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- PDB-8fti: Cryo-EM structure of the Cas13bt3-crRNA-target RNA ternary comple... -

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Basic information

Entry
Database: PDB / ID: 8fti
TitleCryo-EM structure of the Cas13bt3-crRNA-target RNA ternary complex in activated state
Components
  • Integrase
  • crRNA-linker-target hairpin RNA
KeywordsRNA BINDING PROTEIN/RNA / Cas13bt3-crRNA-target RNA ternary complex / activated state / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyRNA / RNA (> 10) / RNA (> 100) / Uncharacterized protein
Function and homology information
Biological speciesPlanctomycetota bacterium (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.5 Å
AuthorsGao, Y. / Deng, X.
Funding support United States, 1items
OrganizationGrant numberCountry
Cancer Prevention and Research Institute of Texas (CPRIT)RR190046 United States
CitationJournal: Nat Commun / Year: 2023
Title: Structural basis for the activation of a compact CRISPR-Cas13 nuclease.
Authors: Xiangyu Deng / Emmanuel Osikpa / Jie Yang / Seye J Oladeji / Jamie Smith / Xue Gao / Yang Gao /
Abstract: The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 ...The CRISPR-Cas13 ribonucleases have been widely applied for RNA knockdown and transcriptional modulation owing to their high programmability and specificity. However, the large size of Cas13 effectors and their non-specific RNA cleavage upon target activation limit the adeno-associated virus based delivery of Cas13 systems for therapeutic applications. Herein, we report detailed biochemical and structural characterizations of a compact Cas13 (Cas13bt3) suitable for adeno-associated virus delivery. Distinct from many other Cas13 systems, Cas13bt3 cleaves the target and other nonspecific RNA at internal "UC" sites and is activated in a target length-dependent manner. The cryo-electron microscope structure of Cas13bt3 in a fully active state illustrates the structural basis of Cas13bt3 activation. Guided by the structure, we obtain engineered Cas13bt3 variants with minimal off-target cleavage yet maintained target cleavage activities. In conclusion, our biochemical and structural data illustrate a distinct mechanism for Cas13bt3 activation and guide the engineering of Cas13bt3 applications.
History
DepositionJan 12, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Sep 27, 2023Provider: repository / Type: Initial release
Revision 1.1May 1, 2024Group: Database references / Category: citation
Item: _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Integrase
B: crRNA-linker-target hairpin RNA


Theoretical massNumber of molelcules
Total (without water)123,3902
Polymers123,3902
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 Integrase


Mass: 90384.680 Da / Num. of mol.: 1 / Mutation: R84A, H89A, R739A, H744A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Planctomycetota bacterium (bacteria) / Gene: DRP66_05270 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A660UUL5
#2: RNA chain crRNA-linker-target hairpin RNA / hpRNA


Mass: 33005.457 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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

Component
IDNameTypeEntity IDParent-IDSource
1Ternary complex of Cas13bt3 in complex with crRNA and 30 nt target RNACOMPLEXall0MULTIPLE SOURCES
2IntegraseCOMPLEX#11RECOMBINANT
3crRNA-linker-target hairpin RNACOMPLEX#21SYNTHETIC
Molecular weightValue: 0.1237 MDa / Experimental value: NO
Source (natural)Organism: Planctomycetota bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenConc.: 0.8 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 3000 nm / Nominal defocus min: 600 nm / C2 aperture diameter: 100 µm
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 37228 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0028492
ELECTRON MICROSCOPYf_angle_d0.46711885
ELECTRON MICROSCOPYf_dihedral_angle_d13.7113497
ELECTRON MICROSCOPYf_chiral_restr0.0321354
ELECTRON MICROSCOPYf_plane_restr0.0031162

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