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- EMDB-4440: Structure of head fiber and inner core protein gp22 of native bac... -

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Basic information

Entry
Database: EMDB / ID: EMD-4440
TitleStructure of head fiber and inner core protein gp22 of native bacteriophage P68
Map dataMap of head fiber and gp22 bound to hexon adjacent to portal, masked from five-fold cryoEM reconstruction of native bacteriophage P68 capsid
Sample
  • Virus: Staphylococcus phage P68 (virus)
    • Protein or peptide: Major head protein
    • Protein or peptide: Arstotzka protein
    • Protein or peptide: Head fiber
    • Protein or peptide: inner core proteinEarth's inner core
Function / homologyUncharacterized protein / Major head protein
Function and homology information
Biological speciesStaphylococcus phage P68 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsHrebik D / Skubnik K / Fuzik T / Plevka P
Funding support Czech Republic, 3 items
OrganizationGrant numberCountry
Czech Science Foundation15-21631Y Czech Republic
European Molecular Biology Organization3041 Czech Republic
Czech Science Foundation18-17810S Czech Republic
CitationJournal: Sci Adv / Year: 2019
Title: Structure and genome ejection mechanism of phage P68.
Authors: Dominik Hrebík / Dana Štveráková / Karel Škubník / Tibor Füzik / Roman Pantůček / Pavel Plevka /
Abstract: Phages infecting can be used as therapeutics against antibiotic-resistant bacterial infections. However, there is limited information about the mechanism of genome delivery of phages that infect ...Phages infecting can be used as therapeutics against antibiotic-resistant bacterial infections. However, there is limited information about the mechanism of genome delivery of phages that infect Gram-positive bacteria. Here, we present the structures of native phage P68, genome ejection intermediate, and empty particle. The P68 head contains 72 subunits of inner core protein, 15 of which bind to and alter the structure of adjacent major capsid proteins and thus specify attachment sites for head fibers. Unlike in the previously studied phages, the head fibers of P68 enable its virion to position itself at the cell surface for genome delivery. The unique interaction of one end of P68 DNA with one of the 12 portal protein subunits is disrupted before the genome ejection. The inner core proteins are released together with the DNA and enable the translocation of phage genome across the bacterial membrane into the cytoplasm.
History
DepositionNov 27, 2018-
Header (metadata) releaseNov 6, 2019-
Map releaseNov 6, 2019-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.67
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 1.67
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6iaw
  • Surface level: 1.67
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_4440.png

Downloads & links

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Map

FileDownload / File: emd_4440.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap of head fiber and gp22 bound to hexon adjacent to portal, masked from five-fold cryoEM reconstruction of native bacteriophage P68 capsid
Voxel sizeX=Y=Z: 1.063 Å
Density
Contour LevelBy AUTHOR: 1.67 / Movie #1: 1.67
Minimum - Maximum-5.177223 - 9.956803
Average (Standard dev.)-0.0000000068 (±0.6600001)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 212.59999 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0631.0631.063
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z212.600212.600212.600
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-5.1779.957-0.000

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Supplemental data

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Sample components

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Entire : Staphylococcus phage P68

EntireName: Staphylococcus phage P68 (virus)
Components
  • Virus: Staphylococcus phage P68 (virus)
    • Protein or peptide: Major head protein
    • Protein or peptide: Arstotzka protein
    • Protein or peptide: Head fiber
    • Protein or peptide: inner core proteinEarth's inner core

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Supramolecule #1: Staphylococcus phage P68

SupramoleculeName: Staphylococcus phage P68 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all / NCBI-ID: 204090 / Sci species name: Staphylococcus phage P68 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Staphylococcus aureus (bacteria) / Strain: dTarM 4220
Molecular weightTheoretical: 19.7 MDa
Virus shellShell ID: 1 / Name: Capsid / Diameter: 480.0 Å / T number (triangulation number): 4

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Macromolecule #1: Major head protein

