Computational protocol: MCM ring hexamerization is a prerequisite for DNA-binding

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Protocol publication

[…] Diffraction data were collected at SER-CAT beam lines 22-ID (PfMCMN-ΔZFD and PfMCMN-F179A) and 22-BM (PfMCMN-βT) at the Advanced Photon Source at Argonne National Lab at 1.0 Å wavelength, 100 K. Data were integrated and scaled with the HKL-2000 package (). Figures were prepared with variety of software (–). Statistics for the crystal structures are provided in Supplementary Table S3.Crystals of PfMCMN-F179A were grown by hanging drop vapor diffusion in a ratio of 1:1 (protein:well solution). The stock protein solution was 340 μM with the well solution consisting of 50 mM sodium cacodylate, pH 6.5, 1 mM spermine, 35 mM MgCl2, 2.45 M ammonium sulfate. Crystals were cryoprotected in a 2.1 M lithium sulfate/well solution (2:1) and flash frozen. Data were collected in 0.25° oscillations for a total of 180° of crystal rotation and were integrated and scaled to 3.20 Å resolution. Phaser () placed 10 copies of a monomer of PfMCMN-WT () as a central pentameric ring with five exterior monomers. The model was refined with CNS (,) and with Refmac5 (). Following coordinate and group B-factor refinement with CNS (,), the final coordinate refinement was carried out with Refmac5 (). A Ramachandran plot calculated by Procheck () indicated the following statistics: core: 2040 (90.3%); allowed: 204 (9.0%); generously allowed: 8 (0.4%); disallowed: 8 (0.4%). Each of the eight disallowed residues is one of the copies of D209, which resides at the end of a tight structural turn of an ‘Allosteric Communication Loop’ (ACL) (,). The D209 phi/psi angles are just outside the allowed region of the plot (chain A-J phi/psi = 55.1/−116.2; 57.1/−116.0; 56.2/−115.6; 55.0/−116.9; 56.1/−115.2; 51.1/−116.8; 52.4/−120.8; 52.4/−117.5; 50.1/−121.4; 55.1/−117.5), very close to the angles observed, in the allowed region, for the wild-type hexamer (PDB 4POF chain A-F phi/psi = 53.1/−121.6; 55.1/−124.4; 52.5/−123.1; 49.8/−122.9; 49.4/−123.9; 47.4/−126.1) ().Crystals of PfMCMN-βT were grown by hanging drop vapor diffusion. The stock protein (340 μM) was mixed with the well solution (100 mM Bis–Tris, pH 5.5, 1.75 M ammonium sulfate) in a ratio of 2:1. Crystals were cryoprotected in a solution composed of one-third well solution and two-thirds 2.5 M ammonium sulfate, 25% glycerol solution and flash frozen. Data were collected in 0.5° oscillations for a total of 180° of crystal rotation. Data were integrated and scaled to 3.20 Å resolution. The PfMCMN-βT dataset was placed in the same setting and assigned the same test set as the roughly isomorphic PfMCMN-F179A dataset. Phaser () placed 10 copies of a monomer of PfMCMN-WT () as a central pentameric ring with five exterior monomers. The model was refined with CNS (,) and with Refmac5 (). Following coordinate and group B-factor refinement with CNS (,), the final coordinate refinement was carried out with Refmac5 (). A Ramachandran plot calculated by Procheck () indicated the following statistics: core: 2033 (89.8%); allowed: 222 (9.8%); generously allowed: 0 (0%); disallowed: 10 (0.4%). The 10 disallowed residues consist of the 10 individual copies of residue D209. This residue is at the end of a tight structural turn of the ‘Allosteric Communication Loop’ (ACL) (,) with phi/psi angles placing it at the border of the allowed and disallowed regions of the plot (chain A-J phi/psi = 54.6/−116.3; 53.8/−114.9; 54.3/−115.3; 54.4/−115.0; 57.2/−112.9; 42.0/−116.9; 39.8/−118.8; 43.7/−118.2; 41.4/−117.5; 45.0/−117.9)—very close to the angles observed, in the allowed region, for the wild-type hexamer (PDB 4POF chain A-F phi/psi = 53.1/−121.6; 55.1/−124.4; 52.5/−123.1; 49.8/−122.9; 49.4/−123.9; 47.4/−126.1) ().Crystals of PfMCMN-ΔZFD were grown by hanging drop vapor diffusion. The stock protein (425 μM) was mixed with well solution (50 mM ammonium fluoride, 21% PEG 3350) in a ratio of 1:2, respectively. Crystals were cryoprotected in 25% ethylene glycol and flash frozen. Data were collected in 0.5° oscillations for a total of 180° of crystal rotation and were integrated and scaled to 1.55 Å resolution. Phaser () placed two copies of a monomer of PfMCMN-WT with the Zn-binding domain removed. The model was refined with CNS (,) and with Refmac5 (). The final refinement was carried out with Refmac5 () with individual atomic B-factors. A Ramachandran plot calculated by Procheck () indicated the following statistics: core: 302 (93.8%); allowed: 20 (6.2%); generously allowed: 0 (0%); disallowed: 0 (0%). […]

Pipeline specifications

Software tools HKL-2000, CNS, REFMAC5, PROCHECK
Application Protein structure analysis
Organisms Mus musculus
Diseases Sleep Initiation and Maintenance Disorders, Neoplasms