+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-26348 | |||||||||
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Title | I-F3b Cascade-TniQ full R-loop complex | |||||||||
Map data | combined map of local-resolution filtered maps from full refinement and focused refinement | |||||||||
Sample |
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Keywords | CRISPR-Cas / Transposon / CAST / Cascade / I-F / I-F3 / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA-RNA complex | |||||||||
Biological species | Aeromonas salmonicida (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.52 Å | |||||||||
Authors | Park JU / Mehrotra E / Kellogg EH | |||||||||
Funding support | United States, 2 items
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Citation | Journal: Mol Cell / Year: 2023 Title: Multiple adaptations underly co-option of a CRISPR surveillance complex for RNA-guided DNA transposition. Authors: Jung-Un Park / Michael T Petassi / Shan-Chi Hsieh / Eshan Mehrotra / Gabriel Schuler / Jagat Budhathoki / Vinh H Truong / Summer B Thyme / Ailong Ke / Elizabeth H Kellogg / Joseph E Peters / Abstract: CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA- ...CRISPR-associated transposons (CASTs) are natural RNA-directed transposition systems. We demonstrate that transposon protein TniQ plays a central role in promoting R-loop formation by RNA-guided DNA-targeting modules. TniQ residues, proximal to CRISPR RNA (crRNA), are required for recognizing different crRNA categories, revealing an unappreciated role of TniQ to direct transposition into different classes of crRNA targets. To investigate adaptations allowing CAST elements to utilize attachment sites inaccessible to CRISPR-Cas surveillance complexes, we compared and contrasted PAM sequence requirements in both I-F3b CAST and I-F1 CRISPR-Cas systems. We identify specific amino acids that enable a wider range of PAM sequences to be accommodated in I-F3b CAST elements compared with I-F1 CRISPR-Cas, enabling CAST elements to access attachment sites as sequences drift and evade host surveillance. Together, this evidence points to the central role of TniQ in facilitating the acquisition of CRISPR effector complexes for RNA-guided DNA transposition. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_26348.map.gz | 7.9 MB | EMDB map data format | |
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Header (meta data) | emd-26348-v30.xml emd-26348.xml | 31.7 KB 31.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_26348_fsc.xml | 15 KB | Display | FSC data file |
Images | emd_26348.png | 81.8 KB | ||
Filedesc metadata | emd-26348.cif.gz | 7.7 KB | ||
Others | emd_26348_additional_1.map.gz emd_26348_additional_2.map.gz emd_26348_additional_3.map.gz emd_26348_additional_4.map.gz emd_26348_half_map_1.map.gz emd_26348_half_map_2.map.gz | 225.1 MB 51.6 MB 51.9 MB 51.9 MB 225.5 MB 225.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-26348 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-26348 | HTTPS FTP |
-Validation report
Summary document | emd_26348_validation.pdf.gz | 881.2 KB | Display | EMDB validaton report |
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Full document | emd_26348_full_validation.pdf.gz | 880.8 KB | Display | |
Data in XML | emd_26348_validation.xml.gz | 21.9 KB | Display | |
Data in CIF | emd_26348_validation.cif.gz | 28.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26348 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-26348 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_26348.map.gz / Format: CCP4 / Size: 282.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Annotation | combined map of local-resolution filtered maps from full refinement and focused refinement | ||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.873 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: unsharpened map of the full structure
