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- EMDB-22441: The bacteriophage Phi-29 viral genome packaging motor assembly -

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Basic information

Entry
Database: EMDB / ID: EMD-22441
TitleThe bacteriophage Phi-29 viral genome packaging motor assembly
Map dataFocused asymmetric reconstruction of the bacteriophage phi29 dsDNA packaging motor assembly at 4.1 A.
SampleBacillus virus phi29 viral genome packaging motor assembly != Bacillus virus phi29

Bacillus virus phi29 viral genome packaging motor assembly

  • Virus: Bacillus virus phi29
    • Protein or peptide: DNA packaging protein
    • RNA: pRNA (117-MER)
    • DNA: DNA (60-MER)
    • DNA: DNA (60-MER)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
Keywordspackaging motor / ATPase / MOTOR PROTEIN
Function / homology
Function and homology information


viral DNA genome packaging / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / ATP hydrolysis activity / DNA binding / RNA binding / ATP binding
Similarity search - Function
Podovirus DNA packaging protein / Podovirus DNA encapsidation protein (Gp16) / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA packaging protein
Similarity search - Component
Biological speciesBacillus phage phi29 (virus) / Bacillus virus phi29
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsWhite MA / Woodson M
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM122979 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM127365 United States
CitationJournal: Sci Adv / Year: 2021
Title: A viral genome packaging motor transitions between cyclic and helical symmetry to translocate dsDNA.
Authors: Michael Woodson / Joshua Pajak / Bryon P Mahler / Wei Zhao / Wei Zhang / Gaurav Arya / Mark A White / Paul J Jardine / Marc C Morais /
Abstract: Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring ...Molecular segregation and biopolymer manipulation require the action of molecular motors to do work by applying directional forces to macromolecules. The additional strand conserved E (ASCE) ring motors are an ancient family of molecular motors responsible for diverse biological polymer manipulation tasks. Viruses use ASCE segregation motors to package their genomes into their protein capsids and provide accessible experimental systems due to their relative simplicity. We show by cryo-EM-focused image reconstruction that ASCE ATPases in viral double-stranded DNA (dsDNA) packaging motors adopt helical symmetry complementary to their dsDNA substrates. Together with previous data, our results suggest that these motors cycle between helical and planar configurations, providing a possible mechanism for directional translocation of DNA. Similar changes in quaternary structure have been observed for proteasome and helicase motors, suggesting an ancient and common mechanism of force generation that has been adapted for specific tasks over the course of evolution.
History
DepositionAug 11, 2020-
Header (metadata) releaseMay 19, 2021-
Map releaseMay 19, 2021-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0114
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.0114
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jqq
  • Surface level: 0.0114
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-7jqq
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22441.png

Downloads & links

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Map

FileDownload / File: emd_22441.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFocused asymmetric reconstruction of the bacteriophage phi29 dsDNA packaging motor assembly at 4.1 A.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 320 pix.
= 342.4 Å		1.07 Å/pix.
x 320 pix.
= 342.4 Å		1.07 Å/pix.
x 320 pix.
= 342.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.0114 / Movie #1: 0.0114
Minimum - Maximum-0.04517124 - 0.064411804
Average (Standard dev.)-0.000038320406 (±0.00528637)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 342.40002 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z342.400342.400342.400
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0450.064-0.000

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Supplemental data

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Sample components

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Entire : Bacillus virus phi29 viral genome packaging motor assembly

EntireName: Bacillus virus phi29 viral genome packaging motor assembly
Components
  • Virus: Bacillus virus phi29
    • Protein or peptide: DNA packaging protein
    • RNA: pRNA (117-MER)
    • DNA: DNA (60-MER)
    • DNA: DNA (60-MER)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Bacillus virus phi29

SupramoleculeName: Bacillus virus phi29 / type: virus / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / NCBI-ID: 10756 / Sci species name: Bacillus virus phi29 / Virus type: VIRION / Virus isolate: SPECIES / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Bacillus subtilis (bacteria) / Strain: A12

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Macromolecule #1: DNA packaging protein

