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- PDB-9dig: Rous sarcoma virus frameshifting pseudoknot RNA EM straight dimer -

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Basic information

Entry
Database: PDB / ID: 9dig
TitleRous sarcoma virus frameshifting pseudoknot RNA EM straight dimer
Componentsframeshifting pseudoknot RNA
KeywordsRNA / pseudoknot / retroviral RNA / frameshifting / translation regulation
Function / homology: / : / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesRous sarcoma virus - Prague C
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsJones, C.P. / Ferre-D'Amare, A.R.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI) United States
CitationJournal: Proc Natl Acad Sci U S A / Year: 2025
Title: Structural switching dynamically controls the doubly pseudoknotted Rous sarcoma virus-programmed ribosomal frameshifting element.
Authors: Christopher P Jones / Adrian R Ferré-D'Amaré /
Abstract: A hallmark of retrovirus replication is the translation of two different polyproteins from one RNA through programmed -1 frameshifting. This is a mechanism in which the actively translating ribosome ...A hallmark of retrovirus replication is the translation of two different polyproteins from one RNA through programmed -1 frameshifting. This is a mechanism in which the actively translating ribosome is induced to slip in the 5' direction at a defined codon and then continues translating in the new reading frame. Programmed frameshifting controls the stoichiometry of viral proteins and is therefore under stringent evolutionary selection. Forty years ago, the first frameshifting stimulatory element was discovered in the Rous sarcoma virus. The ~120 nt RNA segment was predicted to contain a pseudoknot, but its 3D structure has remained elusive. Now, we have determined cryoEM and X-ray crystallographic structures of this classic retroviral element, finding that it adopts a butterfly-like double-pseudoknot fold. One "wing" contains a dynamic pyrimidine-rich helix, observed crystallographically in two conformations and in a third conformation via cryoEM. The other wing encompasses the predicted pseudoknot, which interacts with a second unexpected pseudoknot through a toggle residue, A2546. This key purine switches conformations between structural states and tunes the stability of interacting residues in the two wings. We find that its mutation can modulate frameshifting by as much as 50-fold, likely by altering the relative abundance of different structural states in the conformational ensemble of the RNA. Taken together, our structure-function analyses reveal how a dynamic double pseudoknot junction stimulates frameshifting by taking advantage of conformational heterogeneity, supporting a multistate model in which high Shannon entropy enhances frameshifting efficiency.
History
DepositionSep 5, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 9, 2025Provider: repository / Type: Initial release
Revision 1.1Apr 16, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: frameshifting pseudoknot RNA
B: frameshifting pseudoknot RNA
hetero molecules


Theoretical massNumber of molelcules
Total (without water)71,5198
Polymers71,3142
Non-polymers2056
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, Folded RNA forms a dimer in an ~1:5 dimer:monomer ratio
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain frameshifting pseudoknot RNA


Mass: 35657.125 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Rous sarcoma virus - Prague C
Production host: in vitro transcription vector pT7-TP(deltai) (others)
References: GenBank: 210171
#2: Chemical
ChemComp-K / POTASSIUM ION


Mass: 39.098 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: K
#3: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN
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: Dimeric RNA / Type: COMPLEX
Details: Folded RNA specimen prepared by in vitro transcription
Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Rous sarcoma virus - Prague C
Source (recombinant)Organism: in vitro transcription vector pT7-TP(deltai) (others)
Buffer solutionpH: 7.4 / Details: 25 mM HEPES-KOH, pH 7.4, 150 mM KCl, 10 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Pelco easiGlow from Ted Pella (Redding, CA) / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK I / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 54.4 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 7874 / Details: 7874 curated micrographs

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2SerialEMimage acquisition
4CTFFIND4.1CTF correction
7Cootmodel fitting
9cryoSPARCinitial Euler assignment
10cryoSPARCfinal Euler assignment
11RELIONclassification
12cryoSPARC3D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3400000
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 88523 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingB value: 116.7 / Protocol: OTHER / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Phenix real-space refinement, using real-space refinement, simulated annealing on the first step, B-factor, occupancy, and global minimization with NCS and secondary structure restraints
Atomic model buildingPDB-ID: 9DIB

9dib


Pdb chain-ID: B / Accession code: 9DIB / Details: Two copies of B were placed / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0074526
ELECTRON MICROSCOPYf_angle_d0.7587034
ELECTRON MICROSCOPYf_dihedral_angle_d15.8882276
ELECTRON MICROSCOPYf_chiral_restr0.034956
ELECTRON MICROSCOPYf_plane_restr0.004192

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