|Entry||Database: PDB / ID: 6nm9|
|Title||CryoEM structure of the LbCas12a-crRNA-AcrVA4 dimer|
|Keywords||UNKNOWN FUNCTION/RNA / UNKNOWN FUNCTION-RNA complex|
|Function / homology||CRISPR-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 species||Moraxella bovoculi (bacteria)|
Lachnospiraceae bacterium ND2006 (bacteria)
|Method||ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.38 Å|
|Authors||Chang, L. / Li, Z. / Zhang, H.|
|Citation||Journal: 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.
SummaryFull reportAbout validation report
|Structure viewer||Molecule: |
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G: RNA (25-MER)
E: RNA (25-MER)
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
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|
Mass: 12879.634 Da / Num. of mol.: 2 / Source method: obtained synthetically
Source: (synth.) Lachnospiraceae bacterium ND2006 (bacteria)
|Experiment||Method: ELECTRON MICROSCOPY|
|EM experiment||Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction|
|Component||Name: protein complex 5 / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES|
|Source (natural)||Organism: Lachnospiraceae bacterium ND2006 (bacteria)|
|Buffer solution||pH: 7.5|
|Specimen||Conc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES|
|Specimen support||Details: unspecified|
|Vitrification||Cryogen name: ETHANE|
-Electron microscopy imaging
Model: Titan Krios / Image courtesy: FEI Company
|Microscopy||Model: FEI TITAN KRIOS|
|Electron gun||Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: SPOT SCAN|
|Electron lens||Mode: BRIGHT FIELDBright-field microscopy|
|Image recording||Electron dose: 35 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)|
|Software||Name: PHENIX / Version: 1.15.2_3472: / Classification: refinement|
|EM software||Name: cisTEM / Version: 1.0.0 / Category: 3D reconstruction|
|CTF correction||Type: PHASE FLIPPING ONLY|
|Symmetry||Point symmetry: C2 (2 fold cyclic)|
|3D reconstruction||Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 47609 / Symmetry type: POINT|
|Refine LS restraints|
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