MacromoleculeName: DNA packaging protein / type: protein_or_peptide / ID: 1 / Number of copies: 5 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Bacillus phage phi29 (virus)
Molecular weightTheoretical: 39.010406 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MDKSLFYNPQ KMLSYDRILN FVIGARGIGK SYAMKVYPIN RFIKYGEQFI YVRRYKPELA KVSNYFNDVA QEFPDHELVV KGRRFYIDG KLAGWAIPLS VWQSEKSNAY PNVSTIVFDE FIREKDNSNY IPNEVSALLN LMDTVFRNRE RVRCICLSNA V SVVNPYFL ...String:
MDKSLFYNPQ KMLSYDRILN FVIGARGIGK SYAMKVYPIN RFIKYGEQFI YVRRYKPELA KVSNYFNDVA QEFPDHELVV KGRRFYIDG KLAGWAIPLS VWQSEKSNAY PNVSTIVFDE FIREKDNSNY IPNEVSALLN LMDTVFRNRE RVRCICLSNA V SVVNPYFL FFNLVPDVNK RFNVYDDALI EIPDSLDFSS ERRKTRFGRL IDGTEYGEMS LDNQFIGDSQ VFIEKRSKDS KF VFSIVYN GFTLGVWVDV NQGLMYIDTA HDPSTKNVYT LTTDDLNENM MLITNYKNNY HLRKLASAFM NGYLRFDNQV IRN IAYELF RKMRIQ

UniProtKB: DNA packaging protein

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Macromolecule #2: pRNA (117-MER)

MacromoleculeName: pRNA (117-MER) / type: rna / ID: 2 / Number of copies: 5
Source (natural)Organism: Bacillus virus phi29
Molecular weightTheoretical: 37.469012 KDa
SequenceString:
GGAAUGGUAC GGUACUUCCA UUGUCAUGUG UAUGUUGGGG AUUAAACCCU GAUUGAGUUC AGCCCACAUA CUUUGUUGAU UGGUUGUCA AUCAUGGCAA AAGUGCACGC UACUUUCC

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Macromolecule #3: DNA (60-MER)

MacromoleculeName: DNA (60-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bacillus virus phi29
Molecular weightTheoretical: 18.491848 KDa
SequenceString: (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT) (DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA) (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG) (DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DC) (DA) (DG)(DT)(DC)(DA)(DG)(DT) ...String:
(DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT) (DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA) (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG) (DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT)(DC) (DA) (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA) (DG)(DT)(DC)(DA)(DG)(DT)(DC)(DA)(DG)(DT) (DC)(DA)

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Macromolecule #4: DNA (60-MER)

MacromoleculeName: DNA (60-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Bacillus virus phi29
Molecular weightTheoretical: 18.49185 KDa
SequenceString: (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG) (DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT) (DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)(DA) (DC) (DT)(DG)(DA)(DC)(DT)(DG) ...String:
(DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG) (DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT) (DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG)(DA) (DC) (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC) (DT)(DG)(DA)(DC)(DT)(DG)(DA)(DC)(DT)(DG) (DA)(DC)

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Macromolecule #5: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 3 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 3 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

BufferpH: 7.8
Component:
ConcentrationFormulaName
25.0 mM(HOCH2)3CNH2Tris
5.0 mMMgCl2magnesium chloride
50.0 mMNaClsodium chloride
GridModel: Quantifoil / Material: COPPER / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF 2002
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Dimensions - Width: 7676 pixel / Digitization - Dimensions - Height: 7420 pixel / Digitization - Frames/image: 0-44 / Average electron dose: 41.4 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated defocus max: 4.2 µm / Calibrated defocus min: 1.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 4.2 µm / Nominal defocus min: 1.0 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: OTHER
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.10)
Details: The final step before reconstruction was classification without alignment, which was done with even/odd sets mixed together, but even/odd were completely independent for all angular assignment steps
Number images used: 12526
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.03)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.03)
Details: subparticles were extracted with the localized reconstruction functions in SCIPION 1.2.1, then aligned in RELION 2.03
Final 3D classificationNumber classes: 5 / Avg.num./class: 12500 / Software - Name: RELION (ver. 2.03)
FSC plot (resolution estimation)

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