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- EMDB-0065: Transition state structure of Cpf1(Cas12a) I4 conformation -

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Basic information

Entry
Database: EMDB / ID: EMD-0065
TitleTransition state structure of Cpf1(Cas12a) I4 conformation
Map data
Sample
  • Complex: Transition state complex I4 conformation
    • Complex: CRISPR-associated endonuclease Cas12a
      • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • Complex: RNA (28-mer)
      • RNA: RNA (40-MER)
    • Complex: DNA
      • DNA: DNA (32-MER)
      • DNA: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water
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
CRISPR-associated endonuclease Cas12a
Similarity search - Component
Biological speciesFrancisella tularensis subsp. novicida U112 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.27 Å
AuthorsMesa P / Montoya G / Stella S
Funding support Denmark, 1 items
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, 2018-
Header (metadata) releaseAug 22, 2018-
Map releaseDec 19, 2018-
UpdateDec 18, 2019-
Current statusDec 18, 2019Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6gtg
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0065.png

Downloads & links

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Map

FileDownload / File: emd_0065.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.832 Å
Density
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.3589212 - 0.50456303
Average (Standard dev.)0.00009434421 (±0.012955901)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 199.68001 Å
α=β=γ: 90.0 °

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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS240240240
D min/max/mean-0.3590.5050.000

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Supplemental data

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Sample components

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Entire : Transition state complex I4 conformation

EntireName: Transition state complex I4 conformation
Components
  • Complex: Transition state complex I4 conformation
    • Complex: CRISPR-associated endonuclease Cas12a
      • Protein or peptide: CRISPR-associated endonuclease Cas12a
    • Complex: RNA (28-mer)
      • RNA: RNA (40-MER)
    • Complex: DNA
      • DNA: DNA (32-MER)
      • DNA: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
  • Ligand: MAGNESIUM ION
  • Ligand: water

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Supramolecule #1: Transition state complex I4 conformation

SupramoleculeName: Transition state complex I4 conformation / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Molecular weightExperimental: 180 KDa

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Supramolecule #2: CRISPR-associated endonuclease Cas12a

SupramoleculeName: CRISPR-associated endonuclease Cas12a / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)

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Supramolecule #3: RNA (28-mer)

SupramoleculeName: RNA (28-mer) / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Recombinant expressionOrganism: synthetic construct (others)

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Supramolecule #4: DNA

SupramoleculeName: DNA / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #3-#4
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Recombinant expressionOrganism: synthetic construct (others)

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Macromolecule #1: CRISPR-associated endonuclease Cas12a

MacromoleculeName: CRISPR-associated endonuclease Cas12a / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: deoxyribonuclease I
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Molecular weightTheoretical: 155.639828 KDa
Recombinant expressionOrganism: Escherichia coli BL21 (bacteria)
SequenceString: MSIYQEFVNK YSLSKTLRFE LIPQGKTLEN IKARGLILDD EKRAKDYKKA KQIIDKYHQF FIEEILSSVC ISEDLLQNYS DVYFKLKKS DDDNLQKDFK SAKDTIKKQI SEYIKDSEKF KNLFNQNLID AKKGQESDLI LWLKQSKDNG IELFKANSDI T DIDEALEI ...String:
MSIYQEFVNK YSLSKTLRFE LIPQGKTLEN IKARGLILDD EKRAKDYKKA KQIIDKYHQF FIEEILSSVC ISEDLLQNYS DVYFKLKKS DDDNLQKDFK SAKDTIKKQI SEYIKDSEKF KNLFNQNLID AKKGQESDLI LWLKQSKDNG IELFKANSDI T DIDEALEI IKSFKGWTTY FKGFHENRKN VYSSNDIPTS IIYRIVDDNL PKFLENKAKY ESLKDKAPEA INYEQIKKDL AE ELTFDID YKTSEVNQRV FSLDEVFEIA NFNNYLNQSG ITKFNTIIGG KFVNGENTKR KGINEYINLY SQQINDKTLK KYK MSVLFK QILSDTESKS FVIDKLEDDS DVVTTMQSFY EQIAAFKTVE EKSIKETLSL LFDDLKAQKL DLSKIYFKND KSLT DLSQQ VFDDYSVIGT AVLEYITQQI APKNLDNPSK KEQELIAKKT EKAKYLSLET IKLALEEFNK HRDIDKQCRF EEILA NFAA IPMIFDEIAQ NKDNLAQISI KYQNQGKKDL LQASAEDDVK AIKDLLDQTN NLLHKLKIFH ISQSEDKANI LDKDEH FYL VFEECYFELA NIVPLYNKIR NYITQKPYSD EKFKLNFENS TLANGWDKNK EPDNTAILFI KDDKYYLGVM NKKNNKI FD DKAIKENKGE GYKKIVYKLL PGANKMLPKV FFSAKSIKFY NPSEDILRIR NHSTHTKNGS PQKGYEKFEF NIEDCRKF I DFYKQSISKH PEWKDFGFRF SDTQRYNSID EFYREVENQG YKLTFENISE SYIDSVVNQG KLYLFQIYNK DFSAYSKGR PNLHTLYWKA LFDERNLQDV VYKLNGEAEL FYRKQSIPKK ITHPAKEAIA NKNKDNPKKE SVFEYDLIKD KRFTEDKFFF HCPITINFK SSGANKFNDE INLLLKEKAN DVHILSIDRG ERHLAYYTLV DGKGNIIKQD TFNIIGNDRM KTNYHDKLAA I EKDRDSAR KDWKKINNIK EMKEGYLSQV VHEIAKLVIE YNAIVVFQDL NFGFKRGRFK VEKQVYQKLE KMLIEKLNYL VF KDNEFDK TGGVLRAYQL TAPFETFKKM GKQTGIIYYV PAGFTSKICP VTGFVNQLYP KYESVSKSQE FFSKFDKICY NLD KGYFEF SFDYKNFGDK AAKGKWTIAS FGSRLINFRN SDKNHNWDTR EVYPTKELEK LLKDYSIEYG HGECIKAAIC GESD KKFFA KLTSVLNTIL QMRNSKTGTE LDYLISPVAD VNGNFFDSRQ APKNMPQDAD ANGAYHIGLK GLMLLGRIKN NQEGK KLNL VIKNEEYFEF VQNRNNGSEF ELENLYFQGE LRRQASALEH HHHHH

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Macromolecule #2: RNA (40-MER)

MacromoleculeName: RNA (40-MER) / type: rna / ID: 2 / Number of copies: 1
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Molecular weightTheoretical: 13.749146 KDa
SequenceString:
AAUUUCUACU GUUGUAGAUG AGAAGUCAUU UAAUAAGGCC ACU

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Macromolecule #3: DNA (32-MER)

MacromoleculeName: DNA (32-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Molecular weightTheoretical: 16.898826 KDa
SequenceString: (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) ...String:
(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)

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Macromolecule #4: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')

MacromoleculeName: DNA (5'-D(P*CP*GP*AP*GP*CP*TP*CP*GP*TP*TP*AP*GP*AP*GP*AP*AP*GP*T)-3')
type: dna / ID: 4 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: Francisella tularensis subsp. novicida U112 (bacteria)
Molecular weightTheoretical: 16.992936 KDa
SequenceString: (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) ...String:
(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)

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Macromolecule #5: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 4 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #6: water

MacromoleculeName: water / type: ligand / ID: 6 / Number of copies: 3 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 6
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Average electron dose: 40.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

CTF correctionSoftware - Name: MotionCorr2
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.27 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION / Number images used: 358
FSC plot (resolution estimation)

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