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- PDB-6vrc: Cryo-EM structure of Cas13(crRNA) -

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Basic information

Entry
Database: PDB / ID: 6vrc
TitleCryo-EM structure of Cas13(crRNA)
Components
  • CRISPR-associated endoribonuclease Cas13a
  • RNA (51-MER)
KeywordsIMMUNE SYSTEM / CRISPR-Cas system / Cas13 / TypeVI
Function / homology: / endonuclease activity / defense response to virus / Hydrolases; Acting on ester bonds / RNA binding / : / RNA / RNA (> 10) / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Biological speciesListeria seeligeri serovar 1/2b
Listeria seeligeri serovar 1/2b str. SLCC3954 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsJia, N. / Meeske, A.J. / Marraffini, L.A. / Patel, D.J.
CitationJournal: Science / Year: 2020
Title: A phage-encoded anti-CRISPR enables complete evasion of type VI-A CRISPR-Cas immunity.
Authors: Alexander J Meeske / Ning Jia / Alice K Cassel / Albina Kozlova / Jingqiu Liao / Martin Wiedmann / Dinshaw J Patel / Luciano A Marraffini /
Abstract: The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. ...The CRISPR RNA (crRNA)-guided nuclease Cas13 recognizes complementary viral transcripts to trigger the degradation of both host and viral RNA during the type VI CRISPR-Cas antiviral response. However, how viruses can counteract this immunity is not known. We describe a listeriaphage (ϕLS46) encoding an anti-CRISPR protein (AcrVIA1) that inactivates the type VI-A CRISPR system of Using genetics, biochemistry, and structural biology, we found that AcrVIA1 interacts with the guide-exposed face of Cas13a, preventing access to the target RNA and the conformational changes required for nuclease activation. Unlike inhibitors of DNA-cleaving Cas nucleases, which cause limited immunosuppression and require multiple infections to bypass CRISPR defenses, a single dose of AcrVIA1 delivered by an individual virion completely dismantles type VI-A CRISPR-mediated immunity.
History
DepositionFeb 7, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 10, 2020Provider: repository / Type: Initial release
Revision 1.1Jul 22, 2020Group: 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
Revision 1.2Mar 6, 2024Group: Data collection / Database references / Category: chem_comp_atom / chem_comp_bond / database_2
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession

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Structure visualization

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Assembly

Deposited unit
A: CRISPR-associated endoribonuclease Cas13a
B: RNA (51-MER)


Theoretical massNumber of molelcules
Total (without water)152,3452
Polymers152,3452
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11880 Å2
ΔGint-82 kcal/mol
Surface area56480 Å2

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Components

#1: Protein CRISPR-associated endoribonuclease Cas13a / CRISPR-associated endoribonuclease C2c2 / EndoRNase / LseC2c2


Mass: 133295.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria seeligeri serovar 1/2b (strain ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954) (bacteria)
Strain: ATCC 35967 / DSM 20751 / CIP 100100 / SLCC 3954 / Gene: cas13, c2c2, lse_1149 / Production host: Listeria seeligeri (bacteria)
References: UniProt: P0DPB8, Hydrolases; Acting on ester bonds
#2: RNA chain RNA (51-MER)


Mass: 19049.383 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Listeria seeligeri serovar 1/2b str. SLCC3954 (bacteria)
Production host: Listeria seeligeri (bacteria) / References: GenBank: 289169617

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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: Cas13-crRNA complex / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.13 MDa / Experimental value: YES
Source (natural)Organism: Listeria seeligeri (bacteria)
Source (recombinant)Organism: Listeria seeligeri (bacteria)
Buffer solutionpH: 7.5 / Details: 20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM DTT
Buffer componentFormula: Tris
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / 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: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD
Image recordingElectron dose: 1.23 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM softwareName: RELION / Version: 2.1 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 190543 / Symmetry type: POINT

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