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- EMDB-0062: Transient state structure of CRISPR-Cpf1 (Cas12a) I2 conformation -

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Basic information

Entry
Database: EMDB / ID: 0062
TitleTransient state structure of CRISPR-Cpf1 (Cas12a) I2 conformation
Map dataTransition state CRISPR-Cpf1 (Cas12a)I2 conformation
SampleTRansition state complex I2 conformation
  • (CRISPR-associated endonuclease ...) x 2
  • Nucleic acidsNucleic acid
  • (nucleic-acidNucleic acid) x 3
Function / homologyCas12a, REC1 domain / CRISPR-associated endonuclease Cas12a / Alpha helical recognition lobe domain / Bacillus subtilis ribonuclease / Bacillus subtilis ribonuclease activity / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / RNA binding / DNA binding / CRISPR-associated endonuclease Cas12a
Function and homology information
SourceFrancisella tularensis subsp. novicida U112 (bacteria) / Francisella tularensis subsp. novicida (strain U112) (bacteria)
Methodsingle particle reconstruction / cryo EM / 4.24 Å resolution
AuthorsMontoya G / Mesa P
CitationJournal: Cell / Year: 2018
Title: Conformational Activation Promotes CRISPR-Cas12a Catalysis and Resetting of the Endonuclease Activity.
Authors: Stefano Stella / Pablo Mesa / Johannes Thomsen / Bijoya Paul / Pablo Alcón / Simon B Jensen / Bhargav Saligram / Matias E Moses / Nikos S Hatzakis / Guillermo Montoya
Abstract: Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures ...Cas12a, also known as Cpf1, is a type V-A CRISPR-Cas RNA-guided endonuclease that is used for genome editing based on its ability to generate specific dsDNA breaks. Here, we show cryo-EM structures of intermediates of the cleavage reaction, thus visualizing three protein regions that sense the crRNA-DNA hybrid assembly triggering the catalytic activation of Cas12a. Single-molecule FRET provides the thermodynamics and kinetics of the conformational activation leading to phosphodiester bond hydrolysis. These findings illustrate why Cas12a cuts its target DNA and unleashes unspecific cleavage activity, degrading ssDNA molecules after activation. In addition, we show that other crRNAs are able to displace the R-loop inside the protein after target DNA cleavage, terminating indiscriminate ssDNA degradation. We propose a model whereby the conformational activation of the enzyme results in indiscriminate ssDNA cleavage. The displacement of the R-loop by a new crRNA molecule will reset Cas12a specificity, targeting new DNAs.
Validation ReportPDB-ID: 6gtd

SummaryFull reportAbout validation report
DateDeposition: Jun 18, 2018 / Header (metadata) release: Aug 22, 2018 / Map release: Dec 19, 2018 / Last update: Dec 26, 2018

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.025
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: : PDB-6gtd
  • Surface level: 0.025
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

Fileemd_0062.map.gz (map file in CCP4 format, 55297 KB)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
240 pix
0.83 Å/pix.
= 199.68 Å
240 pix
0.83 Å/pix.
= 199.68 Å
240 pix
0.83 Å/pix.
= 199.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.832 Å
Density
Contour Level:0.03 (by author), 0.025 (movie #1):
Minimum - Maximum-0.09460564 - 0.18113866
Average (Standard dev.)0.00014667785 (0.0054226513)
Details

EMDB XML:

Space Group Number1
Map Geometry
Axis orderXYZ
Dimensions240240240
Origin0.00.00.0
Limit239.0239.0239.0
Spacing240240240
CellA=B=C: 199.68001 Å
α=β=γ: 90.0 deg.

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8320.8320.832
M x/y/z240240240
origin x/y/z0.0000.0000.000
length x/y/z199.680199.680199.680
α/β/γ90.00090.00090.000
start NX/NY/NZ
NX/NY/NZ
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.0950.1810.000

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Supplemental data

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Sample components

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Entire TRansition state complex I2 conformation

EntireName: TRansition state complex I2 conformation / Number of components: 7

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Component #1: protein, TRansition state complex I2 conformation

ProteinName: TRansition state complex I2 conformation / Recombinant expression: No
MassExperimental: 180 kDa

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Component #2: protein, CRISPR-associated endonuclease Cas12a

ProteinName: CRISPR-associated endonuclease Cas12a / Recombinant expression: No
SourceSpecies: Francisella tularensis subsp. novicida U112 (bacteria)
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #3: protein, Nucleic acids

ProteinName: Nucleic acidsNucleic acid / Recombinant expression: No
SourceSpecies: Francisella tularensis subsp. novicida U112 (bacteria)
Source (engineered)Expression System: synthetic construct (others)

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Component #4: protein, CRISPR-associated endonuclease Cas12a

ProteinName: CRISPR-associated endonuclease Cas12a / Number of Copies: 1 / Recombinant expression: No
MassTheoretical: 155.639828 kDa
SourceSpecies: Francisella tularensis subsp. novicida (strain U112) (bacteria)
Strain: U112
Source (engineered)Expression System: Escherichia coli BL21 (bacteria)

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Component #5: nucleic-acid, RNA (28-MER)

Nucleic-acidName: RNA (28-MER) / Class: RNA / Structure: OTHER / Synthetic: No
Sequence:
AAUUUCUACU GUUGUAGAUG AGAAGUCAUU UAAUAAGGCC ACU
MassTheoretical: 13.749146 kDa
SourceSpecies: Francisella tularensis subsp. novicida U112 (bacteria)

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Component #6: nucleic-acid, DNA (5'-D(P*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*AP*AP*CP*...

Nucleic-acidName: DNA (5'-D(P*TP*GP*AP*CP*TP*TP*CP*TP*CP*TP*AP*AP*CP*AP*AP*GP*CP*TP*CP*G)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DA)(DT)(DT)(DG)(DC)(DT)(DT)(DG)(DC)(DT) (DC)(DG)(DA)(DT)(DG)(DC)(DA)(DT)(DG)(DC) (DA)(DG)(DT)(DG)(DG)(DC)(DC)(DT)(DT)(DA) (DT)(DT)(DA)(DA)(DA)(DT)(DG)(DA)(DC)(DT) (DT)(DC)(DT)(DC)(DT)(DA)(DA)(DC)(DG)(DA) (DG)(DC)(DT)(DC)(DG)
MassTheoretical: 16.898826 kDa
SourceSpecies: Francisella tularensis subsp. novicida U112 (bacteria)

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Component #7: nucleic-acid, DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*...

Nucleic-acidName: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
Class: DNA / Structure: OTHER / Synthetic: No
Sequence:
(DC)(DG)(DA)(DG)(DC)(DT)(DC)(DG)(DT)(DT) (DA)(DG)(DA)(DG)(DA)(DA)(DG)(DT)(DC)(DA) (DT)(DT)(DT)(DA)(DA)(DT)(DA)(DA)(DG)(DG) (DC)(DC)(DA)(DC)(DT)(DG)(DC)(DA)(DT)(DG) (DC)(DA)(DT)(DC)(DG)(DA)(DG)(DC)(DA)(DA) (DG)(DC)(DA)(DA)(DT)
MassTheoretical: 16.992936 kDa
SourceSpecies: Francisella tularensis subsp. novicida U112 (bacteria)

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Experimental details

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Sample preparation

SpecimenSpecimen state: particle / Method: cryo EM
Sample solutionpH: 7
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen 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: 4 e/Å2 / Illumination mode: SPOT SCAN
LensImaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: FEI FALCON III (4k x 4k)

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 124000
3D reconstructionResolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF
FSC plot
(resolution estimation)

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Atomic model buiding

Output model

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