PMC:4528928 / 5276-7026 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4528928","sourcedb":"PMC","sourceid":"4528928","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4528928","text":"2.2. Crystallization and structure determination   \nPurified MLH1 protein (10 mg ml−1) was mixed with ADP at a 1:5 molar ratio of protein:ligand and crystallized using the sitting-drop vapor-diffusion method by mixing 1 µl protein solution with 1 µl reservoir solution consisting of 20% PEG 4000, 10% 2-propanol, 0.1 M HEPES pH 7.5.\nDiffraction data were collected on beamline 19ID at the Advanced Photon Source, Argonne National Laboratory. Reflection intensities from 150 1° diffraction images were initially integrated and scaled using HKL-3000 (Minor et al., 2006 ▸). Using the crystal structure of E. coli MutL (PDB entry 1b62; 36% amino-acid sequence identity; Ban et al., 1999 ▸; Johnson et al., 2008 ▸) as the search model, the structure was solved by molecular replacement with MOLREP (Vagin \u0026 Teplyakov, 2010 ▸). The initial refinement alternated cycles of restrained refinement including TLS parameterization in REFMAC (Murshudov et al., 2011 ▸; Winn et al., 2001 ▸) with interactive rebuilding in Coot (Emsley et al., 2010 ▸). After renewed processing of the same diffraction images with XDS (Kabsch, 2010 ▸) and additional scaling with AIMLESS (Evans \u0026 Murshudov, 2013 ▸), the model was further refined using autoBUSTER (Blanc et al., 2004 ▸; Bricogne et al., 2011 ▸) and REFMAC interspersed with interactive rebuilding.\nThe MolProbity statistics of the model compared favorably with a set of reference structures with similar data resolution (MolProbity server v.4.1-537). The model was deposited in the PDB using the PDB_EXTRACT tool (Yang et al., 2004 ▸) with accession code 4p7a. Data-collection, model-refinement and validation statistics are summarized in Table 1 ▸. All figures were prepared using PyMOL (v.1.5.0.4; Schrödinger).","divisions":[{"label":"Title","span":{"begin":6,"end":52}}],"tracks":[]}