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- EMDB-71789: Cryo-EM structure of ATPgammaS-bound Vientovirus FB Rep double he... -

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Basic information

Entry
Database: EMDB / ID: EMD-71789
TitleCryo-EM structure of ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry
Map datasharpened map of 3D auto-refined map from Relion (primary map)
Sample
  • Complex: ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry
    • Protein or peptide: Replication-associated protein
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION
KeywordsSF3 helicase / DNA binding protein / Replication / Viral protein
Function / homology
Function and homology information


nucleotidyltransferase activity / endonuclease activity / DNA replication / RNA helicase activity / hydrolase activity / host cell nucleus / DNA binding / RNA binding / ATP binding / metal ion binding
Similarity search - Function
: / CRESS-DNA virus replication initiator protein (Rep) endonuclease domain profile. / Helicase, superfamily 3, single-stranded DNA/RNA virus / RNA helicase / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Replication-associated protein
Similarity search - Component
Biological speciesVientovirus FB / Human lung-associated vientovirus FB
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsMontermoso S / Gupta K / Pumroy RA / Moiseenkova-Bell V / Bushman FD / Van Duyne GD
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P30AI045008 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM108751 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)T32AI007632 United States
Department of Health & Human Services (HHS)S10OD018483 United States
National Science Foundation (NSF, United States)DMR2342336 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1P30GM124166 United States
CitationJournal: PLoS Pathog / Year: 2026
Title: Structures of nucleotide-bound Redondovirus Rep protein link conformation and function.
Authors: Saira Montermoso / Kushol Gupta / Ruth Anne Pumroy / Vera Moiseenkova-Bell / Frederic D Bushman / Gregory D Van Duyne /
Abstract: Circular Rep-encoding single-stranded DNA (CRESS-DNA) virus Rep proteins are multidomain enzymes that mediate viral DNA rolling-circle replication. Reps nick viral DNA to expose a 3' end for ...Circular Rep-encoding single-stranded DNA (CRESS-DNA) virus Rep proteins are multidomain enzymes that mediate viral DNA rolling-circle replication. Reps nick viral DNA to expose a 3' end for polymerase extension, provide an NTP-dependent helicase activity for DNA unwinding, and join nicked ends to form circular viral genomes. Here, we present the first structures of a Rep protein from the Redondoviridae family, a newly discovered family of human-associated CRESS-DNA viruses that replicates within the oral protozoan Entamoeba gingivalis. Using cryo-EM, we characterized the hexameric structures of a Redondovirus Rep helicase bound with ATPγS, representing the initial ATP-bound state, and with ADP, reflecting the protein state after hydrolysis. The ADP state, but not the ATP state of Rep shows a staircase arrangement of DNA-binding loops that plays a central role in current models for SF3 helicase function. Additionally, we determined a head-to-tail dodecameric structure of ATPγS-bound Rep, in which both the helicase and endonuclease domains are ordered. Conservation of residues involved in stabilizing the dodecamer suggest that this assembly may be functionally relevant for many CRESS-DNA viruses. The positioning of endonuclease domains in the Rep hexamer, combined with our biophysical analyses of Rep oligomerization, provide new insights into Rep function during viral replication.
History
DepositionJul 23, 2025-
Header (metadata) releaseFeb 25, 2026-
Map releaseFeb 25, 2026-
UpdateMar 18, 2026-
Current statusMar 18, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_71789.png

Downloads & links

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Map

FileDownload / File: emd_71789.map.gz / Format: CCP4 / Size: 325 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationsharpened map of 3D auto-refined map from Relion (primary map)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.84 Å/pix.
x 440 pix.
= 369.6 Å		0.84 Å/pix.
x 440 pix.
= 369.6 Å		0.84 Å/pix.
x 440 pix.
= 369.6 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.84 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 5.4
Minimum - Maximum-19.043275999999999 - 44.614803000000002
Average (Standard dev.)-0.000000000000825 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions440440440
Spacing440440440
CellA=B=C: 369.59998 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: 3D auto-refined map from Relion (raw map)

