Computational protocol: FlaF Is a β-Sandwich Protein that Anchors the Archaellum in the Archaeal Cell Envelope by Binding the S-Layer Protein

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Protocol publication

[…] sFlaF protein (residues 35–164 with an N-terminal His-tag [MGSSHHHHHHSQDPNS]), 6 mg/ml in 50 mM Tris (pH 8.0), 150 mM NaCl, and 3% glycerol, was mixed with an equal volume of the reservoir using the hanging drop or sitting drop vapor diffusion method. sFlaF was crystallized in three different conditions. For sFlaF native 1, the reservoir contained 3 M potassium chloride and 50 mM HEPES (pH 7.0); for sFlaF native 2, the reservoir contained 35% Tacsimate (pH 7.0); and for sFlaF derivative, protein was co-crystallized with 2 mM potassium tetrachloroplatinate(II) hydrate and the reservoir contained 3 M sodium acetate (pH 7.0). The crystals grew in 2–3 days at 15°C in all the conditions. Native 1 and derivative crystals were transferred to a reservoir including 15% glycerol as a cryoprotectant; native 2 crystals were transferred to a reservoir including 30% sucrose as a cryoprotectant for about 30 s and then flash cooled in liquid nitrogen for data collection.The X-ray diffraction data for sFlaF were collected at the SIBYLS beamline 12.3.1 at Advanced Light Source (ALS) (). Both sFlaF native 1 and derivative datasets were integrated and scaled using the HKL2000 program package (). sFlaF native 2 was integrated using XDS () and scaled using AIMLESS () in the CCP4 suite (). The dataset for the sFlaF derivative with K2PtCl4 was used for SIRAS phasing. The native 1 structure of sFlaF was solved using the phenix.autosol program in the Phenix suite (). The sFlaF native 2 structure was solved by molecular replacement using the native 1 sFlaF structure as a search model using Phaser in the Phenix suite. Both sFlaF native 1 and 2 structures were fitted manually using Coot () and refined using the Phenix refine program. Rwork/Rfree were refined to 0.158/0.196 and 0.173/0.200 for the native 1 and native 2 sFlaF structures, respectively (). The His-tag was not visible in the electron density map in both native structures. Molecular graphics and analyses were performed with the UCSF Chimera package (). […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, UCSF Chimera
Applications Small-angle scattering, Protein structure analysis
Organisms Sulfolobus acidocaldarius, Dipturus trachyderma
Diseases Spinocerebellar Ataxias
Chemicals Hydrogen