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Lessons Learned from Crystallising Protein DNA Complexes. Synergy between High-Throughput and Novel Crystallisation Techniques. Astbury Centre, University of Leeds. Jonathan Hadden [email protected] Astbury Centre for Structural Molecular Biology University of Leeds. Overview of Talk. - PowerPoint PPT Presentation
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Jonathan Hadden
Astbury Centre for Structural Molecular BiologyUniversity of Leeds
Lessons Learned from Crystallising
Protein DNA Complexes. Synergy
between High-Throughput and Novel
Crystallisation Techniques
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Overview of Talk
•Introduction to the project
•The High-throughput approach we took
(twist due to funding)
•Problems we encountered
•Optimisation approaches used to overcome the problems
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Personal perspective on crystallisation
•Increasing throughput has very much driven developments in protein crystallisation over past 10 years
•Fantastic benefits: faster setup, less sample required, more experiments
•Increasing throughput usually means significant miniaturisation
•Resultant scale-up required during optimisation
•Scale-up particularly important for poorly diffracting crystals or those sensitive to radiation damage. E.g. Protein/DNA complexes or Membrane proteins. Where some of the largest challengers are
•BIG CRYSTALS REQUIRED – BUT OUR DROPS ARE GETTING SMALLER!
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Trying to Crystallise a Holliday Junction Resolving enzyme complex since 1984
A Holliday junction is a four-way DNA junction and is a central intermediate in recombination
Recombination (a ubiquitous biological process)The exchange of one DNA sequence with an other or The incorporation of one DNA sequence into an other
Many examples of recombination in natureExchange of homologous DNA during meiosis in diploid organismsGeneration of diversity in Immunoglobulin genesIntegration of bacteriophage DNA into host DNA
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Strand invasion
Branch migration
or
Junction resolution
Endo I
Strands nicked
Patch Recombination
Splice Recombination
Holliday Junction
Classical (Holliday) scheme for homologous recombination
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Structure of a Holliday junction
Structure of a Resolving enzyme
Hadden et al. 2002, EMBO J. 21, 3505-3515
Endo ICut Endo I
Cut
Ortiz-Lombardia et al. 1999, NSB 6, 913-917
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The Approach
•Purify protein DNA complexes – limited quantities for crystallisation
•Use a mutant Endo I that binds junction but does not cleave
•High-throughput crystallisation – 960 condition screen
•Protein constant
•Varied the DNA sequence – up to 100 sequences
•Potentially 96,000 trials!
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Problem
Prepare 250nl + 250nl crystallisation experiments in 96 well vapour diffusion plates at
minimal cost with technology available 8 years ago
Solution
Use a Douglas instruments ORYX 6 robot
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Another problem The robot is comparatively slow - 18 minutes to set up a tray
Evaporation of 500nl drops is a big problem
Another solution Cover drops with light (1cS) silicone oil. The oil will initially protect the drop but eventually evaporate into the sealed atmosphere
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Add screening solutions to plate using 8-channel
pipette
Aspirate screening solution, protein already
loaded
Dispense 0.25µl of protein and screening solution
Dispense 5 µl of light silicone oil onto drop
Seal with film and wait for oil to evaporate
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Some hits!
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We needed to grow much larger crystals
• Optimise in 24-well plates 1μl+1μl or 2μl+2μl drops
• Quite often conventional methods failed dismally!
• “All or nothing effect” (lots of small crystals or non at all)
• Used two methods to successfully overcome these problems - on most occasions
Equilibration rate controlSeeding
• However, neither performed conventionally
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Equilibration rate control
•Modify the rate of equilibration in a vapour diffusion experiment
•Traditionally – TemperatureSize of drop
•More recently- Use a barrier between drop and reservoir
•Bobs gadget (Equilipro)
•Silicone fluid on reservoir (Naomi Chaen)
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Equilibration rate control – silicone fluid
•Silicone fluid on reservoirEarly stages - Equilibration of drop / unsaturated airspace
SiliconeFluid
Significantly better results
•Silicone fluid on dropEarly stages – protected drop
SiliconeFluid
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Equilibration rate control
Variables
•Viscosity of fluid – 1cS or 5cS works well
•Volume of fluid - 20μl is good in a standard
microbridge with a 2μl-4μl drop
•Pre-equilibration time (time between setting up drop
and covering drop with oil - 0 Hours)
•PPT concentration – usually +0 to +3% works wellSIGNIFICANTLY LARGER CRYSTALS
FOR ~80% OF TARGETS!
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The remaining 20% - Seeding
Higher-throughput seeding
•Simple approach - streak seeding
•Fast turn round - find optimal conditions quickly
•Used drop pre-incubation times to control crystal size, quality, numbers.
•Well solutions and protein concentration identical to those used to grow original hits
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Streak Seeding - Practical Setup
Start with conditions that originally grew original hits
Setup a whole tray of drops – two drops per well
At the pre-defined times manually streak seed into the four drops sequentially
The numbers indicate drop pre-incubation times in hours
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24 hour pre-incubation + 12 hours growth
T7 endonuclease I / Holliday junction complex
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4 hour pre-incubation + 12 hours growth
NB 3 hour pre-incubation no crystals!
T7 endonuclease I / Holliday junction complex
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4 hour pre-incubation + 72 hours growth
T7 endonuclease I / Holliday junction complex
300μm
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Conclusions
• Vapour diffusion using Oryx 6 + silicone fluid works exceptionally well
• Most ‘hits’ can be easily optimised and scaled-up using
Equilbration rate diffusion
or
Seeding
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Endonuclease I Holliday Junction Complex Structure, 3.1Å
To be Published in Nature 4th October 2007
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AcknowledgementsLeeds and Dundee
Anne-Cécile Déclais
Steve Carr
David Lilley
Simon Phillips
Synchrotrons
ESRF Grenoble
SRS Daresbury
Support
Wellcome Trust
Cancer Research UK
BBSRC
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What is silicone oil / fluid?
Polydimethylsiloxane
Defined by viscosity measured in centistokes
The viscosity / volatility can be varied by changing n
CH3-Si-O -Si-O -Si-CH3
CH3
CH3 n
CH3CH3
CH3 CH3
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Arm B
Arm H
Arm R
Arm X
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Oligo Z 3’ Oligo Y 5’
Oligo Z 5’
Cut
Cut
Unequal armLengthThe Approach
• Many DNA variations – cater for up to 100
• Use Vapour diffusion
• 960 condition screen
• Started project about 8 years ago
• Finance for robotics about £30,000
Sequence
Hairpinloops
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Seeding
Higher-throughput seeding
•About 3/4 of targets optimised by setting up one tray!
•3-4 hours pre-incubation often best
QUICK, EASY, FAST