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- EMDB-29902: bacteriophage T4 stalled primosome with mutant gp41-E227Q -

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Basic information

Entry
Database: EMDB / ID: EMD-29902
Titlebacteriophage T4 stalled primosome with mutant gp41-E227Q
Map data
Sample
  • Complex: complex of T4 primosome with mutant helicase gp41
    • Protein or peptide: DnaB-like replicative helicase
    • Protein or peptide: DNA primasePrimase
    • DNA: DNA (70-mer)
    • RNA: RNA (5'-D(*(GTP))-R(P*CP*CP*GP*A)-3')
    • DNA: DNA (70-mer)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION
Keywordsphage / complex / helicase / REPLICATION
Function / homology
Function and homology information


DNA primase activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / single-stranded DNA helicase activity / DNA replication, synthesis of primer / DNA helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / hydrolase activity / DNA binding ...DNA primase activity / bidirectional double-stranded viral DNA replication / viral DNA genome replication / single-stranded DNA helicase activity / DNA replication, synthesis of primer / DNA helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / hydrolase activity / DNA binding / ATP binding / metal ion binding
Similarity search - Function
DNA primase, bacteriophage T4 / Bacteriophage T4 DnaB-like replicative helicase / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA primase / DnaB-like replicative helicase
Similarity search - Component
Biological speciesEscherichia phage T4 (virus) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 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
DepositionFeb 23, 2023-
Header (metadata) releaseJun 7, 2023-
Map releaseJun 7, 2023-
UpdateAug 23, 2023-
Current statusAug 23, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29902.png

Downloads & links

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Map

FileDownload / File: emd_29902.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.029 Å
Density
Contour LevelBy AUTHOR: 0.15
Minimum - Maximum0.0 - 9.197062000000001
Average (Standard dev.)0.050407592 (±0.3865427)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 205.8 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : complex of T4 primosome with mutant helicase gp41

EntireName: complex of T4 primosome with mutant helicase gp41
Components
  • Complex: complex of T4 primosome with mutant helicase gp41
    • Protein or peptide: DnaB-like replicative helicase
    • Protein or peptide: DNA primasePrimase
    • DNA: DNA (70-mer)
    • RNA: RNA (5'-D(*(GTP))-R(P*CP*CP*GP*A)-3')
    • DNA: DNA (70-mer)
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
  • Ligand: ZINC ION

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Supramolecule #1: complex of T4 primosome with mutant helicase gp41

SupramoleculeName: complex of T4 primosome with mutant helicase gp41 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Escherichia phage T4 (virus)

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Macromolecule #1: DnaB-like replicative helicase

MacromoleculeName: DnaB-like replicative helicase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
Source (natural)Organism: Escherichia phage T4 (virus)
Molecular weightTheoretical: 48.796711 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MVEIILSHLI FDQAYFSKVW PYMDSEYFES GPAKNTFKLI KSHVNEYHSV PSINALNVAL ENSSFTETEY SGVKTLISKL ADSPEDHSW LVKETEKYVQ QRAMFNATSK IIEIQTNAEL PPEKRNKKMP DVGAIPDIMR QALSISFDSY VGHDWMDDYE A RWLSYMNK ...String:
MVEIILSHLI FDQAYFSKVW PYMDSEYFES GPAKNTFKLI KSHVNEYHSV PSINALNVAL ENSSFTETEY SGVKTLISKL ADSPEDHSW LVKETEKYVQ QRAMFNATSK IIEIQTNAEL PPEKRNKKMP DVGAIPDIMR QALSISFDSY VGHDWMDDYE A RWLSYMNK ARKVPFKLRI LNKITKGGAE TGTLNVLMAG VNVGKSLGLC SLAADYLQLG HNVLYISMQM AEEVCAKRID AN MLDVSLD DIDDGHISYA EYKGKMEKWR EKSTLGRLIV KQYPTGGADA NTFRSLLNEL KLKKNFVPTI IIVDYLGICK SCR IRVYSE NSYTTVKAIA EELRALAVET ETVLWTAAQV GKQAWDSSDV NMSDIAESAG LPATADFMLA VIETEELAAA EQQL IKQIK SRYGDKNKWN KFLMGVQKGN QKWVEIE

UniProtKB: DnaB-like replicative helicase

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Macromolecule #2: DNA primase

MacromoleculeName: DNA primase / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Escherichia phage T4 (virus)
Molecular weightTheoretical: 39.510613 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SIPWIDNEFA YRALAHLPKF TQVNNSSTFK LRFRCPVCGD SKTDQNKARG WYYGDNNEGN IHCYNCNYHA PIGIYLKEFE PDLYREYIF EIRKEKGKSR PIEKPKELPK QPEKKIIKSL PSCVRLDKLA EDHPIIKYVK ARCIPKDKWK YLWFTTEWPK L VNSIAPGT ...String:
SIPWIDNEFA YRALAHLPKF TQVNNSSTFK LRFRCPVCGD SKTDQNKARG WYYGDNNEGN IHCYNCNYHA PIGIYLKEFE PDLYREYIF EIRKEKGKSR PIEKPKELPK QPEKKIIKSL PSCVRLDKLA EDHPIIKYVK ARCIPKDKWK YLWFTTEWPK L VNSIAPGT YKKEISEPRL VIPIYNANGK AESFQGRALK KDAPQKYITI EAYPEATKIY GVERVKDGDV YVLEGPIDSL FI ENGIAIT GGQLDLEVVP FKDRRVWVLD NEPRHPDTIK RMTKLVDAGE RVMFWDKSPW KSKDVNDMIR KEGATPEQIM EYM KNNIAQ GLMAKMRLSK YAK

UniProtKB: DNA primase

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Macromolecule #3: DNA (70-mer)

MacromoleculeName: DNA (70-mer) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 3.605356 KDa
SequenceString:
(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)

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Macromolecule #5: DNA (70-mer)

MacromoleculeName: DNA (70-mer) / type: dna / ID: 5 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
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 #4: RNA (5'-D(*(GTP))-R(P*CP*CP*GP*A)-3')

MacromoleculeName: RNA (5'-D(*(GTP))-R(P*CP*CP*GP*A)-3') / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 1.744983 KDa
SequenceString:
(GTP)CCGA

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Macromolecule #6: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 6 / Number of copies: 5 / 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 #7: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 7 / Number of copies: 5 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #8: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 8 / 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:

6qem

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: 4.1 Å / Resolution method: OTHER / Software - Name: RELION (ver. 3.1)
Details: lowest resolution in two combined focus refined maps
Number images used: 272868

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER
Output model

PDB-8gao:
bacteriophage T4 stalled primosome with mutant gp41-E227Q

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