Computational protocol: Insights into the RecQ helicase mechanism revealed by the structure of the helicase domain of human RECQL5

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Protocol publication

[…] For crystallization of the APO form (construct RECQL5 11–526) proteins were concentrated to 20 mg/ml using a 50 000 MWCO centrifugal concentrator and crystals were obtained by hanging drop vapor diffusion in conditions containing 17.5% PEG 3350, 0.2 M potassium thiocyanate, 0.1 M HEPES pH 7.0, 10% ethylene glycol. ADP and MgCl2 were added at a final concentration of 5 mM and the monoclinic form (construct RECQL5 11–453) was crystallized at 15 mg/ml by sitting drop vapor diffusion from conditions containing 19% PEG 6K, 0.25 M lithium chloride and 10% ethylene glycol. Crystals of the triclinic form were obtained in conditions containing 0.1 M Tris pH 7.5, 23% PEG 3350 and 0.1 M NaCl. The APO and monoclinic ADP form crystals were cryo-protected by transferring to a solution of mother liquor supplemented with 20% ethylene glycol, whilst the triclinic ADP form was cryo-protected in a solution containing well solution supplemented with 10% DMSO and 10% ethylene glycol. Data were collected at Diamond Light Source beamlines I04 (APO form), I04-1 (triclinic ADP form) and I03 (monoclinic ADP form). Diffraction data were processed with the program XDS (), and the structures were solved by molecular replacement using the program PHASER () with the BLM helicase (,) structure as a starting model. Model building and real space refinement were performed in COOT () and the structures refined using BUSTER (APO and triclinic ADP form) and PHENIX REFINE () (monoclinic ADP form). [...] Small angle X-ray scattering measurements of RECQL5 in solution were performed at Diamond Light Source beamline B21 using a BIOSAXS robot for sample loading. Measurements were made using protein concentrations of 4, 2 and 1 mg/ml in a buffer comprising 20 mM HEPES pH 7.5, 250 mM NaCl, 1 mM TCEP. Nucleotides were added to a final concentration of 1 mM, and DNA substrates (single stranded 19 mer) were added in a 1:1.2 molar ratio of Protein:DNA. The data were reduced and buffer contributions subtracted with the DawnDiamond software suite and analysed using the program SCATTER ( Scattering profiles of atomic models were calculated from atomic models using CRYSOL () and the agreement between theoretical and experimental scattering profiles was evaluated using the χ2 free procedure () implemented in the program SCATTER. Distance distribution functions were calculated using SCATTER and used for subsequent ab inito shape reconstructions using the program DAMMIF (). The results of 13 parallel reconstructions were compared using the program SUPCOMB () and the model with the lowest normalized spatial discrepancy was chosen for analysis. Atomic models were fit to the SAXS envelopes using the program SUPCOMB. […]

Pipeline specifications

Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma, Homo sapiens