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- EMDB-16653: HK97 portal/large terminase complex -

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Basic information

Entry
Database: EMDB / ID: EMD-16653
TitleHK97 portal/large terminase complex
Map dataPortal and large terminase complex from HK97 DNA packaging complex
Sample
  • Virus: Hendrixvirus
    • Other: Large terminase complex from HK97
    • Protein or peptide: Portal protein from HK97
KeywordsHK97 / bacteriophage / packaging motor / DNA complex / portal protein / VIRAL PROTEIN
Biological speciesHendrixvirus
Methodsingle particle reconstruction / cryo EM / Resolution: 7.44 Å
AuthorsHawkins DEDP / Antson AA
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research Council (BBSRC)AC014501 United Kingdom
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Insights into a viral motor: the structure of the HK97 packaging termination assembly.
Authors: Dorothy E D P Hawkins / Oliver W Bayfield / Herman K H Fung / Daniel N Grba / Alexis Huet / James F Conway / Alfred A Antson /
Abstract: Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each ...Double-stranded DNA viruses utilise machinery, made of terminase proteins, to package viral DNA into the capsid. For cos bacteriophage, a defined signal, recognised by small terminase, flanks each genome unit. Here we present the first structural data for a cos virus DNA packaging motor, assembled from the bacteriophage HK97 terminase proteins, procapsids encompassing the portal protein, and DNA containing a cos site. The cryo-EM structure is consistent with the packaging termination state adopted after DNA cleavage, with DNA density within the large terminase assembly ending abruptly at the portal protein entrance. Retention of the large terminase complex after cleavage of the short DNA substrate suggests that motor dissociation from the capsid requires headful pressure, in common with pac viruses. Interestingly, the clip domain of the 12-subunit portal protein does not adhere to C12 symmetry, indicating asymmetry induced by binding of the large terminase/DNA. The motor assembly is also highly asymmetric, showing a ring of 5 large terminase monomers, tilted against the portal. Variable degrees of extension between N- and C-terminal domains of individual subunits suggest a mechanism of DNA translocation driven by inter-domain contraction and relaxation.
History
DepositionFeb 6, 2023-
Header (metadata) releaseMay 24, 2023-
Map releaseMay 24, 2023-
UpdateAug 2, 2023-
Current statusAug 2, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_16653.png

Downloads & links

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Map

FileDownload / File: emd_16653.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPortal and large terminase complex from HK97 DNA packaging complex
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.62 Å/pix.
x 128 pix.
= 335. Å		2.62 Å/pix.
x 128 pix.
= 335. Å		2.62 Å/pix.
x 128 pix.
= 335. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.61719 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.039
Minimum - Maximum-0.06897739 - 0.14026053
Average (Standard dev.)0.0014812264 (±0.01099976)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 335.00006 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_16653_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Portal and large terminase complex from HK97 DNA packaging complex

Fileemd_16653_half_map_1.map
AnnotationPortal and large terminase complex from HK97 DNA packaging complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Portal and large terminase complex from HK97 DNA packaging complex

Fileemd_16653_half_map_2.map
AnnotationPortal and large terminase complex from HK97 DNA packaging complex
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Hendrixvirus

EntireName: Hendrixvirus
Components
  • Virus: Hendrixvirus
    • Other: Large terminase complex from HK97
    • Protein or peptide: Portal protein from HK97

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Supramolecule #1: Hendrixvirus

SupramoleculeName: Hendrixvirus / type: virus / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Proheads were produced by infection of E. coli 594 cells with HK97 amber mutant amC2, propagated using Escherichia LE392 cells
NCBI-ID: 2169654 / Sci species name: Hendrixvirus / Sci species strain: HK97 / Virus type: VIRION / Virus isolate: STRAIN / Virus enveloped: No / Virus empty: No
Host (natural)Organism: Escherichia phage EcSzw-2 (virus) / Strain: 594
Molecular weightTheoretical: 560 KDa

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Macromolecule #1: Large terminase complex from HK97

