Journal of Structural and Functional Genomics

Fully automated high-quality NMR structure determination of small (2)H-enriched proteins.

Publication Date: 2010 Aug 24 PMID: 20734145
Authors: Tang, Y. - Schneider, W. M. - Shen, Y. - Raman, S. - Inouye, M. - Baker, D. - Roth, M. J. - Montelione, G. T.
Journal: J Struct Funct Genomics

Determination of high-quality small protein structures by nuclear magnetic resonance (NMR) methods generally requires acquisition and analysis of an extensive set of structural constraints. The process generally demands extensive backbone and sidechain resonance assignments, and weeks or even months of data collection and interpretation. Here we demonstrate rapid and high-quality protein NMR structure generation using CS-Rosetta with a perdeuterated protein sample made at a significantly reduced cost using new bacterial culture condensation methods. Our strategy provides the basis for a high-throughput approach for routine, rapid, high-quality structure determination of small proteins. As an example, we demonstrate the determination of a high-quality 3D structure of a small 8 kDa protein, E. coli cold shock protein A (CspA), using <4 days of data collection and fully automated data analysis methods together with CS-Rosetta. The resulting CspA structure is highly converged and in excellent agreement with the published crystal structure, with a backbone RMSD value of 0.5 A, an all atom RMSD value of 1.2 A to the crystal structure for well-defined regions, and RMSD value of 1.1 A to crystal structure for core, non-solvent exposed sidechain atoms. Cross validation of the structure with (15)N- and (13)C-edited NOESY data obtained with a perdeuterated (15)N, (13)C-enriched (13)CH(3) methyl protonated CspA sample confirms that essentially all of these independently-interpreted NOE-based constraints are already satisfied in each of the 10 CS-Rosetta structures. By these criteria, the CS-Rosetta structure generated by fully automated analysis of data for a perdeuterated sample provides an accurate structure of CspA. This represents a general approach for rapid, automated structure determination of small proteins by NMR.

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The New York Consortium on Membrane Protein Structure (NYCOMPS): a high-throughput platform for structural genomics of integral membrane proteins.

Publication Date: 2010 Sep PMID: 20690043
Authors: Love, J. - Mancia, F. - Shapiro, L. - Punta, M. - Rost, B. - Girvin, M. - Wang, D. N. - Zhou, M. - Hunt, J. F. - Szyperski, T. - Gouaux, E. - MacKinnon, R. - McDermott, A. - Honig, B. - Inouye, M. - Montelione, G. - Hendrickson, W. A.
Journal: J Struct Funct Genomics

The New York Consortium on Membrane Protein Structure (NYCOMPS) was formed to accelerate the acquisition of structural information on membrane proteins by applying a structural genomics approach. NYCOMPS comprises a bioinformatics group, a centralized facility operating a high-throughput cloning and screening pipeline, a set of associated wet labs that perform high-level protein production and structure determination by x-ray crystallography and NMR, and a set of investigators focused on methods development. In the first three years of operation, the NYCOMPS pipeline has so far produced and screened 7,250 expression constructs for 8,045 target proteins. Approximately 600 of these verified targets were scaled up to levels required for structural studies, so far yielding 24 membrane protein crystals. Here we describe the overall structure of NYCOMPS and provide details on the high-throughput pipeline.

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Engineering of a wheat germ expression system to provide compatibility with a high throughput pET-based cloning platform.

Publication Date: 2010 Sep PMID: 20574660
Authors: Zhao, L. - Zhao, K. Q. - Hurst, R. - Slater, M. R. - Acton, T. B. - Swapna, G. V. - Shastry, R. - Kornhaber, G. J. - Montelione, G. T.
Journal: J Struct Funct Genomics

Wheat germ cell-free methods provide an important approach for the production of eukaryotic proteins. We have developed a protein expression vector for the TNT((R)) SP6 High-Yield Wheat Germ Cell-Free (TNT WGCF) expression system (Promega) that is also compatible with our T7-based Escherichia coli intracellular expression vector pET15_NESG. This allows cloning of the same PCR product into either one of several pET_NESG vectors and this modified WGCF vector (pWGHisAmp) by In-Fusion LIC cloning (Zhu et al. in Biotechniques 43:354-359, 2007). Integration of these two vector systems allowed us to explore the efficacy of the TNT WGCF system by comparing the expression and solubility characteristics of 59 human protein constructs in both WGCF and pET15_NESG E. coli intracellular expression. While only 30% of these human proteins could be produced in soluble form using the pET15_NESG based system, some 70% could be produced in soluble form using the TNT WGCF system. This high success rate underscores the importance of eukaryotic expression host systems like the TNT WGCF system for eukaryotic protein production in a structural genomics sample production pipeline. To further demonstrate the value of this WGCF system in producing protein suitable for structural studies, we scaled up, purified, and analyzed by 2D NMR two (15)N-, (13)C-enriched human proteins. The results of this study indicate that the TNT WGCF system is a successful salvage pathway for producing samples of difficult-to-express small human proteins for NMR studies, providing an important complementary pathway for eukaryotic sample production in the NESG NMR structure production pipeline.

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To automate or not to automate: this is the question.

Publication Date: 2010 Sep PMID: 20526815
Authors: Cymborowski, M. - Klimecka, M. - Chruszcz, M. - Zimmerman, M. D. - Shumilin, I. A. - Borek, D. - Lazarski, K. - Joachimiak, A. - Otwinowski, Z. - Anderson, W. - Minor, W.
Journal: J Struct Funct Genomics

New protocols and instrumentation significantly boost the outcome of structural biology, which has resulted in significant growth in the number of deposited Protein Data Bank structures. However, even an enormous increase of the productivity of a single step of the structure determination process may not significantly shorten the time between clone and deposition or publication. For example, in a medium size laboratory equipped with the LabDB and HKL-3000 systems, we show that automation of some (and integration of all) steps of the X-ray structure determination pathway is critical for laboratory productivity. Moreover, we show that the lag period after which the impact of a technology change is observed is longer than expected.

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