Computational protocol: TubZ filament assembly dynamics requires the flexible C-terminal tail

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Protocol publication

[…] Samples from polymerization experiments were adsorbed onto carbon-coated grids and negatively stained with 2% uranyl acetate. Images were taken at various magnifications with a JEOL 1230 electron microscope equipped with a 16 Mpixel CMOS TVIPS Temcam-F416 camera and operated at 100 kV. For image processing micrographs were captured at 40,000x nominal magnification (2.84 Å/pixel). For filament structural analysis, straight segments were manually selected and boxed at 200 × 200 and 128 × 128 pixels using EMAN. For each sample, the extracted particles (CbTubZ GTP ~ 8000, CbTubZ GTP- γ-S ~ 4600, CbTubZ GDP ~ 7500, CbTubZE200A GTP ~ 20,000 and CbTubZT100A GTP ~ 12,500) were classified and averaged using the CL2D algorithm. Helical reconstruction was performed with CbTubZ GTP data (128 × 128 pixels per particles) using the Iterative Helical Real Space Reconstruction Protocol (IHRSR). An initial non-symmetrized three-dimensional reconstruction was performed using EMAN to obtain an initial estimation of the helical parameters, rise, and angle between monomers. A featureless cylinder of the appropriate diameter was used as starting model. Different values around the estimated ones were used as starting points in the IHRSR scripts to analyze parameter convergences. All runs converged on a similar final reconstruction. Rings were obtained from assembled TubZ350 in the presence of GTP and magnesium acetate after the addition of TubY226 (in a 1:1 ratio) to the solution. Images were selected manually and subsequently classified and averaged using XMIPP. [...] TubZ316 crystals were grown at 22 °C in reservoir solution containing 0.1 M Na-citrate [pH 5.5], 0.1 M NaCl, 12% PEG4000 (Molecular Dimensions/MemStart-MemSys screen) in the presence of a peptide containing the last 8 residues of TubZ (NDFFSKYM) using the sitting-drop vapor-diffusion technique. Crystals were cryo-protected with 22% glycerol before being mounted in a nylon loop and flash-cooled in liquid nitrogen. X-ray diffraction data were collected at beamline PROXIMA 1 beamline (SOLEIL Synchrotron, France) using a wavelength 0.98011 Å and a DECTRIS PILATUS 6 M detector. Datasets were indexed, integrated, and scaled using XDS and the HKL reflection file was converted to a MTZ file using XDSCONV and the CCP4 suite supported program CAD. Initial coordinates were determined by molecular replacement using PHASER and the coordinates for Apo-TubZ (Protein Data Bank (PDB) ID 3V3T). Ligand GDP coordinates and restrains were generated using ELBOW and fitted into the electron density maps using LigandFit. No extra density was observed for the peptide. This was followed by several cycles of refinement using PHENIX, which also was used for water picking, and iterative model building with COOT. The final model was validated using MolProbidity. Figures were generated using Pymol (Schrödinger). […]

Pipeline specifications

Software tools XDS, CCP4, PHENIX, Coot, PyMOL
Applications Small-angle scattering, Protein structure analysis
Organisms Bacteria
Chemicals Guanosine Triphosphate, Nucleotides