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Open data
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Basic information
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Title | SpCas12Cas12f1 in complex with sgRNA and cognate DNA | |||||||||
![]() | Output of phenix.auto_sharpen_1.21.2-5419 with b_iso_to_d_cut | |||||||||
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![]() | CRISPR-Cas / Cas12 / Cas12f1 / RNA BINDING PROTEIN | |||||||||
Function / homology | Transposase 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![]() | |||||||||
Biological species | ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.89 Å | |||||||||
![]() | Sasnauskas G / Baltramonaitis M / Karvelis T / Siksnys V | |||||||||
Funding support | Lithuania, 1 items
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![]() | ![]() 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 |
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Structure visualization
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 26.8 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 22.4 KB 22.4 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.1 KB | Display | ![]() |
Images | ![]() | 162.9 KB | ||
Filedesc metadata | ![]() | 7.2 KB | ||
Others | ![]() ![]() ![]() | 14.9 MB 28.1 MB 28.1 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Validation report
Summary document | ![]() | 776.8 KB | Display | ![]() |
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Full document | ![]() | 776.4 KB | Display | |
Data in XML | ![]() | 12.8 KB | Display | |
Data in CIF | ![]() | 17.4 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 9i8yMC M: atomic model generated by this map C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | Output of phenix.auto_sharpen_1.21.2-5419 with b_iso_to_d_cut | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.1 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: Unsharpened map
File | emd_52745_additional_1.map | ||||||||||||
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Annotation | Unsharpened map | ||||||||||||
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Density Histograms |
-Half map: Half map 1
File | emd_52745_half_map_1.map | ||||||||||||
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Annotation | Half map 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 2
File | emd_52745_half_map_2.map | ||||||||||||
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Annotation | Half map 2 | ||||||||||||
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Density Histograms |
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Sample components
-Entire : SpCas12f1 in complex with sgRNA and cognate DNA
Entire | Name: SpCas12f1 in complex with sgRNA and cognate DNA |
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Components |
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-Supramolecule #1: SpCas12f1 in complex with sgRNA and cognate DNA
Supramolecule | Name: 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 |
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Source (natural) | Organism: ![]() |
-Macromolecule #1: CRISPR-associated endodeoxyribonuclease Cas12f1
Macromolecule | Name: CRISPR-associated endodeoxyribonuclease Cas12f1 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: Hydrolases; Acting on ester bonds |
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Source (natural) | Organism: ![]() |
Molecular weight | Theoretical: 57.259922 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: DNA target strand
Macromolecule | Name: DNA target strand / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 9.891367 KDa |
Sequence | String: (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) |
-Macromolecule #3: DNA non-target strand
Macromolecule | Name: DNA non-target strand / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 5.795758 KDa |
Sequence | String: (DG)(DC)(DG)(DT)(DA)(DG)(DC)(DG)(DT)(DA) (DT)(DT)(DT)(DC)(DT)(DC)(DA)(DA)(DC) |
-Macromolecule #4: sgRNA (single-guide RNA)
Macromolecule | Name: sgRNA (single-guide RNA) / type: rna / ID: 4 / Number of copies: 1 |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 67.845352 KDa |
Sequence | String: 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 |
-Macromolecule #5: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 5 / Number of copies: 3 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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![]() | single particle reconstruction |
Aggregation state | particle |
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Sample preparation
Buffer | pH: 7.5 Component:
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Grid | Model: 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 | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
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Electron microscopy
Microscope | TFS GLACIOS |
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Image recording | Film 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 beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 1.0 µm |
Sample stage | Cooling holder cryogen: NITROGEN |
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Image processing
-Atomic model buiding 1
Initial model | Chain - Source name: AlphaFold / Chain - Initial model type: in silico model |
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Output model | ![]() PDB-9i8y: |