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- PDB-8d49: Structure of Cas12a2 binary complex -

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Basic information

Entry
Database: PDB / ID: 8d49
TitleStructure of Cas12a2 binary complex
Components
  • OrfB_Zn_ribbon domain-containing protein
  • RNA (26-MER)
KeywordsDNA Binding Protein/RNA / Cas12a2 / CRISPR / Nuclease / DNA Binding Protein-RNA complex
Function / homologyTransposase IS605, OrfB, C-terminal / Putative transposase DNA-binding domain / DNA binding / RNA / RNA (> 10) / Cas12f1-like TNB domain-containing protein
Function and homology information
Biological speciesSulfuricurvum sp. PC08-66 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsBravo, J.P.K. / Taylor, D.W.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
Welch FoundationF-1938 United States
Cancer Prevention and Research Institute of Texas (CPRIT)RR160088 United States
CitationJournal: Nature / Year: 2023
Title: RNA targeting unleashes indiscriminate nuclease activity of CRISPR-Cas12a2.
Authors: Jack P K Bravo / Thomson Hallmark / Bronson Naegle / Chase L Beisel / Ryan N Jackson / David W Taylor /
Abstract: Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a ...Cas12a2 is a CRISPR-associated nuclease that performs RNA-guided, sequence-nonspecific degradation of single-stranded RNA, single-stranded DNA and double-stranded DNA following recognition of a complementary RNA target, culminating in abortive infection. Here we report structures of Cas12a2 in binary, ternary and quaternary complexes to reveal a complete activation pathway. Our structures reveal that Cas12a2 is autoinhibited until binding a cognate RNA target, which exposes the RuvC active site within a large, positively charged cleft. Double-stranded DNA substrates are captured through duplex distortion and local melting, stabilized by pairs of 'aromatic clamp' residues that are crucial for double-stranded DNA degradation and in vivo immune system function. Our work provides a structural basis for this mechanism of abortive infection to achieve population-level immunity, which can be leveraged to create rational mutants that degrade a spectrum of collateral substrates.
History
DepositionJun 1, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 18, 2023Provider: repository / Type: Initial release
Revision 1.1Feb 1, 2023Group: 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 1.2Jun 12, 2024Group: Data collection / Category: chem_comp_atom / chem_comp_bond

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: OrfB_Zn_ribbon domain-containing protein
B: RNA (26-MER)


Theoretical massNumber of molelcules
Total (without water)151,2782
Polymers151,2782
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein OrfB_Zn_ribbon domain-containing protein


Mass: 143172.797 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Sulfuricurvum sp. PC08-66 (bacteria) / Gene: KU37_03970 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0C2W1L1
#2: RNA chain RNA (26-MER)


Mass: 8105.683 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

ComponentName: Cas12a2 nuclease / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Sulfuricurvum sp. PC08-66 (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.5
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 40 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97470 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0099486
ELECTRON MICROSCOPYf_angle_d0.70812878
ELECTRON MICROSCOPYf_dihedral_angle_d12.1111462
ELECTRON MICROSCOPYf_chiral_restr0.0471428
ELECTRON MICROSCOPYf_plane_restr0.0051568

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