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- PDB-8tjx: Tetrahymena Ribozyme cryo-EM scaffold -

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Basic information

Entry
Database: PDB / ID: 8tjx
TitleTetrahymena Ribozyme cryo-EM scaffold
ComponentsRNA (440-MER)
KeywordsRNA / Ribozyme / Scaffold
Function / homologyRNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.44 Å
AuthorsLangeberg, C.J. / Kieft, J.S.
Funding support United States, 1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 1, 2023Provider: repository / Type: Initial release
Revision 1.1Nov 22, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
N: RNA (440-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)143,01328
Polymers142,3571
Non-polymers65627
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (440-MER)


Mass: 142357.188 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena (eukaryote)
Production host: in vitro transcription vector pT7-Fluc(deltai) (others)
#2: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 27 / Source method: obtained synthetically / Formula: Mg
Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Tetrahymena Ribozyme cryo-EM scaffold / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Source (natural)Organism: Tetrahymena (eukaryote)
Source (recombinant)Organism: in vitro transcription vector pT7-Fluc(deltai) (others)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTrisC4H11NO31
210 mMMagnesium ChlorideMgCl21
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 50 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 2.44 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 570576 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.01610553
ELECTRON MICROSCOPYf_angle_d1.21916450
ELECTRON MICROSCOPYf_dihedral_angle_d19.2575271
ELECTRON MICROSCOPYf_chiral_restr0.0632200
ELECTRON MICROSCOPYf_plane_restr0.008440

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