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- PDB-8ewg: Cryo-EM structure of a riboendonclease -

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Basic information

Entry
Database: PDB / ID: 8ewg
TitleCryo-EM structure of a riboendonclease
Components
  • CRISPR-associated endonuclease Cas9
  • RNA (56-MER)
KeywordsHydrolase/RNA / riboendonuclease / RNA / Hydrolase-RNA complex
Function / homology: / endonuclease activity / defense response to virus / RNA binding / RNA / RNA (> 10) / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Biological speciesThermoclostridium caenicola (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.9 Å
AuthorsLi, Z. / Wang, F.
Funding support1items
OrganizationGrant numberCountry
Not funded
CitationJournal: J Mol Biol / Year: 2023
Title: Structural Basis for the Ribonuclease Activity of a Thermostable CRISPR-Cas13a from Thermoclostridium caenicola.
Authors: Feng Wang / Chendi Zhang / Haijiang Xu / Wanting Zeng / Lixin Ma / Zhuang Li /
Abstract: The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from ...The RNA-targeting type VI CRISPR-Cas effector complexes are widely used in biotechnology applications such as gene knockdown, RNA editing, and molecular diagnostics. Compared with Cas13a from mesophilic organisms, a newly discovered Cas13a from thermophilic bacteria Thermoclostridium caenicola (TccCas13a) shows low sequence similarity, high thermostability, and lacks pre-crRNA processing activity. The thermostability of TccCas13a has been harnessed to make a sensitive and robust tool for nucleic acid detection. Here we present the structures of TccCas13a-crRNA binary complex at 2.8 Å, and TccCas13a at 3.5 Å. Although TccCas13a shares a similarly bilobed architecture with other mesophilic organism-derived Cas13a proteins, TccCas13a displayed distinct structure features. Specifically, it holds a long crRNA 5'-flank, forming extensive polar contacts with Helical-1 and HEPN2 domains. The detailed analysis of the interaction between crRNA 5'-flank and TccCas13a suggested lack of suitable nucleophile to attack the 2'-OH of crRNA 5'-flank may explain why TccCas13a fails to cleave pre-crRNA. The stem-loop segment of crRNA spacer toggles between double-stranded and single-stranded conformational states, suggesting a potential safeguard mechanism for target recognition. Superimposition of the structures of TccCas13a and TccCas13a-crRNA revealed several conformational changes required for crRNA loading, including dramatic movement of Helical-2 domain. Collectively, these structural insights expand our understanding into type VI CRISPR-Cas effectors, and would facilitate the development of TccCas13a-based applications.
History
DepositionOct 23, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Aug 30, 2023Provider: repository / Type: Initial release
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Revision 1.0Aug 30, 2023Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Aug 30, 2023Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
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Revision 1.1Mar 13, 2024Group: Database references / Category: citation / citation_author
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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas9
B: RNA (56-MER)


Theoretical massNumber of molelcules
Total (without water)162,8842
Polymers162,8842
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein CRISPR-associated endonuclease Cas9


Mass: 143582.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermoclostridium caenicola (bacteria) / Gene: SAMN05444373_102315 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A1M6GDI0
#2: RNA chain RNA (56-MER)


Mass: 19301.541 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Thermoclostridium caenicola (bacteria)
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: Binary complex of a riboendonuclease with its cognate RNA
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Thermoclostridium caenicola (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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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 defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 54 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.20.1_4487: / Classification: refinement
EM softwareName: PHENIX / Category: model refinement
CTF correctionType: NONE
3D reconstructionResolution: 2.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 561050 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0049967
ELECTRON MICROSCOPYf_angle_d0.85213669
ELECTRON MICROSCOPYf_dihedral_angle_d16.5441813
ELECTRON MICROSCOPYf_chiral_restr0.0491514
ELECTRON MICROSCOPYf_plane_restr0.0061559

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