EMD-2364 Experiments and Validation

CryoEM reconstruction of the bacteriophage phi6 procapsid to the near-atomic resolution

Single particle reconstruction
Overview of EMD-2364
Sample name: Wildtype P1247 procapsid of bacteriophage phi6
Organism: Pseudomonas phage phi6
Fitted atomic model: 4btg

Map parameters

Recommended contour level: 2.00 (author)
Number of grid points: 420 × 420 × 420
Voxel size: 1.40 × 1.40 × 1.40 Å
Minimum density: -8.041
Maximum density: 21.125
Average density: 0.00
Standard deviation: 1.00

Sample information

Sample name: Wildtype P1247 procapsid of bacteriophage phi6
Virus: Pseudomonas phage phi6

Validation information

Experimental information

 

Image processing

Applied symmetry: I
Software: EMAN, BSOFT
Number of particles: 18326
CTF correction: Particles from each micrograph

Fitting

Initial atomic model: 4K7H
Used chain: A
Fitting software: Chimera, MDFF, Coot
Fitting protocol: flexible
Refinement space: REAL
Details: the P1 crystal structure was rigid body-fitted into the procapsid map using Chimera. Regions where the crystal structure deviated significantly from the EM density were roughly adjusted in Coot and then the P1A and P1B structures were flexibly fitted using the MDFF package. Finally, both structures were visually inspected and refined in Coot.

Segmentation and Mask

Mask 1
Mask name: emd_2364_msk.map
Annotation: This mask represents the P1A subunit
Mask 2
Mask name: emd_2364_msk_1.map
Annotation: This mask represents the P1B subunit

Imaging

Session
Microscope model: FEI TITAN KRIOS
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 15.0
Nominal CS (mm): 2.70
Nominal magnification: 46000.0
Defocus max (nm): 2,200.00
Defocus min (nm): 800.00
Tilt max (degrees): 0.00
Tilt min (degrees): 0.00
Holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Acquisition
Number of images: 220
Scanner model: NIKON SUPER COOLSCAN 9000
Sampling interval (μm): 6.3499999999999996

Specimen

Preparation
Specimen state: particle
Specimen concentration (mg/mL): 10.00
Buffer: 10 mM potassium phosphate, 5 mM MgCl2
Specimen support: 400 mesh C-flat holey carbon grid
Vitrification
Apparatus: FEI VITROBOT MARK I
Cryogen: ETHANE
Humidity: 80
Protocol: Blot for 2 seconds before plunging.