1. Protein Production and Crystallization Workshop Structural Genomics of Pathogenic Protozoa (SGPP) Crystal Growth Lab Lori Anderson www.sgpp.org

2. SGPP Crystal Growth Pipeline Protein Production Unit University of Washington Seattle Washington Protein Production Unit University of Rochester Rochester New York Crystal Screening Hauptman Woodward Medical Research Institute Buffalo New York Crystal Growth University of Washington Seattle Washington Structure Determination Unit 1 University of Washington Seattle Washington Structure Determination Unit 2 University of Washington Seattle Washington Flash Frozen Protein Images Crystals

3. 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. aethiopica4400 L. braziliensis2411 L. donovani81022 L. guyaneis1111 L. infantum4700 L. major1392425061 L. mexicana5822 L. tarentolae3300 L. tropica4700 P. falciparum224769 Totals1923336276

4. 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

5. 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 Units Larry Desoto

6. 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:

7. Alchemist I Designed by Robodesign Anticipated arrival April 2004 Set-up 96 deep well plate in 20 minutes

8. 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

9. 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

10. 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

11. 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

12. Co-crystallants and Crystallization (III) 0.1 M Ammonium Sulfate 0.1 MOPS pH 6.5 25% PEG 3350 Drop size 1ul + 1ul Pfal008421WES NativePfal008421WES Native plus Acyclovir 0.2 M Ammonium Sulfate 0.1 M HEPES pH 7.5 25% PEG 3350 2.1mM Acyclovir Drop Size 0.4ul + 0.4ul UNMOUNTABLEMOUNTABLE

13. SGPP Crystal Growth Lab: Special Projects DLS and Proteolysis –Problem: Is there any way to predict which proteins will crystallize?

14. DLS and Limited Proteolysis (LP): Definitions (1) Good Medium Bad NS-nonstable protein SF-stable fragment SP-stable protein SSF-superstable fragment SSP-superstable protein NSSFSPSSFSSP 0 1 24 DLS Limited Proteolysis Oleksandr Kalyuzhniy

15. DLS and Limited Proteolysis (LP) as a Predictor of Crystallization (2) 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%) 166 samples, 141 ORFs96 samples, 86 ORFs DLSLimited Proteolysis Oleksandr Kalyuzhniy

16. DLS and LP as a Predictor for Good Diffraction (3) Seattle Optimization Results: Harvestable Crystals-- 24 Targets Good diffractors-- 11 Targets DLS Limited Proteolysis Oleksandr Kalyuzhniy

17. 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

18. 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