Computational protocol: Solution NMR Structure of Hypothetical Protein CV_2116 Encoded by a Viral Prophage Element in Chromobacterium violaceum

Similar protocols

Protocol publication

[…] NMR spectra were acquired on 250 μL NC and NC7 samples in 5 mm Shigemi NMR tubes at 293 K on Varian Inova 600 and 750 MHz NMR spectrometers. Backbone and sidechain resonance assignments were obtained from the analysis of 15N-HSQC (), 13C-HSQC, HNCO, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH, C(CO)NH, HC(CO)NH, HBHA(CO)NH, HCCH-COSY, HCCH-TOCSY and (H)CCH-TOCSY spectra. Stereospecific assignments of isopropyl methyl groups of Val and Leu residues were determined from characteristic 1H-13C coupling patterns in a high resolution 1H-13C HSQC of the NC7 sample []. NOE distance restraints were derived from a 15N-edited NOESY-HSQC (mixing time τm = 70 ms) and two 13C-edited NOESY-HSQC (τm = 70 ms) optimized for either aliphatic or aromatic carbon detection. Additional NOEs were assigned from a 4D 13C-13C-HMQC-NOESY-HMQC (τm = 70 ms) recorded after lyophilization and dissolution into pure D2O. Spectra were processed by NMRPipe and analyzed with Sparky 3.110 []. Nearly complete resonance assignments (99.3%) were determined. NMR assignments, raw NOESY FIDs, and NOESY peak lists have been deposited in the BioMagResBank (BMRB accession number 16,521). [...] The solution NMR structure of CV_2116 was initially calculated using both AutoStructure [] and CYANA 2.1 []. Inputs for calculation were resonance assignments, NOESY peak lists from the four NOESY spectra that included peak intensities, and TALOS-derived dihedral restraints for ϕ and ψ dihedral angles. NOE assignments were initially made automatically in AutoStructure and CYANA and the assignments refined manually. NMR RPF quality assessment scores were used to assess “goodness of fit” between calculated structures and NOESY peaks lists and were used to guide manual and iterative refinement of NOESY peak picking. The NOE-derived distance constraints, dihedral constraints, and hydrogen-bond constraints derived from AutoStructure were converted to Xplor/CNS format using PDBStat and the upper bounds for the NOEs were increased by 10%. These constraints were used to calculate 150 structures using Xplor-NIH (version 2.25) with a standard simulated annealing protocol followed by refinement of the 20 lowest energy structure using the Xplor-NIH protocol [,]. For the final NMR ensemble, the 20 lowest energy structures were deposited in the Protein Data Bank (PDB ID, 2KON). NMR structure quality analysis and structural statistics were performed using the PSVS [] and RPF [] software packages. Structural statistics for CV_2116 are presented in . […]

Pipeline specifications

Software tools Sparky, CYANA, RPF, Xplor-NIH, PSVS
Databases BMRB
Applications NMR-based proteomics analysis, Protein structure analysis
Organisms Chromobacterium violaceum, Escherichia coli, Thiocystis violascens
Diseases Infection