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- EMDB-45994: Small dataset reconstruction of the RaiA RNA motif -

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Basic information

Entry
Database: EMDB / ID: EMD-45994
TitleSmall dataset reconstruction of the RaiA RNA motif
Map data
Sample
  • Organelle or cellular component: Bacterial RaiA RNA motif
Keywordsnon-coding RNA / cryo-EM / structural biology / RNA
Biological speciesClostridium acetobutylicum (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.78 Å
AuthorsRudolfs B / Haack DB / Toor N
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM141706 United States
CitationJournal: bioRxiv / Year: 2024
Title: Scaffold-enabled high-resolution cryo-EM structure determination of RNA.
Authors: Daniel B Haack / Boris Rudolfs / Shouhong Jin / 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, which we demonstrate with the 86-nucleotide thiamine pyrophosphate (TPP) riboswitch, and visualizing 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 undergoes a large-scale conformational change upon ligand binding, illustrating how small molecule binding to an RNA can induce large effects on gene expression. This study both sets a new standard for cryo-EM riboswitch visualization and offers a versatile strategy applicable to a broad range of small to moderate-sized RNAs, which were previously intractable for high-resolution cryo-EM studies.
History
DepositionJul 31, 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_45994.png

Downloads & links

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Map

FileDownload / File: emd_45994.map.gz / Format: CCP4 / Size: 129.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
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AxesZ (Sec.)Y (Row.)X (Col.)
1.34 Å/pix.
x 324 pix.
= 434.176 Å		1.34 Å/pix.
x 324 pix.
= 434.176 Å		1.34 Å/pix.
x 324 pix.
= 434.176 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 1.34005 Å
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Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.389
Minimum - Maximum-0.27892146 - 0.97391856
Average (Standard dev.)-0.00041357268 (±0.01321949)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions324324324
Spacing324324324
CellA=B=C: 434.176 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Density modified map in Phenix

Fileemd_45994_additional_1.map
AnnotationDensity modified map in Phenix
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AxesZYX

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Additional map: EMReady Sharpened

Fileemd_45994_additional_2.map
AnnotationEMReady Sharpened
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Additional map: CryoSPARC sharpened

Fileemd_45994_additional_3.map
AnnotationCryoSPARC sharpened
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AxesZYX

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Half map: #2

Fileemd_45994_half_map_1.map
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Half map: #1

Fileemd_45994_half_map_2.map
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Sample components

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Entire : Bacterial RaiA RNA motif

EntireName: Bacterial RaiA RNA motif
Components
  • Organelle or cellular component: Bacterial RaiA RNA motif

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Supramolecule #1: Bacterial RaiA RNA motif

SupramoleculeName: Bacterial RaiA RNA motif / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Clostridium acetobutylicum (bacteria) / Synthetically produced: Yes

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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: FLOOD BEAM / 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

Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.78 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 34517
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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