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- PDB-8vbx: C6 nozzle and fibre complex of the mature bacteriophage PhiM1 particle -

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Basic information

Entry
Database: PDB / ID: 8vbx
TitleC6 nozzle and fibre complex of the mature bacteriophage PhiM1 particle
Components
  • Tail fiber (gp47)
  • Tail nozzle (gp51)
KeywordsVIRAL PROTEIN / Tail / fiber / fibre / complex / podophage / nozzle / protein / autographiviridae / assembly
Function / homologyBacteriophage T7 tail fibre protein / Phage T7 tail fibre protein, N-terminal domain / virus tail / Putative tail fiber protein / Putative tail tubular protein B
Function and homology information
Biological speciesPectobacterium phage PhiM1 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.23 Å
AuthorsHodgkinson-Bean, J. / Eruera, A.
Funding support New Zealand, 1items
OrganizationGrant numberCountry
Other private New Zealand
CitationJournal: PNAS Nexus / Year: 2024
Title: Ejectosome of bacteriophage ΦM1.
Authors: Alice-Roza Eruera / James Hodgkinson-Bean / Georgia L Rutter / Francesca R Hills / Rosheny Kumaran / Alexander J M Crowe / Nickhil Jadav / Fangfang Chang / Klemens McJarrow-Keller / Fátima ...Authors: Alice-Roza Eruera / James Hodgkinson-Bean / Georgia L Rutter / Francesca R Hills / Rosheny Kumaran / Alexander J M Crowe / Nickhil Jadav / Fangfang Chang / Klemens McJarrow-Keller / Fátima Jorge / Jaekyung Hyun / Hyejin Kim / Bumhan Ryu / Mihnea Bostina /
Abstract: Podophages that infect gram-negative bacteria, such as pathogen ΦM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a ...Podophages that infect gram-negative bacteria, such as pathogen ΦM1, encode tail assemblies too short to extend across the complex gram-negative cell wall. To overcome this, podophages encode a large protein complex (ejectosome) packaged inside the viral capsid and correspondingly ejected during infection to form a transient channel that spans the periplasmic space. Here, we describe the ejectosome of bacteriophage ΦM1 to a resolution of 3.32 Å by single-particle cryo-electron microscopy (cryo-EM). The core consists of tetrameric and octameric ejection proteins which form a ∼1.5-MDa ejectosome that must transition through the ∼30 Å aperture created by the short tail nozzle assembly that acts as the conduit for the passage of DNA during infection. The ejectosome forms several grooves into which coils of genomic DNA are fit before the DNA sharply turns and goes down the tunnel and into the portal. In addition, we reconstructed the icosahedral capsid and hybrid tail apparatus to resolutions between 3.04 and 3.23 Å, and note an uncommon fold adopted by the dimerized decoration proteins which further emphasize the structural diversity of podophages. These reconstructions have allowed the generation of a complete atomic model of the ΦM1, uncovering two distinct decoration proteins and highlighting the exquisite structural diversity of tailed bacteriophages.
History
DepositionDec 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Oct 23, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tail nozzle (gp51)
R: Tail fiber (gp47)
Q: Tail fiber (gp47)
P: Tail fiber (gp47)
O: Tail fiber (gp47)
N: Tail fiber (gp47)
M: Tail fiber (gp47)
X: Tail fiber (gp47)
W: Tail fiber (gp47)
V: Tail fiber (gp47)
U: Tail fiber (gp47)
T: Tail fiber (gp47)
S: Tail fiber (gp47)
L: Tail fiber (gp47)
K: Tail fiber (gp47)
J: Tail fiber (gp47)
I: Tail fiber (gp47)
H: Tail fiber (gp47)
G: Tail fiber (gp47)
B: Tail nozzle (gp51)
C: Tail nozzle (gp51)
D: Tail nozzle (gp51)
E: Tail nozzle (gp51)
F: Tail nozzle (gp51)


Theoretical massNumber of molelcules
Total (without water)1,512,19424
Polymers1,512,19424
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, Structure solved in situ.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Tail nozzle (gp51)


Mass: 84475.789 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Source: (natural) Pectobacterium phage PhiM1 (virus) / References: UniProt: A0A1P7WFX2
#2: Protein
Tail fiber (gp47)


Mass: 55852.188 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Source: (natural) Pectobacterium phage PhiM1 (virus) / References: UniProt: A0A1P7WFW3
Has protein modificationN

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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: Pectobacterium phage PhiM1 / Type: VIRUS
Details: Host Pectobacterium atrocepticum strain SCRI1043 bacteria were infected with WT Pectobacterium phage PhiM1. Sample was purified from overnight lysates.
Entity ID: all / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Pectobacterium phage PhiM1 (virus)
Details of virusEmpty: NO / Enveloped: NO / Isolate: SPECIES / Type: VIRION
Natural hostOrganism: Pectobacterium atrocepticum / Strain: SCRI1043
Virus shellName: capsid / Diameter: 635 nm / Triangulation number (T number): 7
Buffer solutionpH: 7.4
Details: 10 mM Tris HCl pH 7.4, 10 mM MgSO4 and 0.01% w/v gelatin
SpecimenConc.: 20 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Sample had a tendency to sit at the edges of holes, or where ice was slightly thicker.
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal magnification: 64000 X / Calibrated magnification: 36765 X / Nominal defocus max: 2400 nm / Nominal defocus min: 800 nm / Calibrated defocus min: 800 nm / Calibrated defocus max: 2400 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 12.02 sec. / Electron dose: 53.9 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 4429

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Processing

EM software
IDNameVersionCategory
1cryoSPARC4.2.1particle selection
2EPUimage acquisition
4cryoSPARC4.2.1CTF correction
7UCSF ChimeraX1.6model fitting
9ISOLDE1.6model refinement
10Coot0.9.5model refinement
11cryoSPARC4.2.1initial Euler assignment
12cryoSPARC4.2.1final Euler assignment
13cryoSPARC4.2.1classification
14cryoSPARC3.2.13D reconstruction
CTF correctionDetails: Patch based CTF correction. / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C6 (6 fold cyclic)
3D reconstructionResolution: 3.23 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17132 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Details: Initial models were developed using AlphaFold-2, and were docked into EM maps. Models were then flexibly fit using chimeraX, followed by manual refinement using a combination of ISOLDE ...Details: Initial models were developed using AlphaFold-2, and were docked into EM maps. Models were then flexibly fit using chimeraX, followed by manual refinement using a combination of ISOLDE (ChimeraX) and coot. Automatic refinements were performed in PHENIX real space refine.
Atomic model buildingSource name: AlphaFold / Type: in silico model

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