Computational protocol: Crystal Structure of the FeS Cluster–Containing Nucleotide Excision Repair Helicase XPD

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Protocol publication

[…] XPD crystals were grown by vapor diffusion in hanging drops containing equal volumes of protein in 20 mM Tris/HCl (pH 8.0) and 500 mM NaCl at a concentration of 5 mg/ml, and a reservoir solution consisting of 200 mM MgCl2, 100 mM Hepes (pH 8), and 5%–10% PEG 400 equilibrated against the reservoir solution. Crystals grew within 7 d at 20 °C to a maximum size of 100 × 50 × 50 μm3. Prior to data collection, the crystals were cryocooled by sequential transfer into mother liquor containing increasing amounts of glycerol in 5% steps to a final concentration of 30%.The crystals were flash cooled in liquid nitrogen, and data collection was performed at 100 K. Data sets were collected at beamline BM14 (European Synchrotron Radiation Facility [ESRF]) at wavelengths of 1.0 Å, 1.7 Å, 1.7367 Å, and 1.7419 Å. All data were indexed and processed using Moslfm and Scala [,]. The crystals belong to space group P65 with unit cell dimensions of a = b = 78.9 Å, c = 174.0 Å. Structure solution was achieved utilizing the anomalous signal of the endogenous Fe belonging to the 4Fe4S cluster by MAD data collection at the Fe edge. The peak and inflection datasets were obtained from one crystal and were merged with a highly isomorphous dataset collected at the remote wavelength. The Fe sites were located using ShelxD [], and phase improvement was achieved with Sharp []. Substructure solution and refinement was carried out at 4 Å resolution, and the 4Fe4S cluster was treated as a “super” atom for phasing. The initial maps were subjected to solvent flattening and phase extension to 3.6 Å using the programs Solomon [] and Pirate []. The solvent-flattened maps were autotraced using the low-resolution quick-build option in ARP/WARP [] and further extended manually using the programs O and Coot [,]. After assigning the maximum number of residues and side chains possible, the model was subjected to phase-restrained simulated annealing and maximum likelihood refinement using the program phenix.refine []. Refinement was carried out against the highest resolution dataset up to 2.9 Å. The model was further improved by alternating rounds of refinement and manual model building. When the model was sufficiently complete, refinement continued with TLS and restraint maximum likelihood refinement using Refmac5 []. The final model contains 586 out of 602 amino acid residues, the 4Fe-4S cluster, one calcium ion, and one water molecule. […]

Pipeline specifications

Software tools CCP4, SHELX, ARP/wARP, Coot, PHENIX, REFMAC5
Application Protein structure analysis
Organisms Homo sapiens
Diseases Cockayne Syndrome, Xeroderma Pigmentosum
Chemicals Lead, Nucleotides