Protein Production and Crystallization Workshop

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

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

SGPP Crystal Growth Protein Totals • 333 Protein Samples Received of 192 Different Targets, one antibody and one target-antibody complex

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

Acapella Oryx-6 Hydra II Plus 1 RoboDesign MicroScope II Robots in Seattle SCU Initial Optimization or Screen Refined Optimization Matrices Made by Hand Crystallographer Review Manual Scoring Database and Image Archive Harvestable Crystals Structure Determination Units Larry 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

Acapella Hydra II Plus 1 Oryx-6 RoboDesign MicroScope II Where Will We Be? Initial Optimization or Screen Refined Optimization Matrices Made by Hand Crystal Track Designs Matrices Crystallographer Review Manual Scoring Database and Image Archive Harvestable Crystals Structure Determination Units Larry Desoto

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) Pfal008421WES Native Pfal008421WES 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 0.1 M Ammonium Sulfate 0.1 MOPS pH 6.5 25% PEG 3350 Drop size 1ul + 1ul 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) DLS Limited Proteolysis 0 1 24 0 1 24 0 1 24 0 1 24 0 1 24 Good Medium NS SF SP SSF SSP NS-nonstable protein SF-stable fragment SP-stable protein SSF-superstable fragment SSP-superstable protein Bad Oleksandr Kalyuzhniy

DLS and Limited Proteolysis (LP) as a Predictor of Crystallization (2) DLS Limited Proteolysis 166 samples, 141 ORFs 96 samples, 86 ORFs 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%) Oleksandr Kalyuzhniy

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

Protein Production and Crystallization Workshop