Computational protocol: Structures of type IV pilins from Thermus thermophilus demonstrate similarities with type II secretion system pseudopilins

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Protocol publication

[…] Crystallization trials were carried out in MRC 2-well plates using a Mosquito robot (TTP Labtech), with a 1:1 drop ratio and incubated at 20 °C. Eight commercially available crystallization screens (Molecular Dimensions and Microlytic) were used for initial trials. Tt121836-123 native crystals were grown by mixing 400 nl of protein (7 mg/ml) with 400 nl of well solution, which comprised 0.1 M Hepes pH 7, 1 M succinic acid and 1% PEG (w/v) 2000 MME. Selenomethionine Tt121836-123 crystals were grown by the same method, except that the well solution contained 0.2 M LiCl, 0.1 M Hepes pH 7 and 20% (w/v) PEG 6000. Crystals were cryoprotected by washing for 5 min in well solution supplemented with 20% glycerol (v/v), before flash cooling in liquid nitrogen. Tt121836-123 crystals, both native and selenomethionine, initially diffracted X-rays to a resolution of 4–6 Å, but annealing the crystals for 2–4 s at the beamlines improved the resolution considerably. Tt121933-236 crystals were grown by mixing 400 nl of protein (8 mg/ml) with 400 nl 0.01 M zinc chloride, 0.1 M sodium acetate pH 5 and 20% (w/v) PEG 6000. A native dataset to 1.85 Å resolution was collected, in a P21 spacegroup crystal form. A second crystal form was identified, in spacegroup C2, which diffracted to a lower resolution (2.3 Å). The crystals were cryoprotected in a similar manner to the procedure described for Tt121836-123. For phasing of Tt121933-236, crystals were incubated for 10 min in the well solution supplemented with freshly prepared 0.4 M KI, which was also included in the cryoprotectant. Tt122238-123 crystals were grown by mixing 200 nl of protein solution (7–10 mg/ml) with 200 nl of well solution, which comprised 0.1 M sodium acetate pH 4.6 and 2 M ammonium sulfate. Repeated attempts to derivatize Tt122238-123 crystals with heavy metals using platinum, mercury and gold salts were unsuccessful; fortunately, incubation of the crystals with 0.4 M KI, in a similar manner to the procedure for Tt121933-236, yielded an iodide derivative which permitted effective phasing. Diffraction data were collected at Diamond Light Source beamlines I02, I04 and I24. Datasets were processed by automated pipeline implemented in xia2 (), using XDS (), and relevant data collection statistics are summarised in , , . [...] In the case of Tt121933-236, experimental phases were derived from data from four different crystals, which were merged and scaled using Aimless (), as implemented in the CCP4 suite (). For all three pilin structures, automated heavy atom substructure identification was combined with experimental phasing and model building using Autosol (), as implemented in PHENIX (). For Tt121933-236, phase calculation was assisted with autoSHARP (). In each case the partially completed pilin structures were used as search models for molecular replacement, against their respective higher resolution native datasets, using Phaser (). Automated model building by Autobuild () built majority of the residues. The structures were completed by manual model building in Coot () and refined using phenix.refine () and refmac (). The structures were validated using Molprobity () and PDB_REDO (). The relevant phasing statistics and refinement parameters are detailed in , , . Coordinates and structure factors have been deposited in the Protein Data Bank, with the following accession codes: 5G2F (Tt122238-123), 5G25 (Tt121836-123), 5G23 (Tt121933-236, P21 form), 5G24 (Tt121933-236, C2 form). […]

Pipeline specifications

Software tools xia2, XDS, CCP4, PHENIX, Coot, MolProbity
Databases PDB_REDO
Applications Small-angle scattering, Protein structure analysis
Organisms Thermus thermophilus, Bacteria
Diseases Mastocytosis, Systemic