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- PDB-9cbu: Tetrahymena ribozyme with consensus water and magnesium ions -

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Basic information

Entry
Database: PDB / ID: 9cbu
TitleTetrahymena ribozyme with consensus water and magnesium ions
ComponentsRNA (387-MER)
KeywordsRNA / Ribozyme / Magnesium ions / Water
Function / homology: / RNA / RNA (> 10) / RNA (> 100)
Function and homology information
Biological speciesTetrahymena thermophila (eukaryote)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.2 Å
AuthorsKretsch, R.C. / Li, S. / Pintilie, G. / Palo, M.Z. / Case, D.A. / Das, R. / Zhang, K. / Chiu, W.
Funding support United States, China, 10items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM122579 United States
Howard Hughes Medical Institute (HHMI) United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)U24GM129541 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM079429 United States
Chinese Academy of SciencesXDB0490000 China
Other governmentCenter for Advanced Interdisciplinary Science and Biomedicine of IHM QYPY20220019
Other governmentNational Key R&D Program of China 2022YFC2303700
Other governmentNational Key R&D Program of China 2022YFA1302700
Other governmentFundamental Research Funds for the Central Universities WK9100000032
Other governmentFundamental Research Funds for the Central Universities WK9100000044
CitationJournal: Nature / Year: 2025
Title: Complex water networks visualized by cryogenic electron microscopy of RNA.
Authors: Rachael C Kretsch / Shanshan Li / Grigore Pintilie / Michael Z Palo / David A Case / Rhiju Das / Kaiming Zhang / Wah Chiu /
Abstract: The stability and function of biomolecules are directly influenced by their myriad interactions with water. Here we investigated water through cryogenic electron microscopy (cryo-EM) on a highly ...The stability and function of biomolecules are directly influenced by their myriad interactions with water. Here we investigated water through cryogenic electron microscopy (cryo-EM) on a highly solvated molecule: the Tetrahymena ribozyme. By using segmentation-guided water and ion modelling (SWIM), an approach combining resolvability and chemical parameters, we automatically modelled and cross-validated water molecules and Mg ions in the ribozyme core, revealing the extensive involvement of water in mediating RNA non-canonical interactions. Unexpectedly, in regions where SWIM does not model ordered water, we observed highly similar densities in both cryo-EM maps. In many of these regions, the cryo-EM densities superimpose with complex water networks predicted by molecular dynamics, supporting their assignment as water and suggesting a biophysical explanation for their elusiveness to conventional atomic coordinate modelling. Our study demonstrates an approach to unveil both rigid and flexible waters that surround biomolecules through cryo-EM map densities, statistical and chemical metrics, and molecular dynamics simulations.
History
DepositionJun 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 20, 2024Provider: repository / Type: Initial release
Revision 2.0Apr 9, 2025Group: Advisory / Atomic model ...Advisory / Atomic model / Data collection / Database references / Derived calculations / Structure summary
Category: atom_site / citation ...atom_site / citation / citation_author / em_imaging / em_software / entity / pdbx_contact_author / pdbx_nonpoly_scheme / pdbx_struct_assembly_gen / pdbx_struct_conn_angle / pdbx_validate_close_contact / struct_asym / struct_conn
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_imaging.microscope_model / _entity.pdbx_number_of_molecules / _pdbx_struct_assembly_gen.asym_id_list
Description: Atomic clashes
Details: Revised method for water and magnesium ion placement to for example prevent two magnesium ions being placed near each other.
Provider: author / Type: Coordinate replacement
Revision 2.1Jun 18, 2025Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
N: RNA (387-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)125,70426
Polymers125,0971
Non-polymers60825
Water2,486138
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: RNA chain RNA (387-MER)


Mass: 125096.773 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Tetrahymena thermophila (eukaryote) / References: GenBank: 10832
#2: Chemical...
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 25 / Source method: obtained synthetically / Formula: Mg
#3: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 138 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestN
Has protein modificationN

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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: Apo L-21 ScaI Tetrahymena ribozyme with consensus water and magnesium ions
Type: COMPLEX / Entity ID: #1 / Source: SYNTHETIC
Molecular weightValue: 0.125 MDa / Experimental value: NO
Source (natural)Organism: Tetrahymena (eukaryote)
Buffer solutionpH: 8
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMSodium HEPES1
210 mMMagnesium ChlorideMgCl21
SpecimenConc.: 3.125 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R2/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 278 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal magnification: 105000 X / Nominal defocus max: 2000 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.5 sec. / Electron dose: 57.25 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of real images: 18365
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1EMAN2particle selectionNeuralNet
2RELIONparticle selection3 rounds of 2D classification
3cryoSPARCparticle selectionAb Initio reconstruction multiclass
4EPUimage acquisition
6CTFFIND4CTF correction
11cryoSPARCinitial Euler assignmentAb Initio reconstruction multiclass
12cryoSPARCfinal Euler assignmentNon-uniform Refinement
13cryoSPARCclassification3D variability
14cryoSPARC3D reconstructionNon-uniform Refinement
15PHENIXmodel refinementreal_space_refine
16ISOLDEmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3804753
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 708006 / Algorithm: FOURIER SPACE / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 7EZ0

7ez0


Accession code: 7EZ0 / Source name: PDB / Type: experimental model

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