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- EMDB-43112: C12 portal and adaptor complex of the mature bacteriophage PhiM1 ... -

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Basic information

Entry
Database: EMDB / ID: EMD-43112
TitleC12 portal and adaptor complex of the mature bacteriophage PhiM1 particle
Map dataReconstruction of the bacteriophage PhiM1 portal with C12 symmetry imposed.
Sample
  • Virus: Pectobacterium phage PhiM1 (virus)
    • Protein or peptide: Portal protein (gp35)
    • Protein or peptide: Adaptor protein (gp52)
Keywordsportal / bacteriophage / phage / phage portal / portal complex / adaptor protein / portal adaptor complex / podophage / autographiviridae / VIRAL PROTEIN
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.98 Å
AuthorsHodgkinson-Bean J / Eruera A
Funding support New Zealand, 1 items
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, 2023-
Header (metadata) releaseOct 23, 2024-
Map releaseOct 23, 2024-
UpdateOct 23, 2024-
Current statusOct 23, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43112.png

Downloads & links

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Map

FileDownload / File: emd_43112.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationReconstruction of the bacteriophage PhiM1 portal with C12 symmetry imposed.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.4 Å/pix.
x 384 pix.
= 537.6 Å		1.4 Å/pix.
x 384 pix.
= 537.6 Å		1.4 Å/pix.
x 384 pix.
= 537.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.4 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy EMDB: 0.4
Minimum - Maximum-1.0925357 - 1.8549212
Average (Standard dev.)0.0065237773 (±0.06548944)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 537.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Half map reconstruction of the bacteriophage PhiM1 portal...

Fileemd_43112_half_map_1.map
AnnotationHalf map reconstruction of the bacteriophage PhiM1 portal with C12 symmetry imposed. HALF A.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map reconstruction of the bacteriophage PhiM1 portal...

Fileemd_43112_half_map_2.map
AnnotationHalf map reconstruction of the bacteriophage PhiM1 portal with C12 symmetry imposed. HALF B.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Pectobacterium phage PhiM1

EntireName: Pectobacterium phage PhiM1 (virus)
Components
  • Virus: Pectobacterium phage PhiM1 (virus)
    • Protein or peptide: Portal protein (gp35)
    • Protein or peptide: Adaptor protein (gp52)

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Supramolecule #1: Pectobacterium phage PhiM1

SupramoleculeName: Pectobacterium phage PhiM1 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Host Pectobacterium atrocepticum strain SCRI1043 bacteria were infected with WT Pectobacterium phage PhiM1. Sample was purified from overnight lysates.
NCBI-ID: 1211386 / Sci species name: Pectobacterium phage PhiM1 / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Pectobacterium atrocepticum / Strain: SCRI1043
Virus shellShell ID: 1 / Name: capsid / Diameter: 635.0 Å / T number (triangulation number): 7

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Macromolecule #1: Portal protein (gp35)

MacromoleculeName: Portal protein (gp35) / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium phage PhiM1 (virus)
Molecular weightTheoretical: 55.668926 KDa
SequenceString: MYTSSMTYES LYTKYRDDSA ILKTEDYAHW TLPTVYADPD LREGKRVNVR RDYQSVGAVY VNTLSAKLAQ VLFPANQAFF RIDSTGDAA QLAEAMGAES ADLANGLAEL ENTAFRRIFL KSSYHQLVHA MKLLIITGNV LLYRDSNTGN MHAYSIRQYS V LRDGGGKV ...String:
MYTSSMTYES LYTKYRDDSA ILKTEDYAHW TLPTVYADPD LREGKRVNVR RDYQSVGAVY VNTLSAKLAQ VLFPANQAFF RIDSTGDAA QLAEAMGAES ADLANGLAEL ENTAFRRIFL KSSYHQLVHA MKLLIITGNV LLYRDSNTGN MHAYSIRQYS V LRDGGGKV LDMVLKERTV ISELPVEARI KYRNRKQDDC ICLYTRIKRE RRAVGEVFVV TQQLEDGLML DNLEVYPEAI CP FIPAVWN LVTGETYGRG LVEDYAGDLA KLSALSEALA LYEIEACRVL HMAKPGSQID VDSMAERESG AWVAGDPNGV AAY EAGDYN KIIALTQEIQ SIAARLAPAF MYAQNQRNAE RVTAEEIRQN AEEAELALGG VYSVIADTLH IPLAHILCWE VNQQ FINEL LSNGLTLSVL TGVAALSRST DVNKLIQAAQ SLSVILPVFQ NTPRVDPEKI LDMVLTGFGI NTKDLYRTEE QLQAL QAAQ APVTPDLANV AGTINETGL

UniProtKB: Putative head-tail connector protein

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Macromolecule #2: Adaptor protein (gp52)

MacromoleculeName: Adaptor protein (gp52) / type: protein_or_peptide / ID: 2 / Number of copies: 12 / Enantiomer: LEVO
Source (natural)Organism: Pectobacterium phage PhiM1 (virus)
Molecular weightTheoretical: 21.075852 KDa
SequenceString:
MELLDAVNTC LTALGEARVT STDTRHPSVA LILQTLATKQ KLLLERGWWF NTQDEEMFPD LLGRIPYPAA SISVESLDGY NIYSKRNNF LFNNTCNTMY FTGPVCIRVT YNLDFEDLPE SVATVITYRA ARAVYVGDLG NDASVQDLVL NEQQAMLLVE E QHMRNKKH STRRRRPWGK YQNALSG

UniProtKB: Putative tail tubular protein A

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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

Concentration20 mg/mL
BufferpH: 7.4
Details: 10 mM Tris HCl pH 7.4, 10 mM MgSO4 and 0.01% w/v gelatin
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Details: Negative hydrophilicity.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV
DetailsSample had a tendency to sit at the edges of holes, or where ice was slightly thicker.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number real images: 4429 / Average exposure time: 12.02 sec. / Average electron dose: 53.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 2.4 µm / Calibrated defocus min: 0.8 µm / Calibrated magnification: 36765 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.4 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 64000
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionApplied symmetry - Point group: C12 (12 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.98 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.2.1) / Number images used: 17729
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.2.1)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
DetailsInitial 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.
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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