File | emd_26348_additional_1.map | ||||||||||||
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Annotation | unsharpened map of the full structure | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: Unsharpened map from focused refinement
File | emd_26348_additional_2.map | ||||||||||||
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Annotation | Unsharpened map from focused refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: halfmap 1 from focused refinement
File | emd_26348_additional_3.map | ||||||||||||
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Annotation | halfmap 1 from focused refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: halfmap 2 from focused refinement
File | emd_26348_additional_4.map | ||||||||||||
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Annotation | halfmap 2 from focused refinement | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Halfmap 2 of the full structure
File | emd_26348_half_map_1.map | ||||||||||||
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Annotation | Halfmap 2 of the full structure | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Halfmap 1 of the full structure
File | emd_26348_half_map_2.map | ||||||||||||
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Annotation | Halfmap 1 of the full structure | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : I-F3b Cascade-TniQ Full R-loop complex
Entire | Name: I-F3b Cascade-TniQ Full R-loop complex |
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Components |
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-Supramolecule #1: I-F3b Cascade-TniQ Full R-loop complex
Supramolecule | Name: I-F3b Cascade-TniQ Full R-loop complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 450 KDa |
-Macromolecule #1: crRNA
Macromolecule | Name: crRNA / type: rna / ID: 1 / Number of copies: 1 |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 19.399604 KDa |
Sequence | String: CCAAGAAAAG GACUGGAAGA AAUCAUCCAA GUUGGGGACU AUUUUCUGCC GUAUAGGCAG |
-Macromolecule #2: Target strand DNA
Macromolecule | Name: Target strand DNA / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 35.61368 KDa |
Sequence | String: (DC)(DT)(DG)(DG)(DC)(DT)(DG)(DG)(DC)(DG) (DA)(DA)(DC)(DG)(DA)(DG)(DC)(DG)(DC)(DA) (DA)(DG)(DG)(DT)(DG)(DG)(DT)(DG)(DG) (DC)(DC)(DC)(DC)(DA)(DT)(DC)(DA)(DG)(DC) (DC) (DA)(DC)(DA)(DT)(DC)(DC) ...String: (DC)(DT)(DG)(DG)(DC)(DT)(DG)(DG)(DC)(DG) (DA)(DA)(DC)(DG)(DA)(DG)(DC)(DG)(DC)(DA) (DA)(DG)(DG)(DT)(DG)(DG)(DT)(DG)(DG) (DC)(DC)(DC)(DC)(DA)(DT)(DC)(DA)(DG)(DC) (DC) (DA)(DC)(DA)(DT)(DC)(DC)(DC)(DG) (DG)(DC)(DA)(DC)(DT)(DC)(DG)(DA)(DA)(DG) (DT)(DC) (DC)(DC)(DC)(DA)(DA)(DC)(DT) (DT)(DG)(DG)(DA)(DT)(DG)(DA)(DT)(DT)(DT) (DC)(DT)(DT) (DC)(DC)(DA)(DG)(DT)(DC) (DC)(DT)(DG)(DG)(DT)(DA)(DA)(DG)(DC)(DA) (DC)(DC)(DC)(DG) (DA)(DA)(DT)(DC)(DA) (DT)(DC)(DC)(DT)(DC)(DT)(DT)(DG)(DC)(DG) (DG) |
-Macromolecule #3: Non-target strand DNA
Macromolecule | Name: Non-target strand DNA / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 35.737723 KDa |
Sequence | String: (DC)(DC)(DG)(DC)(DA)(DA)(DG)(DA)(DG)(DG) (DA)(DT)(DG)(DA)(DT)(DT)(DC)(DG)(DG)(DG) (DT)(DG)(DC)(DT)(DT)(DA)(DC)(DC)(DT) (DC)(DC)(DT)(DG)(DA)(DC)(DC)(DT)(DT)(DC) (DT) (DT)(DT)(DA)(DG)(DT)(DA) ...String: (DC)(DC)(DG)(DC)(DA)(DA)(DG)(DA)(DG)(DG) (DA)(DT)(DG)(DA)(DT)(DT)(DC)(DG)(DG)(DG) (DT)(DG)(DC)(DT)(DT)(DA)(DC)(DC)(DT) (DC)(DC)(DT)(DG)(DA)(DC)(DC)(DT)(DT)(DC) (DT) (DT)(DT)(DA)(DG)(DT)(DA)(DG)(DG) (DT)(DT)(DC)(DA)(DA)(DC)(DC)(DC)(DC)(DT) (DG)(DA) (DT)(DC)(DG)(DA)(DG)(DT)(DG) (DC)(DC)(DG)(DG)(DG)(DA)(DT)(DG)(DT)(DG) (DG)(DC)(DT) (DG)(DA)(DT)(DG)(DG)(DG) (DG)(DC)(DC)(DA)(DC)(DC)(DA)(DC)(DC)(DT) (DT)(DG)(DC)(DG) (DC)(DT)(DC)(DG)(DT) (DT)(DC)(DG)(DC)(DC)(DA)(DG)(DC)(DC)(DA) (DG) |
