EMD-22933 Experiments and Validation

Negative stain electro microscopy reconstruction of cross-reactive RBD-directed Fab DH1047 in complex with hexapro SARS-CoV-2 spike ectodomain

Single particle reconstruction
Overview of EMD-22933
Sample name: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ectodomain
Organism: Severe acute respiratory syndrome coronavirus 2

Map parameters

Recommended contour level: 0.03 (author)
Number of grid points: 96 × 96 × 96
Voxel size: 4.02 × 4.02 × 4.02 Å
Minimum density: -0.088
Maximum density: 0.139
Average density: 0.00
Standard deviation: 0.011

Sample information

Sample name: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ectodomain
Protein: Complex of RBD-directed Fab DH1042 with hexapro SARS-CoV-2 spike ectodomain

Validation information

Experimental information

 

Image processing

Applied symmetry: C3
Software: RELION
Number of particles: 40046
Reconstruction protocol: BACK PROJECTION

Imaging

Session
Microscope model: FEI/PHILIPS EM420
Electron source: LAB6
Electron dose (e/A**2): 32.0
Nominal CS (mm): 2.00
Nominal magnification: 82000.0
Defocus max (nm): 1,500.00
Defocus min (nm): 400.00
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Number of images: 220
Sampling interval (μm): 33.0

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 0.10
Staining procedure: Samples were diluted to 0.1 mg/mL in 20 mM HEPES buffer, pH 7.4, with 5% glycerol, 150 mM NaCl, and 7.5 mM glutaraldehyde. After 5 minute incubation at room temperature, sufficient 1 M Tris stock, pH 7.4, was added to a final concentration of 75 mM Tris to quench unreacted glutaraldehyde, and was then incubated 5 minutes. Sample was then applied to a carbon film over 400 mesh copper EM grids that had been glow-discharged, incubated 1 minute, and then stained with 2% uranyl formate.
Vitrification
Cryogen: NONE