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- EMDB-41354: Cryo-EM structure of RNA device 43 truncation mutant 3 (U100C), a... -

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Basic information

Entry
Database: EMDB / ID: EMD-41354
TitleCryo-EM structure of RNA device 43 truncation mutant 3 (U100C), apo state
Map dataunsharpened
Sample
  • Complex: Synthetic RNA device containing hammerhead ribozyme and tetracycline-binding aptamer
    • RNA: RNA device 43 truncation mutant 3 (U100C)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
KeywordsRNA device / RNA
Biological speciessynthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.37 Å
AuthorsStagno JR / Deme JC / Lee Y-T / Wang Y-X / Lea SM
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI) United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural investigation of an RNA device that regulates PD-1 expression in mammalian cells.
Authors: Jason R Stagno / Justin C Deme / Vibha Dwivedi / Yun-Tzai Lee / Hyun Kyung Lee / Ping Yu / Szu-Yun Chen / Lixin Fan / Maximilia F S Degenhardt / Raj Chari / Howard A Young / Susan M Lea / Yun-Xing Wang
Abstract: Synthetic RNA devices are engineered to control gene expression and offer great potential in both biotechnology and clinical applications. Here, we present multidisciplinary structural and ...Synthetic RNA devices are engineered to control gene expression and offer great potential in both biotechnology and clinical applications. Here, we present multidisciplinary structural and biochemical data for a tetracycline (Tc)-responsive RNA device (D43) in both ligand-free and bound states, providing a structure-dynamical basis for signal transmission. Activation of self-cleavage is achieved via ligand-induced conformational and dynamical changes that stabilize the elongated bridging helix harboring the communication module, which drives proper coordination of the catalytic residues. We then show the utility of CRISPR-integrated D43 in EL4 lymphocytes to regulate programmed cell death protein 1 (PD-1), a key receptor of immune checkpoints. Treatment of these cells with Tc showed a dose-dependent reduction in PD-1 by immunostaining and a decrease in messenger RNA levels by quantitative PCR as compared with wild type. PD-1 expression was recoverable upon removal of Tc. These results provide mechanistic insight into RNA devices with potential for cancer immunotherapy or other applications.
History
DepositionJul 25, 2023-
Header (metadata) releaseFeb 19, 2025-
Map releaseFeb 19, 2025-
UpdateMar 4, 2026-
Current statusMar 4, 2026Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41354.png

Downloads & links

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Map

FileDownload / File: emd_41354.map.gz / Format: CCP4 / Size: 229.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotationunsharpened
Projections & slices

Image control

Size
Brightness
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AxesZ (Sec.)Y (Row.)X (Col.)
0.69 Å/pix.
x 392 pix.
= 271.656 Å		0.69 Å/pix.
x 392 pix.
= 271.656 Å		0.69 Å/pix.
x 392 pix.
= 271.656 Å

Surface

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Slices (1/3)

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.693 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.32
Minimum - Maximum-0.38477743 - 1.028521
Average (Standard dev.)0.00011912428 (±0.014945127)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions392392392
Spacing392392392
CellA=B=C: 271.656 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_41354_msk_1.map
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Additional map: b-factor sharpened

Fileemd_41354_additional_1.map
Annotationb-factor sharpened
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Additional map: deepEMhancer postprocessed

Fileemd_41354_additional_2.map
AnnotationdeepEMhancer postprocessed
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Half map: half map 1

Fileemd_41354_half_map_1.map
Annotationhalf map 1
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Half map: half map 2

Fileemd_41354_half_map_2.map
Annotationhalf map 2
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Sample components

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Entire : Synthetic RNA device containing hammerhead ribozyme and tetracycl...

EntireName: Synthetic RNA device containing hammerhead ribozyme and tetracycline-binding aptamer
Components
  • Complex: Synthetic RNA device containing hammerhead ribozyme and tetracycline-binding aptamer
    • RNA: RNA device 43 truncation mutant 3 (U100C)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Synthetic RNA device containing hammerhead ribozyme and tetracycl...

SupramoleculeName: Synthetic RNA device containing hammerhead ribozyme and tetracycline-binding aptamer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 35.086 KDa

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Macromolecule #1: RNA device 43 truncation mutant 3 (U100C)

MacromoleculeName: RNA device 43 truncation mutant 3 (U100C) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 34.516688 KDa
SequenceString:
CAGGUACAUC CAGCUGAUGA GUCCCAAAUA GGACAAAAAG GGAGAGGUGA AGAAUACGAC CACCUAGGCU CGAAAGAGCC UAAAACAUA CCCUUCCUGG AUUCCUGC

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Macromolecule #2: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 2 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM

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Macromolecule #3: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 3 / Number of copies: 10 / 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

Concentration1.2 mg/mL
BufferpH: 6.8
Component:
ConcentrationName
10.0 mMBis-Tris
100.0 mMpotassium chloride
1.0 mMmagnesium chloride
GridModel: Quantifoil / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Details: 30s at 30mA on each side
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 284 K / Instrument: FEI VITROBOT MARK IV
Detailsin vitro transcribed RNA

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Electron microscopy

MicroscopeTFS KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Average electron dose: 54.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.37 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 404781
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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