Computational protocol: Structure and DNA binding activity of the mouse condensin hinge domain highlight common and diverse features of SMC proteins

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Protocol publication

[…] To prepare samples suitable for small-angle X-ray scattering (SAXS) measurements, proteins were additionally purified via gel filtration on a Superdex 200 column (GE Healthcare), and concentrated to yield samples in concentration ranges from 2 to 20 mg/ml in buffer C. The flowthrough of the concentration step was used as buffer reference for SAXS measurements. SAXS data were collected at beamline X33 at EMBL/DESY, Hamburg. Scattering profiles of BSA and lysozyme were measured as reference for molecular mass determination. The ATSAS package () was used to process and analyze data. Theoretical scattering profiles from atomic resolution models were calculated and fitted to measured profiles with CRYSOL. Ab initio models of mSMC2h4h-l were reconstructed from the experimental data using the program GASBORp without imposing any symmetry or other restrictions on possible models. Ten independently reconstructed envelopes were aligned and averaged with SUPCOMB and DAMAVER. Envelope representations were calculated using the Situs package (), which was also used to dock atomic resolution models into the envelope. [...] The short mouse condensin hinge construct mSMC2h4h-s was crystallized by vapor diffusion in the hanging-drop setup at 20°C. Crystallization was optimized with selenomethionine-labelled protein. The refined crystallization condition contained 15% (w/v) PEG 4000, 5% (v/v) isopropanol, 20% (v/v) glycerol and 100 mM Tris–HCl pH 8.5. For data collection, crystals were flash-frozen in liquid nitrogen without additional cryoprotection. Data were collected at beamline PXI of the Swiss Light Source (SLS, Villigen, Switzerland). The crystals belong to space group P21 and contain one molecule each of the SMC2 and SMC4 subunits in the asymmetric unit (see Supplementary Table S1 for crystallographic data, phasing and refinement statistics). The structure was determined by single-wavelength anomalous dispersion (SAD) phasing from a peak wavelength dataset of the selenomethionine-labelled protein crystals. Data were indexed and integrated using the XDS package (). Phases were calculated with AutoSHARP (). The model was largely automatically built with ARP/wARP () and completed by manual model building in Coot (). Initial refinement was carried out with CNS (), followed by several rounds of refinement with phenix.refine () and rebuilding in Coot. Refinement included simulated annealing in initial cycles, individual atomic coordinate and anisotropic B factor refinement, and bulk solvent corrections. Solvent molecules were added with phenix.refine and manually. The Rfree factor was calculated from 10% of the data which were removed at random before the structure was refined. The structure was validated using MolProbity () and PROCHECK (). The electrostatic surface potential was calculated with the Adaptive Poisson–Boltzmann Solver (APBS) (). All figures were prepared with PyMOL (DeLano, W.L. The PyMOL Molecular Graphics System. (2008) DeLano Scientific LLC, Palo Alto, CA, USA. http://www.pymol.org). Coordinates and structure factors were deposited at the Protein Data Bank (PDB) with accession number 3L51. […]

Pipeline specifications

Software tools ATSAS, CRYSOL, Situs, ARP/wARP, Coot, CNS, MolProbity, PROCHECK, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Mus musculus