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- PDB-8sfh: WT CRISPR-Cas12a with a 5bp R-loop -

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Basic information

Entry
Database: PDB / ID: 8sfh
TitleWT CRISPR-Cas12a with a 5bp R-loop
Components
  • (DNA (32-MER)) x 2
  • CRISPR-associated endonuclease Cas12a
  • RNA (27-MER)
KeywordsDNA BINDING PROTEIN/DNA/RNA / CRISPR / R-loop / endonuclease / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA 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: 3.4 Å
AuthorsStrohkendl, I. / Taylor, D.W.
Funding support United States, 1items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM138348 United States
CitationJournal: Mol Cell / Year: 2024
Title: Cas12a domain flexibility guides R-loop formation and forces RuvC resetting.
Authors: Isabel Strohkendl / Aakash Saha / Catherine Moy / Alexander-Hoi Nguyen / Mohd Ahsan / Rick Russell / Giulia Palermo / David W Taylor /
Abstract: The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables ...The specific nature of CRISPR-Cas12a makes it a desirable RNA-guided endonuclease for biotechnology and therapeutic applications. To understand how R-loop formation within the compact Cas12a enables target recognition and nuclease activation, we used cryo-electron microscopy to capture wild-type Acidaminococcus sp. Cas12a R-loop intermediates and DNA delivery into the RuvC active site. Stages of Cas12a R-loop formation-starting from a 5-bp seed-are marked by distinct REC domain arrangements. Dramatic domain flexibility limits contacts until nearly complete R-loop formation, when the non-target strand is pulled across the RuvC nuclease and coordinated domain docking promotes efficient cleavage. Next, substantial domain movements enable target strand repositioning into the RuvC active site. Between cleavage events, the RuvC lid conformationally resets to occlude the active site, requiring re-activation. These snapshots build a structural model depicting Cas12a DNA targeting that rationalizes observed specificity and highlights mechanistic comparisons to other class 2 effectors.
History
DepositionApr 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release
Revision 1.1Jan 22, 2025Group: Data collection / Database references / Structure summary
Category: citation / citation_author ...citation / citation_author / em_admin / pdbx_entry_details
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas12a
B: RNA (27-MER)
C: DNA (32-MER)
D: DNA (32-MER)


Theoretical massNumber of molelcules
Total (without water)179,6974
Polymers179,6974
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein CRISPR-associated endonuclease Cas12a / AsCpf1 / CRISPR-associated endonuclease Cpf1


Mass: 151418.953 Da / Num. of mol.: 1
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
#2: RNA chain RNA (27-MER)


Mass: 8598.088 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (32-MER)


Mass: 9882.405 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (32-MER)


Mass: 9797.312 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
Has protein modificationN

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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

ComponentName: WT AsCas12a incubated with 8bp-complementary target DNA.
Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Source (natural)Organism: Acidaminococcus sp. BV3L6 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Nominal defocus max: 2500 nm / Nominal defocus min: 1500 nm
Image recordingElectron dose: 80 e/Å2 / Film or detector model: GATAN K3 (6k x 4k)

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Processing

EM software
IDNameCategory
1cryoSPARCparticle selection
2SerialEMimage acquisition
4cryoSPARCCTF correction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
3D reconstructionResolution: 3.4 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 60595 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00612867
ELECTRON MICROSCOPYf_angle_d0.97617759
ELECTRON MICROSCOPYf_dihedral_angle_d18.8642347
ELECTRON MICROSCOPYf_chiral_restr0.0541955
ELECTRON MICROSCOPYf_plane_restr0.0071959

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