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- PDB-9pqq: Cryo-EM structure of ATPgammaS-bound Vientovirus FB Rep double he... -

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Basic information

Entry
Database: PDB / ID: 9pqq
TitleCryo-EM structure of ATPgammaS-bound Vientovirus FB Rep double hexamer with C1 symmetry
ComponentsReplication-associated protein
KeywordsREPLICATION / SF3 helicase / DNA binding protein / 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
PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / Replication-associated protein
Similarity search - Component
Biological speciesHuman lung-associated vientovirus FB
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.32 Å
AuthorsMontermoso, S. / Gupta, K. / Pumroy, R.A. / Moiseenkova-Bell, V. / Bushman, F.D. / Van Duyne, G.D.
Funding support United States, 6items
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, 2025Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 25, 2026Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Feb 25, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Mar 18, 2026Group: Data collection / Database references / Category: citation / em_admin
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Replication-associated protein
B: Replication-associated protein
C: Replication-associated protein
D: Replication-associated protein
E: Replication-associated protein
F: Replication-associated protein
G: Replication-associated protein
H: Replication-associated protein
I: Replication-associated protein
J: Replication-associated protein
K: Replication-associated protein
L: Replication-associated protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)462,61742
Polymers455,90112
Non-polymers6,71630
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Replication-associated protein / ATP-dependent helicase Rep / RepP


Mass: 37991.738 Da / Num. of mol.: 12 / Mutation: T2S
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Human lung-associated vientovirus FB / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: A0A4D6K5Y8, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'- ...References: UniProt: A0A4D6K5Y8, Transferases; Transferring phosphorus-containing groups; Nucleotidyltransferases, Hydrolases; Acting on ester bonds; Endodeoxyribonucleases producing 5'-phosphomonoesters, Hydrolases; Acting on acid anhydrides; In phosphorus-containing anhydrides
#2: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 18 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical
ChemComp-AGS / PHOSPHOTHIOPHOSPHORIC ACID-ADENYLATE ESTER / ATP-GAMMA-S / ADENOSINE 5'-(3-THIOTRIPHOSPHATE) / ADENOSINE 5'-(GAMMA-THIOTRIPHOSPHATE) / ADENOSINE-5'-DIPHOSPHATE MONOTHIOPHOSPHATE


Mass: 523.247 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: C10H16N5O12P3S / Feature type: SUBJECT OF INVESTIGATION / Comment: ATP-gamma-S, energy-carrying molecule analogue*YM
Has ligand of interestY
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: ATPgammaS-bound Vientovirus FB Rep double hexamer with C1 symmetry
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Vientovirus FB
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
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
Buffer component
IDConc.NameFormulaBuffer-ID
120 mM4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acidHEPES1
2300 mMsodium chlorideNaCl1
30.1 mMtris(2-carboxyethyl)phosphineTCEP1
42.5 %glycerolC3H8O31
55 mMmagnesium chlorideMgCl21
65 mMAdenosine 5'-O-(3-thiotriphosphateC10H16N5O12P3S1
SpecimenConc.: 0.2 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 100 µm
Image recordingAverage exposure time: 1.42 sec. / Electron dose: 41.3 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5605

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.5.3particle selectionManual picking for initial particle picking
2Topazparticle selectionTopaz training and picking
5cryoSPARC4.5.3CTF correctionPatch CTF estimation
6RELION5.0-betaCTF correctionCTFFind-4.1.14
9UCSF ChimeraX1.9model fitting
10Coot0.9.8.96model fitting
12cryoSPARC4.5.3initial Euler assignmentnonuniform refinement
13RELION5.0-betafinal Euler assignment3D auto-refinement
14RELION5.0-betaclassification
15RELION5.0-beta3D reconstruction
16PHENIX1.21_5207model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 138554
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.32 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 28346 / Symmetry type: POINT
Atomic model buildingB value: 49.35 / Protocol: RIGID BODY FIT / Space: REAL
Details: Initial rigid body fitting was done in ChimeraX followed by manual building and flexible fitting in coot
Atomic model buildingPDB-ID: 9PQJ

9pqj


Accession code: 9PQJ / Chain residue range: 121-327 / Pdb chain residue range: 121-327 / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00327324
ELECTRON MICROSCOPYf_angle_d0.56836966
ELECTRON MICROSCOPYf_dihedral_angle_d10.6063708
ELECTRON MICROSCOPYf_chiral_restr0.0423990
ELECTRON MICROSCOPYf_plane_restr0.0044632

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