+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26994 | |||||||||
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Title | IscB and wRNA bound to Target DNA (locked state) | |||||||||
Map data | ||||||||||
Sample |
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Keywords | CRISPR / IscB / HEARO RNA / omega RNA / RNA BINDING PROTEIN-RNA-DNA complex | |||||||||
Biological species | synthetic construct (others) / unidentified (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.1 Å | |||||||||
Authors | Schuler GA / Hu C / Ke A | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Science / Year: 2022 Title: Structural basis for RNA-guided DNA cleavage by IscB-ωRNA and mechanistic comparison with Cas9. Authors: Gabriel Schuler / Chunyi Hu / Ailong Ke / Abstract: Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ...Class 2 CRISPR effectors Cas9 and Cas12 may have evolved from nucleases in IS200/IS605 transposons. IscB is about two-fifths the size of Cas9 but shares a similar domain organization. The associated ωRNA plays the combined role of CRISPR RNA (crRNA) and trans-activating CRISPR RNA tracrRNA) to guide double-stranded DNA (dsDNA) cleavage. Here we report a 2.78-angstrom cryo-electron microscopy structure of IscB-ωRNA bound to a dsDNA target, revealing the architectural and mechanistic similarities between IscB and Cas9 ribonucleoproteins. Target-adjacent motif recognition, R-loop formation, and DNA cleavage mechanisms are explained at high resolution. ωRNA plays the equivalent function of REC domains in Cas9 and contacts the RNA-DNA heteroduplex. The IscB-specific PLMP domain is dispensable for RNA-guided DNA cleavage. The transition from ancestral IscB to Cas9 involved dwarfing the ωRNA and introducing protein domain replacements. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26994.map.gz | 79.2 MB | EMDB map data format | |
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Header (meta data) | emd-26994-v30.xml emd-26994.xml | 17.8 KB 17.8 KB | Display Display | EMDB header |
Images | emd_26994.png | 101.2 KB | ||
Filedesc metadata | emd-26994.cif.gz | 6.2 KB | ||
Others | emd_26994_half_map_1.map.gz emd_26994_half_map_2.map.gz | 77.8 MB 77.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26994 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26994 | HTTPS FTP |
-Validation report
Summary document | emd_26994_validation.pdf.gz | 1.1 MB | Display | EMDB validaton report |
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Full document | emd_26994_full_validation.pdf.gz | 1.1 MB | Display | |
Data in XML | emd_26994_validation.xml.gz | 12.7 KB | Display | |
Data in CIF | emd_26994_validation.cif.gz | 14.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26994 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26994 | HTTPS FTP |
-Related structure data
Related structure data | 8ctlMC 7utnC 8cszC M: atomic model generated by this map C: citing same article (ref.) |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_26994.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.07 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Half map: #2
File | emd_26994_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_26994_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Cryo-EM structure of IscB in complex with RNA and target DNA
Entire | Name: Cryo-EM structure of IscB in complex with RNA and target DNA |
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Components |
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-Supramolecule #1: Cryo-EM structure of IscB in complex with RNA and target DNA
Supramolecule | Name: Cryo-EM structure of IscB in complex with RNA and target DNA type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 190 kDa/nm |
