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- EMDB-41312: Tetrahymena Ribozyme scaffolded Fluoride riboswitch -

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Basic information

Entry
Database: EMDB / ID: EMD-41312
TitleTetrahymena Ribozyme scaffolded Fluoride riboswitch
Map data
Sample
  • Complex: Tetrahymena Ribozyme scaffolded Fluoride riboswitch
    • RNA: RNA (417-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: FLUORIDE ION
  • Ligand: POTASSIUM ION
KeywordsRibozyme / xrRNA / Scaffold / RNA
Biological speciesTetrahymena (eukaryote) / Thermotoga petrophila (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.99 Å
AuthorsLangeberg CJ / Kieft JS
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)7R35GM118070-08 United States
CitationJournal: Nucleic Acids Res / Year: 2023
Title: A generalizable scaffold-based approach for structure determination of RNAs by cryo-EM.
Authors: Conner J Langeberg / Jeffrey S Kieft /
Abstract: Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules (≤50 kDa) remain challenging targets due to their ...Single-particle cryo-electron microscopy (cryo-EM) can reveal the structures of large and often dynamic molecules, but smaller biomolecules (≤50 kDa) remain challenging targets due to their intrinsic low signal to noise ratio. Methods to help resolve small proteins have been applied but development of similar approaches to aid in structural determination of small, structured RNA elements have lagged. Here, we present a scaffold-based approach that we used to recover maps of sub-25 kDa RNA domains to 4.5-5.0 Å. While lacking the detail of true high-resolution maps, these maps are suitable for model building and preliminary structure determination. We demonstrate this method helped faithfully recover the structure of several RNA elements of known structure, and that it promises to be generalized to other RNAs without disturbing their native fold. This approach may streamline the sample preparation process and reduce the optimization required for data collection. This first-generation scaffold approach provides a robust system to aid in RNA structure determination by cryo-EM and lays the groundwork for further scaffold optimization to achieve higher resolution.
History
DepositionJul 24, 2023-
Header (metadata) releaseNov 1, 2023-
Map releaseNov 1, 2023-
UpdateNov 22, 2023-
Current statusNov 22, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_41312.png

Downloads & links

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Map

FileDownload / File: emd_41312.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.85 Å/pix.
x 512 pix.
= 433.357 Å		0.85 Å/pix.
x 512 pix.
= 433.357 Å		0.85 Å/pix.
x 512 pix.
= 433.357 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.8464 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.50123125 - 1.7542869
Average (Standard dev.)0.00019116004 (±0.020791803)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 433.3568 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_41312_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_41312_half_map_2.map
Projections & Slices
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Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrahymena Ribozyme scaffolded Fluoride riboswitch

EntireName: Tetrahymena Ribozyme scaffolded Fluoride riboswitch
Components
  • Complex: Tetrahymena Ribozyme scaffolded Fluoride riboswitch
    • RNA: RNA (417-MER)
  • Ligand: MAGNESIUM ION
  • Ligand: FLUORIDE ION
  • Ligand: POTASSIUM ION

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Supramolecule #1: Tetrahymena Ribozyme scaffolded Fluoride riboswitch

SupramoleculeName: Tetrahymena Ribozyme scaffolded Fluoride riboswitch / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Tetrahymena (eukaryote)

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Macromolecule #1: RNA (417-MER)

MacromoleculeName: RNA (417-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Thermotoga petrophila (bacteria)
Molecular weightTheoretical: 134.830641 KDa
SequenceString: GGGCGAUGAG GCCCGCCCAA ACUGCCCUAU AUGGAUGCAG UUCACAGACU AAAUGUCGGU CGGGGAAGAU GUAUUCUUCU CAUAAGAUA UAGUCGGACC UCUCCUUAAU GGGAGCUAGC GGAUGAAGUG AUGCAACACU GGAGCCGCUG GGAACUAAUU U GUAUGCGA ...String:
GGGCGAUGAG GCCCGCCCAA ACUGCCCUAU AUGGAUGCAG UUCACAGACU AAAUGUCGGU CGGGGAAGAU GUAUUCUUCU CAUAAGAUA UAGUCGGACC UCUCCUUAAU GGGAGCUAGC GGAUGAAGUG AUGCAACACU GGAGCCGCUG GGAACUAAUU U GUAUGCGA AAGUAUAUUG AUUAGUUUUG GAGGAGGGAA AAGUUAUCAG GCAUGCACCU GGUAGCUAGU CUUUAAACCA AU AGAUUGC AUCGGUUUAA AAGGCAAGAC CGUCAAAUUG CGGGAAAGGG GUCAACAGCC GUUCAGUACC AAGUCUCAGG GGA AACUUU GAGAUGGCCU UGCAAAGGGU AUGGUAAUAA GCUGACGGAC AUGGUCCUAA CCACGCAGCC AAGUCCUAAG UAGG GCUGA UGGCCUCUAC UG

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 30 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #3: FLUORIDE ION

MacromoleculeName: FLUORIDE ION / type: ligand / ID: 3 / Number of copies: 1 / Formula: F
Molecular weightTheoretical: 18.998 Da
Chemical component information

ChemComp-F:
FLUORIDE ION

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Macromolecule #4: POTASSIUM ION

MacromoleculeName: POTASSIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: K
Molecular weightTheoretical: 39.098 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: 7.5
Component:
ConcentrationFormulaName
50.0 mMC4H11NO3Tris
10.0 mMMgCl2Magnesium Chloride
GridModel: C-flat-1.2/1.3 / Material: COPPER / Mesh: 50 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 6 sec. / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE

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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: PDB ENTRY
PDB model - PDB ID:

7ez0

Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.99 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 238627
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
FSC plot (resolution estimation)

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