Crystallization, diffraction data collection, and structure determination
This protocol is extracted from research article:
Structural basis of Stu2 recruitment to yeast kinetochores
eLife, Feb 16, 2021; DOI: 10.7554/eLife.65389

Ndc80cdwarf was concentrated to 15 mg/mL in an Amicon centrifugal concentrator in buffer containing 10 mM HEPES pH 7.0, 100 mM NaCl, and 2 mM TCEP. The Se-Met Stu2 peptide was diluted to 2 mg/mL in the same buffer. The protein was crystallized using hanging-drop vapor diffusion with a well solution containing 1.3 M ammonium sulfate and 0.1 mM HEPES pH 7.3. The hanging drops contained 1 µL each of the Ndc80c, peptide, and well solutions, resulting in an approximately threefold molar excess of peptide over Ndc80cdwarf. Crystals were cryoprotected by a 3–5 min soak in well solution supplemented with 25% glucose and flash-frozen by plunging into liquid N2. The complex crystallized in space group C2221 (a = 190.89 Å, b = 183.3 Å, c = 124.32 Å). Data to a minimum Bragg spacing of 2.7 Å were recorded on the NE-CAT beamline 24-ID-C at the Advanced Photon Source, indexed and integrated using HKL2000, and scaled and merged using Scalepack as implemented in HKL2000 (Otwinowski and Minor, 1997; Supplementary file 3). The structure was determined by molecular replacement in Phenix (Adams et al., 2010), using the Ndc80cdwarf (PDB 5TCS) as a search model, yielding clear density for the Stu2 peptide. Model building was carried out in Coot (Emsley et al., 2010) and refinement, in Phenix (Adams et al., 2010 Supplementary file 3). We used the anomalous signal from a Se-methionine residue to calculate an anomalous difference map, confirming that the Se atom was at a position consistent with the chosen register. Coordinates and diffraction data have been deposited in the protein data bank, PDB ID: 7KDF.

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