Computational protocol: Lengths of the C-Terminus and Interconnecting Loops Impact Stability of Spider-Derived Gating Modifier Toxins

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Protocol publication

[…] Data used for PaurTx-3 structure calculations was acquired on a Bruker Avance 600 MHz NMR spectrometer (Bruker, Billerica, MA, USA) equipped with a cryoprobe. The solution NMR structure for PaurTx-3 was calculated using previously described protocols [], on a Bruker Avance 600 MHz NMR spectrometer equipped with a cryoprobe. CCPNMR Analysis 2.4.1 (CCPN, University of Cambridge, Cambridge, UK) was used for amino acid assignment [,]. The solution NMR structure for PaurTx-3 was calculated as previously described [] using the AUTO and ANNEAL functions in CYANA 3.97 (Güntert Group, Goethe-Univerity Frankfurt, Frankfurt, Germany) [] to refine peak assignments. Dihedral angle restraints were generated using TALOS-N (Bax Group, NIH, Pike Bethseda, MD, USA) []. After initial structure determination on CYANA, protocols on the RECOORD database [], were used to generate 50 structures which were then refined in a water shell [], and a final set of 20 structures was chosen based on the lowest energy, best MolProbity scores [], and fewest distance and dihedral angle violations (). PaurTx-3 structure and restraints have been submitted to the Protein data bank (PDB ID: 5WE3) and the Biomagnetic Resonance Data bank (BMRB: 30317). [...] MOLMOL (Version 2k.2, Institute of Molecular Biology and Biophysics, ETH, Zürich, Switzerland) [] was used to calculate the global atomic RMSDs of the backbone of the GMTs including the amino acid residue following the last cysteine to the C-terminal of each sequence. RMSD values of the amino acids prior to the final cysteine were also calculated. […]

Pipeline specifications

Software tools CcpNmr, CYANA, MolProbity, MOLMOL
Databases BMRB RECOORD
Applications NMR-based proteomics analysis, Protein structure analysis
Chemicals Cystine