MacromoleculeName: Large terminase complex from HK97 / type: other / ID: 1 / Details: Large terminase monomer
Classification: polydeoxyribonucleotide/polyribonucleotide hybrid
Source (natural)Organism: Hendrixvirus
SequenceString: MTRGERVIAF IERFCIVPEG KLIGQPMRLD PFQKDFILAV YDNPAGTDMA ILSIARKNGK TGLIAGILLA HLVGPEAVQN TQIVSGALSR EQAAIVFNLA VKMVNLNPKL QEIVHITPSG KKLIGLPCNV EYKALSAEGK TTHGLSPILA ILDETGQVRG PQDDFIDAIT ...String:
MTRGERVIAF IERFCIVPEG KLIGQPMRLD PFQKDFILAV YDNPAGTDMA ILSIARKNGK TGLIAGILLA HLVGPEAVQN TQIVSGALSR EQAAIVFNLA VKMVNLNPKL QEIVHITPSG KKLIGLPCNV EYKALSAEGK TTHGLSPILA ILDETGQVRG PQDDFIDAIT TAQGAHENPL LIVISTQAAN DADLLSIWID DAVKSKDPHI VCHVYEAPKD ADISKRESWL AANPALGTFR SEKDMARQAE KAGRMPSFEN TFRNLNLNQR VSTVSPFISR SVWELCGEMP INTPRKWYAG LDLSARNDLT ALVIAGEADD GVWDVFPFFW TPQKTLEERT KTDRAPYDVW VREGLLRTTP GASVDYSFVV ADIAEIIGDF DLTSMAFDRW RIDQFRKDAD AIGLSLPLVE FGQGFKDMGP AVDTLESLML NGRVRHGMHP VLTMCAVNAV VVKDAAGNRK LDKSKATGRI DGMVAMTMSV GAANGEVTEQ GGDFDDFIFR PLSM
Recombinant expressionOrganism: Escherichia phage EcSzw-2 (virus)

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Macromolecule #3: Portal protein from HK97

MacromoleculeName: Portal protein from HK97 / type: protein_or_peptide / ID: 3 / Enantiomer: LEVO
Source (natural)Organism: Hendrixvirus
Recombinant expressionOrganism: Escherichia phage EcSzw-2 (virus)
SequenceString: MEEPKYTIDL RTNNGWWARL KSWFVGGRLV TPNQGSQTGP VSAHGYLGDS SINDERILQI STVWRCVSLI STLTACLPLD VFETDQNDNR KKVDLSNPLA RLLRYSPNQY MTAQEFREAM TMQLCFYGNA YALVDRNSAG DVISLLPLQS ANMDVKLVGK KVVYRYQRDS ...String:
MEEPKYTIDL RTNNGWWARL KSWFVGGRLV TPNQGSQTGP VSAHGYLGDS SINDERILQI STVWRCVSLI STLTACLPLD VFETDQNDNR KKVDLSNPLA RLLRYSPNQY MTAQEFREAM TMQLCFYGNA YALVDRNSAG DVISLLPLQS ANMDVKLVGK KVVYRYQRDS EYADFSQKEI FHLKGFGFTG LVGLSPIAFA CKSAGVAVAM EDQQRDFFAN GAKSPQILST GEKVLTEQQR SQVEENFKEI AGGPVKKRLW ILEAGFSTSA IGVTPQDAEM MASRKFQVSE LARFFGVPPH LVGDVEKSTS WGSGIEQQNL GFLQYTLQPY ISRWENSIQR WLIPSKDVGR LHAEHNLDGL LRGDSASRAA FMKAMGESGL RTINEMRRTD NMPPLPGGDV AMRQAQYVPI TDLGTNKEPR NNGA

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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: 7.5
Details: final buffer = 20 mM Tris pH 7.5, 10 mM MgCl2, 50 mM KGlu, 5 mM ATP-gamma-S
GridModel: Quantifoil R3.5/1 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Support film - Film thickness: 0.2
VitrificationCryogen name: ETHANE / Chamber humidity: 90 % / Instrument: FEI VITROBOT MARK IV
DetailsPackaging reactions 30 nM hk97 proheads, 2.5 micromolar large terminase, 5 micromolar small terminase and 30 nM DNA, 75 micromolar ATP were incubated for 2 mins then 5 micromolar ATP-gamma-S was added.

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 82279
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 7.44 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 17584
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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