Cryo-EM structure of the bacteria-killing type IV secretion system core complex from Xanthomonas citri
ELECTRON MICROSCOPY
Refinement
RMS Deviations
Key
Refinement Restraint Deviation
f_dihedral_angle_d
10.324
f_angle_d
0.892
f_chiral_restr
0.06
f_bond_d
0.008
f_plane_restr
0.007
Sample
Core complex of a bacterial killing type IV secretion system from Xanthomonas
Specimen Preparation
Sample Aggregation State
PARTICLE
Vitrification Instrument
FEI VITROBOT MARK IV
Cryogen Name
ETHANE
Sample Vitrification Details
Blot for 4.5 seconds after 30 seconds of incubation.
3D Reconstruction
Reconstruction Method
SINGLE PARTICLE
Number of Particles
142306
Reported Resolution (Å)
3.28
Resolution Method
FSC 0.143 CUT-OFF
Other Details
Refinement Type
Symmetry Type
POINT
Point Symmetry
C14
Map-Model Fitting and Refinement
Id
1
Refinement Space
REAL
Refinement Protocol
Refinement Target
Cross-correlation coefficient
Overall B Value
138
Fitting Procedure
Details
The electron density was clearly interpretable, which allowed us to build a de novo structural model. This process began by fitting the crystallograph ...
The electron density was clearly interpretable, which allowed us to build a de novo structural model. This process began by fitting the crystallographic model of the X. citri VirB7 C-terminal N0 domain (PDB:3OV5) and the NMR model of the X. citri VirB9CTD-VirB7NTD complex (PDB:2N01) in order to identify the map with the correct handedness. Models were positioned using Fit in map tool in Chimera, and saved relative to the map. Using these as starting points, we were able to manually build the rest of the model for VirB7 and VirB9CTD, and the de novo models for VirB10CTD, VirB10NTD_150-161 and VirB9NTD using Coot. In this manner, we obtained a combined model for a single VirB7-VirB9-VirB10 heterotrimer unit, which was submitted to iterative rounds of real space refinement and building using PHENIX and Coot software, respectively. Thirteen more copies of the refined heterotrimer were then fit into the density map using Chimera and new rounds of real space refinement (now using NCS for the 42 chains contained in the structure) and building using PHENIX and Coot, respectively, were executed until we obtained good parameters for Ramachandran plot and MolProbity. Chimera and PyMol were used for map and model visualization and figure production.
