Computational protocol: Binding of Myomesin to Obscurin-Like-1 at the Muscle M-Band Provides a Strategy for Isoform-Specific Mechanical Protection

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Protocol publication

[…] Crystals were cryo-protected by soaking them in their reservoir solution supplemented with 20% glycerol. For OL3:My4LMy5 a 3.1 Å resolution dataset was collected in space group C2 while My4LH crystallized in the alternative space groups P65 and P2, yielding diffraction data at 2.05 Å and 2.8 Å resolution, respectively. All datasets were collected at Diamond Light Source synchrotron facility (Didcot, Oxfordshire, UK) and processed with the xia2 expert system () using XDS () and AIMLESS () packages. All X-ray structures were solved by the molecular replacement method with the package MOLREP () and refined using the programs REFMAC5 () and BUSTER (). A summary of data collection and refinement statistics is shown in . Further details on the crystallographic methods are available in the . [...] Synchrotron SAXS data for OL3:My4L and OL3:My4LMy5 were collected at the BM29 BioSAXS beamline (ESRF, Grenoble) using a Pilatus 1M detector (Dectris) (). All samples were measured at four concentrations (0.5–4.5 mg/mL in 20 mM HEPES [pH 7.5], 500 mM NaCl, 1 mM DTT buffer) in the range of momentum transfer 0.005 < s < 0.608 Å−1 (s = 4πsinθ/λ, where the wavelength λ is 0.9919 Å and 2θ is the scattering angle). All experiments were performed at 18°C using a sample volume of 30 μL loaded into the flowing measurement cell. Individual frames were processed automatically and independently within the EDNA framework (). Merging of separate concentrations and further analysis steps were performed using a combination of tools from the ATSAS package (). Initial rigid body modeling of the complex was done with CORAL () and domain dynamics of the protein complexes was further explored by generating conformational ensembles using the tCONCOORD () method. Further details are available in the . […]

Pipeline specifications

Software tools xia2, XDS, CCP4, Molrep, REFMAC5, ATSAS, tCONCOORD
Applications Small-angle scattering, Protein structure analysis
Organisms Rattus norvegicus