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- EMDB-41313: Tetrahymena Ribozyme cryo-EM scaffold -

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Basic information

Entry
Database: EMDB / ID: EMD-41313
TitleTetrahymena Ribozyme cryo-EM scaffold
Map data
Sample
  • Complex: Tetrahymena Ribozyme cryo-EM scaffold
    • RNA: RNA (440-MER)
  • Ligand: MAGNESIUM ION
KeywordsRibozyme / Scaffold / RNA
Biological speciesTetrahymena (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.44 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_41313.png

Downloads & links

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Map

FileDownload / File: emd_41313.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.788 Å
Density
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.1281685 - 3.3169823
Average (Standard dev.)0.00016352675 (±0.031226587)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 403.456 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: #1

Fileemd_41313_additional_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_41313_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_41313_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Tetrahymena Ribozyme cryo-EM scaffold

EntireName: Tetrahymena Ribozyme cryo-EM scaffold
Components
  • Complex: Tetrahymena Ribozyme cryo-EM scaffold
    • RNA: RNA (440-MER)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Tetrahymena Ribozyme cryo-EM scaffold

SupramoleculeName: Tetrahymena Ribozyme cryo-EM scaffold / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Tetrahymena (eukaryote)

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Macromolecule #1: RNA (440-MER)

MacromoleculeName: RNA (440-MER) / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Tetrahymena (eukaryote)
Molecular weightTheoretical: 142.357188 KDa
SequenceString: GGGUCAGGCC GGCUGAUAUG GAUGCAGUUC ACAGACUAAA UGUCGGUCGG GGAAGAUGUA UUCUUCUCAU AAGAUAUAGU CGGACCUCU CCUUAAUGGG AGCUAGCGGA UGAAGUGAUG CAACACUGGA GCCGCUGGGA ACUAAUUUGU AUGCGAAAGU A UAUUGAUU ...String:
GGGUCAGGCC GGCUGAUAUG GAUGCAGUUC ACAGACUAAA UGUCGGUCGG GGAAGAUGUA UUCUUCUCAU AAGAUAUAGU CGGACCUCU CCUUAAUGGG AGCUAGCGGA UGAAGUGAUG CAACACUGGA GCCGCUGGGA ACUAAUUUGU AUGCGAAAGU A UAUUGAUU AGUUUUGGAG GAGGGAAAAG UUAUCAGGCA UGCACCUGGU AGCUAGUCUU UAAACCAAUA GAUUGCAUCG GU UUAAAAG GCAAGACCGU CAAAUUGCGG GAAAGGGGUC AACAGCCGUU CAGUACCAAG UCUCAGGGGA AACUUUGAGA UGG CCUUGC AAAGGGUAUG GUAAUAAGCU GACGGACAUG GUCCUAACCA CGCAGCCAAG UCCUAAGUCA GUCGCCACAG UUUG GGGAA AGCUGUGCAG CCUGUAACCC CCCCACGAAA GUGGG

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Macromolecule #2: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 2 / Number of copies: 27 / 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

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
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2
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

Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.44 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 570576
FSC plot (resolution estimation)

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