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- PDB-6iv6: Cryo-EM structure of AcrVA5-acetylated MbCas12a in complex with crRNA -

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Basic information

Entry
Database: PDB / ID: 6iv6
TitleCryo-EM structure of AcrVA5-acetylated MbCas12a in complex with crRNA
Components
  • RNA (59-MER)
  • nuclease
KeywordsIMMUNE SYSTEM/RNA / enzyme / IMMUNE SYSTEM / IMMUNE SYSTEM-RNA complex
Function / homology
Function and homology information


CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
RNA / RNA (> 10) / Type V CRISPR-associated protein Cpf1
Similarity search - Component
Biological speciesMoraxella bovoculi (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsDong, L. / Li, N. / Guan, X. / Zhu, Y. / Gao, N. / Huang, Z.
Funding support China, 2items
OrganizationGrant numberCountry
National Natural Science Foundation of China31825008 China
National Natural Science Foundation of China31422014 China
CitationJournal: Nat Struct Mol Biol / Year: 2019
Title: An anti-CRISPR protein disables type V Cas12a by acetylation.
Authors: Liyong Dong / Xiaoyu Guan / Ningning Li / Fan Zhang / Yuwei Zhu / Kuan Ren / Ling Yu / Fengxia Zhou / Zhifu Han / Ning Gao / Zhiwei Huang /
Abstract: Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained ...Phages use anti-CRISPR proteins to deactivate the CRISPR-Cas system. The mechanisms for the inhibition of type I and type II systems by anti-CRISPRs have been elucidated. However, it has remained unknown how the type V CRISPR-Cas12a (Cpf1) system is inhibited by anti-CRISPRs. Here we identify the anti-CRISPR protein AcrVA5 and report the mechanisms by which it inhibits CRISPR-Cas12a. Our structural and biochemical data show that AcrVA5 functions as an acetyltransferase to modify Moraxella bovoculi (Mb) Cas12a at Lys635, a residue that is required for recognition of the protospacer-adjacent motif. The AcrVA5-mediated modification of MbCas12a results in complete loss of double-stranded DNA (dsDNA)-cleavage activity. In contrast, the Lys635Arg mutation renders MbCas12a completely insensitive to inhibition by AcrVA5. A cryo-EM structure of the AcrVA5-acetylated MbCas12a reveals that Lys635 acetylation provides sufficient steric hindrance to prevent dsDNA substrates from binding to the Cas protein. Our study reveals an unprecedented mechanism of CRISPR-Cas inhibition and suggests an evolutionary arms race between phages and bacteria.
History
DepositionDec 2, 2018Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Apr 10, 2019Provider: repository / Type: Initial release
Revision 1.1Apr 17, 2019Group: Data collection / Database references / Category: citation
Item: _citation.journal_abbrev / _citation.journal_id_CSD ..._citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 6, 2019Group: Data collection / Other / Category: atom_sites / cell
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] ..._atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3] / _cell.Z_PDB

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Structure visualization

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Assembly

Deposited unit
A: nuclease
G: RNA (59-MER)


Theoretical massNumber of molelcules
Total (without water)163,9272
Polymers163,9272
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area5820 Å2
ΔGint-50 kcal/mol
Surface area63100 Å2

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Components

#1: Protein nuclease /


Mass: 145253.391 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Moraxella bovoculi (bacteria) / Gene: AAX07_00205 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0U2B2X7
#2: RNA chain RNA (59-MER)


Mass: 18673.896 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: cpf1-crRNA / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Moraxella bovoculi (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 8
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: NITROGEN

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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
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 81 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 QUANTUM (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.14_3260: / Classification: refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 93000 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00410458
ELECTRON MICROSCOPYf_angle_d0.90714239
ELECTRON MICROSCOPYf_dihedral_angle_d10.9486202
ELECTRON MICROSCOPYf_chiral_restr0.0531569
ELECTRON MICROSCOPYf_plane_restr0.0061753

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