EMD-2555 Experiments and Validation

Negative-stain electron microscopy of E. coli ClpB mutant E432A (BAP form bound to ClpP)

Single particle reconstruction
Overview of EMD-2555
Sample name: ClpB E432A ATPgammaS. BAP variant bound to ClpP.
Organism: Escherichia coli
Fitted atomic model: 4d2q

Map parameters

Recommended contour level: 0.168 (author)
Number of grid points: 256 × 256 × 256
Voxel size: 2.00 × 2.00 × 2.00 Å
Minimum density: -1.155
Maximum density: 9.923
Average density: 0.01
Standard deviation: 0.19

Sample information

Sample name: ClpB E432A ATPgammaS. BAP variant bound to ClpP.
Protein: ClpB

Validation information

Experimental information

 

Image processing

Applied symmetry: C6
Software: IMAGIC, Spider
Number of particles: 11570
Reconstruction protocol: angular reconstitution and projection matching
CTF correction: phase flipping entire frame
Details: Starting models were generated by angular reconstitution and particle orientations were refined by projection matching in SPIDER. Only part of the molecule was refined in the alignment, but the final reconstruction includes the whole molecule.

Fitting

Fitting 1
Initial atomic model: 1KHY
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Details: Fitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit.
Fitting 2
Initial atomic model: 4CIU
Used chain: A
Fitting software: Chimera
Fitting protocol: rigid body
Refinement space: REAL
Details: Fitting of separate domains was performed manually and locally optimised using Chimera. Known domain interfaces were used to guide the fit.

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 20.0
Nominal CS (mm): 2.00
Nominal magnification: 50000.0
Defocus max (nm): 1,200.00
Defocus min (nm): 500.00
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Number of images: 110

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.03
Buffer: 20 mM Tris-HCl, pH 7.5, 20 mM KCl, 15 mM MgCl2, 1 mM DTT, 2 mM ATPgammaS
Staining procedure: protein adsorbed on carbon coated grids pretreated with 0.01% poly lysine chains. Stained with 2% uranyl acetate for 1 minute.
Vitrification
Apparatus: NONE
Cryogen: NONE