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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

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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
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
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
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

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
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
Alchemist I
  • Designed by Robodesign
  • Anticipated arrival April 2004
  • Set-up 96 deep well plate in 20 minutes
where will we be

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
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
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
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
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 projects1
SGPP Crystal Growth Lab:Special Projects
  • DLS and Proteolysis
    • Problem: Is there any way to predict which proteins will crystallize?
slide14

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 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
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
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
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