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- PDB-6n7i: Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase i... -

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Basic information

Entry
Database: PDB / ID: 6n7i
TitleStructure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (gp4(5)-DNA)
Components
  • DNA (25-MER)
  • DNA primase/helicase
KeywordsHYDROLASE / TRANSFERASE/DNA / helicase / ATPase / hexamer / DNA replication / TRANSFERASE-DNA complex
Function / homology
Function and homology information


DNA primase activity / DNA helicase / DNA helicase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / zinc ion binding / ATP binding / identical protein binding
Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / P-loop containing nucleoside triphosphate hydrolase / Zinc-binding domain of primase-helicase / DnaB-like helicase C terminal domain / Archaeal primase DnaG/twinkle, TOPRIM domain / TOPRIM domain / DNA helicase, DnaB-like, C-terminal / P-loop containing nucleotide triphosphate hydrolases / Rossmann fold / 3-Layer(aba) Sandwich / Alpha Beta
DNA primase/helicase
Biological speciesEnterobacteria phage T7 (bacteriophage)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsGao, Y. / Cui, Y. / Zhou, Z. / Yang, W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Human Genome Research Institute (NIH/NHGRI)1S10RR23057 United States
CitationJournal: Science / Year: 2019
Title: Structures and operating principles of the replisome.
Authors: Yang Gao / Yanxiang Cui / Tara Fox / Shiqiang Lin / Huaibin Wang / Natalia de Val / Z Hong Zhou / Wei Yang /
Abstract: Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model ...Visualization in atomic detail of the replisome that performs concerted leading- and lagging-DNA strand synthesis at a replication fork has not been reported. Using bacteriophage T7 as a model system, we determined cryo-electron microscopy structures up to 3.2-angstroms resolution of helicase translocating along DNA and of helicase-polymerase-primase complexes engaging in synthesis of both DNA strands. Each domain of the spiral-shaped hexameric helicase translocates sequentially hand-over-hand along a single-stranded DNA coil, akin to the way AAA+ ATPases (adenosine triphosphatases) unfold peptides. Two lagging-strand polymerases are attached to the primase, ready for Okazaki fragment synthesis in tandem. A β hairpin from the leading-strand polymerase separates two parental DNA strands into a T-shaped fork, thus enabling the closely coupled helicase to advance perpendicular to the downstream DNA duplex. These structures reveal the molecular organization and operating principles of a replisome.
Validation Report
SummaryFull reportAbout validation report
History
DepositionNov 27, 2018Deposition site: RCSB / Processing site: RCSB
Revision 1.0Mar 6, 2019Provider: repository / Type: Initial release
Revision 1.1Dec 18, 2019Group: Author supporting evidence / Category: pdbx_audit_support / Item: _pdbx_audit_support.funding_organization

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Structure visualization

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Assembly

Deposited unit
A: DNA primase/helicase
B: DNA primase/helicase
C: DNA primase/helicase
D: DNA primase/helicase
E: DNA primase/helicase
F: DNA primase/helicase
T: DNA (25-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)386,02715
Polymers384,0017
Non-polymers2,0268
Water181
1


TypeNameSymmetry operationNumber
identity operation1_5551
Buried area25120 Å2
ΔGint-123 kcal/mol
Surface area53800 Å2

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Components

#1: Protein
DNA primase/helicase / Gene product 4 / Gp4


Mass: 62734.430 Da / Num. of mol.: 6 / Mutation: E343Q
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T7 (bacteriophage)
Variant: E343Q / Production host: Escherichia coli (E. coli)
References: UniProt: P03692, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, DNA helicase
#2: DNA chain DNA (25-MER)


Mass: 7594.877 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (bacteriophage)
#3: Chemical
ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE / Thymidine triphosphate


Mass: 482.168 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C10H17N2O14P3
#4: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium


Mass: 24.305 Da / Num. of mol.: 4 / Source method: isolated from a natural source / Formula: Mg
#5: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Formula: H2O

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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: Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex with five helicase subunits ordered
Type: COMPLEX / Entity ID: 1,2 / Source: RECOMBINANT
Source (natural)Organism: Enterobacteria phage T7 (bacteriophage)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
2150 mMpotassium chlorideKCl1
33 mMDTT1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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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 FIELDBright-field microscopy
Image recordingElectron dose: 72 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

EM software
IDNameCategory
7UCSF Chimeramodel fitting
9RELIONinitial Euler assignment
10RELIONfinal Euler assignment
12RELION3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 227594 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1E0J

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