Computational protocol: Structural Insight into the Rotational Switching Mechanism of the Bacterial Flagellar Motor

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Protocol publication

[…] Tm-FliGMC(ΔPEV) was purified as described previously . Crystals of Tm-FliGMC(ΔPEV) were grown at 4°C using the hanging-drop vapor-diffusion method by mixing 1 µl of protein solution with 1 µl of reservoir solution containing 0.1 M sodium phosphate-citrate buffer pH 4.2–4.4, 36%–50% PEG200, and 200 mM NaCl. Initially, we tried to solve the structure by the molecular replacement method using Tm-FliGMC structure (PDB ID: 1 lkv) as a search model. However, no significant solution was obtained, even though individual domains were used as search models. Therefore, we prepared heavy-atom derivative crystals and determined the structure using the anomalous diffraction data from the derivatives.Derivative crystals were prepared by soaking in a reservoir solution containing K2OsCl6 at 50% (v/v) saturation for one day. Crystals of Tm-FliGMC(ΔPEV) and its Os derivatives were soaked in a solution containing 90%(v/v) of the reservoir solution and 10%(v/v) 2-Methyl-2,4-pentanediol for a few seconds, then immediately transferred into liquid nitrogen for freezing. All the X-ray diffraction data were collected at 100 K under nitrogen gas flow at the synchrotron beamline BL41XU of SPring-8 (Harima, Japan), with the approval of the Japan Synchrotron Radiation Research Institute (JASRI) (Proposal No. 2007B2049). The data were processed with MOSFLM and scaled with SCALA . Phase calculation was performed with SOLVE using the anomalous diffraction data from Os-derivative crystals. The best electron-density map was obtained from MAD phases followed by density modification with DM . The model was constructed with Coot and was refined against the native crystal data to 2.3 Å using the program CNS . About 5% of the data were excluded from the data for the R-free calculation. During the refinement process, iterative manual modifications were performed using “omit map.” Data collection and refinement statistics are summarized in and , respectively. […]

Pipeline specifications

Software tools iMosflm, CCP4, Coot, CNS
Applications Small-angle scattering, Protein structure analysis
Organisms Thermotoga maritima, Salmonella enterica, Aquifex aeolicus
Diseases Bacterial Infections, Multiple Sclerosis, Mitochondrial Diseases