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- EMDB-33814: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoi... -

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Entry
Database: EMDB / ID: EMD-33814
TitleCryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the second-step self-splicing
Map data
Sample
  • Complex: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the second-step self-splicing
    • RNA: RNA (5'-R(*CP*CP*CP*UP*CP*UP*UP*AP*AP*CP*C)-3')
    • RNA: RNA (393-MER)
    • RNA: RNA (5'-R(*UP*CP*G)-3')
  • Ligand: MAGNESIUM ION
KeywordsTetrahymena ribozyme / second step of self-splicing / conformation 3 / RNA
Biological speciesTetrahymena (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.62 Å
AuthorsLi S / Michael ZP / Zhang X / Greg P / Zhang K / Liu L
Funding support China, 1 items
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, 2022-
Header (metadata) releaseMar 29, 2023-
Map releaseMar 29, 2023-
UpdateJul 3, 2024-
Current statusJul 3, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_33814.png

Downloads & links

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Map

FileDownload / File: emd_33814.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.82 Å/pix.
x 256 pix.
= 209.92 Å		0.82 Å/pix.
x 256 pix.
= 209.92 Å		0.82 Å/pix.
x 256 pix.
= 209.92 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.82 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.25
Minimum - Maximum-0.5264194 - 1.80125
Average (Standard dev.)0.00420053 (±0.049593795)
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: #1

Fileemd_33814_additional_1.map
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Half map: #2

Fileemd_33814_half_map_1.map
Projections & Slices
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Histogram (log scale)

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Half map: #1

Fileemd_33814_half_map_2.map
Projections & Slices
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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 second-step self-splicing
Components
  • Complex: Cryo-EM structure of Tetrahymena ribozyme conformation 3 undergoing the second-step self-splicing
    • RNA: RNA (5'-R(*CP*CP*CP*UP*CP*UP*UP*AP*AP*CP*C)-3')
    • RNA: RNA (393-MER)
    • RNA: RNA (5'-R(*UP*CP*G)-3')
  • 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 second-step self-splicing
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Tetrahymena (eukaryote)
Molecular weightTheoretical: 130 KDa

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

MacromoleculeName: RNA (5'-R(*CP*CP*CP*UP*CP*UP*UP*AP*AP*CP*C)-3') / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Tetrahymena (eukaryote)
Molecular weightTheoretical: 3.379108 KDa
SequenceString:
CCCUCU(SSU)AAC 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: RNA (5'-R(*UP*CP*G)-3')

MacromoleculeName: RNA (5'-R(*UP*CP*G)-3') / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Tetrahymena (eukaryote)
Molecular weightTheoretical: 911.596 Da
SequenceString:
UCG

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 54 / 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

Concentration2.6 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 52.8 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.5 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 4255846
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.62 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.3) / Number images used: 70589
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD

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