enom - CABM Protein NMR Lab

High-Throughput, Capillary-based Protein Crystallography

Deirdre Meldrum

Genomation Laboratory, Department of Electrical EngineeringDepartment of Computer Science & Engineering

SGPPUniversity of Washington

NIGMS PPCWMarch 29-31, 2004

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Motivation for Protein Crystallography in Capillaries

• Traverse crystallization space in a unique manner for optimum crystal growth

• Crystals remain in plastic capillaries all the way to the synchrotron so fragile crystals are not disturbed∆ Flash freezing to 100 K in plastic capillary demonstrated

• Potential for low (5 nL range) protein & precipitant volumes yield more experiments per volume of reagent

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Traverse Crystallization Space for Optimum Crystal Growth

protein

precipitant air gap

high conc. compound

capillaryProtein Concentration

Pre

cipi

tant

Con

cent

ratio

nPrecipitate

Nucleation Zone

Clear Drop

Free liquid interfacediffusion + vapor diffusion+ direct cryo protocols inplastic capillaries

Phase Diagram

protein

precipitant mixing

OR

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Potential for Low (5 nL) Reagent Volumes: ACAPELLA instrument to prepare samples

for DNA sequencing

• ACAPELLA-5K core processor + thermal cycler∆ Processes 5000 samples in 8 hours∆ Prepares 1 to 2 µL reactions∆ Precision 100 pL dispensing with

piezoelectric reagent dispensers∆ Design & Fabrication Complete∆ α-tests complete with the UW

Genome Center (UWGC)∆ β-tests in progress with UWGC &

WashU Genome Sequencing Center

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ACAPELLA Features

• Small Volumes∆ Precision 100 pL dispensing

with piezoelectric reagent dispensers into capillaries

∆ Retain High Mixing Precision and Quality in Small Volumes

∆ Dispense protein & multiple reagents

• Automated, High-throughput• High Quality Biology

∆ Demonstrated Ability to Achieve High Quality, Reproducible Biology

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Piezoelectric Dispenser Operating Space

[S. McGuire & M. Holl]

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Piezoelectric Reagent Dispenser Performance:Endurance Testing

Shooter 409Firing Taq + 0.8 mg/mL BSAVelocity = 4.2 m/s20kHz / 20 V10,000 drops/µL, 4 s intervals

Fire 10,000 drops/shot10 million drops fire flawlessly!

[S. McGuire & M. Holl]

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CS II Reagents Dispensed with ACAPELLA Piezoelectric Dispensers (1) [L. DeSoto]

YMPD30% v/v

Sodium Acetate trihydrate pH 4.60.1MSodium Chloride0.2M

YSodium Chloride2.0M

Sodium Acetate trihydrate pH 4.60.1MNone

YEthanol10% v/vNoneSodium Chloride1.5M

Polyethylene Glycol 800010% w/v N

Polyethylene Glycol 100010% w/v

NoneNone

YImidazole pH 7.01.0MNoneNone

YIso-Propanol5% v/vNoneAmmonium Sulfate2.0M

YDioxane35% v/vNoneNone

YEthylene Glycol25% v/vNoneNone

Magnesium Chloride hexahydrate0.01M Y

Sodium Chloride0.5M

Hexadecytrimethylam-monium Bromide0.01M

YPEG 600010% w/vNoneSodium Chloride2.0M

ReagentConcReagentConcReagentConc Good

PrecipitantBufferSalt

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CS II Reagents Dispensed with ACAPELLA Piezoelectric Dispensers (2) [L. DeSoto]

YMagnesium Sulfate heptahydrate1.6MMES pH 6.50.1MNone

Y1.6 Hexanedoil2.5Mtri-Sodium Citrate dihydrate pH 5.60.1MNone

YJeffamine M-60010% v/vtri-Sodium Citrate dihydrate pH 5.60.1M

Ferric Chloride hexahydrate0.01M

Ytert-Butanol35% v/vtri-Sodium Citrate dihydrate pH 5.60.1MNone

YEthylene Imine {olymer2% w/vtri-Sodium Citrate dihydrate pH 5.60.1MSodium Chloride0.5M

YLithium Sulfate monohydrate1.0M

tri-Sodium Citrate dihydrate pH 5.60.1MAmmonium Sulfate0.5M

YAmmonium Sulfate2.0Mtri-Sodium Citrate dihydrate pH 5.60.1M

Potassium Sodium Tartrate tetrahydrate0.2M

NPolyethylene Glycol Monomethyl Ether 200030% v/v

Sodium Acetate trihydrate pH 4.60.1MAmmonium Sulfate0.2M

YPolyethylene Glycol 40030% v/v Sodium Acetate trihydrate pH 4.60.1M

Cadmium Chloride dihydrate0.1M

Y1.6 Hexanedoil1.0 MSodium Acetate trihydrate pH 4.60.1M

Cobaltous Chloride hexahydrate0.01M

ReagentConcReagentConcReagentConc Good

PrecipitantBufferSalt

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CS II Reagents Dispensed with ACAPELLA Piezoelectric Dispensers (3) [L. DeSoto]

