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Yorodumi- EMDB-0365: Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-0365 | |||||||||
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Title | Structure of bacteriophage T7 leading-strand DNA polymerase (D5A/E7A)/Trx in complex with a DNA fork and incoming dTTP (from multiple lead complexes) | |||||||||
Map data | D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes) | |||||||||
Sample |
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Keywords | 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 / DNA-directed DNA polymerase ...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 / DNA-directed DNA polymerase / DNA-directed DNA polymerase activity / nucleotide binding / DNA binding / metal ion binding / cytosol / cytoplasm Similarity search - Function | |||||||||
Biological species | Enterobacteria phage T7 (virus) / Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.5 Å | |||||||||
Authors | Gao Y / Fox T | |||||||||
Funding support | United States, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_0365.map.gz | 3.2 MB | EMDB map data format | |
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Header (meta data) | emd-0365-v30.xml emd-0365.xml | 17 KB 17 KB | Display Display | EMDB header |
Images | emd_0365.png | 75.1 KB | ||
Filedesc metadata | emd-0365.cif.gz | 6.7 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-0365 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-0365 | HTTPS FTP |
-Related structure data
Related structure data | 6n7wMC 0357C 0359C 0362C 0363C 0364C 0379C 0380C 0381C 0382C 0386C 0387C 0388C 0389C 0390C 0391C 0392C 0393C 0394C 0395C 6n7iC 6n7nC 6n7sC 6n7tC 6n7vC 6n9uC 6n9vC 6n9wC 6n9xC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_0365.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.72 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, an...
Entire | Name: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes) |
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Components |
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-Supramolecule #1: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, an...
Supramolecule | Name: D5AE7A mutant gp5 DNA polymerase complexed with trx, fork DNA, and incoming dTTP (from gp4-gp5 leading-strand complexes) type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Enterobacteria phage T7 (virus) |
-Macromolecule #1: DNA-directed DNA polymerase
Macromolecule | Name: DNA-directed DNA polymerase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: DNA-directed DNA polymerase |
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Source (natural) | Organism: Enterobacteria phage T7 (virus) |
Molecular weight | Theoretical: 79.805625 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MIVSDIEANA LLESVTKFHC GVIYDYSTAE YVSYRPSDFG AYLDALEAEV ARGGLIVFHN GHKYDVPALT KLAKLQLNRE FHLPRENCI DTLVLSRLIH SNLKDTDMGL LRSGKLPGKR FGSHALEAWG YRLGEMKGEY KDDFKRMLEE QGEEYVDGME W WNFNEEMM ...String: MIVSDIEANA LLESVTKFHC GVIYDYSTAE YVSYRPSDFG AYLDALEAEV ARGGLIVFHN GHKYDVPALT KLAKLQLNRE FHLPRENCI DTLVLSRLIH SNLKDTDMGL LRSGKLPGKR FGSHALEAWG YRLGEMKGEY KDDFKRMLEE QGEEYVDGME W WNFNEEMM DYNVQDVVVT KALLEKLLSD KHYFPPEIDF TDVGYTTFWS ESLEAVDIEH RAAWLLAKQE RNGFPFDTKA IE ELYVELA ARRSELLRKL TETFGSWYQP KGGTEMFCHP RTGKPLPKYP RIKTPKVGGI FKKPKNKAQR EGREPCELDT REY VAGAPY TPVEHVVFNP SSRDHIQKKL QEAGWVPTKY TDKGAPVVDD EVLEGVRVDD PEKQAAIDLI KEYLMIQKRI GQSA EGDKA WLRYVAEDGK IHGSVNPNGA VTGRATHAFP NLAQIPGVRS PYGEQCRAAF GAEHHLDGIT GKPWVQAGID ASGLE LRCL AHFMARFDNG EYAHEILNGD IHTKNQIAAE LPTRDNAKTF IYGFLYGAGD EKIGQIVGAG KERGKELKKK FLENTP AIA ALRESIQQTL VESSQWVAGE QQVKWKRRWI KGLDGRKVHV RSPHAALNTL LQSAGALICK LWIIKTEEML VEKGLKH GW DGDFAYMAWV HDEIQVGCRT EEIAQVVIET AQEAMRWVGD HWNFRCLLDT EGKMGPNWAI CH UniProtKB: DNA-directed DNA polymerase |
-Macromolecule #2: TrxA
Macromolecule | Name: TrxA / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 11.818582 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MSDKIIHLTD DSFDTDVLKA DGAILVDFWA EWCGPCKMIA PILDEIADEY QGKLTVAKLN IDQNPGTAPK YGIRGIPTLL LFKNGEVAA TKVGALSKGQ LKEFLDANLA UniProtKB: Thioredoxin 1 |
-Macromolecule #3: DNA (25-MER)
Macromolecule | Name: DNA (25-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Enterobacteria phage T7 (virus) |
Molecular weight | Theoretical: 7.716011 KDa |
Sequence | String: (DG)(DG)(DT)(DA)(DC)(DA)(DA)(DC)(DT)(DT) (DG)(DA)(DC)(DG)(DA)(DC)(DA)(DT)(DA)(DG) (DC)(DG)(DT)(DG)(2DA) |
-Macromolecule #4: DNA (77-MER)
Macromolecule | Name: DNA (77-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Enterobacteria phage T7 (virus) |
Molecular weight | Theoretical: 23.278844 KDa |
Sequence | String: (DT)(DT)(DT)(DG)(DG)(DT)(DC)(DA)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DA) (DC)(DG)(DG)(DA)(DG)(DT)(DC)(DG)(DT)(DT) (DT) (DC)(DG)(DA)(DC)(DT)(DC) ...String: (DT)(DT)(DT)(DG)(DG)(DT)(DC)(DA)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DA) (DC)(DG)(DG)(DA)(DG)(DT)(DC)(DG)(DT)(DT) (DT) (DC)(DG)(DA)(DC)(DT)(DC)(DC)(DG) (DT)(DT)(DA)(DT)(DC)(DA)(DC)(DG)(DC)(DT) (DA)(DT) (DG)(DT)(DC)(DG)(DT)(DC)(DA) (DA)(DG)(DT)(DT)(DG)(DT)(DA)(DC)(DC) |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 1 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #6: THYMIDINE-5'-TRIPHOSPHATE
Macromolecule | Name: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: TTP |
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Molecular weight | Theoretical: 482.168 Da |
Chemical component information | ChemComp-TTP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Details: unspecified | ||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK I |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2 |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: PDB ENTRY PDB model - PDB ID: |
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Initial angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION |
Final angle assignment | Type: MAXIMUM LIKELIHOOD / Software - Name: RELION |
Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 129003 |
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model |
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Refinement | Space: REAL / Protocol: FLEXIBLE FIT |
Output model | PDB-6n7w: |