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- EMDB-5568: Focused asymmetric reconstruction I (map 3/8): Visualization of U... -

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Basic information

Entry
Database: EMDB / ID: EMD-5568
TitleFocused asymmetric reconstruction I (map 3/8): Visualization of Uncorrelated Tandem Symmetry Mismatches in the Internal Genome Packaging Apparatus of a dsDNA virus
Map dataFocused asymmetric reconstruction (FAR-1) of bacteriophage T710A capsid I state. Display section view z=105 for connector gp8 and z=125 for core protein gp14.
Sample
  • Sample: Bacteriophage T710A capsid I
  • Protein or peptide: gp14
  • Protein or peptide: gp15
  • Protein or peptide: gp16
  • Protein or peptide: gp8
  • Protein or peptide: gp10A
  • Protein or peptide: gp9
Keywordsbacteriophage T7 / procapsid / DNA packaging / portal / core stack / symmetry mismatch / focused asymmetric reconstruction / combinatorial assembly isomerism
Function / homology
Function and homology information


symbiont genome ejection through host cell envelope / host cell periplasmic space / : / symbiont entry into host cell via disruption of host cell wall peptidoglycan / viral portal complex / lytic transglycosylase activity / symbiont genome ejection through host cell envelope, short tail mechanism / viral DNA genome packaging / symbiont entry into host cell via disruption of host cell envelope / peptidoglycan metabolic process ...symbiont genome ejection through host cell envelope / host cell periplasmic space / : / symbiont entry into host cell via disruption of host cell wall peptidoglycan / viral portal complex / lytic transglycosylase activity / symbiont genome ejection through host cell envelope, short tail mechanism / viral DNA genome packaging / symbiont entry into host cell via disruption of host cell envelope / peptidoglycan metabolic process / symbiont entry into host / viral capsid assembly / virion component / viral capsid / killing of cells of another organism / hydrolase activity / defense response to bacterium / viral translational frameshifting / host cell plasma membrane / identical protein binding / membrane
Similarity search - Function
Capsid assembly scaffolding protein-like / Phage T7 capsid assembly protein / Internal virion protein Gp16 / Internal virion protein Gp14 / Internal virion protein Gp15 / Capsid Gp10A/Gp10B / : / Major capsid protein / Portal protein, Caudovirales / Head-to-tail connector protein, podovirus-type ...Capsid assembly scaffolding protein-like / Phage T7 capsid assembly protein / Internal virion protein Gp16 / Internal virion protein Gp14 / Internal virion protein Gp15 / Capsid Gp10A/Gp10B / : / Major capsid protein / Portal protein, Caudovirales / Head-to-tail connector protein, podovirus-type / Bacteriophage head to tail connecting protein / Prokaryotic transglycosylase, active site / Prokaryotic transglycosylases signature. / Transglycosylase SLT domain 1 / Transglycosylase SLT domain / Lysozyme-like domain superfamily
Similarity search - Domain/homology
Capsid assembly scaffolding protein / Internal virion protein gp14 / Internal virion protein gp15 / Peptidoglycan transglycosylase gp16 / Portal protein / Major capsid protein
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 17.0 Å
AuthorsGuo F / Liu Z / Vago F / Ren Y / Wu W / Wright E / Serwer P / Jiang W
CitationJournal: Proc Natl Acad Sci U S A / Year: 2013
Title: Visualization of uncorrelated, tandem symmetry mismatches in the internal genome packaging apparatus of bacteriophage T7.
Authors: Fei Guo / Zheng Liu / Frank Vago / Yue Ren / Weimin Wu / Elena T Wright / Philip Serwer / Wen Jiang /
Abstract: Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle ...Motor-driven packaging of a dsDNA genome into a preformed protein capsid through a unique portal vertex is essential in the life cycle of a large number of dsDNA viruses. We have used single-particle electron cryomicroscopy to study the multilayer structure of the portal vertex of the bacteriophage T7 procapsid, the recipient of T7 DNA in packaging. A focused asymmetric reconstruction method was developed and applied to selectively resolve neighboring pairs of symmetry-mismatched layers of the portal vertex. However, structural features in all layers of the multilayer portal vertex could not be resolved simultaneously. Our results imply that layers with mismatched symmetries can join together in several different relative orientations, and that orientations at different interfaces assort independently to produce structural isomers, a process that we call combinatorial assembly isomerism. This isomerism explains rotational smearing in previously reported asymmetric reconstructions of the portal vertex of T7 and other bacteriophages. Combinatorial assembly isomerism may represent a new regime of structural biology in which globally varying structures assemble from a common set of components. Our reconstructions collectively validate previously proposed symmetries, compositions, and sequential order of T7 portal vertex layers, resolving in tandem the 5-fold gene product 10 (gp10) shell, 12-fold gp8 portal ring, and an internal core stack consisting of 12-fold gp14 adaptor ring, 8-fold bowl-shaped gp15, and 4-fold gp16 tip. We also found a small tilt of the core stack relative to the icosahedral fivefold axis and propose that this tilt assists DNA spooling without tangling during packaging.
History
DepositionJan 9, 2013-
Header (metadata) releaseJan 23, 2013-
Map releaseApr 10, 2013-
UpdateMay 1, 2013-
Current statusMay 1, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 4.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 4.5
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

T710A-CI.ico...

