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- EMDB-33739: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoi... -

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Entry
Database: EMDB / ID: EMD-33739
TitleCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the first-step self-splicing
Map dataCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the the first-step self-splicing--- unmask
Sample
  • Complex: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the first-step self-splicing
    • RNA: RNA (5'-R(*CP*CP*CP*UP*CP*UP*AP*AP*AP*CP*C)-3')
    • RNA: RNA (393-MER)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
KeywordsTetrahymena ribozyme / first step of self-splicing / conformation 3 / RNA
Biological speciesTetrahymena (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.09 Å
AuthorsZhang X / Li S / Pintilie G / Palo MZ / Zhang K / Liu L
Funding support China, 1 items
OrganizationGrant numberCountry
Other government China
CitationJournal: Nucleic Acids Res / Year: 2023
Title: Snapshots of the first-step self-splicing of Tetrahymena ribozyme revealed by cryo-EM.
Authors: Xiaojing Zhang / Shanshan Li / Grigore Pintilie / Michael Z Palo / Kaiming Zhang /
Abstract: Tetrahymena ribozyme is a group I intron, whose self-splicing is the result of two sequential ester-transfer reactions. To understand how it facilitates catalysis in the first self-splicing reaction, ...Tetrahymena ribozyme is a group I intron, whose self-splicing is the result of two sequential ester-transfer reactions. To understand how it facilitates catalysis in the first self-splicing reaction, we used cryogenic electron microscopy (cryo-EM) to resolve the structures of L-16 Tetrahymena ribozyme complexed with a 11-nucleotide 5'-splice site analog substrate. Four conformations were achieved to 4.14, 3.18, 3.09 and 2.98 Å resolutions, respectively, corresponding to different splicing intermediates during the first enzymatic reaction. Comparison of these structures reveals structural alterations, including large conformational changes in IGS/IGSext (P1-P1ext duplex) and J5/4, as well as subtle local rearrangements in the G-binding site. These structural changes are required for the enzymatic activity of the Tetrahymena ribozyme. Our study demonstrates the ability of cryo-EM to capture dynamic RNA structural changes, ushering in a new era in the analysis of RNA structure-function by cryo-EM.
History
DepositionJul 1, 2022-
Header (metadata) releaseJul 5, 2023-
Map releaseJul 5, 2023-
UpdateAug 16, 2023-
Current statusAug 16, 2023Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33739.png

Downloads & links

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Map

FileDownload / File: emd_33739.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the the first-step self-splicing--- unmask
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 0.1
Minimum - Maximum-0.15716569 - 0.5576552
Average (Standard dev.)0.0022094839 (±0.022227282)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 209.92 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing...

Fileemd_33739_additional_1.map
AnnotationCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the the first-step self-splicing--- sharp
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing...

Fileemd_33739_half_map_1.map
AnnotationCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the the first-step self-splicing--- half
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing...

Fileemd_33739_half_map_2.map
AnnotationCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the the first-step self-splicing--- half
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoi...

EntireName: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the first-step self-splicing
Components
  • Complex: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the first-step self-splicing
    • RNA: RNA (5'-R(*CP*CP*CP*UP*CP*UP*AP*AP*AP*CP*C)-3')
    • RNA: RNA (393-MER)
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoi...

SupramoleculeName: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the first-step self-splicing
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Tetrahymena (eukaryote)
Molecular weightTheoretical: 130 KDa

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Macromolecule #1: RNA (5'-R(*CP*CP*CP*UP*CP*UP*AP*AP*AP*CP*C)-3')

MacromoleculeName: RNA (5'-R(*CP*CP*CP*UP*CP*UP*AP*AP*AP*CP*C)-3') / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Tetrahymena (eukaryote)
Molecular weightTheoretical: 3.386082 KDa
SequenceString:
CCCUCUAAAC C

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Macromolecule #2: RNA (393-MER)

MacromoleculeName: RNA (393-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Tetrahymena (eukaryote)
Molecular weightTheoretical: 127.011883 KDa
SequenceString: GGUUUGGAGG GAAAAGUUAU CAGGCAUGCA CCUGGUAGCU AGUCUUUAAA CCAAUAGAUU GCAUCGGUUU AAAAGGCAAG ACCGUCAAA UUGCGGGAAA GGGGUCAACA GCCGUUCAGU ACCAAGUCUC AGGGGAAACU UUGAGAUGGC CUUGCAAAGG G UAUGGUAA ...String:
GGUUUGGAGG GAAAAGUUAU CAGGCAUGCA CCUGGUAGCU AGUCUUUAAA CCAAUAGAUU GCAUCGGUUU AAAAGGCAAG ACCGUCAAA UUGCGGGAAA GGGGUCAACA GCCGUUCAGU ACCAAGUCUC AGGGGAAACU UUGAGAUGGC CUUGCAAAGG G UAUGGUAA UAAGCUGACG GACAUGGUCC UAACCACGCA GCCAAGUCCU AAGUCAACAG AUCUUCUGUU GAUAUGGAUG CA GUUCACA GACUAAAUGU CGGUCGGGGA AGAUGUAUUC UUCUCAUAAG AUAUAGUCGG ACCUCUCCUU AAUGGGAGCU AGC GGAUGA AGUGAUGCAA CACUGGAGCC GCUGGGAACU AAUUUGUAUG CGAAAGUAUA UUGAUUAGUU UUGGAGU

GENBANK: GENBANK: X54512.1

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 3 / 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

Concentration1 mg/mL
BufferpH: 7.5
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: 2.8000000000000003 µm / Nominal defocus min: 0.8 µm
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 51.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3668247
Startup modelType of model: NONE
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.09 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.3) / Number images used: 202401

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