protein and structure cores steve almo enzyme function initiative (efi) advisory committee meeting

Protein and Structure Cores

Steve Almo

Enzyme Function Initiative (EFI)Advisory Committee Meeting

November 30, 2011

• Distribution Logistics - Need to Equitably Service all Bridging Projects and Cores

• Aggressive protein production goals• Aggressive structure determination goals• Functional analysis -- ThermoFluor• cDNA/synthetic gene acquisition• Small molecule libraries for screening• Balance between pipeline technology development and

production efforts • Realize increased efficiencies and cost savings• Local instrumentation and personnel• Providing access to EFI infrastructure to the community

A wealth of experience from PSI

Major Challenges

• ~2500 expression vectors/yr. (dual vector approach)• ~1000 fermentations/yr. (2000-3000 liters of

fermentation/yr)• Deliver at least 400 sulfur-met samples to the Bridging

Projects• Deliver at least 400 samples to the Structure Core• 35-40 Structures/year• 35-40 Liganded Structures/year

– Dockable Structures• ThermoFluor Analysis of all

– Functional insight• Technology/Process Development

Milestones

Biomek Fx -- Beckman

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

Cloning

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

LIC His/Strep“universal”

Cloning Part 2

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

Small-scale Expression

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

Sonication Robot

Small-scale Expression

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

Small-scale Expression

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

Caliper: labchip

AHs – C-terminal His-Strep Tags

HADs – C-terminal His-Strep Tags N-term/cut

GSTs, ISs – N-terminal His-Tag cut

ENs – First C then N then tagless N again!

Initially ALL were non-cleavable C-terminal His-tags

Construct Rescue

300 Constructs tested:

100 AH : 20% Rescue100 EN : 0% Rescue 100 SINO: 20% Rescue

Lysis Screen – .5M NaCl vs .2M AmSO4

Scale-up Expression

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

QA/QC - Every clone sent to large scale fermentation is sent out for sequencing with overnight turnaround

Purification

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

Quality Control

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

MALDI on allPID on allESI only as required

Distribution

Small Scale Expression Evaluation

Scale-up Expression Purification QA/QC DistributionCloning

Clones Soluble Unique Soluble Purified Shipped

AH 23 12 12 75 60

EN 663 320 236 187 106

HAD 225 137 125 170 126

GST 292 147 127 100 80

IS 256 99 75 111 81

Total 1459 715 575 643 453

EFI Protein Production ProgressMay-Oct 2011

502246-L2 (EN)Hit Summary

ThermoFluor-EN

501014-L1 (GST)Hit Summary

ThermoFluor-GST

Art Robbins Phoenixcrystallization

Formulatrix-1000Crystal Plate Imaging

Structure Determination

Targets Trays Initial Screens with

ThermoFluor Hits

Additive Structures

211 713 52 10 ~30

Crystallization StatisticsMay-Oct 2011

New Crystallization Screens

Microlytic MCSG 1-4 (384 conditions)

Based on successful MCSG crystallization hits

Utilization of the NSLS X29 and APS LRL-CAT (mail-in)

Side View

Top View

CRYSTAL STRUCTURE OF A PUTATIVE FARNESYL PYROPHOSPHATE

SYNTHETASE FROM MARINOMONAS sp. MED121 (EFI-501980)

CRYSTAL STRUCTURE OF ISOPRENOID SYNTHASE (EFI-501974) FROM CLOSTRIDIUM PERFRINGENS (3UCA)

IS Structures

IS Inhibitors

GST Structures

Ralstonia solanacearum EFI-501058

with GSH bound (3TOU)

Ralstonia solanacearum EFI-501058

with acetate bound (3TOT)

Methylococcus capsulatus EFI-501774

with GSH bound (3UAR)

Leptospira interrogans EFI-501770)

with GSH bound (3UBL)

Representative Enolase Structures

Agrobacterium EFI-502087

No Mg (3TJ4)

EnterobacterEFI-501662

with bound Mg2+ (3TJI)

Alpha proteobacterium EFI-501650

with bound Mg2+ (XXX)

Sphingomonas sp. EFI -501683

with bound Mg2+ (3THU)

501676 with bound gluconateSubunit A (Red) vs. Subunit B (Yellow)

Catalytic tyrosine brought to active conformation by binding of Mg2+ and acid-sugar

Loop Dynamics in ENs: 501676

Transition from unliganded “open” to liganded “closed”: 501679

Low pH High pHHigh pH+ Ligand

Catalytic tyrosine brought to active conformation by binding of Mg2+ and acid sugar Glycerol mimics sugar bindingto Q45 and D331 and presentshydrophobic face to Leu168 Leu168 queries the distal end of the acid sugar

Overlay of 501676 (green) and 501679 (cyan). Tyr158 and Leu168

CRYSTAL STRUCTURE OF A PUTATIVE NAD(P) DEPENDENT GLUCONATE 5-DEHYDROGENASE FROM BEUTENBERGIA

CAVERNAE (EFI-502044) WITH BOUND NADP (3UF0)

The First Operon Protein Structure Determined by the EFI

Exhibits 2-keto-3-deoxy-gluconate dehydrogenase activity

Community Target Program is 15% of PSI Center Efforts– NYSGRC currently focuses on mechanistically diverse

enzyme superfamilies • PPG on Enolases and Amidohydrolases (Gerlt, Raushel)

– Now including Crotonase & Rubisco superfamilies – Have additional band-width for other superfamilies (Chris

Whitman; UT Austin)• Tautomerase and fumarylacetoacetate hydrolase (FAH)

superfamilies

Leverage and