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- PDB-6nm9: CryoEM structure of the LbCas12a-crRNA-AcrVA4 dimer -

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Database: PDB / ID: 6nm9
TitleCryoEM structure of the LbCas12a-crRNA-AcrVA4 dimer
  • AcrVA4
  • Cpf1
  • RNA (25-MER)
Function / homologyCRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain / Uncharacterized protein / Cpf1
Function and homology information
Biological speciesMoraxella bovoculi (bacteria)
Lachnospiraceae bacterium ND2006 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å
AuthorsChang, L. / Li, Z. / Zhang, H.
CitationJournal: Cell Host Microbe / Year: 2019
Title: Structural Basis for the Inhibition of CRISPR-Cas12a by Anti-CRISPR Proteins.
Authors: Heng Zhang / Zhuang Li / Courtney M Daczkowski / Clinton Gabel / Andrew D Mesecar / Leifu Chang /
Abstract: CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are ...CRISPR-Cas12a (Cpf1), a type V CRISPR-associated nuclease, provides bacterial immunity against bacteriophages and plasmids but also serves as a tool for genome editing. Foreign nucleic acids are integrated into the CRISPR locus, prompting transcription of CRISPR RNAs (crRNAs) that guide Cas12a cleavage of foreign complementary DNA. However, mobile genetic elements counteract Cas12a with inhibitors, notably type V-A anti-CRISPRs (AcrVAs). We present cryoelectron microscopy structures of Cas12a-crRNA bound to AcrVA1 and AcrVA4 at 3.5 and 3.3 Å resolutions, respectively. AcrVA1 is sandwiched between the recognition (REC) and nuclease (NUC) lobes of Cas12a and inserts into the binding pocket for the protospacer-adjacent motif (PAM), a short DNA sequence guiding Cas12a targeting. AcrVA1 cleaves crRNA in a Cas12a-dependent manner, inactivating Cas12a-crRNA complexes. The AcrVA4 dimer is anchored around the crRNA pseudoknot of Cas12a-crRNA, preventing required conformational changes for crRNA-DNA heteroduplex formation. These results uncover molecular mechanisms for CRISPR-Cas12a inhibition, providing insights into bacteria-phage dynamics.
Validation Report
SummaryFull reportAbout validation report
DepositionJan 10, 2019Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jun 12, 2019Provider: repository / Type: Initial release
Revision 1.1Jun 26, 2019Group: Data collection / Database references / Category: citation / Item: _citation.journal_volume / _citation.page_first

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Deposited unit
A: AcrVA4
B: Cpf1
G: RNA (25-MER)
C: AcrVA4
D: Cpf1
E: RNA (25-MER)
hetero molecules

Theoretical massNumber of molelcules
Total (without water)368,09510

TypeNameSymmetry operationNumber
identity operation1_5551


#1: Protein AcrVA4

Mass: 27369.162 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Moraxella bovoculi (bacteria) / Gene: AAX07_09545 / Production host: Escherichia coli (E. coli) / References: UniProt: A0A0U2APF4
#2: Protein Cpf1 /

Mass: 143750.219 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium ND2006 (bacteria)
Production host: Escherichia coli (E. coli) / References: UniProt: A0A182DWE3
#3: RNA chain RNA (25-MER)

Mass: 12879.634 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)
#4: Chemical
ChemComp-MG / MAGNESIUM ION / Magnesium

Mass: 24.305 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Mg

Experimental details


EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

Sample preparation

ComponentName: protein complex 5 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Source (natural)Organism: Lachnospiraceae bacterium ND2006 (bacteria)
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: unspecified
VitrificationCryogen name: ETHANE

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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 35 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)


SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM softwareName: cisTEM / Version: 1.0.0 / Category: 3D reconstruction
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47609 / Symmetry type: POINT
Refine LS restraints
Refinement-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00723488
ELECTRON MICROSCOPYf_angle_d0.71631818
ELECTRON MICROSCOPYf_dihedral_angle_d7.58114140
ELECTRON MICROSCOPYf_chiral_restr0.0483436
ELECTRON MICROSCOPYf_plane_restr0.0043876

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