Çağdaş DAĞ
STRUCTURAL GENOMICS
What is Structural Genomics ?
Is the process of high-throughtput determination of 3-D structures of Biological macromolecules
What is the Goal of Structural Genomics ?
Provision of enough structural templates to facilitate homology modeling of most proteins.
Structural elucidation of complete biological pathway
To determine new protein folds
To determine the functions of unknown proteins
to develop improved methods that will result in high-throughput biology and protein structure determination
What can we learn from structure ?
The structure will reveal the fold of the protein
The structure will reveal the active site
The structure may reveal evolutionary links between proteins lacking sequence similarity
The structure may reveal the function of the protein
Overview Structural Genomics
Target Selection
Complete organisms;thermophilic, mesophilic or halophiliceukaryotic or prokaryotic
HUMAN DISEASETargets in this area of structural genomics are G-protein coupled receptors, ion channels and kinases etc.
The development of techniques to allow over-expression, purification and crystallization of these targets is required and in progress
Cloning, Expression and Purification
Develop technologies necessary for large-scale initiatives
develop high-throughput (HTP) cloning
develop high-throughput expression
develop high-throughput purification
NMR SpectroscopyProtein<20 kDa
N15 C13 Label NMR
Aggregated, Unfolded
Folded
Structure
Protein-ProteinInteractions
Co-Expression
Stable Domain
Insoluble
Protein-ProteinInteractions
Limited Proteolysis
Co-Expression
Soluble
Expression Purification
Crystal Trials
Protein (>20kDa)
CRYSTALLOGRAPHYSTRUCTURE DETERMINATION
STRUCTURAL GENOMIC CENTERS
BIGS - Bacterial Targets at IGS-CNRS, FranceBSGC - Berkeley Structural Genomics Center, USACESG - Center for Eukaryotic Structural Genomics, USAISPC - Israel Structural Proteomics Center, IsrealJCSG - Joint Center for Structural Genomics, USAMCSG - The Midwest Center for Structural Genomics, USA, UK, CanadaNESG - Northeast Structural Genomics Consortium, USANYSGRC - New York SGXRC - New York Structural Genomics Research Consortium, USAOPPF - Oxford Protein Production Facility, UKPSF - Protein Structure Factory, GermanyRIKEN - RIKEN Structural Genomics Initiative, JapanS2F - Structure 2 Function ProjectSECSG - SouthEast Collaboratory for Structural Genomics, USASGPP - Structural Genomics of Pathogenic Protozoa Consortium, USASPEX Db - (BSGI) - Montreal-Kingston Bacterial Structural Genomics Initiative, CanadaSPINE - Structural Proteomics in Europe, EUTB - Mycobacterium tuberculosis (TB) Structural Genomics Consortium, 70 labs from 12 countriesXMTB - Mycobacterium Tuberculosis Structural Proteomics Project, Germany
The first phase of the Protein Structure Initiative (PSI-1) lasted from June 2000 until September 2005, and had a budget of $270 million funded primarily by NIGMS
The second phase of the Protein Structure Initiative (PSI-2) lasted from July 2005 to June 2010PSI-2 had a five-year budget of $325 million provided by NIGMS
PSI grant period LSC ALL-PSI
PSI-2 grant year 2005 390427
PSI-2 grant year 2006 620700
PSI-2 grant year 2007 682718
PSI-2 grant year 2008 796818
PSI-2 grant year 2009 822879
PSI-2 grant extension 217244
PSI-2 total depositions 3527 3786
PSI-3 grant year 2010 280283
PSI-3 total depositions 377381
PSI-1 + PSI-2 + PSI-3 4804 5583
Determinated Protein Structures
The Northeast Structural Genomics (NESG) consortium
Production stage NESG targets
cloned 23,695Expression tested 22,159Expressed (E > 0) 16,888Expressed-Soluble (S > 0) 12,453Expressed-Soluble (ES >= 9) 9,946Fermented 7,271Purified (>= 0.5 mg yield) 4,848HSQC spectra recorded 2,597"Good" HSQC spectra 1,353HTP crystal screening 2,522Crystal hit 1,295X-Ray structures 547
NMR structures 452Total structures 958
Human proteins Production stage NESGtargets
cloned 5,479
Expression tested5,046
Expressed (E > 0)3,986
Expressed-Soluble (S > 0) 2,599
Expressed-Soluble (ES >= 9) 2,051Fermented
1,097Purified (>= 0.5 mg yield) 813
HSQC spectra recorded 422"Good" HSQC spectra 235HTP crystal screening 320Crystal hit
130X-Ray structures
35NMR structures
66Total structures
93
The Midwest Center for Structural Genomics (MCSG)
The Center for Eukaryotic Structural Genomics-National Magnetic Resonance Facility-Madison
University of Wisconsin-Madison