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- EMDB-52745: SpCas12Cas12f1 in complex with sgRNA and cognate DNA -

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Basic information

Entry
Database: EMDB / ID: EMD-52745
TitleSpCas12Cas12f1 in complex with sgRNA and cognate DNA
Map dataOutput of phenix.auto_sharpen_1.21.2-5419 with b_iso_to_d_cut
Sample
  • Complex: SpCas12f1 in complex with sgRNA and cognate DNA
    • Protein or peptide: CRISPR-associated endodeoxyribonuclease Cas12f1
    • DNA: DNA target strand
    • DNA: DNA non-target strand
    • RNA: sgRNA (single-guide RNA)
  • Ligand: ZINC ION
KeywordsCRISPR-Cas / Cas12 / Cas12f1 / RNA BINDING PROTEIN
Function / homologyTransposase IS605, OrfB, C-terminal / Cas12f1-like, TNB domain / endonuclease activity / Hydrolases; Acting on ester bonds / DNA binding / RNA binding / metal ion binding / CRISPR-associated endodeoxyribonuclease Cas12f1
Function and homology information
Biological speciesSyntrophomonas palmitatica JCM 14374 (bacteria) / synthetic construct (others)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.89 Å
AuthorsSasnauskas G / Baltramonaitis M / Karvelis T / Siksnys V
Funding supportLithuania, 1 items
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, 2025-
Header (metadata) releaseJul 23, 2025-
Map releaseJul 23, 2025-
UpdateJul 23, 2025-
Current statusJul 23, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_52745.png

Downloads & links

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Map

FileDownload / File: emd_52745.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationOutput of phenix.auto_sharpen_1.21.2-5419 with b_iso_to_d_cut
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.1 Å/pix.
x 200 pix.
= 220. Å		1.1 Å/pix.
x 200 pix.
= 220. Å		1.1 Å/pix.
x 200 pix.
= 220. Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 6.0
Minimum - Maximum-28.373058 - 43.269329999999997
Average (Standard dev.)0.000000000000216 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 220.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Unsharpened map

Fileemd_52745_additional_1.map
AnnotationUnsharpened map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1

Fileemd_52745_half_map_1.map
AnnotationHalf map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2

Fileemd_52745_half_map_2.map
AnnotationHalf map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : SpCas12f1 in complex with sgRNA and cognate DNA

EntireName: SpCas12f1 in complex with sgRNA and cognate DNA
Components
  • Complex: SpCas12f1 in complex with sgRNA and cognate DNA
    • Protein or peptide: CRISPR-associated endodeoxyribonuclease Cas12f1
    • DNA: DNA target strand
    • DNA: DNA non-target strand
    • RNA: sgRNA (single-guide RNA)
  • Ligand: ZINC ION

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Supramolecule #1: SpCas12f1 in complex with sgRNA and cognate DNA

SupramoleculeName: SpCas12f1 in complex with sgRNA and cognate DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 / Details: SpCas12f1 in complex with sgRNA and cognate DNA
Source (natural)Organism: Syntrophomonas palmitatica JCM 14374 (bacteria)

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Macromolecule #1: CRISPR-associated endodeoxyribonuclease Cas12f1