-Macromolecule #4: Cas8/5
Macromolecule | Name: Cas8/5 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 78.728367 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MVTIMHIEEL LDIEDHGERD RQLRRYLAPY SAEIGVDGAE KMALVVLLNL TLKRDRVESL CDEGLARQLL SDEGHITNCL HTVRWLHTH NLKYPDARVS GERLIINAPP LIPGVISSAG LPMRMGWAHD SSDINLAKLF GTSFRYRDDS TNLALQLVAR S KTWEQALI ...String: MVTIMHIEEL LDIEDHGERD RQLRRYLAPY SAEIGVDGAE KMALVVLLNL TLKRDRVESL CDEGLARQLL SDEGHITNCL HTVRWLHTH NLKYPDARVS GERLIINAPP LIPGVISSAG LPMRMGWAHD SSDINLAKLF GTSFRYRDDS TNLALQLVAR S KTWEQALI GLGLTQQQLD IWCQLLASNL ENNTFPTVVS PFSKQVRFLY QGNYCVVTPV VSHALLAQLQ NVVHEKKLQC TY IHHDHPA SVGSLVGALG GKVAVLDYPP PVSPDKARSF SQARKHRLAN GQSLFDRSVF NDHVFIDALK HVISRPGLTR KQQ RQLRLS ALRYLRRQLA IWLGPIIEWR DEIVSSGRGE PGNLPSGGLE LELITQPKKM LPELMLQVAG RFHLELQNHS AGRR FAFHP ALMAPIKSQI LWLLRQLADD EEKDEPHPPT SCYYLHLSGL TVYDASALAN PYLCGIPSLS ALAGFCHDYE RRLQS LIGQ SVYFRGLAWY LGRYSLVTGK HLPEPSKSAD PKSVSAIRRP GLLDGRYCDL GMDLIIEVHI PTGGSLPFTT CLDLLR VAL PARFAGGCLH PPSLYEEYNW CTVYQDKSTL FTVLSRLPRY GCWIYPSDAD LRSFEELSEA LALDRRLRPV ATGFVFL EE PVERAGSIEG QHVYAESAIG TALCINPVEM RLAGKKRFFG AGFWQLNDAK GAILMNGSAN TG |
-Macromolecule #5: Cas7
Macromolecule | Name: Cas7 / type: protein_or_peptide / ID: 5 / Number of copies: 6 / Enantiomer: LEVO |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 39.105172 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MELCTHLSYS RSLSPGKAVF FYKTAESDFV PLRIEVAKIS GQKCGYTEGF DANLKPKNIE RYELAYSNPQ TIEACYVPPN VDELYCRFS LRVEANSMRP YVCSNPDVLR VMIGLAQAYQ RLGGYNELAR RYSANVLRGI WLWRNQYTQG TKIEIKTSLG S TYHIPDAR ...String: MELCTHLSYS RSLSPGKAVF FYKTAESDFV PLRIEVAKIS GQKCGYTEGF DANLKPKNIE RYELAYSNPQ TIEACYVPPN VDELYCRFS LRVEANSMRP YVCSNPDVLR VMIGLAQAYQ RLGGYNELAR RYSANVLRGI WLWRNQYTQG TKIEIKTSLG S TYHIPDAR RLSWSGDWPE LEQKQLEQLT SEMAKALSQP DIFWFADVTA SLKTGFCQEI FPSQKFTERP DDHSVASRQL AT VECSDGQ LAACINPQKI GAALQKIDDW WANDADLPLR VHEYGANHEA LTALRHPATG QDFYHLLTKA EQFVTVLESS EGG GVELPG EVHYLMAVLV KGGLFQKGKG R |
-Macromolecule #6: Cas6
Macromolecule | Name: Cas6 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 23.640832 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MTENRYFFAI RYLSDDVDCG LLAGRCISIL HGFRQAHPGI QIGVAFPEWS DRDLGRSIAF VSTNKSLLER FRERSYFQVM QADNFFALS LVLEVPDTCQ NVRFIRNQNL AKLFVGERRR RLARAKRRAK ARGEAFQPHM PDETKVVGVF HSVFMQSASS G QSYILHIQ ...String: MTENRYFFAI RYLSDDVDCG LLAGRCISIL HGFRQAHPGI QIGVAFPEWS DRDLGRSIAF VSTNKSLLER FRERSYFQVM QADNFFALS LVLEVPDTCQ NVRFIRNQNL AKLFVGERRR RLARAKRRAK ARGEAFQPHM PDETKVVGVF HSVFMQSASS G QSYILHIQ KHRYERSEDS GYSSYGLASN DLYTGYVPDL GAIFSTLF |
-Macromolecule #7: TniQ
Macromolecule | Name: TniQ / type: protein_or_peptide / ID: 7 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Aeromonas salmonicida (bacteria) |
Molecular weight | Theoretical: 46.366637 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MHLLVRPEPF ADEALESYFL RLSQENGFER YRIFSGSVQD WLHTTDHAAA GAFPLELSRL NIFHASRSSG LRVRALQLVD RLTDGAPFR LLQLALCHSA ISFGNHYKAV HRSGVDIPLS FIRVHQIPCC PDCLRESAYV RQCWHFKPYV GCHRHGGRLI Y SCPACGES ...String: MHLLVRPEPF ADEALESYFL RLSQENGFER YRIFSGSVQD WLHTTDHAAA GAFPLELSRL NIFHASRSSG LRVRALQLVD RLTDGAPFR LLQLALCHSA ISFGNHYKAV HRSGVDIPLS FIRVHQIPCC PDCLRESAYV RQCWHFKPYV GCHRHGGRLI Y SCPACGES LNYLASESIN HCQCGFDLRT ASTVPAQPDE IQLSALAYGC SFESSNPLLA IGSLSARFGA LYWYQQRYLS DH EAVRDDR ALTKAIGHFT AWPDAFWREL QQMVDDALVR QTKPLNHTDF VDVFGSVVAD CRQIPMRNTG QNFILKNLIG FLT DLVARH PQCRVANVGD LLLSAVDAAT LLSTSVEQVR RLHHEGFLPL SIRPASRNTV SPHRAVFHLR HVVELRQARM QSHH DHSST YLPAW |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.9 mg/mL | |||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: COPPER / Pretreatment - Type: GLOW DISCHARGE | |||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 60.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / 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 |