Computational protocol: Crystal Structures of the F and pSLT Plasmid TraJN-Terminal Regions Reveal Similar Homodimeric PAS Folds withFunctional Interchangeability

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Protocol publication

[…] SOLVE located four expected selenium atoms in the asymmetric unit using the 2.3 Å selenomethionyl TraJF1–140 MAD data. The best solution from SOLVE had a Z score of 57.2 and a mean figure of merit of 0.3. The maximum likelihood density modification in RESOLVE was used to improve initial phases, yielding an overall figure of merit of phasing of 0.58. Automatic building by RESOLVE using the protein sequence generated a model with ∼200 residues built in an asymmetric unit, which contain a homodimer of SeMet-TraJF1–140. The model was used for molecular replacement with MOLREP to generate a TraJF11–130 model from the TraJF11–130 native data set at 3 Å, which was used as a starting model against the 1.55 Å TraJF11–130 native data set for automated model building in ARP/wARP. Iterative runs of ARP/wARP combined with manual model building and iterative cycles of refinement in REFMAC were used to complete and refine the model at 1.55 Å resolution. The final model of the crystallographic asymmetric unit contains two TraJF11–130 dimers. There was no interpretable electron density for residues 10, 11, and 128–130. Refinement statistics are summarized in Table .The structure of TraJpSLT1–128 was determined by SAD phasing using the HKL3000 suite. SHELXD was used for heavy atom search, and initial phases were obtained from SHELXE. The heavy atom sites were refined, and improved phases were calculated by iterations of MLPHARE and DM. The initial protein models were built in ARP/wARP. Manual model rebuilding was conducted in COOT, and crystallographic refinement was performed in PHENIX51. The final model refined to 1.67 Å was evaluated by MolProbity and a Ramachandran plot with good R and Rfree values and stereochemistry. There was no interpretable electron density for N-terminal residues 1–13 and C-terminal residues 127 and 128. The details of the data collection, structure refinement, and model quality are shown in Table S2 of the . The molecular structure figures were prepared by using PyMOL (http://www.pymol.org) with surface of internal cavities created using HOLLOW (http://hollow.sourceforge.net). CASTp was used for calculating the area and volume of protein internal cavities. Areaimol was used for calculating the area of the protein contact surface. […]

Pipeline specifications

Software tools Molrep, ARP/wARP, SHELX, Coot, PHENIX, MolProbity, PyMOL, CASTp
Application Protein structure analysis
Organisms Dipturus trachyderma