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- PDB-8vb4: C12 portal and adaptor complex of the mature bacteriophage PhiM1 ... -

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Basic information

Entry
Database: PDB / ID: 8vb4
TitleC12 portal and adaptor complex of the mature bacteriophage PhiM1 particle
Components
  • Adaptor protein (gp52)
  • Portal protein (gp35)
KeywordsVIRAL PROTEIN / portal / bacteriophage / phage / phage portal / portal complex / adaptor protein / portal adaptor complex / podophage / autographiviridae
Function / homologyTail tubular protein Gp11 / Tail tubular protein / Head-to-tail connector protein, podovirus-type / Bacteriophage head to tail connecting protein / symbiont genome ejection through host cell envelope, short tail mechanism / virion component / Putative tail tubular protein A / Putative head-tail connector protein
Function and homology information
Biological speciesPectobacterium phage PhiM1 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.98 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: Portal protein (gp35)
B: Portal protein (gp35)
C: Portal protein (gp35)
D: Portal protein (gp35)
E: Portal protein (gp35)
F: Portal protein (gp35)
G: Portal protein (gp35)
H: Portal protein (gp35)
I: Portal protein (gp35)
J: Portal protein (gp35)
K: Portal protein (gp35)
L: Portal protein (gp35)
M: Adaptor protein (gp52)
N: Adaptor protein (gp52)
O: Adaptor protein (gp52)
P: Adaptor protein (gp52)
Q: Adaptor protein (gp52)
R: Adaptor protein (gp52)
S: Adaptor protein (gp52)
T: Adaptor protein (gp52)
U: Adaptor protein (gp52)
V: Adaptor protein (gp52)
W: Adaptor protein (gp52)
X: Adaptor protein (gp52)


Theoretical massNumber of molelcules
Total (without water)920,93724
Polymers920,93724
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, In situ reconstruction of WT phage particles.
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Portal protein (gp35)


Mass: 55668.926 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Pectobacterium phage PhiM1 (virus) / References: UniProt: A0A1P7WG10
#2: Protein
Adaptor protein (gp52)


Mass: 21075.852 Da / Num. of mol.: 12 / Source method: isolated from a natural source / Source: (natural) Pectobacterium phage PhiM1 (virus) / References: UniProt: A0A1P7WG03
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.
Specimen supportDetails: Negative hydrophilicity. / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
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
9cryoSPARC4.2.1initial Euler assignment
10cryoSPARC4.2.1final Euler assignment
11cryoSPARC4.2.1classification
12cryoSPARC3.2.13D reconstruction
13ISOLDE1.6model refinement
14Coot0.9.5model refinement
CTF correctionDetails: Patch based CTF correction. / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C12 (12 fold cyclic)
3D reconstructionResolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 17729 / 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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