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- PDB-3j7w: Capsid Expansion Mechanism Of Bacteriophage T7 Revealed By Multi-... -

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Entry
Database: PDB / ID: 3j7w
TitleCapsid Expansion Mechanism Of Bacteriophage T7 Revealed By Multi-State Atomic Models Derived From Cryo-EM Reconstructions
ComponentsMajor capsid protein 10A
KeywordsVIRUS / maturation / DNA packaging / Procapsid / Non-covalent topological linking
Function / homologyCapsid Gp10A/Gp10B / : / Major capsid protein / viral capsid / identical protein binding / Major capsid protein
Function and homology information
Biological speciesEnterobacteria phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGuo, F. / Liu, Z. / Fang, P.A. / Zhang, Q. / Wright, E.T. / Wu, W. / Zhang, C. / Vago, F. / Ren, Y. / Jakata, J. ...Guo, F. / Liu, Z. / Fang, P.A. / Zhang, Q. / Wright, E.T. / 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, 2014Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 15, 2014Provider: repository / Type: Initial release
Revision 1.1Oct 29, 2014Group: Database references
Revision 1.2Nov 12, 2014Group: Database references
Revision 1.3Jul 18, 2018Group: Data collection / Category: em_software / Item: _em_software.image_processing_id / _em_software.name
Revision 1.4Feb 21, 2024Group: Data collection / Database references / Derived calculations
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / pdbx_struct_oper_list
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _pdbx_struct_oper_list.name / _pdbx_struct_oper_list.symmetry_operation / _pdbx_struct_oper_list.type

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Structure visualization

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  • Biological unit as complete icosahedral assembly
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  • Biological unit as icosahedral pentamer
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  • Biological unit as icosahedral 23 hexamer
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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
  • EMDB-6035
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  • Superimposition on EM map
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Structure viewerMolecule:
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Assembly

Deposited unit
A: Major capsid protein 10A
B: Major capsid protein 10A
C: Major capsid protein 10A
D: Major capsid protein 10A
E: Major capsid protein 10A
F: Major capsid protein 10A
G: Major capsid protein 10A


Theoretical massNumber of molelcules
Total (without water)256,1277
Polymers256,1277
Non-polymers00
Water0
1
A: Major capsid protein 10A
B: Major capsid protein 10A
C: Major capsid protein 10A
D: Major capsid protein 10A
E: Major capsid protein 10A
F: Major capsid protein 10A
G: Major capsid protein 10A
x 60


Theoretical massNumber of molelcules
Total (without water)15,367,643420
Polymers15,367,643420
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation59
2


  • Idetical with deposited unit
  • icosahedral asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3
A: Major capsid protein 10A
B: Major capsid protein 10A
C: Major capsid protein 10A
D: Major capsid protein 10A
E: Major capsid protein 10A
F: Major capsid protein 10A
G: Major capsid protein 10A
x 5


  • icosahedral pentamer
  • 1.28 MDa, 35 polymers
Theoretical massNumber of molelcules
Total (without water)1,280,63735
Polymers1,280,63735
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation4
4
A: Major capsid protein 10A
B: Major capsid protein 10A
C: Major capsid protein 10A
D: Major capsid protein 10A
E: Major capsid protein 10A
F: Major capsid protein 10A
G: Major capsid protein 10A
x 6


  • icosahedral 23 hexamer
  • 1.54 MDa, 42 polymers
Theoretical massNumber of molelcules
Total (without water)1,536,76442
Polymers1,536,76442
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation5
5


  • Idetical with deposited unit in distinct coordinate
  • icosahedral asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: I (icosahedral))

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Components

#1: Protein
Major capsid protein 10A / Gene product 10A / Gp10A


Mass: 36589.625 Da / Num. of mol.: 7 / Source method: isolated from a natural source / Source: (natural) Enterobacteria phage T7 (virus) / References: UniProt: P19726

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Bacteriophage T7 MLD capsid II / Type: VIRUS / Details: 415 copies of gp10A form T=7 icosaheral shell
Molecular weightValue: 15.1 MDa / Experimental value: NO
Details of virusEmpty: YES / Enveloped: NO / Host category: BACTERIA(EUBACTERIA) / Isolate: SPECIES / Type: VIRION
Natural hostOrganism: Escherichia coli
Buffer solutionName: T/M buffer / pH: 7.4 / Details: 200 mM NaCl, 10 mM Tris-HCl, 1 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 400 mesh copper grid with one lacy carbon layer and one layer of ultra-thin continuous carbon film on top. The grid is then coated with poly-lysine.
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Temp: 120 K / Humidity: 90 %
Details: Blot for 2 seconds twice with 2 mm offset before plunging into liquid ethane (FEI VITROBOT MARK I).
Method: Blot for 2 seconds twice with 2 mm offset before plunging.

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS / Date: Jan 7, 2011
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 59000 X / Calibrated magnification: 57727 X / Nominal defocus max: 3300 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / Camera length: 0 mm
Specimen holderSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Specimen holder type: Liquid nitrogen-cooled / Temperature: 95 K / Temperature (max): 100 K / Temperature (min): 80 K
Image recordingElectron dose: 25 e/Å2 / Film or detector model: KODAK SO-163 FILM
Image scansNum. digital images: 1368
RadiationProtocol: SINGLE WAVELENGTH / Monochromatic (M) / Laue (L): M / Scattering type: x-ray
Radiation wavelengthRelative weight: 1

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Processing

EM software
IDNameVersionCategory
1EMAN13D reconstruction
2EMAN23D reconstruction
3jspr3D reconstruction
CTF correctionDetails: Each particle
SymmetryPoint symmetry: I (icosahedral)
3D reconstructionMethod: Projection matching / Resolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 43417 / Nominal pixel size: 1.1 Å / Actual pixel size: 1.1 Å
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 ...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.
Symmetry type: POINT
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms17829 0 0 0 17829

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