Computational protocol: Insights into the structure and assembly of the Bacillus subtilis clamp-loader complex and its interaction with the replicative helicase

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Protocol publication

[…] The structure of the B. subtilis clamp-loader was predicted using the iterative threading assembly refinement (I-TASSER) server, a platform for automated protein structure prediction (). For the individual δ and δ′ subunits, each sequence was separately submitted and the highest confidence score models were chosen. To model the τ subunit, the Cτ domain sequence was submitted separately and the highest confidence score models were chosen as before. The full τ sequence was then submitted using the Cτ domain models as structural template for the folding of the C-terminal region, while the remaining domains were modelled by the automated structure prediction. The obtained full τ models were analysed according to the confidence score of each model and topological location of the Cτ domain. The model with the best confidence score and satisfying the spatial restraints on combination with adjacent subunits was selected. These τ, δ and δ′ models were aligned with their equivalents within the E. coli clamp-loader crystal structure to obtain an approximate model of the full B. subtilis clamp-loader. [...] Before data collection at 100 K, crystals were mounted in LithoLoops (Molecular Dimensions Ltd) and soaked briefly in a solution of 30% (v/v) glycerol, 1 M (NH4)2SO4, 0.1 M Bis-Tris, pH 5.5, and 6% (w/v) PEG 3350. Diffraction from native and Se-Met δ crystals was measured with synchrotron radiation at the Diamond Light Source (DLS) and extended to 2.1 and 3.3 Å, respectively. The native datasets were collected on beamline I-02, while the multiwavelength anomalous dispersion (MAD) data collection strategy was used with the Se-Met crystals, and data at three wavelengths were collected on beamline I-04.The data from native and Se-Met crystals were processed using iMOSFLM () and analysed, scaled and merged using POINTLESS and SCALA in the CCP4 software suite () and shown to belong to space group P43212. The asymmetric unit was predicted to contain one copy of δ with a solvent content of 62%. An initial set of phases were obtained from the Se-Met data using SOLVE () followed by density modification using RESOLVE () in the PHENIX suite (). The model building was carried out using BUCANEER (). Further iterations of manual building using the COOT program () were followed by maximum likelihood refinement against the native data using phenix.refine in the PHENIX suite. Water molecules were added in the final refinement cycles. Model validity and quality were assessed using MOLPROBITY ().A summary of the relevant data statistics is shown in . Structure factors and coordinates have been deposited at the protein data bank (PDB) with the accession code 3zh9. All structure figures were generated using PyMOL (Schrödinger, LLC). […]

Pipeline specifications

Software tools I-TASSER, iMosflm, CCP4, PHENIX, Coot, MolProbity, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Bacillus subtilis, Escherichia coli, Dipturus trachyderma
Chemicals Adenosine Triphosphate, Amino Acids