Computational protocol: Crystal Structure and Molecular Imaging of the Nav Channel β3 Subunit Indicates a Trimeric Assembly*

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Protocol publication

[…] The x-ray data collection experiments were performed at a temperature of 100 K at the Diamond Light Source Synchrotron Science Facility (Oxford, UK), beamline I04. The crystals diffracted to a maximum resolution of 2.5 Å (the resolution cutoff level was set to the resolution shell where the average I/σ of the reflections is still greater than 2). A total of 100° of data were collected at a 1.0° oscillation angle. The crystals belonged to the P3121 space group and contained three molecules of the β3 Ig domain in the asymmetric unit. This results in ∼50% crystal solvent content (Matthews coefficient () of 2.47). All diffraction data were indexed, scaled, and merged using XDS software suite (). The crystallographic data collection statistics are shown in . [...] The structure of the β3 Ig domain was solved by the molecular replacement (MR) method. The positions of the three β3 Ig domain molecules within the asymmetric part of the unit cell were identified. All MR calculations were performed in PHASER, part of the PHENIX crystallographic software suite (). For the identification of the MR search probe, the homolog search was performed using the FUGUE sequence-structure homology recognition server (). The top three structurally homologous candidates (Protein Data Bank codes 2X1X (), 1I8L (), and 1F97 ()) were clearly separated from the rest of the homologous structures, based on their Z-scores. The homologous regions of the structures corresponding to the β3 Ig domain sequence were cut out (residues 226–326 in 2X1X, residues 1–104 in 1I8L, and residues 29–129 in 1F97) and superposed onto the highest ranking homolog, i.e. 2X1X. The superposition resulted in a root mean square deviation of 1.8 Å between equivalent Cα atoms of fragments from 2X1X and 1I8L and 1.6 Å between 2X1X and 1F97. The combined superposition of these three structures was used as the MR search probe. The use of this probe allowed unambiguous determination of the positions of two of the molecules of β3 Ig domain in the asymmetric unit. The translation function Z-score value for this solution was 10.7. The position of the third molecule could not be identified clearly at this stage. The crystallographic refinement and automatic model building of the MR solution obtained were performed using the PHENIX software suite; the coordinates of only the 2X1X portion of the MR probe were used in calculations. These calculations caused a significant drop in Rcryst/Rfree values by more than 12% each, reaching values of 31.0 and 35.7%, respectively, thus indicating the correctness of the MR solution obtained for the two molecules of the β3 Ig domain. Re-running the MR calculations using one of the refined molecules from the previous step as the MR search probe identified the position of the third molecule.The next round of refinement and auto-building calculations dropped the Rcryst/Rfree values even further to 25.1 and 28.4%, respectively. The model was then subjected to several rounds of alternating manual rebuilding performed in molecular graphics software suite COOT () and crystallographic refinement calculations with either PHENIX or REFMAC software (). The NCS restraints were used at the initial stages of the refinement except for the last two rounds of refinement. The Rcryst and Rfree converged to the values of 20.2 and 23.8%, respectively. The structural validation was also performed on the final model; the crystallographic statistics are shown in . Examination of the trimer interface was performed using PISA (), and the results are shown in and . […]

Pipeline specifications

Software tools XDS, PHENIX, FUGUE, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Homo sapiens
Chemicals Cysteine, Sodium