Computational protocol: Insights into Mad2 Regulation in the Spindle Checkpoint Revealed by the Crystal Structure of the Symmetric Mad2 Dimer

Similar protocols

Protocol publication

[…] The Mad2 L13A dimer was crystallized at 20 °C using the sitting-drop vapor-diffusion method. Drops were formed by mixing 1 μl of protein and 1 μl of reservoir solution that contained 19% (w/v) PEG 2000, 16% (v/v) glycerol, 100 mM Tris (pH 8.0), and 0.3 M MgCl2. Larger crystals were obtained by seeding using the same conditions. The crystals were cryoprotected with reservoir solution and then flash-cooled in liquid propane. Crystals diffracted to a minimum Bragg spacing (d min) of about 1.9 Å. At lower resolution, the diffraction data are compatible with an orthorhombic crystal symmetry. However, at higher resolution, the crystals exhibited the symmetry of space group C2 with cell dimensions of a = 109 Å, b = 191 Å, c = 154 Å and β = 90.02° with 12 molecules per asymmetric unit.Diffraction data were collected at beamline 19-ID (SBC-CAT) at the Advanced Photon Source (Argonne National Laboratory, Argonne, Illinois, United States) and processed with HKL2000 []. The Mad2L13A dimer structure was determined by the molecular replacement method with the program Phaser [] using the Mad2 core (residues 12–36, 58–158, and 177–205) from the structure of Mad2–MBP1 as the search model. Refinement was performed with REFMAC5 [] from the CCP4 package [] using diffraction data to a resolution of 1.95 Å, interspersed with manual rebuilding using the program Coot []. The 12 molecules in the asymmetric unit are arranged in two sets of six molecules related by almost perfect translational symmetry. No noncrystallographic symmetry restraints were used during refinement. Between one and four residues per Mad2 molecule were disordered and were not included in the model. The final model (R work = 21.2% and R free = 24.7%) contains 2,464 residues, 1,342 water molecules, eight magnesium ions, 32 chloride ions, as well as ten short PEG molecules. All but two residues are in the favored region of the Ramachandran plot. The two residues in the disallowed region are located at surface loops and are associated with weak electron density. Data collection and structure refinement statistics are summarized in . […]

Pipeline specifications

Software tools REFMAC5, CCP4, Coot
Application Protein structure analysis