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- PDB-6n7w: Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/... -

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Basic information

Entry
Database: PDB / ID: 6n7w
TitleStructure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx in complex with a DNA fork and incoming dTTP (from multiple lead complexes)
Components
  • DNA (25-MER)
  • DNA (77-MER)
  • DNA-directed DNA polymerase
  • TrxA
KeywordsTRANSFERASE/DNA / DNA polymerase / helicase / DNA replication / replisome / TRANSFERASE-DNA complex
Function / homology
Function and homology information


DNA synthesis involved in DNA replication / DNA exonuclease activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / protein-disulfide reductase activity / 3'-5' exonuclease activity / cell redox homeostasis / DNA-templated DNA replication / double-strand break repair ...DNA synthesis involved in DNA replication / DNA exonuclease activity / viral DNA genome replication / Hydrolases; Acting on ester bonds; Exodeoxyribonucleases producing 5'-phosphomonoesters / DNA polymerase processivity factor activity / protein-disulfide reductase activity / 3'-5' exonuclease activity / cell redox homeostasis / DNA-templated DNA replication / double-strand break repair / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding / cytosol / cytoplasm
Similarity search - Function
DNA-directed DNA polymerase T7 / Thioredoxin / 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 / Thioredoxin / Thioredoxin, conserved site ...DNA-directed DNA polymerase T7 / Thioredoxin / 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 / Thioredoxin / Thioredoxin, conserved site / Thioredoxin family active site. / Thioredoxin domain profile. / Thioredoxin domain / Thioredoxin-like superfamily / Ribonuclease H superfamily / Ribonuclease H-like superfamily / DNA/RNA polymerase superfamily
Similarity search - Domain/homology
THYMIDINE-5'-TRIPHOSPHATE / DNA / DNA (> 10) / DNA-directed DNA polymerase / Thioredoxin 1 / Thioredoxin
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsGao, Y. / Fox, T. / Val, N. / 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
DepositionNov 28, 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 1.2Mar 20, 2024Group: Data collection / Database references ...Data collection / Database references / Derived calculations / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model / 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 / _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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Structure viewerMolecule:
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Assembly

Deposited unit
H: DNA-directed DNA polymerase
I: TrxA
P: DNA (25-MER)
T: DNA (77-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)123,1266
Polymers122,6194
Non-polymers5062
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area10230 Å2
ΔGint-57 kcal/mol
Surface area50060 Å2

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Components

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Protein , 2 types, 2 molecules HI

#1: 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
#2: Protein TrxA / Thioredoxin 1


Mass: 11818.582 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Gene: trxA / Production host: Escherichia coli (E. coli) / References: UniProt: Q14F07, UniProt: P0AA25*PLUS

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DNA chain , 2 types, 2 molecules PT

#3: DNA chain DNA (25-MER)


Mass: 7716.011 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (virus)
#4: DNA chain DNA (77-MER)


Mass: 23278.844 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Enterobacteria phage T7 (virus)

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Non-polymers , 2 types, 2 molecules

#5: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Mg
#6: Chemical ChemComp-TTP / THYMIDINE-5'-TRIPHOSPHATE


Mass: 482.168 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C10H17N2O14P3

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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: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes)
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: COUNTING / 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
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 129003 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 1T8E

1t8e


Accession code: 1T8E / Source name: PDB / Type: experimental model

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