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- PDB-9i8y: SpCas12Cas12f1 in complex with sgRNA and cognate DNA -

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Basic information

Entry
Database: PDB / ID: 9i8y
TitleSpCas12Cas12f1 in complex with sgRNA and cognate DNA
Components
  • CRISPR-associated endodeoxyribonuclease Cas12f1
  • DNA non-target strand
  • DNA target strand
  • sgRNA (single-guide RNA)
KeywordsRNA BINDING PROTEIN / CRISPR-Cas / Cas12 / Cas12f1
Function / homology
Function and homology information


endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding
Similarity search - Function
Transposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain
Similarity search - Domain/homology
DNA / DNA (> 10) / RNA / RNA (> 10) / RNA (> 100) / CRISPR-associated endodeoxyribonuclease Cas12f1
Similarity search - Component
Biological speciesSyntrophomonas palmitatica JCM 14374 (bacteria)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsSasnauskas, G. / Baltramonaitis, M. / Karvelis, T. / Siksnys, V.
Funding supportLithuania, 1items
OrganizationGrant numberCountry
Research Council of LithuaniaS-MIP-19-32Lithuania
CitationJournal: Nucleic Acids Res / Year: 2025
Title: Structural and mechanistic insights into the sequential dsDNA cleavage by SpCas12f1.
Authors: Julene Madariaga-Marcos / Marius Baltramonaitis / Selgar Henkel-Heinecke / Dominik J Kauert / Patrick Irmisch / Greta Bigelyte-Stankeviciene / Arunas Silanskas / Tautvydas Karvelis / ...Authors: Julene Madariaga-Marcos / Marius Baltramonaitis / Selgar Henkel-Heinecke / Dominik J Kauert / Patrick Irmisch / Greta Bigelyte-Stankeviciene / Arunas Silanskas / Tautvydas Karvelis / Virginijus Siksnys / Giedrius Sasnauskas / Ralf Seidel /
Abstract: Miniature CRISPR-Cas12f1 effector complexes have recently attracted considerable interest for genome engineering applications due to their compact size. Unlike other Class 2 effectors, Cas12f1 ...Miniature CRISPR-Cas12f1 effector complexes have recently attracted considerable interest for genome engineering applications due to their compact size. Unlike other Class 2 effectors, Cas12f1 functions as a homodimer bound to a single ∼200 nt RNA. While the basic biochemical properties of Cas12f1, such as its use of a single catalytic center for catalysis, have been characterized, the orchestration of the different events occurring during Cas12f1 reactions remained little explored. To gain insights into the dynamics and mechanisms involved in DNA recognition and cleavage by Cas12f1 from Syntrophomonas palmitatica (SpCas12f1), we solved the structure of SpCas12f1 bound to target DNA and employed single-molecule magnetic tweezers measurements in combination with ensemble kinetic measurements. Our data indicate that SpCas12f1 forms 18 bp R-loops, in which local contacts of the protein to the R-loop stabilize R-loop intermediates. DNA cleavage is catalyzed by a single SpCas12f1 catalytic center, which first rapidly degrades a ∼11 bp region on the nontarget strand by cutting at random sites. Subsequent target strand cleavage is slower and requires at least a nick in the nontarget strand.
History
DepositionFeb 6, 2025Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 23, 2025Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: CRISPR-associated endodeoxyribonuclease Cas12f1
B: CRISPR-associated endodeoxyribonuclease Cas12f1
C: DNA target strand
D: DNA non-target strand
E: sgRNA (single-guide RNA)
hetero molecules


Theoretical massNumber of molelcules
Total (without water)198,2498
Polymers198,0525
Non-polymers1963
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: Protein CRISPR-associated endodeoxyribonuclease Cas12f1 / SpCas12f1 / CRISPR-associated endonuclease C2c10


Mass: 57259.922 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Syntrophomonas palmitatica JCM 14374 (bacteria)
Gene: cas12f1 / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): Arctic Express
References: UniProt: P0DW62, Hydrolases; Acting on ester bonds
#2: DNA chain DNA target strand


Mass: 9891.367 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#3: DNA chain DNA non-target strand


Mass: 5795.758 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#4: RNA chain sgRNA (single-guide RNA)


Mass: 67845.352 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) synthetic construct (others)
#5: Chemical ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: Zn
Has ligand of interestN
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: SpCas12f1 in complex with sgRNA and cognate DNA / Type: COMPLEX / Details: SpCas12f1 in complex with sgRNA and cognate DNA / Entity ID: #1-#4 / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Syntrophomonas palmitatica JCM 14374 (bacteria)
Source (recombinant)Organism: Escherichia coli BL21 (bacteria) / Strain: Arctic Express
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1150 mMsodium chlorideNaCl1
240 mMTris-HClTris-HCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Glowcube plus 45 s @20 mA / Grid material: COPPER / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 277 K

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Electron microscopy imaging

MicroscopyModel: TFS GLACIOS
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: 1000 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN
Image recordingElectron dose: 30 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON III (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 1983
Image scansWidth: 4000 / Height: 4000

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Processing

EM softwareName: PHENIX / Version: 1.21.2_5419 / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 1520039
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 462111 / Symmetry type: POINT
Atomic model buildingSource name: AlphaFold / Type: in silico model
RefinementCross valid method: NONE

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