Computational protocol: Structure determination of a major facilitator peptide transporter: Inward facing PepTSt from Streptococcus thermophilus crystallized in space group P3121

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Protocol publication

[…] Four data sets were used for structure determination: a native one used for refinement, and another native as well as two derivatives used for MIRAS phasing (). All data sets were processed using the XDS package []. As the diffraction displayed severe anisotropy, the native data sets were furthermore subjected to anisotropic scaling and truncation using the Diffraction Anisotropy Server [] (). Initial phasing was carried out using a trimmed version of the PepTSo structure (PDB: 2XUT) as a molecular replacement search model in Phaser [] from the PHENIX suite [], but the map was not of high quality, and we therefore decided to pursue experimental phases. The derivatives used for phasing were a SeMet labeled F338M crystal and a wild-type crystal soaked with KAu(CN)2. Soaking was carried out by adding 1.5 μL of a solution containing 90% of the original crystallant and 1 mM KAu(CN)2 to a crystallization drop with a well-formed crystal, which was then removed and flash frozen in liquid nitrogen after an incubation time of nine minutes. Scaling, site finding and initial MIRAS phasing was carried out with AutoSHARP [], while final sub-structure refinement, phasing and density modification was carried out with SHARP [] (). We used an initial resolution range of 48.94–4.10 Å, corresponding to the full range of the SeMet data set, and also close to that of the KAu(CN)2 data set. This was however extended to the full range of the native data set, 48.94–3.60 Å, during density modification. FOM was 0.401 for acentric reflections and 0.258 for centric reflections before density modification, and respectively 0.281 and 0.204, after. [...] Model building and rebuilding was carried out in Coot []. We initially relied only on the experimental map, but later also used the P212121 and C2221 structures for guidance as they became available (Solcan et al., 2012; Lyons et al., 2014). Refinement was carried out with PHENIX refine [] using one translation libration screw (TLS) group (). In order to ensure good geometry of the model, both secondary structure and reference restraints were used. This was found to greatly improve the Ramachandran plot and to reduce the number of rotamer outliers. For the last rounds of refinement, the X-ray/stereochemistry and X-ray/ADP weights were furthermore optimized, which helped improve RMS bonds and angles, as well as the Molprobity clash score []. The final clash score was 6.8 and the overall Molprobity score was 1.4 (both are in the 100th percentile of structures with similar resolution). Structural alignments were made using the DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/start) [], and structure figures were generated using PyMol []. […]

Pipeline specifications

Software tools XDS, PHENIX, Coot, MolProbity, DALI, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Streptococcus thermophilus