EMD-5678 Experiments and Validation

Validated Near-Atomic Resolution Structure of Bacteriophage Epsilon15 Derived from Cryo-EM and Modeling

Single particle reconstruction
Overview of EMD-5678
Sample name: Bacteriophage epsilon15
Organism: Salmonella phage epsilon15
Fitted atomic model: 3j40
Publicly available raw data: http://ncmi.bcm.edu

Map parameters

Recommended contour level: 5.20 (author)
Number of grid points: 720 × 720 × 720
Voxel size: 1.19 × 1.19 × 1.19 Å
Minimum density: -15.007
Maximum density: 24.633
Average density: 0.009
Standard deviation: 1.422

Sample information

Sample name: Bacteriophage epsilon15
Virus: Salmonella phage epsilon15

Validation information

Experimental information

 

Image processing

Applied symmetry: I
Software: jspr, EMAN2, EMAN
Number of particles: 14000
Reconstruction protocol: icosahedral
CTF correction: per particle
Details: The gold standard definition for the resolution estimate was adopted whereby the particle images were split into two subsets at the onset of image processing and the datasets were individually reconstructed and then combined after determination of the resolution estimate. Independent initial models were built de novo and used for the subsequent particle refinements in each of the two subsets of particle images. The Fourier Shell Correlation (FSC) between the two independently determined reconstructions was computed and indicated a resolution 4.5 Angstrom using the 0.143 threshold for the combined dataset. Individual particles (720x720 pixels) were first automatically selected using the ethan method followed by manual screening using EMAN boxer program. A total of 54161 particles were selected for initial processing. The selected particles within a micrograph were incoherently averaged to generate 2D power spectra for contrast transfer function (CTF) parameter determination. CTF parameters were first automatically estimated and then visually verified using the EMAN1 ctfit program. Defocus values range from 0.5 to 2.5 um. The data set was divided into two data subsets for the following reconstruction steps. The particle images were first binned 4x for initial model building and initial determination of orientation and center parameters. The initial model was built de novo by iterative refinement of a subset of 300 particles randomly selected from the half data set with randomly assigned initial orientations. The initial orientations of all particles in each of the half data sets were determined using the EMAN1 projection matching program classesbymra with an angular projection step size of 3 degrees. The orientations were then refined to higher accuracy using the program jalign, which is based on simplex optimization of matching between the particle image and model projections. The particle orientation parameters were then transferred to particles binned at 2x and ultimately to particles without binning for further refinements. In the last stage of refinement, magnification, astigmatism, and defocus parameters were also included. 3D maps with icosahedral symmetry enforcement were reconstructed using a newly developed program j3dr using EMAN2 library and parallelized with message passing interface (MPI) to speed up the reconstruction process. These steps were iterated until the refinement converged. The map for each data subset was reconstructed from ~7000 particles by removing particles with poor alignment scores and unstable alignment parameters. The resolution of the map was evaluated using the Fourier Shell Correlation (FSC). Only the icosahedral shell region was included in this FSC analysis by masking out the external background noises and the internal DNA densities using soft masks with a half width of 6A. The final map of the entire dataset was then built from ~14000 particles by combining these two subsets of particles.

Imaging

Session
Microscope model: JEOL 3200FSC
Electron source: FIELD EMISSION GUN
Electron dose (e/A**2): 17.0
Energy filter: in-column filter
Energy window (eV): 0.00
Nominal CS (mm): 4.10
Nominal magnification: 50000.0
Defocus max (nm): 2,700.00
Defocus min (nm): 400.00
Holder model: JEOL 3200FSC CRYOHOLDER
Acquisition
Number of images: 1309
Scanner model: NIKON SUPER COOLSCAN 9000
Sampling interval (μm): 6.3499999999999996
Details: Digitized using Nikon Super CoolScan 9000 ED at 6.35 um/pixel

Specimen

Preparation
Specimen state: particle
Buffer: 50 mM Tris-HCl, pH 7.5, 25 mM NaCl, 5 mM MgCl2
Specimen support: Quantifoil R2/2 grid
Vitrification
Apparatus: FEI VITROBOT MARK II
Cryogen: ETHANE
Humidity: 90
Protocol: Blot before plunging.