GeNMR: a web server for rapid NMR-based protein structure determination

GeNMR (GEnerate NMR structures) is a web server for rapidly generating accurate 3D protein structures using sequence data, NOE-based distance restraints and/or NMR chemical shifts as input. GeNMR accepts distance restraints in XPLOR or CYANA format as well as chemical shift files in either SHIFTY or BMRB formats. The web server produces an ensemble of PDB coordinates for the protein within 15–25 min, depending on model complexity and completeness of experimental restraints. GeNMR uses a pipeline of several pre-existing programs and servers to calculate the actual protein structure. In particular, GeNMR combines genetic algorithms for structure optimization along with homology modeling, chemical shift threading, torsion angle and distance predictions from chemical shifts/NOEs as well as ROSETTA-based structure generation and simulated annealing with XPLOR-NIH to generate and/or refine protein coordinates. GeNMR greatly simplifies the task of protein structure determination as users do not have to install or become familiar with complex stand-alone programs or obscure format conversion utilities. Tests conducted on a sample of 90 proteins from the BioMagResBank indicate that GeNMR produces high-quality models for all protein queries, regardless of the type of NMR input data. GeNMR was developed to facilitate rapid, user-friendly structure determination of protein structures via NMR spectroscopy. GeNMR is accessible at http://www.genmr.ca

NMR Constraints Analyser: a web-server for the graphical analysis of NMR experimental constraints

Nuclear magnetic resonance (NMR) spectroscopy together with X-ray crystallography, are the main techniques used for the determination of high-resolution 3D structures of biological molecules. The output of an NMR experiment includes a set of lower and upper limits for the distances (constraints) between pairs of atoms. If the number of constraints is high enough, there will be a finite number of possible conformations (models) of the macromolecule satisfying the data. Thus, the more constraints are measured, the better defined these structures will be. The availability of a user-friendly tool able to help in the analysis and interpretation of the number of experimental constraints per residue, is thus of valuable importance when assessing the levels of structure definition of NMR solved biological macromolecules, in particular, when high-quality structures are needed in techniques such as, computational biology approaches, site-directed mutagenesis experiments and/or drug design. Here, we present a free publicly available web-server, i.e. NMR Constraints Analyser, which is aimed at providing an automatic graphical analysis of the NMR experimental constraints atom by atom. The NMR Constraints Analyser server is available from the web-page http://molsim.sci.univr.it/constrain

Engineering a two-helix bundle protein for folding studies

The SAP domain from the Saccharomyces cerevisiae THO1 protein contains a hydrophobic core and just two α-helices. It could provide a system for studying protein folding that bridges the gap between studies on isolated helices and those on larger protein domains. We have engineered the SAP domain for protein folding studies by inserting a tryptophan residue into the hydrophobic core (L31W) and solved its structure. The helical regions had a backbone root mean-squared deviation of 0.9 Å from those of wild type. The mutation L31W destabilised wild type by 0.8 ± 0.1 kcal mol−1. The mutant folded in a reversible, apparent two-state manner with a microscopic folding rate constant of around 3700 s−1 and is suitable for extended studies of folding

PROSESS: a protein structure evaluation suite and server

PROSESS (PROtein Structure Evaluation Suite and Server) is a web server designed to evaluate and validate protein structures generated by X-ray crystallography, NMR spectroscopy or computational modeling. While many structure evaluation packages have been developed over the past 20 years, PROSESS is unique in its comprehensiveness, its capacity to evaluate X-ray, NMR and predicted structures as well as its ability to evaluate a variety of experimental NMR data. PROSESS integrates a variety of previously developed, well-known and thoroughly tested methods to evaluate both global and residue specific: (i) covalent and geometric quality; (ii) non-bonded/packing quality; (iii) torsion angle quality; (iv) chemical shift quality and (v) NOE quality. In particular, PROSESS uses VADAR for coordinate, packing, H-bond, secondary structure and geometric analysis, GeNMR for calculating folding, threading and solvent energetics, ShiftX for calculating chemical shift correlations, RCI for correlating structure mobility to chemical shift and PREDITOR for calculating torsion angle-chemical shifts agreement. PROSESS also incorporates several other programs including MolProbity to assess atomic clashes, Xplor-NIH to identify and quantify NOE restraint violations and NAMD to assess structure energetics. PROSESS produces detailed tables, explanations, structural images and graphs that summarize the results and compare them to values observed in high-quality or high-resolution protein structures. Using a simplified red–amber–green coloring scheme PROSESS also alerts users about both general and residue-specific structural problems. PROSESS is intended to serve as a tool that can be used by structure biologists as well as database curators to assess and validate newly determined protein structures. PROSESS is freely available at http://www.prosess.ca

PERMOL: Restraint-based protein homology modeling using DYANA or CNS.

PERMOL is a new restraint-based program for homology modeling of proteins. Restraints are generated from the information contained in structures of homologous template proteins. Employing the restraints generated by PERMOL, three-dimensional structures are obtained using MD programs such as DYANA or CNS. In contrast to other programs PERMOL is mainly based on the use of dihedral angle information which is optimally suited to preserve the local secondary structure. The global arrangement of these elements is then facilitated by a small number of distance restraints. Using PERMOL homology, models of high quality are obtained. A key advantage of the proposed method is its flexibility, which allows the inclusion of data from other sources, such as experimental restraints and the use of modern molecular dynamics programs to calculate structures

The metal reductase activity of some multiheme cytochromes c: NMR structural characterization of the reduction of chromium(VI) to chromium(III) by cytochrome c(7)

The redox reaction between CrO [Formula: see text] and the fully reduced three-heme cytochrome c(7) from Desulfuromonas acetoxidans to give chromium(III) and the fully oxidized protein has been followed by NMR spectroscopy. The hyperfine coupling between the oxidized protein protons and chromium(III), which remains bound to the protein, gives rise to line-broadening effects on the NMR resonances that can be transformed into proton-metal distance restraints. Structure calculations based on these unconventional constraints allowed us to demonstrate that chromium(III) binds at a unique site and to locate it on the protein surface. The metal ion is located 7.9 ± 0.4 Å from the iron of heme IV, 16.3 ± 0.7 Å from the iron of heme III, and 22.5 ± 0.5 Å from the iron of heme I. Shift changes caused by the presence of unreactive MoO [Formula: see text] , a CrO [Formula: see text] analogue, indicate the involvement of the same protein area in the anion binding. The titration of the oxidation of cytochrome c(7) shows a detailed mechanism of action. The presence of a specific binding site supports the hypothesis of the biological role of this cytochrome as a metal reductase