protein and structure cores steve almo enzyme function initiative (efi) advisory committee meeting
DESCRIPTION
Protein and Structure Cores Steve Almo Enzyme Function Initiative (EFI) Advisory Committee Meeting November 30, 2011. Major Challenges. Distribution Logistics - Need to Equitably Service all Bridging Projects and Cores Aggressive protein production goals - PowerPoint PPT PresentationTRANSCRIPT
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