#1 Bottleneck is Psychological!!! Structural Biology in the 3rd Millennium Structural Genomics Structural Proteomics Beyond Structure—Function and Mechanism! #1 Bottleneck is Psychological!!!
High-Throughput Structure Discovery
• Synchrotron Radiation 1960 Today PDB Entries ~20500 1 Mb Hb Lyso • Molecular Biology • Detectors (IP, CCD) • Software • Synchrotron Radiation
1999 Protein Structure Initiative (PSI) 9 National Centers with the mission to solve 10,000 structures in the next 10 years (5+5). ~20,500 Structures 4,000 unique (<90%) 1,500 Families ~700 distinct folds PDB contains: Vastly increase # of unique structures and families Approach 2-3K distinct folds in Nature *** 10K new structures: (if selected from distinct families)
NY Structural Genomics Research Consortium www.nysgrc.org Albert Einstein College of Medicine Brookhaven National Laboratory Columbia University Weill Medical College of Cornell University UCSF Structural GenomiX Inc.***
SUMMARY OF NYSGRC PROGRESS!! ~60 Structures Solved ~2500 Models ~45% of ALL Structures at X9A/B Goal: 100-200 structures/yr.
Swaminathan, et al.
• Mevalonate pyrophosphate decarboxylase ~3,000 Å2 interface Burley, et al.
MPD Sequence and Secondary Structure Alignment white green 30% 100%
Detection of a Conserved Cleft Dimer Interface white green 30% 100%
Opportunities/Target Selection: Functional Studies: Kinases (122) Phosphatases (~30) Regulatory Domains Nucleic acid modifying Enzymes Biologically Driven: Neurobiology Motile Processes Immunology Pathogens and Virulence Factors Individual Pieces Fit into the Larger Complex Puzzle
function with putative essential roles Systematic examination of essential genes in pathogens: 259 ORFs of unknown function with putative essential roles
Cell Motility/Actin Cytoskeleton
Las17 Arp2/3 Pan1 Abp1 Ent1 Ent2 End3 Yap180 Yor284w Srv2 Rvs167 Cla4 JCB 154, 549-571 (2001) Ent1 Ent2 End3 Yap180 Yor284w Srv2 Rvs167 Cla4 Ynl094w Sla1 Yhr133c Ypl246c Arp2/3 Pan1 Abp1 Las17
ABP1 ?????? Cofilin Homology SH3 Strokopytov, et al
Srv2/Cyclase Associated Protein (CAP) PPPPP 1 168 370 526 Adenyl Cyclase SH3 Actin/Oligomerization Fedorov, et al.
Las17 Arp2/3 Pan1 Abp1 Ypl246c Sla1 Yhr133c Rvs167 Yap180 Ynl094w Srv2 ? Aip1 Ent1 Ent2 End3 Yap180 Yor284w Srv2 Rvs167 Cla4 Ynl094w Sla1 Yhr133c Ypl246c Pan1 Abp1 Las17 Arp2/3
Aip1 Nine WD40 Motifs
1.6 Å: Solved in 12 Hours! Vorobiev, et al.
ABP1 ? ? ? ABP1 ABP1 Arp2/3 CAP CAP CAP F - Actin C N C N C N N N C C Cofilin HD ? ? ? F - Actin Cofilin HD Cofilin HD Cofilin HD Cofilin Cofilin Cofilin Cofilin HD HD HD HD ABP1 ABP1 Arp2/3 SH3 SH3 SH3 SH3 C N PP C N PP CAP PP PP SH3 SH3 C N N N PP PP CAP CAP Cofilin C C C C C N N N N Aip1 Adenylate Adenylate Cyclase Cyclase Actin Actin Actin Ras Ras
High-Throughput Complex Analysis
Tight, stoichiometric complexes Rout & Akey; 98
New Crystallization Platforms INDUSTRIAL SCALE PROCESS! Protein limited: 10-100 mg from 10s of liters of yeast New Crystallization Platforms and/or INDUSTRIAL SCALE PROCESS! Absolute requirement for dedicated Synchrotron access!!!
RNAP-DNA Complex ~6.5 Å Darst, et al. Tubulin-Stathmin ~4.0 Å Knossow, et al. F1F0 ATP Synthase ~4.0 Å Leslie, Walker, et al.
Range of Resolutions High Resolution (X-ray) 3.0 Å or better Intermediate Resolution (X-ray/EM) 4-10 Å Modest Resolution (EM) 10-30 Å
Footprinting of Partial Complexes
#1 Bottleneck is Psychological!!! Structural Biology in the 3rd Millennium Structural Genomics Structural Proteomics In vivo structure function relationships #1 Bottleneck is Psychological!!!