Fileemd_71789_additional_1.map
Annotation3D auto-refined map from Relion (raw map)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 1 from 3D auto-refinement

Fileemd_71789_half_map_1.map
Annotationhalf map 1 from 3D auto-refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: half map 2 from 3D auto-refinement

Fileemd_71789_half_map_2.map
Annotationhalf map 2 from 3D auto-refinement
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry

EntireName: ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry
Components
  • Complex: ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry
    • Protein or peptide: Replication-associated protein
  • Ligand: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER
  • Ligand: MAGNESIUM ION

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Supramolecule #1: ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry

SupramoleculeName: ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Vientovirus FB

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Macromolecule #1: Replication-associated protein

MacromoleculeName: Replication-associated protein / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO
EC number: Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases
Source (natural)Organism: Human lung-associated vientovirus FB
Molecular weightTheoretical: 37.991738 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SLRLQNKNLY LTYLDTEERI FSELILITEL AERLDKYGVK YAIACKEVAP STGTVHYHCL ICCENVISTR NGKELLTIEN IMPHVGRIQ NNLVNIVNYI KKDGSFAEVN KENAPKKKEM EKREKAELMI NGNLRELFME GVLGAIDIIR AEKLRNIFQV Q APADKYQK ...String:
SLRLQNKNLY LTYLDTEERI FSELILITEL AERLDKYGVK YAIACKEVAP STGTVHYHCL ICCENVISTR NGKELLTIEN IMPHVGRIQ NNLVNIVNYI KKDGSFAEVN KENAPKKKEM EKREKAELMI NGNLRELFME GVLGAIDIIR AEKLRNIFQV Q APADKYQK KLILWFKGET GEGKTKTAVD IAERFFNGEY WMSNETLKWF DGYRGQPVAI IDDFRKSMLT EWSFLLRLLD GY NLIVQVK GGFVKWQPKV IIVTSPASPS EAFQWVNKEG EIIEWDKQVQ LTRRLTYEDE EQIYNFPLWN QDQLKLENTI RKY LGLAPM

UniProtKB: Replication-associated protein

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Macromolecule #2: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER

MacromoleculeName: PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / type: ligand / ID: 2 / Number of copies: 12 / Formula: AGS
Molecular weightTheoretical: 523.247 Da
Chemical component information

ChemComp-AGS:
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-gamma-S, energy-carrying molecule analogue*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 18 / 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

Concentration0.2 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
20.0 mMHEPES4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid
300.0 mMNaClsodium chloride
0.1 mMTCEPtris(2-carboxyethyl)phosphine
2.5 %C3H8O3glycerol
5.0 mMMgCl2magnesium chloride
5.0 mMC10H16N5O12P3SAdenosine 5'-O-(3-thiotriphosphate

Details: 20 mM HEPES-NaOH pH 7.5, 300 mM NaCl, 0.1 mM TCEP, 2.5% glycerol added with 5 mM MgCl2 and 5mM ATPgammaS
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.03 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 5605 / Average exposure time: 1.42 sec. / Average electron dose: 41.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 138554
CTF correctionSoftware:
Namedetails
cryoSPARC (ver. 4.5.3)Patch CTF estimation
RELION (ver. 5.0-beta)CTFFind-4.1.14

Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C6 (6 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 5.0-beta) / Number images used: 28346
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.5.3) / Software - details: nonuniform refinement
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 5.0-beta) / Software - details: 3D auto-refinement

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Atomic model buiding 1

Initial modelPDB ID:

9pqj


Chain - Residue range: 121-327 / Chain - Source name: PDB / Chain - Initial model type: experimental model
DetailsInitial rigid body fitting was done in ChimeraX followed by manual building and flexible fitting in coot
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 41.66
Output model

PDB-9pqr:
Cryo-EM structure of ATPgammaS-bound Vientovirus FB Rep double hexamer with C6 symmetry

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