Computational protocol: Modulation of MICAL Monooxygenase Activity by its Calponin Homology Domain: Structuraland Mechanistic Insights

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Protocol publication

[…] Cell parameter variations among crystals obtained in the same conditions during the search for diffraction quality crystals were frequently observed (data not shown). Two crystal forms showing differences in the c cell-dimension and β angle were selected for analysis. Diffraction data from both crystal forms (native 1 and native 2) frozen in the their mother liquors, with 20% v/v glycerol added as cryoprotectant, were collected using a Saturn 944 + CCD as a detector. The source was an FR-E + Super-BrightTM copper rotating anode x-ray generator equipped with VariMAX™ mirrors for monocromatization and collimation (Rigaku Americas Corporation, The Woodlands, TX). Data were processed with HKL2000 (HKL Research Inc.). The structure of native 1 was determined by molecular replacement with the program MOLREP as implemented in the CCP4 Suite, using the previously determined structure of MICALMO (residues 1–484) as a search model (PDB 2BRA). The model of the CH domain was built manually with the program COOT on the unassigned electron density of a sigmaA-weighted difference Fourier map (mFO-DFC) calculated with phases from the MO domain alone. The final structure was refined using the program REFMAC in CCP4 Suite). The structure of the second crystal was determined by molecular replacement using the two domains of the first crystal as separate search models. The models were refined by rigid body refinement, followed by restrained refinement and TLS/Restrained refinement (). All data collection and refinement statistics are presented in . [...] SAXS data were collected at the SIBYLS beam line (B12.3.1) of the ALS for q values in the range 0.0128–0.3253 Å−1 using three protein concentrations (2, 4 and 7 mg/ml) in a buffer containing 50 mM Tris HCl pH 7.0, 200 mM NaCl, 2 mM DTT, and 2% v/v glycerol (). The particle molecular mass was estimated by three independent methods ( and ): a) using the relation between the intensities of the scattering at zero angle [I(0)] of the MICALMO-CH sample and that of a standard -dimeric bovine serum albumin (BSA; Mw: 132 kDa, SIGMA Inc.)- at the same three concentrations used for the protein sample; b) using the saxsmow web server estimator (http://www.ifsc.usp.br/~saxs/saxsmow.html); and c) using the relation between the molecular weight and the ratio of the square of the correlation volume (Vc) to Rg as implemented in Scatter 2.0. An ab-initio low-resolution model of MICALMO-CH was obtained by a two-step procedure. In the first step, twenty envelopes were generated by DAMMIN (ATSAS) and averaged with DAMAVER (ATSAS) to provide a low-resolution dummy atom model (DAM) with an overall normalized spatial discrepancy (NSD) of 0.7. In a second step, the averaged DAM generated by DAMAVER was used as a starting model for a final run of DAMMIN. The final low-resolution model () has an averaged NSD of 0.47 with a standard deviation of 0.08. In addition, an all atom-model, obtained by rigid-body refinement of the two separated MICALMO-CH domains against the experimental scattering using the program SASREF, reached a χ2 of ≈0.69 (). […]

Pipeline specifications

Software tools Molrep, CCP4, Coot, DAMMIN, ATSAS, SASREF
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma