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- PDB-7ez0: Apo L-21 ScaI Tetrahymena ribozyme -

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Basic information

Entry
Database: PDB / ID: 7ez0
TitleApo L-21 ScaI Tetrahymena ribozyme
ComponentsApo L-21 ScaI Tetrahymena ribozyme
KeywordsRNA / RNA structure / Tetrahymena ribozyme
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.14 Å
AuthorsSu, Z. / Zhang, K. / Kappel, K. / Luo, B. / Das, R. / Chiu, W.
Funding support China, United States, 6items
OrganizationGrant numberCountry
National Science Foundation (NSF, China)32070049 China
National Science Foundation (NSF, China)82041016 China
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)P41GM103832 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)P01AI120943 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)S10OD021600 United States
CitationJournal: Nature / Year: 2021
Title: Cryo-EM structures of full-length Tetrahymena ribozyme at 3.1 Å resolution.
Authors: Zhaoming Su / Kaiming Zhang / Kalli Kappel / Shanshan Li / Michael Z Palo / Grigore D Pintilie / Ramya Rangan / Bingnan Luo / Yuquan Wei / Rhiju Das / Wah Chiu /
Abstract: Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies of protein-free RNA are in ...Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.
History
DepositionJun 1, 2021Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Aug 25, 2021Provider: repository / Type: Initial release
Revision 1.1Feb 16, 2022Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

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Assembly

Deposited unit
N: Apo L-21 ScaI Tetrahymena ribozyme
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,75328
Polymers125,0971
Non-polymers65627
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2780 Å2
ΔGint-212 kcal/mol
Surface area59830 Å2

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Components

#1: RNA chain Apo L-21 ScaI Tetrahymena ribozyme


Mass: 125096.773 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Tetrahymena thermophila (eukaryote) / Production host: synthetic construct (others) / References: GenBank: 10832
#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: Apo L-21 ScaI Tetrahymena ribozyme with metal ions. / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Source (recombinant)Organism: synthetic construct (others)
Buffer solutionpH: 8.1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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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 FIELDBright-field microscopy / Nominal magnification: 215000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 300 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Temperature (min): 78.2 K
Image recordingElectron dose: 75 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of real images: 7577
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Width: 3838 / Height: 3710 / Movie frames/image: 25 / Used frames/image: 1-25

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Processing

EM software
IDNameVersionCategory
2EPU2.5image acquisition
4CTFFIND4.1.13CTF correction
5RELION3CTF correction
8UCSF Chimera1.14model fitting
10PHENIX1.15.2-3472model refinement
11Coot0.8.7model refinement
12RELION3initial Euler assignment
13RELION3final Euler assignment
14RELION3classification
15RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1658961
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.14 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 415918 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Atomic model buildingPDB-ID: 6WLS

6wls


Pdb chain-ID: A / Pdb chain residue range: 22-409

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