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- EMDB-29744: local refinement of the primase/DNA/primer region in mutant T4 pr... -

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Basic information

Entry
Database: EMDB / ID: EMD-29744
Titlelocal refinement of the primase/DNA/primer region in mutant T4 primosome
Map datafocus refinement of the primase/DNA-RNA region
Sample
  • Complex: Complex of mutated T4 bacteriophage helicase gp41 with ssDNA/RNA hybrid
    • Protein or peptide: Bacteriophage T4 DNA primase
    • DNA: DNA (70-mer)
    • RNA: RNA (5'-R(*(GTP)P*CP*CP*GP*A)-3')
Keywordsphage / complex / helicase / REPLICATION
Biological speciesEscherichia phage T4 (virus)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.1 Å
AuthorsFeng X / Li H
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM131754 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM013306 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis of the T4 bacteriophage primosome assembly and primer synthesis.
Authors: Xiang Feng / Michelle M Spiering / Ruda de Luna Almeida Santos / Stephen J Benkovic / Huilin Li /
Abstract: The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer ...The T4 bacteriophage gp41 helicase and gp61 primase assemble into a primosome to couple DNA unwinding with RNA primer synthesis for DNA replication. How the primosome is assembled and how the primer length is defined are unclear. Here we report a series of cryo-EM structures of T4 primosome assembly intermediates. We show that gp41 alone is an open spiral, and ssDNA binding triggers a large-scale scissor-like conformational change that drives the ring closure and activates the helicase. Helicase activation exposes a cryptic hydrophobic surface to recruit the gp61 primase. The primase binds the helicase in a bipartite mode in which the N-terminal Zn-binding domain and the C-terminal RNA polymerase domain each contain a helicase-interacting motif that bind to separate gp41 N-terminal hairpin dimers, leading to the assembly of one primase on the helicase hexamer. Our study reveals the T4 primosome assembly process and sheds light on the RNA primer synthesis mechanism.
History
DepositionFeb 13, 2023-
Header (metadata) releaseAug 9, 2023-
Map releaseAug 9, 2023-
UpdateAug 9, 2023-
Current statusAug 9, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_29744.png

Downloads & links

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Map

FileDownload / File: emd_29744.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationfocus refinement of the primase/DNA-RNA region
Voxel sizeX=Y=Z: 1.029 Å
Density
Contour LevelBy AUTHOR: 0.29
Minimum - Maximum-2.531926 - 5.335367
Average (Standard dev.)-0.0013680188 (±0.029434057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 329.28003 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_29744_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_29744_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Complex of mutated T4 bacteriophage helicase gp41 with ssDNA/RNA ...

EntireName: Complex of mutated T4 bacteriophage helicase gp41 with ssDNA/RNA hybrid
Components
  • Complex: Complex of mutated T4 bacteriophage helicase gp41 with ssDNA/RNA hybrid
    • Protein or peptide: Bacteriophage T4 DNA primase
    • DNA: DNA (70-mer)
    • RNA: RNA (5'-R(*(GTP)P*CP*CP*GP*A)-3')

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Supramolecule #1: Complex of mutated T4 bacteriophage helicase gp41 with ssDNA/RNA ...

SupramoleculeName: Complex of mutated T4 bacteriophage helicase gp41 with ssDNA/RNA hybrid
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia phage T4 (virus)

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Macromolecule #1: Bacteriophage T4 DNA primase

MacromoleculeName: Bacteriophage T4 DNA primase / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Escherichia phage T4 (virus)
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSSIPWIDNE FAYRALAHLP KFTQVNNSST FKLRFRCPVC GDSKTDQNKA RGWYYGDNNE GNIHCYNCN YHAPIGIYLK EFEPDLYREY IFEIRKEKGK SRPIEKPKEL PKQPEKKIIK S LPSCVRLD KLAEDHPIIK YVKARCIPKD KWKYLWFTTE WPKLVNSIAP ...String:
MSSIPWIDNE FAYRALAHLP KFTQVNNSST FKLRFRCPVC GDSKTDQNKA RGWYYGDNNE GNIHCYNCN YHAPIGIYLK EFEPDLYREY IFEIRKEKGK SRPIEKPKEL PKQPEKKIIK S LPSCVRLD KLAEDHPIIK YVKARCIPKD KWKYLWFTTE WPKLVNSIAP GTYKKEISEP RL VIPIYNA NGKAESFQGR ALKKDAPQKY ITIEAYPEAT KIYGVERVKD GDVYVLEGPI DSL FIENGI AITGGQLDLE VVPFKDRRVW VLDNEPRHPD TIKRMTKLVD AGERVMFWDK SPWK SKDVN DMIRKEGATP EQIMEYMKNN IAQGLMAKMR LSKYAKI

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Macromolecule #2: DNA (70-mer)

MacromoleculeName: DNA (70-mer) / type: dna / ID: 2 / Classification: DNA
Source (natural)Organism: Escherichia phage T4 (virus)
SequenceString: (DG)(DA)(DA)(DT)(DG)(DA)(DG)(DG)(DA)(DG) (DT)(DA)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DA) (DT)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC) (DT)(DG)(DG)(DT)(DC)(DT) ...String:
(DG)(DA)(DA)(DT)(DG)(DA)(DG)(DG)(DA)(DG) (DT)(DA)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DA) (DT)(DG)(DT)(DA)(DG)(DT)(DG)(DA)(DG) (DG)(DT)(DA)(DA)(DT)(DA)(DT)(DC)(DG)(DG) (DC) (DT)(DG)(DG)(DT)(DC)(DT)(DG)(DG) (DT)(DC)(DT)(DG)(DT)(DG)(DC)(DC)(DA)(DA) (DG)(DT) (DT)(DG)(DC)(DT)(DG)(DC)(DA) (DA)(DA)(DA)

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Macromolecule #3: RNA (5'-R(*(GTP)P*CP*CP*GP*A)-3')

MacromoleculeName: RNA (5'-R(*(GTP)P*CP*CP*GP*A)-3') / type: rna / ID: 3
Source (natural)Organism: Escherichia phage T4 (virus)
SequenceString:
(GTP)CCGA

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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: 7.8
Component:
ConcentrationFormulaName
20.0 mMHEPESHEPES
100.0 mMNaClSodium chloridesodium chloride
10.0 mMMgCl2Magnesium chloride
2.0 mMDTTDTT

Details: 20 mM HEPES pH 7.8, 100 mM NaCl, 10 mM MgCl2 and 2 mM DTT
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 66.0 e/Å2
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:

6qem

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 3 / Avg.num./class: 800000 / Software - Name: RELION (ver. 3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1)
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 4.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1) / Number images used: 157809
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: OTHER

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