Computational protocol: The Escherichia coli Clamp Loader Can Actively Pry Open the β-Sliding Clamp*

Similar protocols

Protocol publication

[…] Crystals were grown using the hanging drop vapor diffusion method at room temperature. Purified protein, β-AF4882 (1.8 mg/ml), was mixed with an equal volume of the precipitant solution (100 mm MES, pH 6.0, 125 mm calcium chloride, 30% PEG 400) for a final volume of 4 μl (). The drops were equilibrated against 0.5 ml of precipitant solution, and crystals were observed after about 3 days. The crystals were quick-dipped into a cryoprotectant solution of precipitant solution with 25% (v/v) glycerol before being flash-cooled to 100 K for data collection. At the beginning of data collection, crystals were bright green due to AF488, but by the end the crystals were colorless, indicating that the fluorophore was bleached during data collection.Diffraction data were collected in-house using a Rigaku RU-H3R rotating anode equipped with VariMax HR optic and R-AXIS IV++ image plate detector. Data were indexed and scaled using HKL2000 (), and the structure was solved using molecular replacement (PDB ID 1MMI ()). Refinement was initiated using PHENIX () and changes to the model between rounds of refinement were made using Coot (). PROCHECK was used to check the final geometry of the model (). Data collection and refinement statistics are given in . […]

Pipeline specifications

Software tools SHELX, PHENIX, Coot, PROCHECK
Applications Small-angle scattering, Protein structure analysis
Organisms Escherichia coli
Chemicals Adenosine Triphosphate