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- PDB-7dmq: Cryo-EM structure of LshCas13a-crRNA-anti-tag RNA complex -

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Basic information

Entry
Database: PDB / ID: 7dmq
TitleCryo-EM structure of LshCas13a-crRNA-anti-tag RNA complex
Components
  • Anti-tag target RNA
  • CRISPR RNA
  • CRISPR/Cas system Cas13a
KeywordsIMMUNE SYSTEM/RNA / Type VI-A CRISPR-Cas system / Cas13a / anti-tag RNA / inhibition / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA complex
Function / homologydefense response to virus / endonuclease activity / Hydrolases; Acting on ester bonds / RNA binding / RNA / RNA (> 10) / CRISPR/Cas system Cas13a / CRISPR-associated endoribonuclease Cas13a
Function and homology information
Biological speciesLeptotrichia shahii (bacteria)
Escherichia coli (E. coli)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsWang, B. / Zhang, T. / Ding, J. / Patel, D.J. / Yang, H.
Funding support China, 1items
OrganizationGrant numberCountry
National Natural Science Foundation of China (NSFC)31971135 China
CitationJournal: Mol Cell / Year: 2021
Title: Structural basis for self-cleavage prevention by tag:anti-tag pairing complementarity in type VI Cas13 CRISPR systems.
Authors: Beibei Wang / Tianlong Zhang / Jun Yin / You Yu / Wenhao Xu / Jianping Ding / Dinshaw J Patel / Hui Yang /
Abstract: Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, ...Bacteria and archaea apply CRISPR-Cas surveillance complexes to defend against foreign invaders. These invading genetic elements are captured and integrated into the CRISPR array as spacer elements, guiding sequence-specific DNA/RNA targeting and cleavage. Recently, in vivo studies have shown that target RNAs with extended complementarity with repeat sequences flanking the target element (tag:anti-tag pairing) can dramatically reduce RNA cleavage by the type VI-A Cas13a system. Here, we report the cryo-EM structure of Leptotrichia shahii LshCas13a in complex with target RNA harboring tag:anti-tag pairing complementarity, with the observed conformational changes providing a molecular explanation for inactivation of the composite HEPN domain cleavage activity. These structural insights, together with in vitro biochemical and in vivo cell-based assays on key mutants, define the molecular principles underlying Cas13a's capacity to target and discriminate between self and non-self RNA targets. Our studies illuminate approaches to regulate Cas13a's cleavage activity, thereby influencing Cas13a-mediated biotechnological applications.
History
DepositionDec 5, 2020Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Feb 10, 2021Provider: repository / Type: Initial release
Revision 1.1Mar 24, 2021Group: Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first
Revision 1.2Mar 27, 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/Cas system Cas13a
B: CRISPR RNA
C: Anti-tag target RNA


Theoretical massNumber of molelcules
Total (without water)197,1473
Polymers197,1473
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area11140 Å2
ΔGint-79 kcal/mol
Surface area60210 Å2

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Components

#1: Protein CRISPR/Cas system Cas13a


Mass: 166697.641 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Leptotrichia shahii (bacteria) / Gene: cas13a, JCM16776_0110 / Plasmid: pRSFDuet-SUMO / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: A0A510JNC0, UniProt: P0DOC6*PLUS
#2: RNA chain CRISPR RNA /


Mass: 18565.096 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Leptotrichia shahii (bacteria)
#3: RNA chain Anti-tag target RNA


Mass: 11884.038 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Escherichia coli (E. coli)

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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
1LshCas13a-crRNA-anti-tag RNA complexCOMPLEXall0MULTIPLE SOURCES
2LshCas13aCOMPLEX#11MULTIPLE SOURCES
3Lsh crRNACOMPLEX#21MULTIPLE SOURCES
4Anti-tag target RNACOMPLEX#31MULTIPLE SOURCES
Molecular weightExperimental value: NO
Source (natural)Organism: Leptotrichia shahii (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
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 FIELDBright-field microscopy / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingAverage exposure time: 8 sec. / Electron dose: 64.7 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 212861 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL

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