre for Structural Systems Biology (CSSB) (in preparation). Helmholtz Centre Berlin (HZB) Synchrotron radiation provided by BESSY II. Research Centre Karlsruhe (FZK) Synchrotron radiation provided by ANKA with beamlines for IR and CD spectroscopy, and complementary NMR analysis available on site. Research Centre Geesthacht (GKSS) Access to the small angle X-ray scattering beamlines BioSAXS and MINAXS as well as to micro- and nanoto-mography beamlines at Doris and PETRA III. Neutron Scattering facilities for biological structure research at the FRM-II (REFSANS) and FRG-1 (SANS1, NeRO). Lab facilities for biophysical and biomedical studies in the Engineering Materials Science Centre of GKSS at DESY. Bavarian NMR Centre (HMGU) High field NMR spectrometers for solid and/or liquid state NMR spectroscopy (800 MHz and 900 MHz) Biomolecular NMR Centre (FZJ) High field NMR spectrometers for solid and/or liquid state NMR spectroscopy (800 MHz and 900 MHz) Helmholtz Centre for Infection Research (HZI) and Max -Delbrück -Centre (MDC) Helmholtz Protein Sample Production Facility (PSPF)

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F. Contact

Prof. Dr. Georg Büldt g.bueldt@fz-juelich.de 02461-61-2030 Institute for Structural Biology and Biophysics – Molecular Biophysics (ISB-2) FZJ Forschungszentrum Jülich 52425 Jülich Prof . Dr. Dmitrij Frishman d.frishman@wzw.tum.de 08161-712134 Technische Universitaet Muenchen Wissenschaftszentrum Weihenstephan Am Forum 1 85354 Freising Prof. Dr. Udo Heinemann Heinemann@MDC-Berlin.de 030-9406-3420 MDC Max-Delbrück-Centrum für Molekulare Medizin in der Helmholtz-Gemeinschaft Robert-Rössle-Straße 10 13125 Berlin-Buch Prof. Dr. Dirk Heinz Dirk.Heinz@Helmholtz-hzi.de 0531-6181-7000 Division of Structural Biology HZI Helmholtz Centre for Infection Research Inhoffenstrasse 7 38124 Braunschweig Dr. Harald Herrmann -Lerdon h.herrmann@dkfz.de 06221-42-3512 Div. Cell Biology DKFZ Deutsches Krebsforschungszentrum Im Neuenheimer Feld 280 69120 Heidelberg Prof. Dr. Werner Mewes w.mewes@Helmholtz-muenchen.de 089-3187-3580 Institute of Bioinformatics HMGU Helmholtz Zentrum München / German Research Centre for Environmental Health Ingolstädter Landstraße 1 85764 Neuherberg Dr. Uwe Müller umue@Helmholtz-berlin.de 030-6392-4974 Helmholtz-Zentrum Berlin für Materialien und Energie GmbH Glienicker Straße 100 14109 Berlin Prof. Dr. Michael Sattler sattler@Helmholtz-muenchen.de 089-289-13418 Institute of Structural Biology HMGU Helmholtz Zentrum München / German Research Centre for Environmental Health Ingolstädter Landstr. 1 85764 Neuherberg

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Prof. Dr. Anne Ulrich anne.ulrich@ibg.fzk.de 07247-82-2563 Institut für Biologische Grenzflächen FZK Forschungszentrum Karlsruhe Hermann-von-Helmholtz-Platz 1 76344 Eggenstein-Leopoldshafen Prof. Dr. Edgar Weckert edgar.weckert@desy.de 040-8998-4509 HASYLAB at DESY Notkestraße 85 22607 Hamburg Prof. Dr. Dieter Willbold d.willbold@fz-juelich.de 02461-61-2100 Institute for Structural Biology and Biophysics – Structural Biochemistry (ISB-3) FZJ Forschungszentrum Jülich 52425 Jülich Prof. Dr. Regine Willumeit Regine.willumeit@gkss.de 04152-87-1291 Abteilung “Strukturforschung an Makromolekülen” GKSS Forschungszentrum Geesthacht Max-Planck-Strasse 1 21502 Geesthacht