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- PDB-6gtd: Transient state structure of CRISPR-Cpf1 (Cas12a) I2 conformation -

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Basic information

Entry
Database: PDB / ID: 6gtd
TitleTransient state structure of CRISPR-Cpf1 (Cas12a) I2 conformation
Components
  • CRISPR-associated endonuclease Cas12a
  • DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
  • 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')
  • RNA (28-MER)
KeywordsHYDROLASE / CRISPR genome editing ribonucleoprotein complex
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / Bacillus subtilis ribonuclease activity / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
CRISPR-associated endonuclease Cas12a / Cas12a, REC1 domain / Cas12a, RuvC nuclease domain / Cas12a, nuclease domain / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida (bacteria)
Francisella tularensis subsp. novicida U112 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.24 Å
AuthorsMontoya, G. / Mesa, P.
Funding support Denmark, 1items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001 Denmark
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.
History
DepositionJun 18, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Dec 19, 2018Provider: repository / Type: Initial release
Revision 1.1Dec 26, 2018Group: Data collection / Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: software / Item: _software.name
Revision 1.3Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas12a
B: RNA (28-MER)
C: 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')
D: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')


Theoretical massNumber of molelcules
Total (without water)203,2814
Polymers203,2814
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration, native gel electrophoresis
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
2
B: RNA (28-MER)
C: 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')
D: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')


Theoretical massNumber of molelcules
Total (without water)47,6413
Polymers47,6413
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area2380 Å2
ΔGint-8 kcal/mol
Surface area12620 Å2
MethodPISA

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Components

#1: Protein CRISPR-associated endonuclease Cas12a / CRISPR-associated endonuclease Cpf1 / FnCas12a / FnCpf1


Mass: 155639.828 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Francisella tularensis subsp. novicida (strain U112) (bacteria)
Strain: U112 / Gene: cas12a, cpf1, FTN_1397 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: A0Q7Q2, deoxyribonuclease I, EC: 3.1.27.2
#2: RNA chain RNA (28-MER)


Mass: 13749.146 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)
#3: DNA chain 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')


Mass: 16898.826 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)
#4: DNA chain DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')


Mass: 16992.936 Da / Num. of mol.: 1 / Source method: obtained synthetically
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)

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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

Component
IDNameTypeEntity IDParent-IDSource
1TRansition state complex I2 conformationCOMPLEXall0MULTIPLE SOURCES
2CRISPR-associated endonuclease Cas12aCOMPLEX#11RECOMBINANT
3Nucleic acidsNucleic acidCOMPLEX#2-#41RECOMBINANT
Molecular weightValue: 0.180 MDa / Experimental value: YES
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Francisella tularensis subsp. novicida U112 (bacteria)401614
33Francisella tularensis subsp. novicida U112 (bacteria)401614
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
22Escherichia coli BL21 (bacteria)511693
33synthetic construct (others)32630
Buffer solutionpH: 7
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: C-flat-1.2/1.3 4C
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE

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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: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy
Image recordingElectron dose: 40 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k)

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Processing

Software
NameVersionClassification
PHENIXdev_3071refinement
PHENIXdev_3071refinement
EM software
IDNameCategory
2EPUimage acquisition
4MotionCorr2CTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.24 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 124000 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00866614
ELECTRON MICROSCOPYf_angle_d1.09188713
ELECTRON MICROSCOPYf_chiral_restr0.0822293
ELECTRON MICROSCOPYf_plane_restr0.00511330
ELECTRON MICROSCOPYf_dihedral_angle_d21.72791976

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