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Jeffrey B. Bonanno,
Steven C. Almo,
Anne Bresnick,
Mark R. Chance,
Andras Fiser,
S. Swaminathan,
J. Jiang,
F. William Studier,
Lawrence Shapiro,
Christopher D. Lima, [......],
Kevin Bain,
Ingeborg Feil, Xia Gao,
Don Lorimer,
Aurora Ramos,
J. Michael Sauder,
Steven R. Wasserman,
Spencer Emtage,
Kevin L. D’Amico,
Stephen K. Burley
[show abstract]
[hide abstract]
ABSTRACT: Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the
New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual
core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family
classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies,
structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC
will be capable of producing 100–200 experimentally determined protein structures annually. All Consortium activities can
be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes
both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported
by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal
Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed
of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org)
and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In
addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC
aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry.
The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR
spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural
biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress
toward development of a high-throughput Gene→Structure platform. An analysis of current and projected consortium metrics reflects
progress to date and delineates opportunities for further technology development.
Journal of Structural and Functional Genomics 08/2005; 6(2):225-232.
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Xia Gao,
Kevin Bain,
Jeffery B Bonanno,
Michelle Buchanan,
Davin Henderson,
Don Lorimer,
Curtis Marsh,
Julie A Reynes,
J Michael Sauder,
Ken Schwinn,
Chau Thai,
Stephen K Burley
[show abstract]
[hide abstract]
ABSTRACT: High-resolution structural information is important for improving our understanding of protein function in vitro and in vivo and providing information to enable drug discovery. The process leading to X-ray structure determination is often time consuming and labor intensive. It requires informed decisions in expression construct design, expression host selection, and strategies for protein purification, crystallization and structure determination. Previously published studies have demonstrated that compact globular domains defined by limited proteolysis represent good candidates for production of diffraction quality crystals [1-7]. Integration of mass spectrometry and proteolysis experiments can provide accurate definition of domain boundaries at unprecedented rates. We have conducted a critical evaluation of this approach with 400 target proteins produced by SGX (Structural GenomiX, Inc.) for the New York Structural GenomiX Research Consortium (NYSGXRC; http://www.nysgxrc.org) under the auspices of the National Institute of General Medical Sciences Protein Structure Initiative (http://www.nigms.nih.gov/psi). The objectives of this study were to develop parallel/automated protocols for proteolytic digestion and data acquisition for multiple proteins, and to carry out a systematic study to correlate domain definition via proteolysis with outcomes of crystallization and structure determination attempts. Initial results from this work demonstrate that proteins yielding diffraction quality crystals are typically resistant to proteolysis. Large-scale sub cloning and subsequent testing of expression, solubility, and crystallizability of proteolytically defined truncations is currently underway.
Journal of Structural and Functional Genomics 02/2005; 6(2-3):129-34.
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Jeffrey B Bonanno,
Steven C Almo,
Anne Bresnick,
Mark R Chance,
Andras Fiser,
S Swaminathan,
J Jiang,
F William Studier,
Lawrence Shapiro,
Christopher D Lima, [......],
Kevin Bain,
Ingeborg Feil, Xia Gao,
Don Lorimer,
Aurora Ramos,
J Michael Sauder,
Steven R Wasserman,
Spencer Emtage,
Kevin L D'Amico,
Stephen K Burley
[show abstract]
[hide abstract]
ABSTRACT: Structural GenomiX, Inc. (SGX), four New York area institutions, and two University of California schools have formed the New York Structural GenomiX Research Consortium (NYSGXRC), an industrial/academic Research Consortium that exploits individual core competencies to support all aspects of the NIH-NIGMS funded Protein Structure Initiative (PSI), including protein family classification and target selection, generation of protein for biophysical analyses, sample preparation for structural studies, structure determination and analyses, and dissemination of results. At the end of the PSI Pilot Study Phase (PSI-1), the NYSGXRC will be capable of producing 100-200 experimentally determined protein structures annually. All Consortium activities can be scaled to increase production capacity significantly during the Production Phase of the PSI (PSI-2). The Consortium utilizes both centralized and de-centralized production teams with clearly defined deliverables and hand-off procedures that are supported by a web-based target/sample tracking system (SGX Laboratory Information Data Management System, LIMS, and NYSGXRC Internal Consortium Experimental Database, ICE-DB). Consortium management is provided by an Executive Committee, which is composed of the PI and all Co-PIs. Progress to date is tracked on a publicly available Consortium web site (http://www.nysgxrc.org) and all DNA/protein reagents and experimental protocols are distributed freely from the New York City Area institutions. In addition to meeting the requirements of the Pilot Study Phase and preparing for the Production Phase of the PSI, the NYSGXRC aims to develop modular technologies that are transferable to structural biology laboratories in both academe and industry. The NYSGXRC PI and Co-PIs intend the PSI to have a transforming effect on the disciplines of X-ray crystallography and NMR spectroscopy of biological macromolecules. Working with other PSI-funded Centers, the NYSGXRC seeks to create the structural biology laboratory of the future. Herein, we present an overview of the organization of the NYSGXRC and describe progress toward development of a high-throughput Gene-->Structure platform. An analysis of current and projected consortium metrics reflects progress to date and delineates opportunities for further technology development.
