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- PDB-6n9w: Structure of bacteriophage T7 lagging-strand DNA polymerase (D5A/... -

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Basic information

Entry
Database: PDB / ID: 6n9w
TitleStructure of bacteriophage T7 lagging-strand DNA polymerase (D5A/E7A) and gp4 (helicase/primase) bound to DNA including RNA/DNA hybrid, and an incoming dTTP (LagS2)
Components
  • DNA primase/helicase
  • DNA-directed DNA polymerase
  • Primer
  • Template
KeywordsHYDROLASE / TRANSFERASE/DNA / helicase / ATPase / hexamer / DNA replication / TRANSFERASE-DNA complex
Function / homology
Function and homology information


DNA synthesis involved in DNA replication / DNA exonuclease activity / : / primosome complex / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / 3'-5' exonuclease activity / DNA helicase activity / DNA-templated DNA replication / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases ...DNA synthesis involved in DNA replication / DNA exonuclease activity / : / primosome complex / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / 3'-5' exonuclease activity / DNA helicase activity / DNA-templated DNA replication / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / double-strand break repair / single-stranded DNA binding / 5'-3' DNA helicase activity / DNA helicase / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / ATP hydrolysis activity / DNA binding / zinc ion binding / ATP binding / identical protein binding / metal ion binding
Similarity search - Function
Bacteriophage T7 DNA helicase/primase / : / Bacteriophage T7 DNA helicase/primase, N-terminal a+b fold / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / Zinc-binding domain of primase-helicase / Zinc-binding domain of primase-helicase / DNA-directed DNA polymerase T7 / Twinkle-like protein / Archaeal primase DnaG/twinkle-like, TOPRIM domain / Toprim-like ...Bacteriophage T7 DNA helicase/primase / : / Bacteriophage T7 DNA helicase/primase, N-terminal a+b fold / Bacteriophage T7, Gp4, DNA primase/helicase, N-terminal / Zinc-binding domain of primase-helicase / Zinc-binding domain of primase-helicase / DNA-directed DNA polymerase T7 / Twinkle-like protein / Archaeal primase DnaG/twinkle-like, TOPRIM domain / Toprim-like / DnaB-like helicase C terminal domain / DNA helicase, DnaB-like, C-terminal / Superfamily 4 helicase domain profile. / TOPRIM / DNA polymerase A / DNA polymerase family A / DNA-directed DNA polymerase, family A, conserved site / DNA polymerase family A signature. / DNA-directed DNA polymerase, family A, palm domain / DNA polymerase A domain / Toprim domain profile. / TOPRIM domain / Ribonuclease H-like superfamily/Ribonuclease H / Nucleotidyltransferase; domain 5 / Ribonuclease H superfamily / Ribonuclease H-like superfamily / P-loop containing nucleotide triphosphate hydrolases / DNA/RNA polymerase superfamily / P-loop containing nucleoside triphosphate hydrolase / Rossmann fold / 2-Layer Sandwich / 3-Layer(aba) Sandwich / Alpha Beta
Similarity search - Domain/homology
THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / RNA / DNA-directed DNA polymerase / DNA helicase/primase
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4 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.
History
DepositionDec 4, 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
Revision 1.2Oct 23, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description / Structure summary
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / em_admin / pdbx_entry_details / pdbx_initial_refinement_model / pdbx_modification_feature / struct_conn / struct_conn_type
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type / _em_admin.last_update / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn.ptnr2_label_seq_id / _struct_conn_type.id

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-0381
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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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
H: DNA-directed DNA polymerase
P: Primer
T: Template
hetero molecules


Theoretical massNumber of molelcules
Total (without water)474,07921
Polymers471,4579
Non-polymers2,62212
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area33040 Å2
ΔGint-161 kcal/mol
Surface area109570 Å2

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Components

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Protein , 2 types, 7 molecules ABCDEFH

#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 (virus) / Production host: Escherichia coli (E. coli)
References: UniProt: P03692, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, DNA helicase
#2: Protein DNA-directed DNA polymerase / Gene product 5 / Gp5


Mass: 79805.625 Da / Num. of mol.: 1 / Mutation: D5A, E7A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Enterobacteria phage T7 (virus) / Production host: Escherichia coli (E. coli)
References: UniProt: P00581, DNA-directed DNA polymerase, Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters

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RNA chain / DNA chain , 2 types, 2 molecules PT

#3: RNA chain Primer


Mass: 1842.206 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (virus)
#4: DNA chain Template


Mass: 13402.571 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (virus)

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Non-polymers , 3 types, 12 molecules

#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Zn
#6: Chemical
ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 5 / Source method: obtained synthetically / Formula: C10H17N2O14P3
#7: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: Mg

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Details

Has protein modificationY

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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: gp5 DNA polymerase binding to A and B subunits of gp4 primase-helicase hexamer complexed with trx, RNA/DNA hybrid, and incoming dTTP (LagS2)
Type: COMPLEX / Entity ID: #1-#4 / Source: RECOMBINANT
Source (natural)Organism: Enterobacteria phage T7 (virus)
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 supportDetails: unspecified
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 FIELD
Image recordingElectron dose: 40 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
9PHENIXmodel refinement
10RELIONinitial Euler assignment
11RELIONfinal Euler assignment
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 38760 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1E0J

1e0j


Accession code: 1E0J / Source name: PDB / Type: experimental model

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