Jonathan Hadden [email protected] Astbury Centre for Structural Molecular Biology

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Jonathan Hadden

[email protected]

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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NB 3 hour pre-incubation no crystals!


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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

Jonathan Hadden [email protected] Astbury Centre for Structural Molecular Biology

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Transcript of Jonathan Hadden [email protected] Astbury Centre for Structural Molecular Biology