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- EMDB-45250: Cryo-EM structure of the apo TPP riboswitch embedded in an RNA sc... -

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Basic information

Entry
Database: EMDB / ID: EMD-45250
TitleCryo-EM structure of the apo TPP riboswitch embedded in an RNA scaffold
Map data
Sample
  • Complex: apo TPP riboswitch embedded in an RNA scaffold
    • RNA: apo TPP riboswitch embedded in an RNA scaffold
KeywordsRNA / Riboswitch / Ligand
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.73 Å
AuthorsToor N
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141706 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122532 United States
CitationJournal: Nat Commun / Year: 2025
Title: Scaffold-enabled high-resolution cryo-EM structure determination of RNA.
Authors: Daniel B Haack / Boris Rudolfs / Shouhong Jin / Alexandra Khitun / Kevin M Weeks / Navtej Toor /
Abstract: Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are ...Cryo-EM structure determination of protein-free RNAs has remained difficult with most attempts yielding low to moderate resolution and lacking nucleotide-level detail. These difficulties are compounded for small RNAs as cryo-EM is inherently more difficult for lower molecular weight macromolecules. Here we present a strategy for fusing small RNAs to a group II intron that yields high resolution structures of the appended RNA. We demonstrate this technology by determining the structures of the 86-nucleotide (nt) thiamine pyrophosphate (TPP) riboswitch aptamer domain and the recently described 210-nt raiA bacterial non-coding RNA involved in sporulation and biofilm formation. In the case of the TPP riboswitch aptamer domain, the scaffolding approach allowed visualization of the riboswitch ligand binding pocket at 2.5 Å resolution. We also determined the structure of the ligand-free apo state and observe that the aptamer domain of the riboswitch adopts an open Y-shaped conformation in the absence of ligand. Using this scaffold approach, we determined the structure of raiA at 2.5 Å in the core. Our versatile scaffolding strategy enables efficient RNA structure determination for a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.
History
DepositionJun 7, 2024-
Header (metadata) releaseJan 29, 2025-
Map releaseJan 29, 2025-
UpdateFeb 5, 2025-
Current statusFeb 5, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45250.png

Downloads & links

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Map

FileDownload / File: emd_45250.map.gz / Format: CCP4 / Size: 91.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.26 Å/pix.
x 288 pix.
= 363.341 Å		1.26 Å/pix.
x 288 pix.
= 363.341 Å		1.26 Å/pix.
x 288 pix.
= 363.341 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.2616 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.227
Minimum - Maximum-0.45772824 - 1.3334571
Average (Standard dev.)-0.00041656606 (±0.007810677)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions288288288
Spacing288288288
CellA=B=C: 363.34082 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_45250_additional_1.map
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Half map: #1

Fileemd_45250_half_map_1.map
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Half map: #2

Fileemd_45250_half_map_2.map
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Sample components

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Entire : apo TPP riboswitch embedded in an RNA scaffold

EntireName: apo TPP riboswitch embedded in an RNA scaffold
Components
  • Complex: apo TPP riboswitch embedded in an RNA scaffold
    • RNA: apo TPP riboswitch embedded in an RNA scaffold

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Supramolecule #1: apo TPP riboswitch embedded in an RNA scaffold

SupramoleculeName: apo TPP riboswitch embedded in an RNA scaffold / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 27 KDa

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Macromolecule #1: apo TPP riboswitch embedded in an RNA scaffold

MacromoleculeName: apo TPP riboswitch embedded in an RNA scaffold / type: rna / ID: 1
Source (natural)Organism: Escherichia coli (E. coli)
SequenceString: UUAUGUGUGC CCGGCAUGGG UGCAGUCUAU AGGGUGAGAG UCCCGAACUG UGAAGGCAGA AGUAACAGUU AGCCUAACGC AAGGGUGUCC GUGGCGACAU GGAAUCUGAA GGAAGCGGAC GGCAAACCUU CGGUCUGAGG AACACGAACU UCAUAUGAGG CUAGGUAUCA ...String:
UUAUGUGUGC CCGGCAUGGG UGCAGUCUAU AGGGUGAGAG UCCCGAACUG UGAAGGCAGA AGUAACAGUU AGCCUAACGC AAGGGUGUCC GUGGCGACAU GGAAUCUGAA GGAAGCGGAC GGCAAACCUU CGGUCUGAGG AACACGAACU UCAUAUGAGG CUAGGUAUCA AUGGAUGAGU UUGCAUAACA AAACAAAGUC CUUUCUGCCA AAGUUGGUAC AGAGUAAAUG AAGCAGAUUG AUGAAGGGAA AGACUGCAUU CUUACCCGGG GAGGUCUGGA AACAGAAGUC AGCAGAAGUC AUAGUACCCU CAGUACUCG GGGUGCCCUU CUGCGUGAAG GCUGAGAAAU ACCCGUAUCA CCUGAUCUGG AUAAUGCCAG CGUAGGGAAG UGCUG AGGG GAAGGACGGA ACAAGUAUGG CGUUCGCGCC UAAGCUUGAA CCACCGUAUA CCGAACGGUA CGUACGGUGG UGUGAGAGGA GUUCGCUCUA CUCUAUUAG

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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: 6.5
VitrificationCryogen name: ETHANE-PROPANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
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: 1.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 4.73 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 106248
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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