|Entry||Database: EMDB / ID: 6037|
|Title||Capsid Expansion Mechanism Of Bacteriophage T7 Revealed By Multi-State Atomic Models Derived From Cryo-EM Reconstructions|
|Keywords||Bacteriophage T7 / Maturation / DNA packaging / Procapsid / Non-covalent topological linking / Single particle cryo-EM|
|Sample||Bacteriophage T7 mature phage capsid|
|Source||Enterobacteria phage T7 / virus|
|Map data||Reconstruction of bacteriophage T7 mature capsid with icosahedral symmetry averaging|
|Method||single particle (icosahedral) reconstruction, at 3.6 Å resolution|
|Authors||Guo F / Liu Z / Fang PA / Zhang Q / Wright ET / Wu W / Zhang C / Vago F / Ren Y / Jakata J / Chiu W / Serwer P / Jiang W|
|Citation||Proc. Natl. Acad. Sci. U.S.A., 2014, 111, E4606-E4614|
Proc. Natl. Acad. Sci. U.S.A., 2014, 111, E4606-E4614 Yorodumi Papers
|Validation Report||PDB-ID: 3j7x|
SummaryFull reportAbout validation report
|Date||Deposition: Aug 12, 2014 / Header (metadata) release: Sep 24, 2014 / Map release: Oct 15, 2014 / Last update: Nov 19, 2014|
Downloads & links
|File||emd_6037.map.gz (map file in CCP4 format, 2000001 KB)|
|Projections & slices|
Images are generated by Spider package.
|Voxel size||X=Y=Z: 1.1 Å|
CCP4 map header:
-Entire Bacteriophage T7 mature phage capsid
|Entire||Name: Bacteriophage T7 mature phage capsid / Number of components: 1|
Oligomeric State: 415 copies of gp10A form T=7 icosahedral shell
|Mass||Theoretical: 15.1 MDa|
-Component #1: virus, Enterobacteria phage T7
|Virus||Name: Enterobacteria phage T7 / Class: VIRION / Empty: No / Enveloped: No / Isolate: SPECIES|
|Mass||Theoretical: 15.1 MDa|
|Species||Species: Enterobacteria phage T7 / virus|
|Source (natural)||Host Species: Escherichia coli / bacteria / エシェリキア・コリ, 大腸菌 / |
Host category: BACTERIA(EUBACTERIA)
|Shell #1||Name of element: mature phage capsid / Diameter: 564 Å / T number(triangulation number): 7|
|Sample solution||Buffer solution: 200 mM NaCl, 10 mM Tris-HCl, 1 mM MgCl2 / pH: 7.4|
|Support film||400 mesh copper grid with one lacy carbon layer|
|Vitrification||Instrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temperature: 120 K / Humidity: 90 %|
Method: Blot for 2 seconds twice with 2 mm offset before plunging.
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Imaging||Microscope: FEI TITAN KRIOS / Date: Aug 9, 2010|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 25 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 59000 X (nominal), 57727 X (calibrated) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 600 - 2400 nm|
|Specimen Holder||Holder: Liquid nitrogen-cooled / Model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature: 95 K ( 80 - 100 K)|
|Camera||Detector: KODAK SO-163 FILM|
|Image acquisition||Number of digital images: 364 / Scanner: NIKON SUPER COOLSCAN 9000 / Sampling size: 6.35 microns / Bit depth: 16 / OD range: 1|
|Processing||Method: single particle (icosahedral) reconstruction / Applied symmetry: I (icosahedral) / Number of projections: 33952|
Details: Particles were selected from scanned micrograph images, first automatically by the ethan method and then by manual screening with the boxer program in EMAN. The TEM instrument contrast transfer function parameters were determined automatically using fitctf2.py and were then visually validated using the EMAN ctfit program. The datasets were then divided into two subsets (even and odd) and processed completely independently, including both initial models and refinements. For 3D reconstructions, the whole datasets were divided into even-odd halves and the initial de novo models and subsequent iterative refinements were all independently performed for each half dataset. The images were first binned 4x to obtain initial models and particle parameters assuming icosahedral symmetry. De novo initial models were built using the random model approach. Random subsets of particles were assigned random initial orientations and iteratively refined until convergence. Consistent icosahedral capsid structures (other than occasional differences in handedness) were obtained by repeating the random model process. Particles with inconsistent/unstable view parameters in the initial refinements were excluded in further image processing. The orientation and center parameters were then transferred to the un-binned images for high-resolution refinements which included Simplex method-based orientation/center optimization and grid search-based refinement of defocus, astigmatism, and magnification of the images. All image refinement and reconstructions were performed with in-house developed programs jspr.py (for overall work-flow), jalign (for 2D alignment) and j3dr (for 3D reconstruction), which use EMAN and EMAN2 library functions.
|3D reconstruction||Algorithm: Projection matching / Software: jspr, EMAN, EMAN2 / CTF correction: Each particle|
Details: For 3D reconstruction, whole datasets were divided into even and odd halves and the initial de novo models and subsequent iterative refinements were all independently performed for each half dataset.
Resolution: 3.6 Å / Resolution method: FSC 0.143, gold-standard
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