Downloads & links

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Map

FileDownload / File: emd_5568.map.gz / Format: CCP4 / Size: 173.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused asymmetric reconstruction (FAR-1) of bacteriophage T710A capsid I state. Display section view z=105 for connector gp8 and z=125 for core protein gp14.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.2 Å/pix.
x 360 pix.
= 792. Å		2.2 Å/pix.
x 360 pix.
= 792. Å		2.2 Å/pix.
x 360 pix.
= 792. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.2 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 4.5 / Movie #1: 4.5
Minimum - Maximum-11.9188385 - 19.942455290000002
Average (Standard dev.)0.71645761 (±2.41129756)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-180-180-180
Dimensions360360360
Spacing360360360
CellA=B=C: 792.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.22.22.2
M x/y/z360360360
origin x/y/z0.0000.0000.000
length x/y/z792.000792.000792.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS-180-180-180
NC/NR/NS360360360
D min/max/mean-11.91919.9420.716

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Supplemental data

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Sample components

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Entire : Bacteriophage T710A capsid I

EntireName: Bacteriophage T710A capsid I
Components
  • Sample: Bacteriophage T710A capsid I
  • Protein or peptide: gp14
  • Protein or peptide: gp15
  • Protein or peptide: gp16
  • Protein or peptide: gp8
  • Protein or peptide: gp10A
  • Protein or peptide: gp9

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Supramolecule #1000: Bacteriophage T710A capsid I

SupramoleculeName: Bacteriophage T710A capsid I / type: sample / ID: 1000 / Number unique components: 6

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Macromolecule #1: gp14

MacromoleculeName: gp14 / type: protein_or_peptide / ID: 1 / Name.synonym: Internal virion protein B / Details: core stack protein / Number of copies: 12 / Oligomeric state: dodecamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Enterobacteria phage T7 (virus) / synonym: phage T7
Molecular weightTheoretical: 21 KDa
SequenceUniProtKB: Internal virion protein gp14

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Macromolecule #2: gp15

MacromoleculeName: gp15 / type: protein_or_peptide / ID: 2 / Name.synonym: Internal virion protein C / Details: core stack protein / Number of copies: 8 / Oligomeric state: octamer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Enterobacteria phage T7 (virus) / Strain: T710A / synonym: phage T7
Molecular weightTheoretical: 84 KDa
SequenceUniProtKB: Internal virion protein gp15

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Macromolecule #3: gp16

MacromoleculeName: gp16 / type: protein_or_peptide / ID: 3 / Name.synonym: Internal virion protein D / Details: core stack protein / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Enterobacteria phage T7 (virus) / Strain: T710A / synonym: phage T7
Molecular weightTheoretical: 144 KDa
SequenceUniProtKB: Peptidoglycan transglycosylase gp16

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Macromolecule #4: gp8

MacromoleculeName: gp8 / type: protein_or_peptide / ID: 4 / Name.synonym: Head-to-tail joining protein / Details: connector/portal / Number of copies: 4 / Oligomeric state: tetramer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Enterobacteria phage T7 (virus) / Strain: T710A / synonym: phage T7
Molecular weightTheoretical: 59 KDa
SequenceUniProtKB: Portal protein

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Macromolecule #5: gp10A

MacromoleculeName: gp10A / type: protein_or_peptide / ID: 5 / Name.synonym: Major capsid protein 10A
Details: major capsid protein. The correct copy number is 415.
Oligomeric state: icosahedral (T=7) / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Enterobacteria phage T7 (virus) / Strain: T710A / synonym: phage T7
Molecular weightTheoretical: 36 KDa
SequenceUniProtKB: Major capsid protein

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Macromolecule #6: gp9

MacromoleculeName: gp9 / type: protein_or_peptide / ID: 6 / Name.synonym: Capsid assembly protein / Details: scaffolding protein / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Enterobacteria phage T7 (virus) / Strain: T710A / synonym: phage T7
Molecular weightTheoretical: 34 KDa
SequenceUniProtKB: Capsid assembly scaffolding protein

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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: Ted Pella 01824, Ultrathin Carbon Film on Holey Carbon Support Film, 400 mesh, Copper
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 120 K / Instrument: FEI VITROBOT MARK I / Method: Blot for 2 seconds before plunging

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Electron microscopy #1

Microscopy ID1
MicroscopeFEI TITAN KRIOS
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DetailsParallel beam illumination
DateSep 22, 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: 1270 / Average electron dose: 25 e/Å2 / Od range: 0.6 / 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: 4.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Electron microscopy #2

Microscopy ID2
MicroscopeFEI TITAN KRIOS
TemperatureMin: 80 K / Max: 105 K / Average: 100 K
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 120,000 times magnification
DetailsParallel beam illumination
DateNov 5, 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: 1270 / Average electron dose: 25 e/Å2 / Od range: 0.6 / 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: 4.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected automatically by the ethan method and then by manual screening with the EMAN boxer program. CTF parameters were determined automatically using fitctf2.py and then visually validated using EMAN ctfit program. For 3-D reconstructions, the images were first binned 4x to make initial reconstructions. After alignment parameters and reconstructions converged, the 2x binned images were used for final reconstructions with a sampling of 2.2 A/pixel.
CTF correctionDetails: phase flipping and amplitude weighted; per particle
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.0 Å / Resolution method: OTHER / Software - Name: jspr, EMAN2, EMAN
Details: This is the FAR-I map of a serials of 8 maps (Icos, SAR, FAR-I to FAR-VI). Only the major capsid protein gp10, the connector gp8, and the core stack protein gp14 are resolved. All other ...Details: This is the FAR-I map of a serials of 8 maps (Icos, SAR, FAR-I to FAR-VI). Only the major capsid protein gp10, the connector gp8, and the core stack protein gp14 are resolved. All other proteins (gp9, gp15/16) are smeared.
Number images used: 12360

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