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- PDB-8sfn: WT CRISPR-Cas12a with a 16bp R-loop and nontarget strand in the R... -

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Basic information

Entry
Database: PDB / ID: 8sfn
TitleWT CRISPR-Cas12a with a 16bp R-loop and nontarget strand in the RuvC active site.
Components
  • CRISPR-associated endonuclease Cas12a
  • DNA (30-MER)
  • DNA (31-MER)
  • RNA (36-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: 3.3 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: To Be Published
Title: WT CRISPR-Cas12a with a 16bp R-loop and nontarget strand in the RuvC active site.
Authors: Strohkendl, I. / Taylor, D.W.
History
DepositionApr 11, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jul 3, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endonuclease Cas12a
B: RNA (36-MER)
C: DNA (31-MER)
D: DNA (30-MER)


Theoretical massNumber of molelcules
Total (without water)201,5674
Polymers201,5674
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: 151705.234 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 (36-MER)


Mass: 15351.995 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA (31-MER)


Mass: 17211.082 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: DNA chain DNA (30-MER)


Mass: 17299.061 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)

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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 16bp-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: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 97787 / Symmetry type: POINT
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00513304
ELECTRON MICROSCOPYf_angle_d1.03718408
ELECTRON MICROSCOPYf_dihedral_angle_d20.4232516
ELECTRON MICROSCOPYf_chiral_restr0.0522026
ELECTRON MICROSCOPYf_plane_restr0.0081998

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