Computational protocol: Rapid cadmium SAD phasing at the standard wavelength (1 Å)

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Protocol publication

[…] A single crystal was selected for each sample and X-ray diffraction data sets were collected on beamline P11 at PETRA III, DESY for HEWL and the PfActI-G1 complex and on beamline X06DA at the Swiss Light Source (SLS) for ETR1. Data-collection details are summarized in Table 1. The diffraction data sets were processed using XDS and scaled with XSCALE (Kabsch, 2010). The (hkl) and (−h −k −l) reflections were treated separately in all steps, including scaling and merging. In the integration and scaling steps, the parameters FRIDEL’S_LAW=FALSE and STRICT_ABSORPTION_CORRECTION=TRUE were used. To determine the minimum amount of data required for successful phasing and model building, a data set from a single data-collection run was divided into smaller parts and labelled according to the total rotation range. Initial SAD phasing protocols were carried out using SHELXC/D/E (Sheldrick, 2010) through HKL2MAP (Pape & Schneider, 2004). Further analysis was performed with a shell script using various combinations of parameters as discussed below. Heavy-atom sites were compared with their respective reference sets from the final refined model with SITCOM (Dall’Antonia & Schneider, 2006). For the low-resolution cutoff analysis, data sets were truncated at the scaling step with XSCALE. Successful SHELXE models were fed into phenix.autobuild to complete the automatic building (Adams et al., 2010). Some of the low-resolution SHELXE-built models were further optimized with Auto-Rickshaw (Panjikar et al., 2005) using the MRSAD protocol, in which several programs such as Phaser (McCoy et al., 2007), ARP/wARP (Langer et al., 2008), RESOLVE (Terwilliger, 2000) and Buccaneer (Cowtan, 2006) are used. Models were manually analyzed and built using Coot (Emsley & Cowtan, 2004) and final refinements were performed using phenix.refine (Afonine et al., 2012) and the PDB_REDO server (Joosten et al., 2012). Final refinement statistics are provided in Table 2. The coordinates and diffraction data have been deposited in the Protein Data Bank (PDB) as entries 5myy for the HEWL–cadmium complex structure and 5mvv for the PfActI-G1–CdATP complex structure. The ETR1 crystal structure was initially determined by molecular replacement and has been described elsewhere (PDB entry 4pl9; Mayerhofer et al., 2015). The figures were generated using PyMOL v1.7.6.0 (http://www.pymol.org). […]

Pipeline specifications

Software tools SHELX, HKL2MAP, ARP/wARP, Buccaneer, PyMOL
Applications Small-angle scattering, Protein structure analysis
Chemicals Cadmium