EMD-4038 Experiments and Validation

Cryo-EM structure of RecBCD+DNA complex revealing activated nuclease domain

Single particle reconstruction
Overview of EMD-4038
Sample name: Ternary complex of RecBCD with forked DNA substrate and ADPNP
Organisms: Escherichia coli K12, Escherichia coli (strain K12), Endothia gyrosa
Fitted atomic model: 5ld2

Map parameters

Recommended contour level: 0.02 (annotator)
Number of grid points: 180 × 180 × 180
Voxel size: 1.34 × 1.34 × 1.34 Å
Minimum density: -0.059
Maximum density: 0.145
Average density: 0.00
Standard deviation: 0.009

Sample information

Sample name: Ternary complex of RecBCD with forked DNA substrate and ADPNP
Ligands: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER, MAGNESIUM ION
Nucleic acid: Fork-Hairpin DNA (70-MER)
Proteins: Ternary complex of RecBCD with forked DNA substrate and ADPNP, RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB,RecBCD enzyme subunit RecB, RecBCD enzyme subunit RecC, RecBCD enzyme subunit RecD

Validation information

Experimental information

 

Image processing

Software: RELION
Number of particles: 74656

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 1.2
Energy filter: GIF
Nominal CS (mm): 2.70
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 1674
Sampling interval (μm): 5.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 5.00
Specimen support: Glow discharge was used to thin the carbon film, but allowed to dissipate prior to use. Detergent or amphipols treatment was used to render carbon hydrophilic. See article for details.
Vitrification
Apparatus: FEI VITROBOT MARK III
Cryogen: ETHANE