MacromoleculeName: Major head protein / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus phage P68 (virus)
Molecular weightTheoretical: 46.954941 KDa
SequenceString: MAQQSTKNET ALLVAKSAKS ALQDFNHDYS KSWTFGDKWD NSNTMFETFV NKYLFPKINE TLLIDIALGN RFNWLAKEQD FIGQYSEEY VIMDTVPINM DLSKNEELML KRNYPRMATK LYGNGIVKKQ KFTLNNNDTR FNFQTLADAT NYALGVYKKK I SDINVLEE ...String:
MAQQSTKNET ALLVAKSAKS ALQDFNHDYS KSWTFGDKWD NSNTMFETFV NKYLFPKINE TLLIDIALGN RFNWLAKEQD FIGQYSEEY VIMDTVPINM DLSKNEELML KRNYPRMATK LYGNGIVKKQ KFTLNNNDTR FNFQTLADAT NYALGVYKKK I SDINVLEE KEMRAMLVDY SLNQLSETNV RKATSKEDLA SKVFEAILNL QNNSAKYNEV HRASGGAIGQ YTTVSKLKDI VI LTTDSLK SYLLDTKIAN TFQIAGIDFT DHVISFDDLG GVFKVTKEFK LQNQDSIDFL RAYGDYQSQL GDTIPVGAVF TYD VSKLKE FTGNVEEIKP KSDLYAFILD INSIKYKRYT KGMLKPPFHN PEFDEVTHWI HYYSFKAISP FFNKILITDQ DVNP KPEEE LQE

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Macromolecule #2: Arstotzka protein

MacromoleculeName: Arstotzka protein / type: protein_or_peptide / ID: 2 / Number of copies: 6 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus phage P68 (virus)
Molecular weightTheoretical: 6.922464 KDa
SequenceString:
MYEGNNMRSM MGTSYEDSRL NKRTELNENM SIDTNKSEDS YGVQIHSLSK QSFTGDVEEE

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Macromolecule #3: Head fiber

MacromoleculeName: Head fiber / type: protein_or_peptide / ID: 3
Details: Poly alanine chain fitted to the density of head-fiber
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus phage P68 (virus)
Molecular weightTheoretical: 5.720042 KDa
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)

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Macromolecule #4: inner core protein

MacromoleculeName: inner core protein / type: protein_or_peptide / ID: 4
Details: Poly alanine chain fitted to the density of the inner core protein bound to hexon adjacent to portal
Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Staphylococcus phage P68 (virus)
Molecular weightTheoretical: 3.507314 KDa
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)

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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

Concentration2 mg/mL
BufferpH: 8
Component:
ConcentrationFormulaName
50.0 mMTristris(hydroxymethyl)aminomethane
10.0 mMCaClcalcium chloride
10.0 mMNaClSodium chloridesodium chloride
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: NITROGEN
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: blot time 2s; blot force -2; 3.6 ul of sample.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 0.003 µm / Nominal defocus min: 0.001 µm / Nominal magnification: 75000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-7 / Number grids imaged: 2 / Number real images: 2891 / Average exposure time: 1.0 sec. / Average electron dose: 21.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 37218
CTF correctionSoftware - Name: CTFFIND (ver. 4.0)
Startup modelType of model: EMDB MAP
EMDB ID:

1222


Details: The initial model was scaled and clipped in EMAN2 to match the dimensions of phage P68. command: e2proc3d.py --clip=600 --scale=0.73
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 11210 / Software - Name: RELION (ver. 2.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C5 (5 fold cyclic) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Number images used: 28826

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Atomic model buiding 1

Initial modelPDB ID:

6iat

DetailsThe refinement was conducted on rigid body fitted major capsid proteins and Arstotzka proteins obtained from related structure of 6IAT; EMD-4438. The chains were fitted into hexon adjacent to portal of P68 capsid obtained from 5-fold symmetrized reconstruction. Subsequently, poly-alanine chains were manually built into densities corresponding to N-terminal part of head-fiber (chain IDs H,K,L) and C-terminal part of inner core protein (chain IDs X,Y,Z). The map was masked according to the related pdb in chimera, normalized and put into several rounds of a real space refinement in Phenix.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6iaw:
Structure of head fiber and inner core protein gp22 of native bacteriophage P68

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