Computational protocol: The latch modulates nucleotide and DNA binding to the helicase-like domain of Thermotoga maritima reverse gyrase and is required for positive DNA supercoiling

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Protocol publication

[…] Diffraction quality crystals for rgyr_hel_Δlatch were obtained at 25°C from the PEG/ion screen (Hampton). 20 mg ml−1 protein was mixed 1:1 (v/v) with reservoir consisting of 0.2 M magnesium formate, 20% PEG 3350. Crystals were cryo-protected with paraffin oil, mounted in an arbitrary orientation and data were collected at SLS beamline X06DA on a MAR-CCD detector. Data were indexed in a primitive orthorhombic lattice, integrated with XDS (), scaled with MOSFLM () and corrected for anisotropy with XPREP (Bruker). Data statistics are collected in . Systematic absences identified space group P212121. A Matthews coefficient of 2.8 Å3/Da corresponding to 56% solvent content indicated two rgyr_hel_Δlatch molecules per asymmetric unit. The structure was determined by molecular replacement using the truncated (termini and loops were removed, the sequence of conserved residues was adjusted to match T. maritima reverse gyrase) H1 and H2 domains of A. fulgidus reverse gyrase as separate search models with PHASER (). Both H2 domains, but only one H1 domain, were found. Refinement of this model with BUSTER () resulted in spurious electron density for the second H1 domain, which could be placed manually. Further refinement using PHENIX () and rebuilding in COOT () enabled tracing of the connections between the H1 and H2 domains to generate a model of two complete helicase modules. Refinement statistics are summarized in . The coordinates and structure factors have been deposited in the Protein Data Bank (accession code 3oiy). […]

Pipeline specifications

Software tools XDS, iMosflm, PHENIX, Coot
Applications Small-angle scattering, Protein structure analysis
Organisms Thermotoga maritima
Chemicals Adenosine Triphosphate