MacromoleculeName: CRISPR-associated endodeoxyribonuclease Cas12f1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds
Source (natural)Organism: Syntrophomonas palmitatica JCM 14374 (bacteria)
Molecular weightTheoretical: 57.259922 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: SNAMGESVKA IKLKILDMFL DPECTKQDDN WRKDLSTMSR FCAEAGNMCL RDLYNYFSMP KEDRISSKDL YNAMYHKTKL LHPELPGKV ANQIVNHAKD VWKRNAKLIY RNQISMPTYK ITTAPIRLQN NIYKLIKNKN KYIIDVQLYS KEYSKDSGKG T HRYFLVAV ...String:
SNAMGESVKA IKLKILDMFL DPECTKQDDN WRKDLSTMSR FCAEAGNMCL RDLYNYFSMP KEDRISSKDL YNAMYHKTKL LHPELPGKV ANQIVNHAKD VWKRNAKLIY RNQISMPTYK ITTAPIRLQN NIYKLIKNKN KYIIDVQLYS KEYSKDSGKG T HRYFLVAV RDSSTRMIFD RIMSKDHIDS SKSYTQGQLQ IKKDHQGKWY CIIPYTFPTH ETVLDPDKVM GVDLGVAKAV YW AFNSSYK RGCIDGGEIE HFRKMIRARR VSIQNQIKHS GDARKGHGRK RALKPIETLS EKEKNFRDTI NHRYANRIVE AAI KQGCGT IQIENLEGIA DTTGSKFLKN WPYYDLQTKI VNKAKEHGIT VVAINPQYTS QRCSMCGYIE KTNRSSQAVF ECKQ CGYGS RTICINCRHV QVSGDVCEEC GGIVKKENVN ADYNAAKNIS TPYIDQIIME KCLELGIPYR SITCKECGHI QASGN TCEV CGSTNILKPK KIRKAK

UniProtKB: CRISPR-associated endodeoxyribonuclease Cas12f1

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Macromolecule #2: DNA target strand

MacromoleculeName: DNA target strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 9.891367 KDa
SequenceString:
(DG)(DG)(DC)(DG)(DA)(DC)(DG)(DT)(DT)(DG) (DG)(DG)(DT)(DC)(DA)(DA)(DC)(DT)(DG)(DA) (DA)(DA)(DT)(DA)(DC)(DG)(DC)(DT)(DA) (DC)(DG)(DC)

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Macromolecule #3: DNA non-target strand

MacromoleculeName: DNA non-target strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 5.795758 KDa
SequenceString:
(DG)(DC)(DG)(DT)(DA)(DG)(DC)(DG)(DT)(DA) (DT)(DT)(DT)(DC)(DT)(DC)(DA)(DA)(DC)

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Macromolecule #4: sgRNA (single-guide RNA)

MacromoleculeName: sgRNA (single-guide RNA) / type: rna / ID: 4 / Number of copies: 1
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 67.845352 KDa
SequenceString: GGGAUUUACU CUGUUUCGCG CGCCAGGGCA GUUAGGUGCC CUAAAAGAGC GAAGUGGCCG AAAGGAAAGG CUAACGCUUC UCUAACGCU ACGGCGACCU UGGCGAAAUG CCAUCAAUAC CACGCGGCCC GAAAGGGUUC GCGCGAAACU GAGUAAUGAA A GUCGCAUC ...String:
GGGAUUUACU CUGUUUCGCG CGCCAGGGCA GUUAGGUGCC CUAAAAGAGC GAAGUGGCCG AAAGGAAAGG CUAACGCUUC UCUAACGCU ACGGCGACCU UGGCGAAAUG CCAUCAAUAC CACGCGGCCC GAAAGGGUUC GCGCGAAACU GAGUAAUGAA A GUCGCAUC UUGCGUAAGC GCGUGGAUUG AAACAGUUGA CCCAACGUCG CC

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Macromolecule #5: ZINC ION

MacromoleculeName: ZINC ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: ZN
Molecular weightTheoretical: 65.409 Da

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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: 7.5
Component:
ConcentrationFormulaName
150.0 mMNaClsodium chloride
40.0 mMTris-HClTris-HCl
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 300 / Support film - Material: GRAPHENE OXIDE / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 45 sec. / Pretreatment - Atmosphere: AIR / Details: Glowcube plus 45 s @20 mA
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 4000 pixel / Digitization - Dimensions - Height: 4000 pixel / Number grids imaged: 1 / Number real images: 1983 / Average electron dose: 30.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 1520039
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: INSILICO MODEL / In silico model: Ab-initio model
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.89 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 462111
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
Output model

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

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