-Macromolecule #1: IscB
Macromolecule | Name: IscB / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 56.81968 KDa |
Recombinant expression | Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria) |
Sequence | String: MMAVVYVISK SGKPLMPTTR CGHVRILLKE GKARVVERKP FTIQLTYESA EETQPLVLGI DPGRTNIGMS VVTESGESVF NAQIETRNK DVPKLMKDRK QYRMAHRRLK RRCKRRRRAK AAGTAFEEGE KQRLLPGCFK PITCKSIRNK EARFNNRKRP V GWLTPTAN ...String: MMAVVYVISK SGKPLMPTTR CGHVRILLKE GKARVVERKP FTIQLTYESA EETQPLVLGI DPGRTNIGMS VVTESGESVF NAQIETRNK DVPKLMKDRK QYRMAHRRLK RRCKRRRRAK AAGTAFEEGE KQRLLPGCFK PITCKSIRNK EARFNNRKRP V GWLTPTAN HLLVTHLNVV KKVQKILPVA KVVLELNRFS FMAMNNPKVQ RWQYQRGPLY GKGSVEEAVS MQQDGHCLFC KH GIDHYHH VVPRRKNGSE TLENRVGLCE EHHRLVHTDK EWEANLASKK SGMNKKYHAL SVLNQIIPYL ADQLADMFPG NFC VTSGQD TYLFREEHGI PKDHYLDAYC IACSALTDAK KVSSPKGRPY MVHQFRRHDR QACHKANLNR SYYMGGKLVA TNRH KAMDQ KTDSLEEYRA AHSAADVSKL TVKHPSAQYK DMSRIMPGSI LVSGEGKLFT LSRSEGRNKG QVNYFVSTEG IKYWA RKCQ YLRNNGGLQI YV |
-Macromolecule #2: RNA (222-MER)
Macromolecule | Name: RNA (222-MER) / type: rna / ID: 2 / Number of copies: 1 |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 71.877797 KDa |
Sequence | String: AAAAGAGUGA ACGAGAGGCU CUUCCAACUU UAUGGUUGCG ACCGUAGGUU GAAAGAGCAC AGGCUGAGAC AUUCGUAAGG CCGAAAGAC CGGACGCACC CUGGGAUUUC CCCAGUCCCC GGAACUGCAU AGCGGAUGCC AGUUGAUGGA GCAAUCUAUC A GAUAAGCC ...String: AAAAGAGUGA ACGAGAGGCU CUUCCAACUU UAUGGUUGCG ACCGUAGGUU GAAAGAGCAC AGGCUGAGAC AUUCGUAAGG CCGAAAGAC CGGACGCACC CUGGGAUUUC CCCAGUCCCC GGAACUGCAU AGCGGAUGCC AGUUGAUGGA GCAAUCUAUC A GAUAAGCC AGGGGGAACA AUCACCUCUC UGUAUCAGAG AGAGUUUUAC AAAAGGAGGA ACGG |
-Macromolecule #3: DNA target strand
Macromolecule | Name: DNA 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: 18.305646 KDa |
Sequence | String: (DG)(DC)(DC)(DA)(DC)(DG)(DG)(DG)(DC)(DT) (DG)(DA)(DC)(DC)(DT)(DC)(DG)(DA)(DC)(DT) (DT)(DC)(DT)(DA)(DG)(DT)(DC)(DT)(DC) (DG)(DT)(DT)(DC)(DA)(DC)(DT)(DC)(DT)(DT) (DT) (DT)(DG)(DC)(DC)(DG)(DT) ...String: (DG)(DC)(DC)(DA)(DC)(DG)(DG)(DG)(DC)(DT) (DG)(DA)(DC)(DC)(DT)(DC)(DG)(DA)(DC)(DT) (DT)(DC)(DT)(DA)(DG)(DT)(DC)(DT)(DC) (DG)(DT)(DT)(DC)(DA)(DC)(DT)(DC)(DT)(DT) (DT) (DT)(DG)(DC)(DC)(DG)(DT)(DA)(DC) (DC)(DC)(DT)(DC)(DG)(DT)(DG)(DG)(DG)(DG) (DC)(DC) |
-Macromolecule #4: DNA non-target strand
Macromolecule | Name: DNA non-target strand / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: unidentified (others) |
Molecular weight | Theoretical: 18.557873 KDa |
Sequence | String: (DC)(DG)(DC)(DC)(DC)(DC)(DA)(DC)(DG)(DA) (DG)(DG)(DG)(DT)(DA)(DC)(DG)(DG)(DC)(DA) (DA)(DA)(DA)(DG)(DA)(DG)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DG) (DA) (DA)(DG)(DT)(DC)(DG)(DA) ...String: (DC)(DG)(DC)(DC)(DC)(DC)(DA)(DC)(DG)(DA) (DG)(DG)(DG)(DT)(DA)(DC)(DG)(DG)(DC)(DA) (DA)(DA)(DA)(DG)(DA)(DG)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DA)(DC)(DT)(DA)(DG) (DA) (DA)(DG)(DT)(DC)(DG)(DA)(DG)(DG) (DT)(DC)(DA)(DG)(DC)(DC)(DC)(DG)(DT)(DG) (DG)(DC) |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL | |||||||||||||||
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Buffer | pH: 7.25 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Support film - Material: CARBON / Support film - topology: HOLEY ARRAY / Support film - Film thickness: 100 | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: blot for 6.5 seconds before plunging. |
-Electron microscopy
Microscope | TFS KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
-Image processing
Startup model | Type of model: NONE |
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Final reconstruction | Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 71831 |
Initial angle assignment | Type: NOT APPLICABLE |
Final angle assignment | Type: NOT APPLICABLE |