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- PDB-8dvi: T4 bacteriophage primosome with single strand DNA, State 2 -

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Basic information

Entry
Database: PDB / ID: 8dvi
TitleT4 bacteriophage primosome with single strand DNA, State 2
Components
  • DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
  • DNA (70-MER)
  • DNA primase
  • DnaB-like replicative helicase
KeywordsREPLICATION/DNA/RNA / phage / complex / helicase / REPLICATION-DNA-RNA complex
Function / homology
Function and homology information


bidirectional double-stranded viral DNA replication / DNA primase activity / primosome complex / DNA replication, synthesis of primer / viral DNA genome replication / single-stranded DNA helicase activity / 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 ...bidirectional double-stranded viral DNA replication / DNA primase activity / primosome complex / DNA replication, synthesis of primer / viral DNA genome replication / single-stranded DNA helicase activity / 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / DNA / DNA (> 10) / DNA primase / DnaB-like replicative helicase
Similarity search - Component
Biological speciesEscherichia phage T4 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsFeng, X. / Li, H.
Funding support United States, 2items
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
Citation
Journal: Nat Commun / 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 to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer ...The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
#1: Journal: 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
DepositionJul 29, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 14, 2023Provider: repository / Type: Initial release
Revision 1.1Aug 23, 2023Group: Data collection / Database references
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / citation / citation_author

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: DnaB-like replicative helicase
B: DnaB-like replicative helicase
C: DnaB-like replicative helicase
D: DnaB-like replicative helicase
E: DnaB-like replicative helicase
F: DnaB-like replicative helicase
M: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')
H: DNA primase
T: DNA (70-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)369,69819
Polymers366,9619
Non-polymers2,73810
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 7 molecules ABCDEFH

#1: Protein
DnaB-like replicative helicase / Gene product 41 / Gp41


Mass: 53662.848 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage T4 (virus) / Gene: 41 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P04530, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement
#3: Protein DNA primase / DNA priming protein


Mass: 39842.043 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia phage T4 (virus) / Gene: 61, 58 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: P04520, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases

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DNA chain , 2 types, 2 molecules MT

#2: DNA chain DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3')


Mass: 3605.356 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage T4 (virus)
#4: DNA chain DNA (70-MER)


Mass: 1536.035 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage T4 (virus)

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Non-polymers , 2 types, 10 molecules

#5: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Complex of T4 bacteriophage primosome including helicase gp41, primase gp61 and ssDNA/RNA hybrid
Type: COMPLEX / Entity ID: #1-#4 / Source: MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Escherichia phage T4 (virus)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.8
Details: 20 mM HEPES pH 7.8, 100 mM NaCl, 10 mM MgCl2 and 2 mM DTT
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMHEPESHEPES1
2100 mMsodium chlorideNaCl1
310 mMMagnesium chlorideMgCl21
42 mMDTTDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm
Image recordingElectron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20_4459: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3.1particle selection
2SerialEM3.8image acquisition
4CTFFIND4.1.10CTF correction
7PHENIX1.2model fitting
9PHENIX1.2model refinement
10Coot0.9.6model refinement
11cryoSPARC3.3.1initial Euler assignment
12RELION3.1final Euler assignment
13RELION3.1classification
14RELION3.13D reconstruction
CTF correctionType: NONE
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97947 / Symmetry type: POINT
Atomic model buildingProtocol: OTHER / Space: REAL
Atomic model building
IDPDB-ID 3D fitting-IDAccession codeSource nameType
16QEM

6qem

16QEMPDBexperimental model
26N9U

6n9u

16N9UPDBexperimental model
32AU3

2au3

12AU3PDBexperimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00221368
ELECTRON MICROSCOPYf_angle_d0.4428966
ELECTRON MICROSCOPYf_dihedral_angle_d11.6357956
ELECTRON MICROSCOPYf_chiral_restr0.0393246
ELECTRON MICROSCOPYf_plane_restr0.0033599

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