Computational protocol: Structure of the C-terminal domain of nsp4 from feline coronavirus

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Protocol publication

[…] The structure was solved using the SIRAS protocol of the Auto-Rickshaw automated crystal structure-determination platform (Panjikar et al., 2005). F A values were calculated using the program SHELXC (Sheldrick, 2008). Based on an initial analysis of the data, the maximum resolution for substructure determination and initial phase calculation was set to 3.8 Å. 20 selenium positions were found using the program SHELXD (Sheldrick, 2008). The correct hand of the substructure was determined using the programs ABS (Hao, 2004) and SHELXE (Sheldrick, 2008). The occupancy of all substructure atoms was refined using the program BP3 (Pannu et al., 2003; Pannu & Read, 2004). The initial phases were improved using density modification, noncrystallographic symmetry (NCS) averaging and phase extension using the program RESOLVE (Terwilliger, 2000). A partial α-helical model was produced using the program HELICAP (Morris et al., 2004). The partial model contained 119 of the total of 440 residues expected for four molecules. The initial phases were improved by phase combination of experimental and model phases using the program SIGMAA (Read, 1986). The density modification and fourfold NCS averaging were repeated again as described above. The resultant phases were used to continue model building using the program ARP/wARP (Perrakis et al., 1999), resulting in the placement of 242 residues. The partial models generated in the intermediate steps of ARP/wARP were then used to assemble an almost complete dimer using the graphics program Coot (Emsley & Cowtan, 2004). This dimer was then used to find the second dimer in the electron density using phased molecular-replacement techniques as implemented in MOLREP (Vagin & Teplyakov, 1997). 2F o − F c and F o − F c electron-density maps calculated at this stage showed additional electron density indicating the presence of a fifth molecule in the asymmetric unit. The phased molecular replacement was repeated again to place the fifth molecule in the electron-density map. The resultant model was then used for restrained refinement in REFMAC5 (Murshudov et al., 1997), including use of the translation, libration and screw method (TLS; Schomaker & Trueblood, 1968) for describing group motions.The structure was manually modified, followed by cycles of refinement, using the program Coot. The progress of the refinement was monitored by means of the free R factor (Brünger, 1992). Water molecules were included where clear peaks were present in both the 2F o − F c and F o − F c maps and where appropriate hydrogen bonds could be made to surrounding residues or to other water molecules. The stereochemistry of the model was evaluated with the program MOLPROBITY (Davis et al., 2007).Interfaces between molecules were analyzed with the PISA server (Krissinel & Henrick, 2007). Interactions between molecules were initially evaluated using the CCP4 program CONTACT with a maximum contact distance of 3.6 Å. […]

Pipeline specifications

Software tools SHELX, ARP/wARP, Molrep, REFMAC5, MolProbity
Application Protein structure analysis
Organisms Feline coronavirus, Homo sapiens