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- EMDB-27756: Primase of mutant bacteriophage T4 primosome with single strand D... -

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Basic information

Entry
Database: EMDB / ID: EMD-27756
TitlePrimase of mutant bacteriophage T4 primosome with single strand DNA/RNA primer hybrid in primer exiting state
Map data
Sample
  • Complex: Complex of T4 bacteriophage primosome including helicase gp41, primase gp61 and ssDNA/RNA hybrid
    • RNA: RNA(5'-ppp-GCCGA-3')
    • DNA: DNA (70-MER)
    • Protein or peptide: DNA primasePrimase
  • Ligand: ZINC ION
Keywordsphage / complex / helicase / REPLICATION-DNA-RNA complex
Function / homologyDNA primase, bacteriophage T4 / DNA primase activity / viral DNA genome replication / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / metal ion binding / DNA primase
Function and homology information
Biological speciesEscherichia phage T4 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.9 Å
AuthorsFeng X / Li H
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM013306 United States
CitationJournal: bioRxiv / Year: 2023
Title: Structural basis of the T4 bacteriophage primosome assembly and primer synthesis.
Authors: Xiang Feng / Michelle M Spiering / Ruda de Luna Almeida Santos / Stephen J Benkovic / Huilin Li /
Abstract: The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome complex to couple DNA unwinding with RNA primer synthesis for DNA replication. How a primosome is assembled and how the ...The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome complex to couple DNA unwinding with RNA primer synthesis for DNA replication. How a primosome is assembled and how the length of the RNA primer is defined in the T4 bacteriophage, or in any model system, are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates at resolutions up to 2.7 Å. We show that the gp41 helicase is an open spiral in the absence of ssDNA, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the open spiral to a closed ring that activates the helicase. We found that the activation of the gp41 helicase exposes a cryptic hydrophobic primase-binding surface allowing for the recruitment of the gp61 primase. The primase binds the gp41 helicase in a bipartite mode in which the N-terminal Zn-binding domain (ZBD) and the C-terminal RNA polymerase domain (RPD) each contain a helicase-interacting motif (HIM1 and HIM2, respectively) that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Based on two observed primosome conformations - one in a DNA-scanning mode and the other in a post RNA primer-synthesis mode - we suggest that the linker loop between the gp61 ZBD and RPD contributes to the T4 pentaribonucleotide primer. Our study reveals T4 primosome assembly process and sheds light on RNA primer synthesis mechanism.
History
DepositionAug 1, 2022-
Header (metadata) releaseJun 14, 2023-
Map releaseJun 14, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

Downloads & links

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Map

FileDownload / File: emd_27756.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.029 Å
Density
Contour LevelBy AUTHOR: 0.012
Minimum - Maximum-0.055026572 - 0.091325626
Average (Standard dev.)-0.0000017138516 (±0.0033104403)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 329.28003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_27756_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Half map: #1

Fileemd_27756_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Complex of T4 bacteriophage primosome including helicase gp41, pr...

EntireName: Complex of T4 bacteriophage primosome including helicase gp41, primase gp61 and ssDNA/RNA hybrid
Components
  • Complex: Complex of T4 bacteriophage primosome including helicase gp41, primase gp61 and ssDNA/RNA hybrid
    • RNA: RNA(5'-ppp-GCCGA-3')
    • DNA: DNA (70-MER)
    • Protein or peptide: DNA primasePrimase
  • Ligand: ZINC ION

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Supramolecule #1: Complex of T4 bacteriophage primosome including helicase gp41, pr...

SupramoleculeName: Complex of T4 bacteriophage primosome including helicase gp41, primase gp61 and ssDNA/RNA hybrid
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Escherichia phage T4 (virus)

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Macromolecule #1: RNA(5'-ppp-GCCGA-3')

MacromoleculeName: RNA(5'-ppp-GCCGA-3') / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia phage T4 (virus)
Molecular weightTheoretical: 1.744983 KDa
SequenceString:
(GTP)CCGA

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Macromolecule #2: DNA (70-MER)

MacromoleculeName: DNA (70-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Escherichia phage T4 (virus)
Molecular weightTheoretical: 21.900025 KDa
SequenceString: (DG)(DA)(DA)(DT)(DG)(DA)(DG)(DG)(DA)(DG) (DT)(DA)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DA) (DT)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC) (DT)(DG)(DG)(DT)(DC)(DT) ...String:
(DG)(DA)(DA)(DT)(DG)(DA)(DG)(DG)(DA)(DG) (DT)(DA)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DA) (DT)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC) (DT)(DG)(DG)(DT)(DC)(DT)(DG)(DG) (DT)(DC)(DT)(DG)(DT)(DG)(DC)(DC)(DA)(DA) (DG)(DT) (DT)(DG)(DC)(DT)(DG)(DC)(DA) (DA)(DA)(DA)

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Macromolecule #3: DNA primase

MacromoleculeName: DNA primase / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Escherichia phage T4 (virus)
Molecular weightTheoretical: 39.842043 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSSIPWIDNE FAYRALAHLP KFTQVNNSST FKLRFRCPVC GDSKTDQNKA RGWYYGDNNE GNIHCYNCNY HAPIGIYLKE FEPDLYREY IFEIRKEKGK SRPIEKPKEL PKQPEKKIIK SLPSCVRLDK LAEDHPIIKY VKARCIPKDK WKYLWFTTEW P KLVNSIAP ...String:
MSSIPWIDNE FAYRALAHLP KFTQVNNSST FKLRFRCPVC GDSKTDQNKA RGWYYGDNNE GNIHCYNCNY HAPIGIYLKE FEPDLYREY IFEIRKEKGK SRPIEKPKEL PKQPEKKIIK SLPSCVRLDK LAEDHPIIKY VKARCIPKDK WKYLWFTTEW P KLVNSIAP GTYKKEISEP RLVIPIYNAN GKAESFQGRA LKKDAPQKYI TIEAYPEATK IYGVERVKDG DVYVLEGPID SL FIENGIA ITGGQLDLEV VPFKDRRVWV LDNEPRHPDT IKRMTKLVDA GERVMFWDKS PWKSKDVNDM IRKEGATPEQ IME YMKNNI AQGLMAKMRL SKYAKI

UniProtKB: DNA primase

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Macromolecule #4: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: ZN
Molecular weightTheoretical: 65.409 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
20.0 mMHEPESHEPES
100.0 mMNaClSodium chloridesodium chloride
10.0 mMMgCl2Magnesium chloride
2.0 mMDTTDTT

Details: 20 mM HEPES pH 7.8, 100 mM NaCl, 10 mM MgCl2 and 2 mM DTT
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 800000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 157809
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model(PDB ID:
,
)
RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8dwj:
Primase of mutant bacteriophage T4 primosome with single strand DNA/RNA primer hybrid in primer exiting state

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