rRRSP construct previously designed (13) was expressed in M9 selenomethionine high-yield media according to the vendor’s instructions (MD045004-50L, Orion Enterprise Inc.). Protein expression was induced by adding 1 mM isopropyl-β-d-thiogalactoside (IPTG) at an OD600 (optical density of 600 nm) of 0.8, and growth was continued at 18°C cells for 18 hours. Cells were harvested by centrifugation, and rRRSP was purified as previously described (13). The purified protein was concentrated to 15 mg/ml. Sitting drop crystallization plates were set up at room temperature by mixing 1 μl of rRRSP and 1 μl of reservoir solution with 90 μl of reservoir solution. Crystals were grown at 19°C using a reservoir of 0.1 M 3-(N-morpholino)propanesulfonic acid) (Mops) (pH 6.5), 5% (w/v) polyethylene glycol 400, and 2 M NH4SO4. Harvested crystals were transferred to 4 M formate before being frozen in liquid nitrogen. Diffraction data were collected at 100 K from a single crystal at experimental station 21ID-F of the Life Science Collaborative Access Team (LS-CAT) at the Advanced Photon Source (APS). Crystals belong to the cubic space group I23 with unit cell parameters 247.1, 247.1, 247.1, 90.0, 90.0, and 90.0. Two hundred frames, which correspond to 120° of spindle axes rotation, were collected from a single crystal at the selenium peak (λ = 0.97872). Data were indexed, integrated, and scaled using the HKL3000 suite (54). The structure was solved with PHENIX (55), using the single anomalous dispersion technique. The initial figure of merit was 0.40, and after density modification, 975 of 1036 residues (two protein chains of 494 each and 24 residues of the purification tag) were automatically built into the experimentally obtained electron density maps. The initial model was checked and manually corrected in Coot (56). Another data set was generated for refinement by scaling and merging only the first 100 frames, thus reducing radiation damage and increasing the resolution of the data. The corrected model was refined in Refmac (57), and solvent molecules were added to the model using ARP/wARP (58) followed by several rounds of refinement in Coot. The TLS group correction to the B factors (59) was applied at the final steps of refinement. The structure factors and coordinates of the final model were validated using SFCHECK (60) and MolProbity (61) and deposited to the PDB with PDB ID 5W6L. The data quality and the quality of the model are summarized in Table 1.

Note: The content above has been extracted from a research article, so it may not display correctly.



Q&A
Please log in to submit your questions online.
Your question will be posted on the Bio-101 website. We will send your questions to the authors of this protocol and Bio-protocol community members who are experienced with this method. you will be informed using the email address associated with your Bio-protocol account.



We use cookies on this site to enhance your user experience. By using our website, you are agreeing to allow the storage of cookies on your computer.