RESUMO
Homology modeling is a powerful technique that greatly increases the value of experimental structure determination by using the structural information of one protein to predict the structures of homologous proteins. We have previously described a method of homology modeling by satisfaction of spatial restraints (Li et al., Protein Sci 1997;6:956-970). The Homology Modeling Automatically (HOMA) web site,
Assuntos
Proteínas/química , Software , Modelos Moleculares , Conformação ProteicaRESUMO
Structural genomics projects are providing large quantities of new 3D structural data for proteins. To monitor the quality of these data, we have developed the protein structure validation software suite (PSVS), for assessment of protein structures generated by NMR or X-ray crystallographic methods. PSVS is broadly applicable for structure quality assessment in structural biology projects. The software integrates under a single interface analyses from several widely-used structure quality evaluation tools, including PROCHECK (Laskowski et al., J Appl Crystallog 1993;26:283-291), MolProbity (Lovell et al., Proteins 2003;50:437-450), Verify3D (Luthy et al., Nature 1992;356:83-85), ProsaII (Sippl, Proteins 1993;17: 355-362), the PDB validation software, and various structure-validation tools developed in our own laboratory. PSVS provides standard constraint analyses, statistics on goodness-of-fit between structures and experimental data, and knowledge-based structure quality scores in standardized format suitable for database integration. The analysis provides both global and site-specific measures of protein structure quality. Global quality measures are reported as Z scores, based on calibration with a set of high-resolution X-ray crystal structures. PSVS is particularly useful in assessing protein structures determined by NMR methods, but is also valuable for assessing X-ray crystal structures or homology models. Using these tools, we assessed protein structures generated by the Northeast Structural Genomics Consortium and other international structural genomics projects, over a 5-year period. Protein structures produced from structural genomics projects exhibit quality score distributions similar to those of structures produced in traditional structural biology projects during the same time period. However, while some NMR structures have structure quality scores similar to those seen in higher-resolution X-ray crystal structures, the majority of NMR structures have lower scores. Potential reasons for this "structure quality score gap" between NMR and X-ray crystal structures are discussed.
Assuntos
Proteínas/química , Proteínas/metabolismo , Biologia Computacional , Cristalografia por Raios X , Bases de Dados de Proteínas , Genômica , Humanos , Modelos Moleculares , Ressonância Magnética Nuclear Biomolecular , Estrutura Terciária de Proteína , Proteínas/genética , Design de SoftwareRESUMO
Tropomyosin is a coiled-coil protein that binds head-to-tail along the length of actin filaments in eukaryotic cells, stabilizing them and providing protection from severing proteins. Tropomyosin cooperatively regulates actin's interaction with myosin and mediates the Ca2+ -dependent regulation of contraction by troponin in striated muscles. The N-terminal and C-terminal ends are critical functional determinants that form an "overlap complex". Here we report the solution NMR structure of an overlap complex formed of model peptides. In the complex, the chains of the C-terminal coiled coil spread apart to allow insertion of 11 residues of the N-terminal coiled coil into the resulting cleft. The plane of the N-terminal coiled coil is rotated 90 degrees relative to the plane of the C terminus. A consequence of the geometry is that the orientation of postulated periodic actin binding sites on the coiled-coil surface is retained from one molecule to the next along the actin filament when the overlap complex is modeled into the X-ray structure of tropomyosin determined at 7 Angstroms. Nuclear relaxation NMR data reveal flexibility of the junction, which may function to optimize binding along the helical actin filament and to allow mobility of tropomyosin on the filament surface as it switches between regulatory states.
Assuntos
Actinas/metabolismo , Tropomiosina/química , Sequência de Aminoácidos , Animais , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Complexos Multiproteicos , Ressonância Magnética Nuclear Biomolecular , Peptídeos/química , Peptídeos/genética , Peptídeos/metabolismo , Ligação Proteica , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Ratos , Tropomiosina/metabolismoRESUMO
A standardized protocol enabling rapid NMR data collection for high-quality protein structure determination is presented that allows one to capitalize on high spectrometer sensitivity: a set of five G-matrix Fourier transform NMR experiments for resonance assignment based on highly resolved 4D and 5D spectral information is acquired in conjunction with a single simultaneous 3D 15N,13C(aliphatic),13C(aromatic)-resolved [1H,1H]-NOESY spectrum providing 1H-1H upper distance limit constraints. The protocol was integrated with methodology for semiautomated data analysis and used to solve eight NMR protein structures of the Northeast Structural Genomics Consortium pipeline. The molecular masses of the hypothetical target proteins ranged from 9 to 20 kDa with an average of approximately 14 kDa. Between 1 and 9 days of instrument time were invested per structure, which is less than approximately 10-25% of the measurement time routinely required to date with conventional approaches. The protocol presented here effectively removes data collection as a bottleneck for high-throughput solution structure determination of proteins up to at least approximately 20 kDa, while concurrently providing spectra that are highly amenable to fast and robust analysis.