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- EMDB-46905: Rous sarcoma virus frameshifting pseudoknot RNA EM straight dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-46905
TitleRous sarcoma virus frameshifting pseudoknot RNA EM straight dimer
Map dataRous sarcoma virus frameshifting pseudoknot straight dimer sharpened map
Sample
  • Complex: Dimeric RNA
    • RNA: frameshifting pseudoknot RNA
  • Ligand: POTASSIUM ION
  • Ligand: MAGNESIUM ION
Keywordspseudoknot / retroviral RNA / frameshifting / translation regulation / RNA
Biological speciesRous sarcoma virus - Prague C
Methodsingle particle reconstruction / cryo EM / Resolution: 3.22 Å
AuthorsJones CP / Ferre-D'Amare AR
Funding support United States, 1 items
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, 2024-
Header (metadata) releaseApr 9, 2025-
Map releaseApr 9, 2025-
UpdateApr 16, 2025-
Current statusApr 16, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_46905.png

Downloads & links

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Map

FileDownload / File: emd_46905.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationRous sarcoma virus frameshifting pseudoknot straight dimer sharpened map
Projections & slices

Image control

Size
Brightness
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Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å		0.83 Å/pix.
x 320 pix.
= 265.6 Å

Surface

Projections

Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.2
Minimum - Maximum-0.40144467 - 0.89556324
Average (Standard dev.)0.00027347784 (±0.01854784)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 265.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_46905_msk_1.map
Projections & Slices
AxesZYX

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Additional map: Rous sarcoma virus frameshifting pseudoknot straight dimer unsharpened...

Fileemd_46905_additional_1.map
AnnotationRous sarcoma virus frameshifting pseudoknot straight dimer unsharpened map
Projections & Slices
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Half map: Rous sarcoma virus frameshifting pseudoknot straight dimer half map A

Fileemd_46905_half_map_1.map
AnnotationRous sarcoma virus frameshifting pseudoknot straight dimer half map A
Projections & Slices
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Half map: Rous sarcoma virus frameshifting pseudoknot straight dimer half map B

Fileemd_46905_half_map_2.map
AnnotationRous sarcoma virus frameshifting pseudoknot straight dimer half map B
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Sample components

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Entire : Dimeric RNA

EntireName: Dimeric RNA
Components
  • Complex: Dimeric RNA
    • RNA: frameshifting pseudoknot RNA
  • Ligand: POTASSIUM ION
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Dimeric RNA

SupramoleculeName: Dimeric RNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Details: Folded RNA specimen prepared by in vitro transcription
Source (natural)Organism: Rous sarcoma virus - Prague C

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Macromolecule #1: frameshifting pseudoknot RNA

MacromoleculeName: frameshifting pseudoknot RNA / type: rna / ID: 1 / Number of copies: 2
Source (natural)Organism: Rous sarcoma virus - Prague C
Molecular weightTheoretical: 35.657125 KDa
SequenceString:
GGGCCACUGU UCUCACUGUU GCGCUACAUC UGGCUUUUCG GAAAUGGAAG CCAGACCACA CGAAAGUGUG GAUUGACCAG UGGCCCCUC CCUGAAGGUA AACUUGUAGC GC

GENBANK: GENBANK: J02342.1

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Macromolecule #2: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 2 / Number of copies: 4 / Formula: K
Molecular weightTheoretical: 39.098 Da

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 2 / 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

BufferpH: 7.4 / Details: 25 mM HEPES-KOH, pH 7.4, 150 mM KCl, 10 mM MgCl2
GridModel: Quantifoil / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: Pelco easiGlow from Ted Pella (Redding, CA)
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK I

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 7874 / Average electron dose: 54.4 e/Å2 / Details: 7874 curated micrographs
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3400000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

9dib


Details: Chain B of 9DIB was placed twice into the map and adjusted
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.22 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 88523
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Avg.num./class: 81500 / Software - Name: RELION

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Atomic model buiding 1

Initial modelPDB ID:

9dib


Chain - Chain ID: B / Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: Two copies of B were placed
DetailsPhenix 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
RefinementSpace: REAL / Protocol: OTHER / Overall B value: 116.7 / Target criteria: Cross-correlation coefficient
Output model

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

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