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- PDB-7yga: Cryo-EM structure of Tetrahymena ribozyme conformation 2 undergoi... -

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Basic information

Entry
Database: PDB / ID: 7yga
TitleCryo-EM structure of Tetrahymena ribozyme conformation 2 undergoing the second-step self-splicing
Components
  • RNA (393-MER)
  • RNA (5'-R(*CP*CP*CP*UP*CP*U)-3')
  • RNA (5'-R(*UP*CP*GP*(SSU)P*AP*AP*CP*C)-3')
KeywordsRNA / Tetrahymena ribozyme / second step of self-splicing / conformation 2
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.35 Å
AuthorsLi, S. / Michael, Z.P. / Zhang, X. / Greg, P. / Zhang, K.
Funding support China, 1items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nat Commun / Year: 2023
Title: Snapshots of the second-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM.
Authors: Shanshan Li / Michael Z Palo / Xiaojing Zhang / Grigore Pintilie / Kaiming Zhang /
Abstract: Group I introns are catalytic RNAs that coordinate two consecutive transesterification reactions for self-splicing. To understand how the group I intron promotes catalysis and coordinates self- ...Group I introns are catalytic RNAs that coordinate two consecutive transesterification reactions for self-splicing. To understand how the group I intron promotes catalysis and coordinates self-splicing reactions, we determine the structures of L-16 Tetrahymena ribozyme in complex with a 5'-splice site analog product and a 3'-splice site analog substrate using cryo-EM. We solve six conformations from a single specimen, corresponding to different splicing intermediates after the first ester-transfer reaction. The structures reveal dynamics during self-splicing, including large conformational changes of the internal guide sequence and the J5/4 junction as well as subtle rearrangements of active-site metals and the hydrogen bond formed between the 2'-OH group of A261 and the N2 group of guanosine substrate. These results help complete a detailed structural and mechanistic view of this paradigmatic group I intron undergoing the second step of self-splicing.
History
DepositionJul 11, 2022Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Mar 29, 2023Provider: repository / Type: Initial release
Revision 1.1Jul 3, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond / em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: RNA (5'-R(*UP*CP*GP*(SSU)P*AP*AP*CP*C)-3')
B: RNA (5'-R(*CP*CP*CP*UP*CP*U)-3')
N: RNA (393-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)131,49711
Polymers131,3033
Non-polymers1948
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (5'-R(*UP*CP*GP*(SSU)P*AP*AP*CP*C)-3')


Mass: 2502.603 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena (eukaryote)
#2: RNA chain RNA (5'-R(*CP*CP*CP*UP*CP*U)-3')


Mass: 1788.101 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena (eukaryote)
#3: RNA chain RNA (393-MER)


Mass: 127011.883 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)
References: GenBank: 10832
#4: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg / Feature type: SUBJECT OF INVESTIGATION
Has ligand of interestY

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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: Cryo-EM structure of Tetrahymena ribozyme conformation 2 undergoing the second-step self-splicing
Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: YES
Source (natural)Organism: Tetrahymena (eukaryote)
Source (recombinant)Organism: in vitro transcription vector pT7-Fluc(deltai) (others)
Buffer solutionpH: 8
SpecimenConc.: 2.6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen 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: 2500 nm / Nominal defocus min: 500 nm
Image recordingElectron dose: 52.8 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
2EPUimage acquisition
12cryoSPARC2.3classification
13cryoSPARC2.33D reconstruction
CTF correctionType: NONE
Particle selectionNum. of particles selected: 4255846
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.35 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 835303 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0089718
ELECTRON MICROSCOPYf_angle_d0.93915143
ELECTRON MICROSCOPYf_dihedral_angle_d15.4564866
ELECTRON MICROSCOPYf_chiral_restr0.0382031
ELECTRON MICROSCOPYf_plane_restr0.005407

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