MPD5% v/v

Polyethylene Glycol 600010% w/v

HEPES pH 7.50.1MNone

YMPD30% v/vHEPES pH 7.50.1MAmmonium Sulfate0.5M

Ntri-Sodium Citrate dihydrate

pH 6.51.6MNoneNone

YPolyethylene Glycol

Monomethyl Ether 55025% v/vMES pH 6.50.1MZinc Sulfate

heptahydrate0.01M

NPolyethylene Glycol

Monomethyl Ether 500030% v/vMES pH 6.50.1MAmmonium Sulfate0.2M

YAmmonium Sulfate1.8MMES pH 6.50.1MCobaltous Chloride

hexahydrate0.01M

YJeffamine M-60030% v/vMES pH 6.50.1MCesium Chloride0.05M

YDioxane10% v/vMES pH 6.50.1MAmmonium Sulfate1.6M

Polyethylene Glycol 2000012% w/vMES pH 6.50.1MNone

mono-Potassium dihydrogenPhosphate0.1M YSodium Chloride2.0MMES pH 6.50.1M

Sodium dihydrogenphosphate mono0.1M

ReagentConcReagentConcReagentConc Good

PrecipitantBufferSalt

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Experiments Preparedwith ACAPELLA Piezoelectric Dispensers

plasticCryo6 mg/mlP. falciparum aldolase

PlasticCryo10 mg/mlL. mexicana aldolase + 0.2% Decylmaltoside *

PlasticPEG/ion10 mg/mlGlassPEG/ion10 mg/mlGlassCryo10 mg/mlL. mexicana aldolaseGlassWizard20 mg/mlBovine hemoglobin *GlassWizard5 mg/mlBovine insulin *GlassCryo40 mg/mlGlassCryo20 mg/mlLysozyme *CapillariesScreenConcentrationProtein

* Crystals observed[S. Turley, C. Fisher]

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SGPP Targets Dispensed with ACAPELLA Piezoelectric Dispensers [L. DeSoto]

33

33

5

24

22

14

32

20

44

8

ORF

40

40

12

18

6

21

31

25

77

15

Sample

Good (crystals, plastic caps)20PFAL007201WES

Good, no dilution3PFAL003589AAA_W.SM

Good, no dilution10.2LMAJ00153AAA

Good – shot 20 uL, no spin, no dilution

4.4LMAJ00903AAA_W.SM

Good – shot 30 uL, no spin, no dilution

15LMAJ00822AAA

Good – shot 30 uL, no spin, no dilution

10.3LMAJ00462AAA_R.SM

Failed full conc.9.6PFAL003552AAA

Failed full, 50%, 25% conc.; starts 25%

10.4LMAJ00618AAA_R.SM

Good (crystals)18 LMAJ002144AAA_W.SM

Difficult to start; then ok23.298LMAJ00200AAA_R1

ResultConc.mg/ml

Protein

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Protein Crystallography Results

Pfal007201wesinside plastic capillaries

190 µm i.d.

U6-05-05 U6-15-12 U6-25-08inside glass capillaries

340 µm i.d. [L. DeSoto]

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ACAPELLA-5K for Protein Crystallography with SGPP

• Results of HEW lysozyme crystal grown & frozen in thick-walled polycarbonate-plastic capillaries∆ Cryoprotection: 30% glycerol added in situ∆ Flash frozen in liquid N2

• ALS Berkeley synchrotron demonstrates high resolution and excellent quality data on beam line 5.0.3 ∆ Rmerge 0.036 ( 0.221 for outer shell 1.32-1.25 Ångstroms)

• Wavelength of 1.000 Ångstroms used for X-rays[W.G. Hol, S. Turley, M. Robien]

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Synchrotron Results [M. Robien, S. Turley]

10 second exposure

1.65 Ångstroms edge

1.28 Ångstroms corner

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Synchrotron Results[M. Robien, S. Turley]

13Å----res-----1.25Å

0.23

Rsym

0.03

Lysozyme diffractionNo background problems in indexing/processing

Corner detail circa 3-1.25ÅHigh resolution &low mosaicity (0.15°)

Rsym vs resolutionExcellent merging statisticsRsym: 0.038 overall

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Current Protein Crystallography Process in Capillaries

RoboDesignMicroscope II

ACAPELLA capillary sample preparation Cassette of capillaries

Transferred to RoboDesign

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Summary

• Capillary-based protein crystal growth has potential for automated processing with low (5 nL range) reagent volumes∆ Glass capillary process “ready-to-go”∆ Plastic capillaries – crystals untouched from start to

finish!∆ Vapor diffusion methods in capillaries provide

flexibility & potential for improved crystal growth

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Acknowledgments

• Wim Hol, Larry DeSoto, Stewart Turley, Mark Robien, Oleksandr Kalyuzhniy & members of SGPP

• Eve Riskin, Richard Ladner, Linda Shapiro, Jue Wang of UW EE/CSE

• Mark Holl, Charles Fisher, Shawn McGuire, & Matt Moore of UW EE Genomation Laboratory

• Orca Photonic Systems, Inc.• NIH NHGRI support for development of the

ACAPELLA system for DNA sequencing• NIH NIGMS support for adaptation of

ACAPELLA & new instrumentation for protein crystallography