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- PDB-6gtg: Transition state structure of Cpf1(Cas12a) I4 conformation -

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Basic information

Entry
Database: PDB / ID: 6gtg
TitleTransition state structure of Cpf1(Cas12a) I4 conformation
Components
  • CRISPR-associated endonuclease Cas12a
  • DNA (32-MER)
  • DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
  • RNA (40-MER)
KeywordsHYDROLASE / genome editing CRISPR ribonucleoprotein complex
Function / homologyCas12a, REC1 domain / CRISPR-associated endonuclease Cas12a / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease domain / Bacillus subtilis ribonuclease / Bacillus subtilis ribonuclease activity / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus ...Cas12a, REC1 domain / CRISPR-associated endonuclease Cas12a / Alpha helical recognition lobe domain / Nuclease domain / RuvC nuclease 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
Specimen sourceFrancisella tularensis subsp. novicida U112 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.27 Å resolution
AuthorsMesa, P. / Montoya, G. / Stella, S.
Funding supportDenmark , 1 items
OrganizationGrant numberCountry
Novo Nordisk FoundationNNF14CC0001Denmark
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 Report
SummaryFull reportAbout validation report
DateDeposition: Jun 18, 2018 / Release: Dec 19, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Dec 19, 2018Structure modelrepositoryInitial release
1.1Dec 26, 2018Structure modelData collection / Database referencescitation_citation.journal_volume / _citation.page_first / _citation.page_last

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Structure visualization

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  • Deposited structure unit
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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas12a
B: RNA (40-MER)
C: DNA (32-MER)
D: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
hetero molecules


Theoretical massNumber of molelcules
Total (without water)203,3788
Polyers203,2814
Non-polymers974
Water543
1


  • idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, native gel electrophoresis
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)19450
ΔGint (kcal/M)-152
Surface area (Å2)69440
MethodPISA

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Components

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DNA chain , 2 types, 2 molecules CD

#3: DNA chain DNA (32-MER)


Mass: 16898.826 Da / Num. of mol.: 1
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: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)

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Protein/peptide / RNA chain , 2 types, 2 molecules AB

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


Mass: 155639.828 Da / Num. of mol.: 1 / Mutation: E1006Q
Source: (gene. exp.) Francisella tularensis subsp. novicida U112 (bacteria)
Gene: cas12a, cpf1, FTN_1397 / Production host: Escherichia coli BL21 (bacteria)
References: UniProt: A0Q7Q2, deoxyribonuclease I, Bacillus subtilis ribonuclease
#2: RNA chain RNA (40-MER)


Mass: 13749.146 Da / Num. of mol.: 1
Source: (synth.) Francisella tularensis subsp. novicida U112 (bacteria)

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Non-polymers , 2 types, 7 molecules

#5: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 4 / Formula: Mg / Magnesium
#6: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 3 / Formula: H2O / Water

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / Reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent IDSource
1Transition state complex I4 conformationCOMPLEX1,2,3,40MULTIPLE SOURCES
2CRISPR-associated endonuclease Cas12aCOMPLEX11RECOMBINANT
3RNA (28-mer)COMPLEX21RECOMBINANT
4DNACOMPLEX3,41RECOMBINANT
Molecular weightValue: 0.180 MDa / Experimental value: YES
Source (natural)
IDEntity assembly IDNcbi tax IDOrganism
12401614Francisella tularensis subsp. novicida U112 (bacteria)
23401614Francisella tularensis subsp. novicida U112 (bacteria)
34401614Francisella tularensis subsp. novicida U112 (bacteria)
Source (recombinant)
IDEntity assembly IDNcbi tax IDOrganism
12511693Escherichia coli BL21 (bacteria)
2332630synthetic construct (others)
3432630synthetic construct (others)
Buffer solutionpH: 6
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationCryogen name: ETHANE

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyMicroscope model: 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
phenix.real_space_refinedev_3071refinement
PHENIXdev_3071refinement
EM software
IDNameCategory
2EPUimage acquisition
4MotionCorr2CTF correction
7Cootmodel fitting
9PHENIXmodel refinement
13RELION3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1
3D reconstructionResolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 358 / Symmetry type: POINT
RefineStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.008612971
ELECTRON MICROSCOPYf_angle_d1.055717860
ELECTRON MICROSCOPYf_chiral_restr0.05711960
ELECTRON MICROSCOPYf_plane_restr0.00671969
ELECTRON MICROSCOPYf_dihedral_angle_d10.39877582

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