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- PDB-9cjh: Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, ... -

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Basic information

Entry
Database: PDB / ID: 9cjh
TitleCas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1
Components
  • CRISPR-associated endonuclease Cas12a
  • Non-target DNA strand
  • Target DNA strand
  • guide RNA (25-MER)
KeywordsHYDROLASE/RNA/DNA / DNase / complex / ribonucleoprotein / genome editor / HYDROLASE-RNA complex / HYDROLASE / HYDROLASE-RNA-DNA complex
Function / homology
Function and homology information


Bacillus subtilis ribonuclease / deoxyribonuclease I / deoxyribonuclease I activity / defense response to virus / lyase activity / DNA binding / RNA binding
Similarity search - Function
: / CRISPR-associated endonuclease Cpf1 REC2 domain / 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 speciesAcidaminococcus sp. BV3L6 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.6 Å
AuthorsSoczek, K.M. / Doudna, J.A.
Funding support United States, 6items
OrganizationGrant numberCountry
Howard Hughes Medical Institute (HHMI) United States
National Science Foundation (NSF, United States)2334028 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R21HL173710 United States
National Institutes of Health/National Institute of Neurological Disorders and Stroke (NIH/NINDS)U19NS132303 United States
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)U19AI171110, U54AI170792, U19AI135990, UH3AI150552, U01AI142817 United States
Department of Energy (DOE, United States)DE-AC02-05CH11231, 2553571, B656358 United States
CitationJournal: Nucleic Acids Res / Year: 2025
Title: CRISPR-Cas12a bends DNA to destabilize base pairs during target interrogation.
Authors: Katarzyna M Soczek / Joshua C Cofsky / Owen T Tuck / Honglue Shi / Jennifer A Doudna /
Abstract: RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use ...RNA-guided endonucleases are involved in processes ranging from adaptive immunity to site-specific transposition and have revolutionized genome editing. CRISPR-Cas9, -Cas12 and related proteins use guide RNAs to recognize ∼20-nucleotide target sites within genomic DNA by mechanisms that are not yet fully understood. We used structural and biochemical methods to assess early steps in DNA recognition by Cas12a protein-guide RNA complexes. We show here that Cas12a initiates DNA target recognition by bending DNA to induce transient nucleotide flipping that exposes nucleobases for DNA-RNA hybridization. Cryo-EM structural analysis of a trapped Cas12a-RNA-DNA surveillance complex and fluorescence-based conformational probing show that Cas12a-induced DNA helix destabilization enables target discovery and engagement. This mechanism of initial DNA interrogation resembles that of CRISPR-Cas9 despite distinct evolutionary origins and different RNA-DNA hybridization directionality of these enzyme families. Our findings support a model in which RNA-mediated DNA interference begins with local helix distortion by transient CRISPR-Cas protein binding.
History
DepositionJul 6, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 8, 2025Provider: repository / Type: Initial release
Revision 1.0Jan 8, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
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Revision 1.0Jan 8, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
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Revision 1.0Jan 8, 2025Data content type: Mask / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0Jan 8, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release
Revision 1.1Feb 5, 2025Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.journal_volume / _citation.year ..._citation.journal_volume / _citation.year / _citation_author.identifier_ORCID / _em_admin.last_update
Revision 1.2May 28, 2025Group: Data collection / Category: em_admin / em_software / Item: _em_admin.last_update / _em_software.name
Revision 1.1May 28, 2025Data content type: EM metadata / Data content type: EM metadata / EM metadata / Group: Data processing / Experimental summary / Data content type: EM metadata / EM metadata / Category: em_admin / em_software / Data content type: EM metadata / EM metadata / Item: _em_admin.last_update / _em_software.name

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
B: guide RNA (25-MER)
A: CRISPR-associated endonuclease Cas12a
C: Non-target DNA strand
D: Target DNA strand


Theoretical massNumber of molelcules
Total (without water)184,2244
Polymers184,2244
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: RNA chain guide RNA (25-MER)


Mass: 13418.956 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#2: Protein CRISPR-associated endonuclease Cas12a / AsCpf1 / CRISPR-associated endonuclease Cpf1


Mass: 151680.250 Da / Num. of mol.: 1 / Mutation: N551C
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Acidaminococcus sp. BV3L6 (bacteria) / Gene: cas12a, cpf1, HMPREF1246_0236 / Production host: Escherichia coli BL21(DE3) (bacteria)
References: UniProt: U2UMQ6, deoxyribonuclease I, Bacillus subtilis ribonuclease
#3: DNA chain Non-target DNA strand


Mass: 9567.206 Da / Num. of mol.: 1 / Source method: obtained synthetically
Details: cytidine in position 7 is covalently modified with XL1, forming N4-cystamine-2'-deoxycytidine; the modified base is forming a disulfide bond with cysteine 551 in the protein
Source: (synth.) synthetic construct (others)
#4: DNA chain Target DNA strand


Mass: 9557.205 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has ligand of interestY
Has protein modificationY

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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
1Cas12a:gRNA:DNA (Acidaminococcus sp.) with 0 RNA:DNA base pairs, structure 1COMPLEXall0MULTIPLE SOURCES
2Cas12aCOMPLEX#21RECOMBINANT
3gRNA:DNACOMPLEX#1, #3-#41SYNTHETIC
Molecular weightValue: 0.185 MDa / Experimental value: NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
22Acidaminococcus sp. BV3L6 (bacteria)1111120
33synthetic construct (others)32630
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
120 mMTris-Cl1
2200 mMpotassium chlorideKCl1
30.1 mMdithiothreitol1
45 mMmagnesium chlorideMgCl21
50.25 %glycerol1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: 25 mA / Grid material: COPPER / Grid mesh size: 400 divisions/in. / Grid type: C-flat-1.2/1.3
VitrificationCryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 281 K

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: OTHER
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2000 nm / Nominal defocus min: 800 nm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 50 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4particle selection
2SerialEM3.8.7image acquisition
4cryoSPARC4CTF correctionpatch ctf
8PHENIXmodel refinement
10cryoSPARC4initial Euler assignment
11RELION5-betafinal Euler assignment
12RELION5-betaclassification
13RELION5-beta3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 20031 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00311566
ELECTRON MICROSCOPYf_angle_d0.56315888
ELECTRON MICROSCOPYf_dihedral_angle_d17.4074416
ELECTRON MICROSCOPYf_chiral_restr0.0391773
ELECTRON MICROSCOPYf_plane_restr0.0051836

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