Computational protocol: Structural basis for the bacterial transcription repair coupling factor/RNA polymerase interaction

Similar protocols

Protocol publication

[…] Crystals were grown by hanging-drop vapor diffusion by mixing 2 µl protein solution (10–12 mg/ml in storage buffer) with 1 µl crystallization solution (0.1 M Tris–HCl [pH 7.5 at 22°C], 1.6 M di-potassium ammonium phosphate), and incubating over a well containing crystallization solution. Crystals (10–20 µm octahedra) grew in ∼1 week. The crystals were prepared for cryo-crystallography by stepwise transfer into 0.1 M Tris–HCl [pH 7.5 at 22°C], 100 mM NaCl, 7 M ammonium formate, then frozen in liquid nitrogen. X-ray diffraction data sets () were collected at the Advanced Photon Source (Argonne National Laboratory) NE-CAT 24-ID-E beamline, using an MD-2 microdiffractometer with a 20 µm aperture. Because of the small size of the crystals, the data quality degraded before a full data set from a single crystal could be collected, but partial data sets were collected from nine separate crystals. The crystals appeared to belong to space group P43212, but subsequent analysis indicated the crystals were hemihedrally twinned and belonged to space group P43. One data set was chosen as a reference (crystal a, ). The additional data sets were processed in two orientations (h, k, l and −h, k, −l), and were then combined, one at a time, and kept or discarded, depending on whether the overall data improved, resulting in the final combined data set from partial data sets collected from three separate crystals (). Using the structure of the Taq RNAP-β1 (Taq RNAP-β[17–195 and 334–395]) as a search model, a molecular replacement solution containing four copies in the asymmetric unit was obtained using CNS (). Additional molecular replacement searches to locate the TRCF–RID, using a homology model of the Tth TRCF–RID based on the Eco TRCF–RID () as a search model, using either CNS or BRUTEPTF () were unsuccessful. Nevertheless, maps phased from the Taq RNAP-β1 molecular replacement solution alone, combined with density modification using CNS, revealed clear electron density for the TRCF–RIDs (Supplementary Figure S1A). After iterative rounds of building and minimization to 2.9 Å, the final model was refined to an R/Rfree of 0.228/0.250 using Refmac5 (). Initial rounds of refinement incorporated tight non-crystallographic symmetry (NCS) restraints (with two NCS groups, the TRCF–RID and RNAP-β1). Final rounds kept loose NCS restraints and also utilized TLS refinement (with two TLS groups, the TRCF–RID and RNAP-β1). […]

Pipeline specifications

Software tools CNS, REFMAC5
Databases RID
Applications Small-angle scattering, Protein structure analysis
Organisms Dipturus trachyderma
Diseases Tuberculosis
Chemicals Adenosine Triphosphate