Computational protocol: THE CRYSTAL STRUCTURE OF A VOLTAGE-GATED SODIUM CHANNEL

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Protocol publication

[…] X-ray diffraction data were integrated and scaled with the HKL2000 suite or DENZO/SCALEPACK and, when required, further processed with the CCP4 package. Experimental phases were determined using a 3.4 Å SAD-data set from a Hg-containing NavAb-Ile217Cys crystal. The SOLVE/RESOLVE software were run in a standard setting and the first map, calculated at 3.7 Å, is shown in . Ideal poly-alanine α-helices were manually fitted into this map and the model was subsequently used in combined SAD-molecular replacement (MR) protocols within the Phenix software using a 3.3 Å SAD-data set obtained from a SeMet-labeled NavAb-Met221Cys crystal. SAD-MR and MR-SAD-based maps were calculated and compared, allowing for complete register and amino-acid assignment of the NavAb model. Higher resolution native data sets were ultimately obtained and phased by MR-methods using the CNS suite (although our best native NavAb-Met221Cys data set is actually from a SeMet-containing crystal). Reiterative rounds of model building in O were guided by inspection of omit maps and refinement with CNS was performed with strict NCS-restraints, which were later relaxed during final rounds of refinement. Two strong densities (one per protein chain) assigned as solvent molecules (near the pore turret loop; not discussed in the main text) and all lipid molecules were added to the models at very late stages of refinement. Although trace amounts of digitonin are present in the crystallization condition, digitonin molecules were not readily observed in any electron density map. Refinement statistics, scaling statistics, and overall map quality were ultimately used to assign the NavAb space group as I222, although the data were found to closely mimic I422 (Rwork/Rfree stall at ~32% in I422). [...] The geometry of NavAb structural models was assessed using PROCHECK. The pore radius of NavAb was calculated using standard settings in the MOLE software. Electrostatic surface calculations were performed with the APBS software, calculated with 150 mM NaCl in the solvent. Structural alignments were performed using LSQMAN and O, where all channels were independently aligned onto NavAb based on the amino acid positions at the very beginning (i.e. N-terminal portion) of their P-helices. The superposition of the atomic resolution Na+-complex structure shown in was positioned manually, but the K+-channel and NaK-channel superpositions (, , , ) were obtained by simply aligning P-helices, as described above. All Fo-Fc omit maps shown throughout the main text and supplement have been calculated using standard settings and appropriate buffers in the CNS program. The Fo-Fc omit map shown in specifically derives from the 2.7 Å NaAb-Ile217Cys data set and amino acids 170-183 were omitted from the calculation box. All structural figures were prepared with the PyMol software. […]

Pipeline specifications

Software tools CCP4, PHENIX, CNS, PROCHECK, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Arcobacter butzleri
Chemicals Hydrogen, Potassium, Sodium, Glutamic Acid