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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. Major Challenges. Distribution Logistics - Need to Equitably Service all Bridging Projects and Cores Aggressive protein production goals

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Protein and Structure Cores Steve Almo Enzyme Function Initiative (EFI) Advisory Committee Meeting

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  1. Protein and Structure Cores Steve Almo Enzyme Function Initiative (EFI) Advisory Committee Meeting November 30, 2011

  2. Major Challenges • 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

  3. Milestones • ~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

  4. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Biomek Fx -- Beckman Cloning Distribution

  5. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Cloning Cloning Distribution LIC His/Strep “universal”

  6. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Cloning Part 2 Cloning Distribution

  7. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Small-scale Expression Cloning Distribution

  8. Sonication Robot

  9. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Small-scale Expression Cloning Distribution

  10. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Small-scale Expression Cloning Distribution Caliper: labchip

  11. Construct Rescue Initially ALL were non-cleavable C-terminal His-tags 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!

  12. Lysis Screen – .5M NaClvs .2M AmSO4 300 Constructs tested: 100 AH : 20% Rescue 100 EN : 0% Rescue 100 SINO: 20% Rescue

  13. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Scale-up Expression Cloning Distribution QA/QC - Every clone sent to large scale fermentation is sent out for sequencing with overnight turnaround

  14. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Purification Cloning Distribution

  15. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Quality Control Cloning Distribution MALDI on all PID on all ESI only as required

  16. Small Scale Expression Evaluation Scale-up Expression QA/QC Purification Distribution Cloning Distribution

  17. EFI Protein Production Progress May-Oct 2011

  18. ThermoFluor-EN 502246-L2 (EN) Hit Summary

  19. ThermoFluor-GST 501014-L1 (GST) Hit Summary

  20. Structure Determination Art Robbins Phoenixcrystallization Formulatrix-1000Crystal Plate Imaging

  21. Crystallization Statistics May-Oct 2011 New Crystallization ScreensMicrolytic MCSG 1-4 (384 conditions)Based on successful MCSG crystallization hits Utilization of the NSLS X29 and APS LRL-CAT (mail-in)

  22. IS Structures CRYSTAL STRUCTURE OF A PUTATIVE FARNESYL PYROPHOSPHATE SYNTHETASE FROM MARINOMONAS sp. MED121 (EFI-501980) Crystal structure of isoprenoidsynthase (EFI-501974) from clostridium perfringens(3UCA) Side View Top View

  23. IS Inhibitors

  24. GST Structures Leptospira interrogans EFI-501770) with GSH bound (3UBL) Methylococcus capsulatus EFI-501774 with GSH bound (3UAR) Ralstonia solanacearum EFI-501058 with acetate bound (3TOT) Ralstonia solanacearum EFI-501058 with GSH bound (3TOU)

  25. Representative Enolase Structures Sphingomonas sp. EFI -501683 with bound Mg2+(3THU) Enterobacter EFI-501662 with bound Mg2+(3TJI) Agrobacterium EFI-502087 No Mg (3TJ4) Alpha proteobacterium EFI-501650 with bound Mg2+(XXX)

  26. Loop Dynamics in ENs: 501676 501676 with bound gluconateSubunit A (Red) vs. Subunit B (Yellow) Catalytic tyrosine brought to active conformation by binding of Mg2+ and acid-sugar

  27. Transition from unliganded “open” to liganded “closed”: 501679 High pH+ Ligand Low pH High pH  Catalytic tyrosine brought to active conformation by binding of Mg2+ and acid sugar  Glycerol mimics sugar binding to Q45 and D331 and presents hydrophobic face to Leu168  Leu168 queries the distal end of the acid sugar Overlay of 501676 (green) and 501679 (cyan). Tyr158 and Leu168

  28. 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

  29. Leverage and 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

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