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- PDB-8hd7: The intermediate pre-Tet-S1 state of G264A mutated Tetrahymena gr... -

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Entry
Database: PDB / ID: 8hd7
TitleThe intermediate pre-Tet-S1 state of G264A mutated Tetrahymena group I intron with 6nt 3'/5'-exon and 2-aminopurine nucleoside
ComponentsThe intermediate pre-Tet-S1 state molecule of co-transcriptional folded G264A mutant Tetrahymena group I intron with 6nt 3'/5'-exon and 2-aminopurine nucleoside
KeywordsRNA / Catalytic RNA / G264A mutated Tetrahymena group I intron
Function / homologySPERMIDINE / : / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.52 Å
AuthorsLuo, B. / Zhang, C. / Ling, X. / Mukherjee, S. / Jia, G. / Xie, J. / Jia, X. / Liu, L. / Baulin, E.F. / Luo, Y. ...Luo, B. / Zhang, C. / Ling, X. / Mukherjee, S. / Jia, G. / Xie, J. / Jia, X. / Liu, L. / Baulin, E.F. / Luo, Y. / Jiang, L. / Dong, H. / Wei, X. / Bujnicki, J.M. / Su, Z.
Funding support China, Poland, European Union, 7items
OrganizationGrant numberCountry
Ministry of Science and Technology (MoST, China)2021YFA1301900 China
Ministry of Science and Technology (MoST, China)2022YFC2303700 China
National Natural Science Foundation of China (NSFC)32222040 China
National Natural Science Foundation of China (NSFC)32070049 China
Polish National Science Centre2017/26/A/NZ1/01083 Poland
Polish National Science Centre2021/43/D/NZ1/03360 Poland
European Molecular Biology Organization (EMBO)ALTF 525-2022European Union
CitationJournal: Nat Catal / Year: 2023
Title: Cryo-EM reveals dynamics of Tetrahymena group I intron self-splicing
Authors: Luo, B. / Zhang, C. / Ling, X. / Mukherjee, S. / Jia, G. / Xie, J. / Jia, X. / Liu, L. / Baulin, E.F. / Luo, Y. / Jiang, L. / Dong, H. / Wei, X. / Bujnicki, J.M. / Su, Z.
History
DepositionNov 3, 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

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Assembly

Deposited unit
N: The intermediate pre-Tet-S1 state molecule of co-transcriptional folded G264A mutant Tetrahymena group I intron with 6nt 3'/5'-exon and 2-aminopurine nucleoside
hetero molecules


Theoretical massNumber of molelcules
Total (without water)135,95729
Polymers135,1561
Non-polymers80128
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 The intermediate pre-Tet-S1 state molecule of co-transcriptional folded G264A mutant Tetrahymena group I intron with 6nt 3'/5'-exon and 2-aminopurine nucleoside


Mass: 135155.719 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: JN547815
#2: Chemical ChemComp-SPD / SPERMIDINE / N-(2-AMINO-PROPYL)-1,4-DIAMINOBUTANE / PA(34)


Mass: 145.246 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C7H19N3
#3: 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: Co-transcriptional folded G264A mutated Tetrahymena group I intron with 6-nt 3'/5'-exon and 2-aminopurine nucleoside
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: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 FIELD / Nominal magnification: 165000 X / Nominal defocus max: 3700 nm / Nominal defocus min: 600 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): 79.3 K
Image recordingElectron dose: 55 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 30 / Used frames/image: 1-30

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM software
IDNameVersionCategory
1EMAN22.31particle selection
2EPU2.9image acquisition
4RELION3.1CTF correction
5CTFFIND4CTF correction
8UCSF Chimera1.14model fitting
10PHENIX1.15model refinement
11Coot0.8.7model refinement
12RELION3.1initial Euler assignment
13RELION3.1final Euler assignment
14RELION3.1classification
15RELION3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4012136
3D reconstructionResolution: 3.52 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 111196 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00210022
ELECTRON MICROSCOPYf_angle_d0.61215612
ELECTRON MICROSCOPYf_dihedral_angle_d14.475032
ELECTRON MICROSCOPYf_chiral_restr0.0272095
ELECTRON MICROSCOPYf_plane_restr0.004419

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