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- EMDB-45249: Cryo-EM structure of the TPP riboswitch embedded in an RNA scaffo... -

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Basic information

Entry
Database: EMDB / ID: EMD-45249
TitleCryo-EM structure of the TPP riboswitch embedded in an RNA scaffold bound to thiamine pyrophosphate
Map data
Sample
  • Complex: TPP riboswitch embedded in an RNA scaffold bound with thiamine pyrophosphate
    • RNA: TPP riboswitch embedded in an RNA scaffold bound to thiamine pyrophosphate
  • Ligand: MAGNESIUM ION
  • Ligand: THIAMINE DIPHOSPHATE
  • Ligand: SODIUM ION
KeywordsRNA / Riboswitch / Ligand
Biological speciesEscherichia coli (E. coli)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.94 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) releaseDec 4, 2024-
Map releaseDec 4, 2024-
UpdateMay 14, 2025-
Current statusMay 14, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_45249.png

Downloads & links

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Map

FileDownload / File: emd_45249.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

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AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 400 pix.
= 324.4 Å		0.81 Å/pix.
x 400 pix.
= 324.4 Å		0.81 Å/pix.
x 400 pix.
= 324.4 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.811 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.14
Minimum - Maximum-0.15170471 - 0.521266
Average (Standard dev.)-0.00007471716 (±0.004410476)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 324.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_45249_additional_1.map
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Half map: #2

Fileemd_45249_half_map_1.map
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Half map: #1

Fileemd_45249_half_map_2.map
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Sample components

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Entire : TPP riboswitch embedded in an RNA scaffold bound with thiamine py...

EntireName: TPP riboswitch embedded in an RNA scaffold bound with thiamine pyrophosphate
Components
  • Complex: TPP riboswitch embedded in an RNA scaffold bound with thiamine pyrophosphate
    • RNA: TPP riboswitch embedded in an RNA scaffold bound to thiamine pyrophosphate
  • Ligand: MAGNESIUM ION
  • Ligand: THIAMINE DIPHOSPHATE
  • Ligand: SODIUM ION

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Supramolecule #1: TPP riboswitch embedded in an RNA scaffold bound with thiamine py...

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

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Macromolecule #1: TPP riboswitch embedded in an RNA scaffold bound to thiamine pyro...

MacromoleculeName: TPP riboswitch embedded in an RNA scaffold bound to thiamine pyrophosphate
type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Escherichia coli (E. coli)
Molecular weightTheoretical: 161.102484 KDa
SequenceString: UUAUGUGUGC CCGGCAUGGG UGCAGUCUAU AGGGUGAGAG UCCCGAACUG UGAAGGCAGA AGUAACAGUU AGCCUAACGC AAGGGUGUC CGUGGCGACA UGGAAUCUGA AGGAAGCGGA CGGCAAACCU UCGGUCUGAG GAACACGAAC UUCAUAUGAG G CUAGGUAU ...String:
UUAUGUGUGC CCGGCAUGGG UGCAGUCUAU AGGGUGAGAG UCCCGAACUG UGAAGGCAGA AGUAACAGUU AGCCUAACGC AAGGGUGUC CGUGGCGACA UGGAAUCUGA AGGAAGCGGA CGGCAAACCU UCGGUCUGAG GAACACGAAC UUCAUAUGAG G CUAGGUAU CAAUGGAUGA GUUUGCAUAA CAAAACAAAG UCCUUUCUGC CAAAGUUGGU ACAGAGUAAA UGAAGCAGAU UG AUGAAGG GAAAGACUGC AUUCUUACCC GGGGAGGUCU GGAAACAGAA GUCAGCAGAA GUCAUAGUAC CCUCAGUACU CGG GGUGCC CUUCUGCGUG AAGGCUGAGA AAUACCCGUA UCACCUGAUC UGGAUAAUGC CAGCGUAGGG AAGUGCUGAG GGGA AGGAC GGAACAAGUA UGGCGUUCGC GCCUAAGCUU GAACCACCGU AUACCGAACG GUACGUACGG UGGUGUGAGA GGAGU UCGC UCUACUCUAU UAG

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 6 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: THIAMINE DIPHOSPHATE

MacromoleculeName: THIAMINE DIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: TPP
Molecular weightTheoretical: 425.314 Da
Chemical component information

ChemComp-TPP:
THIAMINE DIPHOSPHATE

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Macromolecule #4: SODIUM ION

MacromoleculeName: SODIUM ION / type: ligand / ID: 4 / Number of copies: 2
Molecular weightTheoretical: 22.99 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: 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

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: OTHER
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.94 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 655558
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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