PMC:4504005 / 33314-34787 JSONTXT

{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4504005","sourcedb":"PMC","sourceid":"4504005","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4504005","text":"Experimental Procedures\n\nProtein Production and Characterization and Peptides\nPlasmid construction, mutagenesis, protein expression, purification, and the AlphaScreen peptide-binding assay were as previously described with minor modifications to the AlphaScreen assay (Hill and Pioszak, 2013; Moad and Pioszak, 2013). Synthetic peptides were from RS Synthesis, Bachem, or were synthesized in-house. Details are in Supplemental Experimental Procedures.\n\nCrystallization, Structure Solution, and Homology Modeling\nThe tethered MBP-RAMP1 ECD-CLR ECD and MBP-RAMP2 ECD [L106R]-CLR ECD proteins were complexed with CGRP(27-37)NH2 [D31, P34, F35] or AM(25-52)NH2 and crystallized with a reservoir solution of 22% PEG3350, 8% Tacsimate (pH 6.0) for the CGRP receptor complex or 19% PEG3350, 0.1 M Tris-HCl (pH 8.3), 225 mM sodium acetate, and 20% ethylene glycol for the AM1 receptor complex. Diffraction data collected at the APS synchrotron were processed with HKL2000 (Otwinowski and Minor, 1997) and the CCP4 suite (Winn et al., 2011). The structures were solved by molecular replacement with Phaser (McCoy et al., 2007), rebuilt with COOT (Emsley et al., 2010), and refined with REFMAC5 (Murshudov et al., 1997). Details and homology modeling are in Supplemental Experimental Procedures.\n\nCell-Based Assays\nTransfection of COS-7 cells, cAMP assay, ELISA for cell surface expression, and data analysis were as previously described (Barwell et al., 2010; Watkins et al., 2014).","divisions":[{"label":"title","span":{"begin":0,"end":23}},{"label":"sec","span":{"begin":25,"end":451}},{"label":"title","span":{"begin":25,"end":77}},{"label":"p","span":{"begin":78,"end":451}},{"label":"sec","span":{"begin":453,"end":1285}},{"label":"title","span":{"begin":453,"end":511}},{"label":"p","span":{"begin":512,"end":1285}},{"label":"title","span":{"begin":1287,"end":1304}}],"tracks":[{"project":"TEST0","denotations":[{"id":"25982113-209-217-8319056","span":{"begin":287,"end":291},"obj":"[\"23247088\", \"23247088\", \"23247088\"]"},{"id":"25982113-233-241-8319057","span":{"begin":311,"end":315},"obj":"[\"24115156\", \"24115156\", 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