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- EMDB-0362: Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase i... -

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Basic information

Entry
Database: EMDB / ID: EMD-0362
TitleStructure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (form II)
Map databacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II
Sample
  • Complex: Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II
    • Protein or peptide: DNA primase/helicase
    • DNA: DNA (25-MER)
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
Keywordshelicase / ATPase / hexamer / DNA replication / HYDROLASE / TRANSFERASE-DNA complex
Function / homology
Function and homology information


DNA primase activity / primosome complex / viral DNA genome replication / DNA helicase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / single-stranded DNA binding / 5'-3' DNA helicase activity / DNA helicase / ATP hydrolysis activity / zinc ion binding ...DNA primase activity / primosome complex / viral DNA genome replication / DNA helicase activity / Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases / single-stranded DNA binding / 5'-3' DNA helicase activity / DNA helicase / ATP hydrolysis activity / zinc ion binding / ATP binding / identical protein 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 / Twinkle-like protein / Archaeal primase DnaG/twinkle-like, TOPRIM domain / Toprim-like / DnaB-like helicase C terminal domain ...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 / 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 / Toprim domain profile. / TOPRIM domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
DNA helicase/primase
Similarity search - Component
Biological speciesEnterobacteria phage T7 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsGao Y / Cui Y
Funding support United States, 1 items
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, 2018-
Header (metadata) releaseFeb 20, 2019-
Map releaseMar 6, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6n7s
  • Surface level: 0.028
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6n7s
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0362.png

Downloads & links

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Map

FileDownload / File: emd_0362.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationbacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å		1.07 Å/pix.
x 256 pix.
= 273.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.07 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.028
Minimum - Maximum-0.08504928 - 0.13022318
Average (Standard dev.)0.0006212913 (±0.004387732)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 273.92 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.071.071.07
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z273.920273.920273.920
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0850.1300.001

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Supplemental data

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Sample components

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Entire : Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II

EntireName: Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II
Components
  • Complex: Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II
    • Protein or peptide: DNA primase/helicase
    • DNA: DNA (25-MER)
  • Ligand: THYMIDINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II

SupramoleculeName: Bacteriophage T7 gene product 4 (gp4) helicase primase DNA complex II
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Enterobacteria phage T7 (virus)

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Macromolecule #1: DNA primase/helicase

MacromoleculeName: DNA primase/helicase / type: protein_or_peptide / ID: 1 / Number of copies: 6 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 62.73443 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MDNSHDSDSV FLYHIPCDNC GSSDGNSLFS DGHTFCYVCE KWTAGNEDTK ERASKRKPSG GKPMTYNVWN FGESNGRYSA LTARGISKE TCQKAGYWIA KVDGVMYQVA DYRDQNGNIV SQKVRDKDKN FKTTGSHKSD ALFGKHLWNG GKKIVVTEGE I DMLTVMEL ...String:
MDNSHDSDSV FLYHIPCDNC GSSDGNSLFS DGHTFCYVCE KWTAGNEDTK ERASKRKPSG GKPMTYNVWN FGESNGRYSA LTARGISKE TCQKAGYWIA KVDGVMYQVA DYRDQNGNIV SQKVRDKDKN FKTTGSHKSD ALFGKHLWNG GKKIVVTEGE I DMLTVMEL QDCKYPVVSL GHGASAAKKT CAANYEYFDQ FEQIILMFDM DEAGRKAVEE AAQVLPAGKV RVAVLPCKDA NE CHLNGHD REIMEQVWNA GPWIPDGVVS ALSLRERIRE HLSSEESVGL LFSGCTGIND KTLGARGGEV IMVTSGSGMG KST FVRQQA LQWGTAMGKK VGLAMLQESV EETAEDLIGL HNRVRLRQSD SLKREIIENG KFDQWFDELF GNDTFHLYDS FAEA ETDRL LAKLAYMRSG LGCDVIILDH ISIVVSASGE SDERKMIDNL MTKLKGFAKS TGVVLVVICH LKNPDKGKAH EEGRP VSIT DLRGSGALRQ LSDTIIALER NQQGDMPNLV LVRILKCRFT GDTGIAGYME YNKETGWLEP SSYSGEEESH SESTDW SND TDF

UniProtKB: DNA helicase/primase

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Macromolecule #2: DNA (25-MER)

MacromoleculeName: DNA (25-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Enterobacteria phage T7 (virus)
Molecular weightTheoretical: 7.594877 KDa
SequenceString:
(DT)(DG)(DG)(DT)(DC)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)

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Macromolecule #3: THYMIDINE-5'-TRIPHOSPHATE

MacromoleculeName: THYMIDINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 4 / Formula: TTP
Molecular weightTheoretical: 482.168 Da
Chemical component information

ChemComp-TTP:
THYMIDINE-5'-TRIPHOSPHATE

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMTris
150.0 mMpotassium chlorideKCl
3.0 mMDTT
GridDetails: unspecified
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Average electron dose: 72.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

1e0j

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 40734
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1e0j


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-6n7s:
Structure of bacteriophage T7 E343Q mutant gp4 helicase-primase in complex with ssDNA, dTTP, AC dinucleotide and CTP (form II)

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