EMD-2380 Experiments and Validation

Structure of herpesvirus fusion glycoprotein B-bilayer complex revealing the protein-membrane and lateral protein-protein interaction

Subtomogram averaging
Overview of EMD-2380
Sample name: HSV-1 glycoprotein B ectodomain lacking the membrane-proximal region bound to a lipid bilayer
Organism: Human herpesvirus 1
Fitted atomic model: 4bom
Related EM entry by publication: EMD-2379

Map parameters

Minimum density: -12.513
Maximum density: 18.674
Average density: 0.00
Standard deviation: 1.00
Recommended contour level: 2.00 (author)

Sample information

Sample name: HSV-1 glycoprotein B ectodomain lacking the membrane-proximal region bound to a lipid bilayer
Protein: Envelope glycoprotein B

Validation information

Experimental information

 

Image processing

Applied symmetry: C3
Software: Jsubtomo
Number of subtomograms averaged: 996
Reconstruction protocol: Subtomogram averaging
CTF correction: Low pass filter to the first zero crossing of the CTF
Details: The best 786 spikes (of 996) were selected based on constrained cross correlation coefficient and by excluding overlaps. All three Euler angles of the spike were refined. The sub-tomograms were picked manually from tomographic reconstructions of 38 liposomes

Fitting

Initial atomic model: 3NWF
Used chains: A, B, C
Fitting software: Flex-EM
Fitting protocol: flexible
Refinement space: REAL

Imaging

Session
Microscope model: FEI TECNAI F20
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 100.0
Nominal CS (mm): 2.00
Defocus max (nm): 2,000.00
Defocus min (nm): 2,000.00
Tilt max (degrees): 60.00
Tilt min (degrees): -60.00
Holder model: SIDE ENTRY, EUCENTRIC
Acquisition
Number of images: 9
Sampling interval (μm): 15.0
Details: The dataset consists of 9 tomograms (containing 38 liposomes with bound gB). Data were binned by factor of 2.

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 1.00
Buffer: PBS with sodium citrate
Specimen support: Cflat
Vitrification
Apparatus: OTHER
Cryogen: ETHANE-PROPANE MIXTURE
Protocol: Blot manually for 3 s before plunging