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- EMDB-6037: Capsid Expansion Mechanism Of Bacteriophage T7 Revealed By Multi-... -

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Entry
Database: EMDB / ID: EMD-6037
TitleCapsid Expansion Mechanism Of Bacteriophage T7 Revealed By Multi-State Atomic Models Derived From Cryo-EM Reconstructions
Map dataReconstruction of bacteriophage T7 mature capsid with icosahedral symmetry averaging
Sample
  • Sample: Bacteriophage T7 mature phage capsid
  • Virus: Enterobacteria phage T7 (virus)
KeywordsBacteriophage T7 / Maturation / DNA packaging / Procapsid / Non-covalent topological linking / Single particle cryo-EM
Function / homologyCapsid Gp10A/Gp10B / : / Major capsid protein / viral capsid / viral translational frameshifting / identical protein binding / Major capsid protein
Function and homology information
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsGuo F / Liu Z / Fang PA / Zhang Q / Wright ET / Wu W / Zhang C / Vago F / Ren Y / Jakata J ...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
CitationJournal: Proc Natl Acad Sci U S A / Year: 2014
Title: Capsid expansion mechanism of bacteriophage T7 revealed by multistate atomic models derived from cryo-EM reconstructions.
Authors: Fei Guo / Zheng Liu / Ping-An Fang / Qinfen Zhang / Elena T Wright / Weimin Wu / Ci Zhang / Frank Vago / Yue Ren / Joanita Jakana / Wah Chiu / Philip Serwer / Wen Jiang /
Abstract: Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For ...Many dsDNA viruses first assemble a DNA-free procapsid, using a scaffolding protein-dependent process. The procapsid, then, undergoes dramatic conformational maturation while packaging DNA. For bacteriophage T7 we report the following four single-particle cryo-EM 3D reconstructions and the derived atomic models: procapsid (4.6-Å resolution), an early-stage DNA packaging intermediate (3.5 Å), a later-stage packaging intermediate (6.6 Å), and the final infectious phage (3.6 Å). In the procapsid, the N terminus of the major capsid protein, gp10, has a six-turn helix at the inner surface of the shell, where each skewed hexamer of gp10 interacts with two scaffolding proteins. With the exit of scaffolding proteins during maturation the gp10 N-terminal helix unfolds and swings through the capsid shell to the outer surface. The refolded N-terminal region has a hairpin that forms a novel noncovalent, joint-like, intercapsomeric interaction with a pocket formed during shell expansion. These large conformational changes also result in a new noncovalent, intracapsomeric topological linking. Both interactions further stabilize the capsids by interlocking all pentameric and hexameric capsomeres in both DNA packaging intermediate and phage. Although the final phage shell has nearly identical structure to the shell of the DNA-free intermediate, surprisingly we found that the icosahedral faces of the phage are slightly (∼4 Å) contracted relative to the faces of the intermediate, despite the internal pressure from the densely packaged DNA genome. These structures provide a basis for understanding the capsid maturation process during DNA packaging that is essential for large numbers of dsDNA viruses.
History
DepositionAug 12, 2014-
Header (metadata) releaseSep 24, 2014-
Map releaseOct 15, 2014-
UpdateNov 19, 2014-
Current statusNov 19, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.6
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 4.6
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-3j7x
  • Surface level: 4.6
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-3j7x
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6037.png

Downloads & links

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Map

FileDownload / File: emd_6037.map.gz / Format: CCP4 / Size: 1.9 GB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of bacteriophage T7 mature capsid with icosahedral symmetry averaging
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.1 Å/pix.
x 800 pix.
= 880. Å		1.1 Å/pix.
x 800 pix.
= 880. Å		1.1 Å/pix.
x 800 pix.
= 880. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.6 / Movie #1: 4.6
Minimum - Maximum-18.852264399999999 - 28.699796679999999
Average (Standard dev.)0.12035374 (±1.19772434)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-400-400-400
Dimensions800800800
Spacing800800800
CellA=B=C: 880.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.11.11.1
M x/y/z800800800
origin x/y/z0.0000.0000.000
length x/y/z880.000880.000880.000
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ969680
MAP C/R/S123
start NC/NR/NS-400-400-400
NC/NR/NS800800800
D min/max/mean-18.85228.7000.120

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Supplemental data

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Sample components

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Entire : Bacteriophage T7 mature phage capsid

EntireName: Bacteriophage T7 mature phage capsid
Components
  • Sample: Bacteriophage T7 mature phage capsid
  • Virus: Enterobacteria phage T7 (virus)

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Supramolecule #1000: Bacteriophage T7 mature phage capsid

SupramoleculeName: Bacteriophage T7 mature phage capsid / type: sample / ID: 1000
Oligomeric state: 415 copies of gp10A form T=7 icosahedral shell
Number unique components: 1
Molecular weightTheoretical: 15.1 MDa

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Supramolecule #1: Enterobacteria phage T7

SupramoleculeName: Enterobacteria phage T7 / type: virus / ID: 1 / NCBI-ID: 10760 / Sci species name: Enterobacteria phage T7 / Database: NCBI / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Escherichia coli (E. coli) / synonym: BACTERIA(EUBACTERIA)
Molecular weightTheoretical: 15.1 MDa
Virus shellShell ID: 1 / Name: mature phage capsid / Diameter: 564 Å / T number (triangulation number): 7

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.4 / Details: 200 mM NaCl, 10 mM Tris-HCl, 1 mM MgCl2
GridDetails: 400 mesh copper grid with one lacy carbon layer
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK I
Method: Blot for 2 seconds twice with 2 mm offset before plunging.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
TemperatureMin: 80 K / Max: 100 K / Average: 95 K
DateAug 9, 2010
Image recordingCategory: FILM / Film or detector model: KODAK SO-163 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Digitization - Sampling interval: 6.35 µm / Number real images: 364 / Average electron dose: 25 e/Å2 / Od range: 1 / Bits/pixel: 16
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 57727 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 59000
Sample stageSpecimen holder: Liquid nitrogen-cooled / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsParticles 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.
CTF correctionDetails: Each particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 3.6 Å / Resolution method: OTHER / Software - Name: jspr, EMAN, EMAN2
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.
Number images used: 33952

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