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- EMDB-0704: Structure of LbCas12a-crRNA complex bound to AcrVA4 (form A complex) -

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Basic information

Entry
Database: EMDB / ID: EMD-0704
TitleStructure of LbCas12a-crRNA complex bound to AcrVA4 (form A complex)
Map data
SampleLbCas12a-crRNA complex bound to AcrVA4 (form A complex):
LbCas12a / AcrVA4 / nucleic-acidNucleic acid / (ligand) x 2
Function / homologyUncharacterized protein
Function and homology information
Biological speciesLachnospiraceae bacterium (bacteria) / Moraxella bovoculi (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.25 Å
AuthorsPeng R / Li Z / Xu Y / He S / Peng Q / Shi Y / Gao GF
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 ReportPDB-ID: 6kl9

SummaryFull reportAbout validation report
History
DepositionJul 30, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 11, 2019-
UpdateOct 2, 2019-
Current statusOct 2, 2019Processing site: PDBj / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: : PDB-6kl9
  • Surface level: 0.016
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_0704.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.08 Å/pix.
x 256 pix.
= 276.48 Å
1.08 Å/pix.
x 256 pix.
= 276.48 Å
1.08 Å/pix.
x 256 pix.
= 276.48 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.08 Å
Density
Contour LevelBy AUTHOR: 0.016 / Movie #1: 0.016
Minimum - Maximum-0.15286848 - 0.21663801
Average (Standard dev.)0.00010925988 (±0.002991805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 276.48 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.081.081.08
M x/y/z256256256
origin x/y/z0.0000.0000.000
length x/y/z276.480276.480276.480
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS256256256
D min/max/mean-0.1530.2170.000

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Supplemental data

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Sample components

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Entire LbCas12a-crRNA complex bound to AcrVA4 (form A complex)

EntireName: LbCas12a-crRNA complex bound to AcrVA4 (form A complex)
Number of components: 6

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Component #1: protein, LbCas12a-crRNA complex bound to AcrVA4 (form A complex)

ProteinName: LbCas12a-crRNA complex bound to AcrVA4 (form A complex)
Recombinant expression: No
MassExperimental: 170 kDa
SourceSpecies: Lachnospiraceae bacterium (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli) / Strain: BL21

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Component #2: protein, LbCas12a

ProteinName: LbCas12a / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 143.888359 kDa
SourceSpecies: Lachnospiraceae bacterium (bacteria)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #3: protein, AcrVA4

ProteinName: AcrVA4 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 27.369162 kDa
SourceSpecies: Moraxella bovoculi (bacteria)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #4: nucleic-acid, RNA (42-MER)

nucleic acidName: RNA (42-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AAUUUCUACU AAGUGUAGAU CGGUCUCGCA AAGAAUGGAU AU
MassTheoretical: 13.48202 kDa
SourceSpecies: synthetic construct (others)

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 2.430505 MDa

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Component #6: ligand, water

LigandName: water / Number of Copies: 6 / Recombinant expression: No
MassTheoretical: 1.801505 MDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1 mg/mL / pH: 7.5
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 60 e/Å2 / Illumination mode: FLOOD BEAM
LensImaging mode: BRIGHT FIELD / Energy filter: GIF Bioquantum
Specimen HolderModel: FEI TITAN KRIOS AUTOGRID HOLDER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 508000
3D reconstructionAlgorithm: FOURIER SPACE / Software: RELION / Resolution: 3.25 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot (resolution estimation)

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Atomic model buiding

Modeling #1Refinement protocol: flexible / Target criteria: Correlation coefficient / Refinement space: REAL
Input PDB model: 5ID6
Output model

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