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- PDB-9im3: The Cryo-EM structure of MPXV E5 head-to-head double hexamer conf... -

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Basic information

Entry
Database: PDB / ID: 9im3
TitleThe Cryo-EM structure of MPXV E5 head-to-head double hexamer conformation
ComponentsPrimase D5
KeywordsVIRAL PROTEIN / complex
Function / homology
Function and homology information


helicase activity / hydrolase activity
Similarity search - Function
DNA primase/nucleoside triphosphatase, C-terminal / Poxvirus D5 protein-like / : / Bacteriophage/plasmid primase, P4, C-terminal / D5 N terminal like / Helicase, superfamily 3, DNA virus / Superfamily 3 helicase of DNA viruses domain profile. / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Uncoating factor OPG117
Similarity search - Component
Biological speciesMonkeypox virus
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.31 Å
AuthorsCheng, Y.X. / Han, P. / Wang, H.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)32270157 China
CitationJournal: Nat Commun / Year: 2025
Title: Assembly and breakage of head-to-head double hexamer reveals mpox virus E5-catalyzed DNA unwinding initiation.
Authors: Yingxian Cheng / Pu Han / Qi Peng / Ruili Liu / Hao Liu / Bin Yuan / Yunfei Zhao / Lu Kuai / Jianxun Qi / Kai Miao / Yi Shi / George Fu Gao / Han Wang /
Abstract: The replicative helicase-catalyzed unwinding of the DNA double helix is the initiation of DNA replication. Helicases and primases are functionally related enzymes that have even been expressed as ...The replicative helicase-catalyzed unwinding of the DNA double helix is the initiation of DNA replication. Helicases and primases are functionally related enzymes that have even been expressed as fusion proteins in some organisms and viruses. However, the mechanism underlying DNA unwinding initiation by these helicase-primase fusion enzymes and the functional association between domains have not been elucidated. Herein, we report the cryo-EM structures of mpox virus E5, the founding member of these helicase-primase enzymes, in various enzymatic stages. Notably, E5 forms a head-to-head double hexamer encircling dsDNA, disrupted by the conformational rearrangement of primase domains upon nucleotide incorporation. Five E5-ssDNA-ATP structures further support an ATP cycle-driven non-classical escort model for E5 translocation. Finally, the helicase domain is found to enhance the primase function as a DNA scaffold. Together, our data shed light on the E5-mediated DNA unwinding model including dsDNA loading, DNA melting, ssDNA translocation, and provide a reasonable interpretation for evolutionary preservation of helicase-primase fusion from a functional perspective.
History
DepositionJul 1, 2024Deposition site: PDBJ / Processing site: PDBC
Revision 1.0Jun 18, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Primase D5
B: Primase D5
C: Primase D5
E: Primase D5
F: Primase D5
J: Primase D5
M: Primase D5
N: Primase D5
O: Primase D5
Q: Primase D5
R: Primase D5
S: Primase D5


Theoretical massNumber of molelcules
Total (without water)1,085,71612
Polymers1,085,71612
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein
Primase D5


Mass: 90476.344 Da / Num. of mol.: 12
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Monkeypox virus
Gene: E5R, MPXV-COP-096, MPXV-M2940_FCT-100, MPXV-M2957_Lagos-100, MPXV-M3021_Delta-100, MPXV-M5320_M15_Bayelsa-093, MPXV-Nig_SEV71_2_82-095, MPXV-PCH-097, MPXV-Singapore-100, MPXV-SL-096, MPXV-UK_P1- ...Gene: E5R, MPXV-COP-096, MPXV-M2940_FCT-100, MPXV-M2957_Lagos-100, MPXV-M3021_Delta-100, MPXV-M5320_M15_Bayelsa-093, MPXV-Nig_SEV71_2_82-095, MPXV-PCH-097, MPXV-Singapore-100, MPXV-SL-096, MPXV-UK_P1-100, MPXV-UK_P2-100, MPXV-UK_P3-100, MPXV-USA2003_099_GR-100, MPXV-USA2003_206_DM-100, MPXV-USA2003_223_RS-100, MPXV-UTC-091, MPXV-W_Nigeria-095, MPXV-WRAIR096, MPXV298464_082, MPXV_LIB1970_184_107, MPXV_USA2003_039_107, MPXV_USA2003_044_107, PDLMKLCO_00105
Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q5IXS3
Has protein modificationN

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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: MPXV E5 in head-to-head double hexamer conformation / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Monkeypox virus
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 / Nominal defocus max: 2300 nm / Nominal defocus min: 1300 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.31 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 311720 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00350796
ELECTRON MICROSCOPYf_angle_d0.57168652
ELECTRON MICROSCOPYf_dihedral_angle_d3.7896588
ELECTRON MICROSCOPYf_chiral_restr0.0427752
ELECTRON MICROSCOPYf_plane_restr0.0048676

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