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- EMDB-9398: CryoEM structure of the LbCas12a-crRNA-AcrVA4 dimer -

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Basic information

Entry
Database: EMDB / ID: EMD-9398
TitleCryoEM structure of the LbCas12a-crRNA-AcrVA4 dimer
Map data
Sampleprotein complex 5:
AcrVA4 / Cpf1 / nucleic-acidNucleic acid / ligand
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 speciesLachnospiraceae bacterium ND2006 (bacteria) / Moraxella bovoculi (bacteria)
Methodsingle 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 ReportPDB-ID: 6nm9

SummaryFull reportAbout validation report
DateDeposition: Jan 10, 2019 / Header (metadata) release: Jan 23, 2019 / Map release: Jun 12, 2019 / Update: Jun 12, 2019

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.0177
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.0177
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6nm9
  • Surface level: 0.0177
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_9398.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.07 Å/pix.
x 240 pix.
= 255.84 Å
1.07 Å/pix.
x 240 pix.
= 255.84 Å
1.07 Å/pix.
x 240 pix.
= 255.84 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.066 Å
Density
Contour LevelBy AUTHOR: 0.0177 / Movie #1: 0.0177
Minimum - Maximum-0.06602688 - 0.11155322
Average (Standard dev.)0.00028914717 (±0.0031515923)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 255.84 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.0661.0661.066
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z255.840255.840255.840
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ192192192
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0660.1120.000

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Supplemental data

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Sample components

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Entire protein complex 5

EntireName: protein complex 5 / Number of components: 5

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Component #1: protein, protein complex 5

ProteinName: protein complex 5 / Recombinant expression: No
SourceSpecies: Lachnospiraceae bacterium ND2006 (bacteria)

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Component #2: 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 (E. coli)

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Component #3: protein, Cpf1

ProteinName: Cpf1 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 143.750219 kDa
SourceSpecies: Lachnospiraceae bacterium ND2006 (bacteria)
Source (engineered)Expression System: Escherichia coli (E. coli)

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Component #4: nucleic-acid, RNA (25-MER)

nucleic acidName: RNA (25-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AAUUUCUACU AAGUGUAGAU GGAAAUUAGG UGCGCUUGGC
MassTheoretical: 12.879634 kDa
SourceSpecies: Lachnospiraceae bacterium ND2006 (bacteria)

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Component #5: ligand, MAGNESIUM ION

LigandName: MAGNESIUM ION / Number of Copies: 4 / Recombinant expression: No
MassTheoretical: 2.430505 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
Support filmunspecified
VitrificationCryogen name: ETHANE

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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: 35 e/Å2 / Illumination mode: SPOT SCAN
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 47609
3D reconstructionSoftware: cisTEM / Resolution: 3.38 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Output model

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