EMD-5607 Experiments and Validation

Structural dynamics and inter-ring communication of the MecA-ClpC complex during active substrate unfolding and translocation revealed by cryo-EM

Single particle reconstruction
Overview of EMD-5607
Sample name: MecA-ClpC(E280A)
Organism: Bacillus subtilis
Fitted atomic model: 3j3t
Related EM entries by publication: EMD-5608, EMD-5609, EMD-5610

Map parameters

Recommended contour level: 1.50 (author)
Number of grid points: 150 × 150 × 150
Voxel size: 1.50 × 1.50 × 1.50 Å
Minimum density: -4.429
Maximum density: 8.116
Average density: 0.002
Standard deviation: 0.997

Sample information

Sample name: MecA-ClpC(E280A)
Proteins: MecA, ClpC

Validation information

Experimental information

 

Image processing

Applied symmetry: C6
Software: SPIDER
Number of particles: 45514
Reconstruction protocol: Reference Projections
CTF correction: each defocus group on 3D level

Fitting

Initial atomic model: 3PXI
Fitting software: MDFF
Fitting protocol: flexible
Target criteria: Cross-correlation
Refinement space: REAL
Details: Protocol: Initial local fitting was done using Chimera and then MDFF was used for flexible fitting. ref: Trabuco, L.G., Villa, E., Mitra, K., Frank, J. and Schulten, K. (2008) Flexible fitting of atomic structures into electron microscopy maps using molecular dynamics.

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal magnification: 59000.0
Defocus max (nm): 3,000.00
Defocus min (nm): 1,500.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 994

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.03
Buffer: 50mM kCl, 10mM Tris-HCL,2mM MgCl2, 2mM ATP
Vitrification
Apparatus: FEI VITROBOT MARK IV
Cryogen: ETHANE
Humidity: 100
Protocol: Blot for 2 seconds before plunging