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- PDB-6kl9: Structure of LbCas12a-crRNA complex bound to AcrVA4 (form A complex) -

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Basic information

Entry
Database: PDB / ID: 6kl9
TitleStructure of LbCas12a-crRNA complex bound to AcrVA4 (form A complex)
Components
  • AcrVA4
  • LbCas12a
  • RNA (42-MER)
KeywordsRNA BINDING PROTEIN/RNA / CRISPR-Cas / anti-CRISPR / RNA BINDING PROTEIN / RNA BINDING PROTEIN-RNA complex
Function / homologyUncharacterized protein
Function and homology information
Biological speciesLachnospiraceae bacterium (bacteria)
Moraxella bovoculi (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsPeng, R. / Li, Z. / Xu, Y. / He, S. / Peng, Q. / Shi, Y. / Gao, G.F.
CitationJournal: Proc. Natl. Acad. Sci. U.S.A. / Year: 2019
Title: Structural insight into multistage inhibition of CRISPR-Cas12a by AcrVA4.
Authors: Ruchao Peng / Zhiteng Li / Ying Xu / Shaoshuai He / Qi Peng / Lian-Ao Wu / Ying Wu / Jianxun Qi / Peiyi Wang / Yi Shi / George F Gao /
Abstract: Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the ...Prokaryotes possess CRISPR-Cas systems to exclude parasitic predators, such as phages and mobile genetic elements (MGEs). These predators, in turn, encode anti-CRISPR (Acr) proteins to evade the CRISPR-Cas immunity. Recently, AcrVA4, an Acr protein inhibiting the CRISPR-Cas12a system, was shown to diminish Cas12a (LbCas12a)-mediated genome editing in human cells, but the underlying mechanisms remain elusive. Here we report the cryo-EM structures of AcrVA4 bound to CRISPR RNA (crRNA)-loaded LbCas12a and found AcrVA4 could inhibit LbCas12a at several stages of the CRISPR-Cas working pathway, different from other characterized type I/II Acr inhibitors which target only 1 stage. First, it locks the conformation of the LbCas12a-crRNA complex to prevent target DNA-crRNA hybridization. Second, it interacts with the LbCas12a-crRNA-dsDNA complex to release the bound DNA before cleavage. Third, AcrVA4 binds the postcleavage LbCas12a complex to possibly block enzyme recycling. These findings highlight the multifunctionality of AcrVA4 and provide clues for developing regulatory genome-editing tools.
Validation Report
SummaryFull reportAbout validation report
History
DepositionJul 30, 2019Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release

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Structure visualization

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Assembly

Deposited unit
A: LbCas12a
B: AcrVA4
C: AcrVA4
G: RNA (42-MER)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)212,1576
Polymers212,1094
Non-polymers492
Water1086
1


TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein/peptide LbCas12a


Mass: 143888.359 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Lachnospiraceae bacterium (bacteria) / Production host: Escherichia coli BL21 (bacteria) / Strain (production host): BL21
#2: Protein/peptide 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 BL21 (bacteria) / Strain (production host): BL21 / References: UniProt: A0A0U2APF4
#3: RNA chain RNA (42-MER)


Mass: 13482.020 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: Chemical ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Mg / Magnesium
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 6 / Source method: isolated from a natural source / Formula: H2O / Water
Has ligand of interestN

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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: LbCas12a-crRNA complex bound to AcrVA4 (form A complex)
Type: COMPLEX / Entity ID: 1,2,3 / Source: RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: YES
Source (natural)Organism: Lachnospiraceae bacterium (bacteria)
Source (recombinant)Organism: Escherichia coli (E. coli) / Strain: BL21
Buffer solutionpH: 7.5
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Homemade
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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 / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 60 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k)
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

SoftwareName: PHENIX / Version: 1.11.1_2575: / Classification: refinement
EM software
IDNameVersionCategory
1RELION3particle selection
2SerialEM3.6image acquisition
4Gctf0.5CTF correction
7UCSF Chimera1.11model fitting
8Coot0.8.9model fitting
10RELION3initial Euler assignment
11RELION3final Euler assignment
12RELION3classification
13RELION33D reconstruction
20PHENIX1.11model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1400000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 508000 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Target criteria: Correlation coefficient
Atomic model buildingPDB-ID: 5ID6
Refine LS restraints

Refinement-ID: ELECTRON MICROSCOPY

TypeDev idealNumber
f_bond_d0.00512478
f_angle_d1.00416915
f_dihedral_angle_d12.4757532
f_chiral_restr0.0631845
f_plane_restr0.0072077

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