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- EMDB-17818: Complex of HK97 small terminase with DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-17818
TitleComplex of HK97 small terminase with DNA
Map dataHK97 small terminase in complex with DNA
Sample
  • Complex: complex of HK97 with DNA
    • Protein or peptide: small terminase
    • DNA: DNA (31-mer)
    • DNA: DNA (31-mer)
KeywordsHK97 small terminase / bacteriophage / protein:DNA complex / cryoEM structure / DNA BINDING PROTEIN
Function / homologyTerminase small subunit
Function and homology information
Biological speciesByrnievirus HK97
Methodsingle particle reconstruction / cryo EM / Resolution: 2.92 Å
AuthorsChechik M / Greive SJ / Antson AA / Jenkins HT
Funding support United Kingdom, 1 items
OrganizationGrant numberCountry
Wellcome Trust206377 United Kingdom
CitationJournal: bioRxiv / Year: 2023
Title: Structure of HK97 small terminase:DNA complex unveils a novel DNA binding mechanism by a circular protein.
Authors: Maria Chechik / Sandra J Greive / Alfred A Antson / Huw T Jenkins /
Abstract: DNA recognition is critical for assembly of double-stranded DNA viruses, in particular for the initiation of packaging the viral genome into the capsid. DNA packaging has been extensively studied for ...DNA recognition is critical for assembly of double-stranded DNA viruses, in particular for the initiation of packaging the viral genome into the capsid. DNA packaging has been extensively studied for three archetypal bacteriophage systems: , and phi29. We identified the minimal site within the region of bacteriophage HK97 specifically recognised by the small terminase and determined a cryoEM structure for the small terminase:DNA complex. This nonameric circular protein utilizes a previously unknown mechanism of DNA binding. While DNA threads through the central tunnel, unexpectedly, DNA-recognition is generated at its exit by a substructure formed by the N- and C-terminal segments of two adjacent protomers of the terminase which are unstructured in the absence of DNA. Such interaction ensures continuous engagement of the small terminase with DNA, allowing sliding along DNA while simultaneously checking the DNA sequence. This mechanism allows locating and instigating packaging initiation and termination precisely at the site.
History
DepositionJul 10, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17818.png

Downloads & links

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Map

FileDownload / File: emd_17818.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHK97 small terminase in complex with DNA
Voxel sizeX=Y=Z: 1.18889 Å
Density
Contour LevelBy AUTHOR: 0.03
Minimum - Maximum-0.110717855 - 0.19165114
Average (Standard dev.)0.00008754204 (±0.012290124)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions128128128
Spacing128128128
CellA=B=C: 152.17792 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17818_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: HK97 small terminase in complex with DNA

Fileemd_17818_half_map_1.map
AnnotationHK97 small terminase in complex with DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: HK97 small terminase in complex with DNA

Fileemd_17818_half_map_2.map
AnnotationHK97 small terminase in complex with DNA
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : complex of HK97 with DNA

EntireName: complex of HK97 with DNA
Components
  • Complex: complex of HK97 with DNA
    • Protein or peptide: small terminase
    • DNA: DNA (31-mer)
    • DNA: DNA (31-mer)

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Supramolecule #1: complex of HK97 with DNA

SupramoleculeName: complex of HK97 with DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Byrnievirus HK97
Molecular weightTheoretical: 185 KDa

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Macromolecule #1: small terminase

MacromoleculeName: small terminase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
Source (natural)Organism: Byrnievirus HK97
Recombinant expressionOrganism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
SequenceString:
MADKRIRSDS SAAAVQAMKN AAVDTIDPPS HAGLEKKAEP FWHDNIRSKA LDSWTPADLL AAVELANNQ LYITVLRKDL RKEERIRGEE RDEGLIKDLR KQIVELQRTI LAQRRDLQIH S HATNGESR DQKKRNQNDR DARNTKNEHQ DQDDNLIAFP KHG

UniProtKB: Terminase small subunit

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Macromolecule #2: DNA (31-mer)

MacromoleculeName: DNA (31-mer) / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: Byrnievirus HK97
SequenceString:
TAAAACTAAA AAAATCGGGT TAGCGTTAAA T

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Macromolecule #3: DNA (31-mer)

MacromoleculeName: DNA (31-mer) / type: dna / ID: 3 / Classification: DNA
Source (natural)Organism: Byrnievirus HK97
SequenceString:
ATTTAACGCT AACCCGATTT TTTTAGTTTT 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

BufferpH: 7.5
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chlorideSodium Chloride
10.0 mMMgCl2Magnesium Chloride
25.0 mMC4H11NO3.HClTris-HClTris
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 180 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV
Detailscomplex was purified on S200 10/300 column

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Electron microscopy

MicroscopeTFS 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: 2.4 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2767 / Average exposure time: 8.0 sec. / Average electron dose: 53.6 e/Å2
Details: Two datasets were collected: 1st dataset: 682 images 2nd dataset: 2085 images
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1144385 / Details: 1st dataset:306866 2nd dataset: 837519
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6z6e

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.92 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 174268
FSC plot (resolution estimation)

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