protein production and crystallization workshop
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Protein Production and Crystallization Workshop
Structural Genomics of Pathogenic Protozoa (SGPP)
Crystal Growth LabLori Anderson
www.sgpp.org
SGPP Crystal Growth Pipeline
Protein Production UnitUniversity of Washington
Seattle Washington
Protein Production UnitUniversity of Rochester
Rochester New York
Crystal ScreeningHauptman Woodward
Medical Research InstituteBuffalo New York
Crystal Growth University of Washington
Seattle Washington
Structure Determination Unit 1University of Washington
Seattle Washington
Structure Determination Unit 2University of Washington
Seattle Washington
Flash Frozen Protein
Images
Crystals
SGPP Crystal Growth Protein Totals
• 333 Protein Samples Received of 192 Different Targets, one antibody and one target-antibody complex
Species # of Targets
Total # of Samples
# of SelMet Targets
# of SelMet Samples
L. aethiopica 4 4 0 0
L. braziliensis 2 4 1 1
L. donovani 8 10 2 2
L. guyaneis 1 1 1 1
L. infantum 4 7 0 0
L. major 139 242 50 61
L. mexicana 5 8 2 2
L. tarentolae 3 3 0 0
L. tropica 4 7 0 0
P. falciparum 22 47 6 9
Totals 192 333 62 76
SGPP Crystal Growth Lab Seattle
• Crystals from 26 targets sent downstream to the Structure Determination Units
• Crystals sent downstream represent 4 different species– 24 L. major – 2 P. falciparum – 1 L. donovani– 1 L. braziliensis
• 8 structures to date – 5 structures deposited into PDB
• 4 L. major • 1 P. falciparum
Robots in Seattle SCU
Oryx-6 Hydra II Plus 1
Acapella
RoboDesign
MicroScope II
Refined Optimization
Matrices Made by Hand
Initial Optimization or Screen
Database and Image Archive
Harvestable Crystals
Crystallographer Review Manual Scoring
Structure Determination UnitsLarry Desoto
SGPP Crystal Growth Bottleneck:Making Solutions
• Optimization matrices made by hand • Hydra II Plus 1 uses 96 deep well plates• 96 well plate set-up takes over 2 hours to prepare
SOLUTION:
Alchemist I
• Designed by Robodesign• Anticipated arrival April 2004• Set-up 96 deep well plate in 20
minutes
Where Will We Be?
Oryx-6 Hydra II Plus 1
Acapella
RoboDesign
MicroScope II
Refined Optimization Matrices Made by Hand
Initial Optimization or Screen
Database and Image Archive
Structure Determination Units
Crystal Track Designs Matrices
Larry Desoto
Crystallographer Review Manual Scoring
Harvestable Crystals
SGPP Crystal Growth Lab:Special Projects
Problem: How do we increase the success for getting crystals?
SGPP Approches
• Small Molecule Co-Crystallants; Erkang Fan, Christophe Verlinde UW
• Antibodies; Mark Sullivan UR
Co-crystallants and Crystallization (I)A Case Study
• Pfal008421WES; Uridine phosphorylase-like– Acyclovir is a potential inhibitor of Uridine phosphorylase-like
• Screening of the SulMet protein in absence of Acyclovir – SulMet produced 132 Hits at HWI– Optimization produced SulMet crystals that were ~35µm X 35µm
• Screening of the SelMet protein in absence Acyclovir – SelMet had 21 Hits at HWI– Optimization produced SelMet crystals that were ~50µm X 50µm– SDU #2 was able to mount SelMet crystals and obtain diffraction
at ALS
• 5 generations of optimization of protein without Acyclovir, DID NOT produce larger crystals
Co-crystallants and Crystallization (II)
• SulMet plus Acyclovir – SulMet-Acyclovir complex produced 235 hits
at HWI– One generation of optimization in Seattle
produced 100µm X 100µm crystals
• The best of the Pfal008421WES/Acyclovir complex hits were unique and DID NOT overlap with the initial SulMet hits
Co-crystallants and Crystallization (III)
0.1 M Ammonium Sulfate0.1 MOPS pH 6.525% PEG 3350Drop size 1ul + 1ul
Pfal008421WES Native Pfal008421WES Native plus Acyclovir
0.2 M Ammonium Sulfate0.1 M HEPES pH 7.525% PEG 33502.1mM AcyclovirDrop Size 0.4ul + 0.4ul
UNMOUNTABLE MOUNTABLE
SGPP Crystal Growth Lab:Special Projects
• DLS and Proteolysis– Problem: Is there any way to predict which
proteins will crystallize?
DLS and Limited Proteolysis (LP): Definitions (1)
Good
Medium
Bad
NS-nonstable proteinSF-stable fragmentSP-stable proteinSSF-superstable fragmentSSP-superstable protein
NS SF SP SSF SSP
0 1 24 0 1 24 0 1 24 0 1 240 1 24
DLS Limited Proteolysis
Oleksandr Kalyuzhniy
DLS and Limited Proteolysis (LP) as a Predictor of Crystallization (2)
0%
20%
40%
60%
80%
100%
Medium
Bad
Good
Medium 6 19 11 36
Bad 10 20 17 47
Good 29 29 25 83
Definite Questionable No hits Total
Hauptman Woodward Institute Results (July 2003-February 2004)Total………………….166 samples (100%)Definite……………….45 samples (27%)Questionable………...68 samples (41%)No hits………………..53 samples (32%)
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
NS
SF
SP
SSF
SSP
NS 6 10 21 37
SF 4 4 5 13
SP 5 6 6 17
SSF 2 6 1 9
SSP 10 5 5 20
Def inite Questionable No hits Total
166 samples, 141 ORFs 96 samples, 86 ORFs
DLS Limited Proteolysis
Oleksandr Kalyuzhniy
DLS and LP as a Predictor for Good Diffraction (3)
0%
20%
40%
60%
80%
100%
Medium
Bad
Good
Medium 1 0
Bad 3 0
Good 20 11
Harvestable Diffractors
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
NS
Stable
NS 1 0
Stable 18 7
Harvestable Diffractors
Seattle Optimization Results:• Harvestable Crystals-- 24 Targets• Good diffractors-- 11 Targets
DLS Limited Proteolysis
Oleksandr Kalyuzhniy
Crystal Growth Summary
• 333 Protein Samples Received– 192 Targets
• 26 targets sent to SDU• Crystal Track Database has been implemented • Alchemist Liquid Handling Gradient Maker
anticipated arrival April 2004• Known co-crystallant greatly improved
crystallization• If your protein has good DLS and is “stable”,
then the crystal is more likely to get good diffraction
Acknowledgments
• Thanks to all SGPP members especially:– The SGPP Protein Production Units– The SGPP HWI Crystal Screening – The SGPP Antibody Unit– The SGPP Co-crystallant Unit – The SGPP Structure Determination Units– NIGMS AND NIAID
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