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- EMDB-31386: Holo L-16 ScaI Tetrahymena ribozyme -

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Basic information

Entry
Database: EMDB / ID: EMD-31386
TitleHolo L-16 ScaI Tetrahymena ribozyme
Map data
Sample
  • Complex: Holo L-16 ScaI Tetrahymena ribozyme with substrates S1 and S2, and metal ions.
    • RNA: Holo L-16 ScaI Tetrahymena ribozyme S1
    • RNA: Holo L-16 ScaI Tetrahymena ribozyme S2
    • RNA: Holo L-16 ScaI Tetrahymena ribozyme
  • Ligand: MAGNESIUM ION
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.05 Å
AuthorsSu Z / Zhang K / Kappel K / Luo B / Das R / Chiu W
Funding support China, United States, 6 items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)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, 2021-
Header (metadata) releaseAug 25, 2021-
Map releaseAug 25, 2021-
UpdateFeb 16, 2022-
Current statusFeb 16, 2022Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7ez2
  • Surface level: 0.02
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_31386.png

Downloads & links

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Map

FileDownload / File: emd_31386.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.86 Å
Density
Contour LevelBy AUTHOR: 0.023 / Movie #1: 0.02
Minimum - Maximum-0.079133704 - 0.1844443
Average (Standard dev.)0.00017411028 (±0.0044977837)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 220.16 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.860.860.86
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z220.160220.160220.160
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ384384384
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.0790.1840.000

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Supplemental data

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Sample components

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Entire : Holo L-16 ScaI Tetrahymena ribozyme with substrates S1 and S2, an...

EntireName: Holo L-16 ScaI Tetrahymena ribozyme with substrates S1 and S2, and metal ions.
Components
  • Complex: Holo L-16 ScaI Tetrahymena ribozyme with substrates S1 and S2, and metal ions.
    • RNA: Holo L-16 ScaI Tetrahymena ribozyme S1
    • RNA: Holo L-16 ScaI Tetrahymena ribozyme S2
    • RNA: Holo L-16 ScaI Tetrahymena ribozyme
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Holo L-16 ScaI Tetrahymena ribozyme with substrates S1 and S2, an...

SupramoleculeName: Holo L-16 ScaI Tetrahymena ribozyme with substrates S1 and S2, and metal ions.
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: Holo L-16 ScaI Tetrahymena ribozyme S1

MacromoleculeName: Holo L-16 ScaI Tetrahymena ribozyme S1 / type: rna / ID: 1 / Number of copies: 1
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 2.502603 KDa
SequenceString:
UCG(SSU)AACC

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Macromolecule #2: Holo L-16 ScaI Tetrahymena ribozyme S2

MacromoleculeName: Holo L-16 ScaI Tetrahymena ribozyme S2 / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 1.788101 KDa
SequenceString:
CCCUCU

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Macromolecule #3: Holo L-16 ScaI Tetrahymena ribozyme

MacromoleculeName: Holo L-16 ScaI Tetrahymena ribozyme / type: rna / ID: 3 / Number of copies: 1
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 126.705711 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 UUGGAG

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 31 / 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

Concentration3 mg/mL
BufferpH: 8
GridModel: Quantifoil R2/1 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.36 kPa
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 278 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
TemperatureMin: 78.2 K
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 5559 / Average electron dose: 50.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 854763
CTF correctionSoftware: (Name: CTFFIND (ver. 4.1.13), RELION (ver. 3.0))
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.05 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 230386

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-7ez2:
Holo L-16 ScaI Tetrahymena ribozyme

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