Cryo EM structure of the E. coli disaggregase ClpB (BAP form, DWB mutant), in the ATPgammaS state
ELECTRON MICROSCOPY
Sample
ClpB (BAP form, double walker B mutant) homohexamer in the ATPgammaS state
Specimen Preparation
Sample Aggregation State
PARTICLE
Vitrification Instrument
FEI VITROBOT MARK III
Cryogen Name
ETHANE
Sample Vitrification Details
3D Reconstruction
Reconstruction Method
SINGLE PARTICLE
Number of Particles
60000
Reported Resolution (Å)
4.5
Resolution Method
FSC 0.143 CUT-OFF
Other Details
Refinement Type
Symmetry Type
POINT
Point Symmetry
C1
Map-Model Fitting and Refinement
Id
1
Refinement Space
REAL
Refinement Protocol
FLEXIBLE FIT
Refinement Target
local correlation coeficient
Overall B Value
Fitting Procedure
Details
Initial models of the BAPDWB monomer were generated using MODELLER v9.17 with previously determined crystal structures of ClpB or ClpB domains as temp ...
Initial models of the BAPDWB monomer were generated using MODELLER v9.17 with previously determined crystal structures of ClpB or ClpB domains as templates (PDB ids 4CIU, 1QVR, 4HSE and 4LJ9). The crystal structure of E. coli ClpB (4CIU) was used as a main template, the crystal structure of T. thermophilus (1QVR) was used to model positions of the NTD and the crystal structures of AAA1 and AAA2 of E. coli ClpB (4HSE and 4LJ9) were used to model the pore loops disordered in other crystal structures. Initial rigid body fitting of the monomers in the map was manually done in Chimera using the Fit-in-Map tool.
iMODFIT was used for fitting involving large domain motions. FlexEM was then used for refinement of secondary structures and loops in the map. Quality and improvement of the fit was assessed with TEMPy using the Segment based Manders Overlap Coefficient (SMOC) scores and segment based cross correlation scores. Rigid body fitting of ATPgammaS (structure extracted from PDB id 3EIH) into the nucleotide binding sites was manually done in Chimera using the Fit-in-Map tool. The target map for fitting of ATPgammaS was the difference map between experimental map and map generated using the nucleotide-free model reveiling nucleotide densities. A round of real-space refinement was performed in phenix using energy minimization to fix the model's geometry and clashes.
