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Yorodumi- PDB-8g0z: Mutant bacteriophage T4 gp41 helicase hexamer bound with single s... -
+Open data
-Basic information
Entry | Database: PDB / ID: 8g0z | |||||||||
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Title | Mutant bacteriophage T4 gp41 helicase hexamer bound with single strand DNA and ATPgammaS in the stalled primosome | |||||||||
Components |
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Keywords | REPLICATION / phage / complex / helicase | |||||||||
Function / homology | Function and homology information viral DNA genome replication / DNA helicase activity / Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement / DNA replication / hydrolase activity / DNA binding / ATP binding Similarity search - Function | |||||||||
Biological species | Escherichia phage T4 (virus) | |||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.61 Å | |||||||||
Authors | Feng, X. / Li, H. | |||||||||
Funding support | United States, 2items
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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 |
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-Structure visualization
Structure viewer | Molecule: MolmilJmol/JSmol |
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-Downloads & links
-Download
PDBx/mmCIF format | 8g0z.cif.gz | 507.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb8g0z.ent.gz | 418.6 KB | Display | PDB format |
PDBx/mmJSON format | 8g0z.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 8g0z_validation.pdf.gz | 1.6 MB | Display | wwPDB validaton report |
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Full document | 8g0z_full_validation.pdf.gz | 1.6 MB | Display | |
Data in XML | 8g0z_validation.xml.gz | 80.3 KB | Display | |
Data in CIF | 8g0z_validation.cif.gz | 118.6 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/g0/8g0z ftp://data.pdbj.org/pub/pdb/validation_reports/g0/8g0z | HTTPS FTP |
-Related structure data
Related structure data | 29658MC 8dtpC 8dueC 8duoC 8dvfC 8dviC 8dw6C 8dwjC 8gaoC C: citing same article (ref.) M: map data used to model this data |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
-Assembly
Deposited unit |
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-Components
#1: Protein | Mass: 48796.711 Da / Num. of mol.: 6 / Mutation: E227Q Source method: isolated from a genetically manipulated source Details: mutant Bacteriophage T4 / Source: (gene. exp.) Escherichia phage T4 (virus) / Gene: 41 / Production host: Escherichia coli BL21(DE3) (bacteria) References: UniProt: A0A7S9SV99, Hydrolases; Acting on acid anhydrides; Acting on acid anhydrides to facilitate cellular and subcellular movement #2: DNA chain | | Mass: 3605.356 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia phage T4 (virus) #3: Chemical | ChemComp-AGS / #4: Chemical | ChemComp-MG / Has ligand of interest | N | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
Component | Name: Complex of mutated T4 bacteriophage helicase gp41 with ssDNA/RNA hybrid Type: COMPLEX / Entity ID: #1-#2 / Source: MULTIPLE SOURCES | |||||||||||||||||||||||||
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Molecular weight | Experimental value: NO | |||||||||||||||||||||||||
Source (natural) | Organism: Escherichia phage T4 (virus) | |||||||||||||||||||||||||
Source (recombinant) | Organism: Escherichia coli BL21(DE3) (bacteria) | |||||||||||||||||||||||||
Buffer solution | pH: 7.8 Details: 20 mM HEPES pH 7.8, 100 mM NaCl, 10 mM MgCl2 and 2 mM DTT | |||||||||||||||||||||||||
Buffer component |
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Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE |
-Electron microscopy imaging
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 1000 nm |
Image recording | Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) |
-Processing
Software | Name: PHENIX / Version: 1.20_4459: / Classification: refinement | ||||||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: NONE | ||||||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 3.61 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 272868 / Algorithm: FOURIER SPACE / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||||||
Atomic model building | Protocol: OTHER / Space: REAL | ||||||||||||||||||||||||||||||||||||||||
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