Journal of Structural and Functional Genomics 01/2005; 6(2-3):225-32.
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Shane Atwell,
Jason M Adams,
John Badger,
Michelle D Buchanan,
Ingeborg K Feil,
Karen J Froning, Xia Gao,
Jörg Hendle,
Kevin Keegan,
Barbara C Leon,
Hans J Müller-Dieckmann,
Vicki L Nienaber,
Brian W Noland,
Kai Post,
K R Rajashankar,
Aurora Ramos,
Marijane Russell,
Stephen K Burley,
Sean G Buchanan
[show abstract]
[hide abstract]
ABSTRACT: Spleen tyrosine kinase (Syk) is a non-receptor tyrosine kinase required for signaling from immunoreceptors in various hematopoietic cells. Phosphorylation of two tyrosine residues in the activation loop of the Syk kinase catalytic domain is necessary for signaling, a phenomenon typical of tyrosine kinase family members. Syk in vitro enzyme activity, however, does not depend on phosphorylation (activation loop tyrosine --> phenylalanine mutants retain catalytic activity). We have determined the x-ray structure of the unphosphorylated form of the kinase catalytic domain of Syk. The enzyme adopts a conformation of the activation loop typically seen only in activated, phosphorylated tyrosine kinases, explaining why Syk does not require phosphorylation for activation. We also demonstrate that Gleevec (STI-571, Imatinib) inhibits the isolated kinase domains of both unphosphorylated Syk and phosphorylated Abl with comparable potency. Gleevec binds Syk in a novel, compact cis-conformation that differs dramatically from the binding mode observed with unphosphorylated Abl, the more Gleevec-sensitive form of Abl. This finding suggests the existence of two distinct Gleevec binding modes: an extended, trans-conformation characteristic of tight binding to the inactive conformation of a protein kinase and a second compact, cis-conformation characteristic of weaker binding to the active conformation. Finally, the Syk-bound cis-conformation of Gleevec bears a striking resemblance to the rigid structure of the nonspecific, natural product kinase inhibitor staurosporine.
Journal of Biological Chemistry 01/2005; 279(53):55827-32. · 4.77 Impact Factor
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Hal A Lewis,
Sean G Buchanan,
Stephen K Burley,
Kris Conners,
Mark Dickey,
Michael Dorwart,
Richard Fowler, Xia Gao,
William B Guggino,
Wayne A Hendrickson, [......],
Kai W Post,
Kanagalaghatta R Rajashankar,
Marc E Rutter,
J Michael Sauder,
Stephanie Shriver,
Patrick H Thibodeau,
Philip J Thomas,
Marie Zhang,
Xun Zhao,
Spencer Emtage
[show abstract]
[hide abstract]
ABSTRACT: Cystic fibrosis transmembrane conductance regulator (CFTR) is an ATP-binding cassette (ABC) transporter that functions as a chloride channel. Nucleotide-binding domain 1 (NBD1), one of two ABC domains in CFTR, also contains sites for the predominant CF-causing mutation and, potentially, for regulatory phosphorylation. We have determined crystal structures for mouse NBD1 in unliganded, ADP- and ATP-bound states, with and without phosphorylation. This NBD1 differs from typical ABC domains in having added regulatory segments, a foreshortened subdomain interconnection, and an unusual nucleotide conformation. Moreover, isolated NBD1 has undetectable ATPase activity and its structure is essentially the same independent of ligand state. Phe508, which is commonly deleted in CF, is exposed at a putative NBD1-transmembrane interface. Our results are consistent with a CFTR mechanism, whereby channel gating occurs through ATP binding in an NBD1-NBD2 nucleotide sandwich that forms upon displacement of NBD1 regulatory segments.
The EMBO Journal 02/2004; 23(2):282-93. · 9.20 Impact Factor
