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- PDB-6nma: CryoEM structure of the LbCas12a-crRNA-AcrVA4 complex -

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Basic information

Entry
Database: PDB / ID: 6nma
TitleCryoEM structure of the LbCas12a-crRNA-AcrVA4 complex
Components
  • AcrVA1
  • Cpf1
  • RNA
KeywordsUNKNOWN FUNCTION/RNA / UNKNOWN FUNCTION-RNA complex
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
DateDeposition: Jan 10, 2019 / Release: Jun 12, 2019
RevisionDateData content typeProviderType
1.0Jun 12, 2019Structure modelrepositoryInitial release

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Assembly

Deposited unit
A: AcrVA1
B: Cpf1
G: RNA
C: AcrVA1
hetero molecules


Theoretical massNumber of molelcules
Total (without water)211,4176
Polymers211,3684
Non-polymers492
Water0
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide AcrVA1


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/peptide Cpf1 /


Mass: 143750.219 Da / Num. of mol.: 1
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 /


Mass: 12879.634 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Magnesium

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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: protein complex 4 / Type: COMPLEX / Entity ID: 1,2,3 / 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
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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 0.85 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)

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Processing

SoftwareName: PHENIX / Version: 1.15.2_3472: / Classification: refinement
EM softwareName: cisTEM / Version: 1.0.0 / Category: 3D reconstruction
CTF correctionType: PHASE FLIPPING ONLY
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 297026 / Symmetry type: POINT
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00912739
f_angle_d0.72217268
f_dihedral_angle_d15.3227684
f_chiral_restr0.0481869
f_plane_restr0.0042118

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