+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23724 | |||||||||
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Title | ATP bound TnsC filament structure from ShCAST system | |||||||||
Map data | Post-processed map (1 layer of filament) | |||||||||
Sample |
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Biological species | Scytonema hofmannii (bacteria) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.7 Å | |||||||||
Authors | Park J / Tsai AWL / Mehrotra E / Kellogg EH | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Science / Year: 2021 Title: Structural basis for target site selection in RNA-guided DNA transposition systems. Authors: Jung-Un Park / Amy Wei-Lun Tsai / Eshan Mehrotra / Michael T Petassi / Shan-Chi Hsieh / Ailong Ke / Joseph E Peters / Elizabeth H Kellogg / Abstract: CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron ...CRISPR-associated transposition systems allow guide RNA-directed integration of a single DNA cargo in one orientation at a fixed distance from a programmable target sequence. We used cryo-electron microscopy (cryo-EM) to define the mechanism that underlies this process by characterizing the transposition regulator, TnsC, from a type V-K CRISPR-transposase system. In this scenario, polymerization of adenosine triphosphate-bound TnsC helical filaments could explain how polarity information is passed to the transposase. TniQ caps the TnsC filament, representing a universal mechanism for target information transfer in Tn7/Tn7-like elements. Transposase-driven disassembly establishes delivery of the element only to unused protospacers. Finally, TnsC transitions to define the fixed point of insertion, as revealed by structures with the transition state mimic ADP•AlF These mechanistic findings provide the underpinnings for engineering CRISPR-associated transposition systems for research and therapeutic applications. | |||||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_23724.map.gz | 4.2 MB | EMDB map data format | |
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Header (meta data) | emd-23724-v30.xml emd-23724.xml | 22.5 KB 22.5 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_23724_fsc.xml | 9.1 KB | Display | FSC data file |
Images | emd_23724.png | 83.9 KB | ||
Masks | emd_23724_msk_1.map | 64 MB | Mask map | |
Others | emd_23724_additional_1.map.gz emd_23724_half_map_1.map.gz emd_23724_half_map_2.map.gz | 49.2 MB 49.6 MB 49.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23724 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23724 | HTTPS FTP |
-Validation report
Summary document | emd_23724_validation.pdf.gz | 488.1 KB | Display | EMDB validaton report |
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Full document | emd_23724_full_validation.pdf.gz | 487.6 KB | Display | |
Data in XML | emd_23724_validation.xml.gz | 15.9 KB | Display | |
Data in CIF | emd_23724_validation.cif.gz | 20.6 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23724 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23724 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_23724.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Post-processed map (1 layer of filament) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.33 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Mask #1
File | emd_23724_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: Full map
File | emd_23724_additional_1.map | ||||||||||||
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Annotation | Full map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: Half map 1
File | emd_23724_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_23724_half_map_2.map | ||||||||||||
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Annotation | Half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Helical filament of TnsC monomers bound to DNA
Entire | Name: Helical filament of TnsC monomers bound to DNA |
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Components |
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-Supramolecule #1: Helical filament of TnsC monomers bound to DNA
Supramolecule | Name: Helical filament of TnsC monomers bound to DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Reconstituted with ATP |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Molecular weight | Theoretical: 45.4 kDa/nm |
-Macromolecule #1: TnsC
Macromolecule | Name: TnsC / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTVP VVYIRPHQKC GPKDLFKKIT EYLKYRVTKG TVSDFRDRTI EVLKGCGVEM LIIDEADRLK PETFADVRDI AEDLGIAVVL ...String: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTVP VVYIRPHQKC GPKDLFKKIT EYLKYRVTKG TVSDFRDRTI EVLKGCGVEM LIIDEADRLK PETFADVRDI AEDLGIAVVL VGTDRLDAVI KRDEQVLERF RAHLRFGKLS GEDFKNTVEM WEQMVLKLPV SSNLKSKEML RILTSATEGY IGRLDEILRE AAIRSLSRGL KKIDKAVLQE VAKEYK |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | filament |
-Sample preparation
Concentration | 1 mg/mL | |||||||||||||||||||||
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Buffer | pH: 7.4 Component:
Details: Sample was diluted into low salt using dialysis. | |||||||||||||||||||||
Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.038 kPa | |||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Blot for 6 seconds before plunging. | |||||||||||||||||||||
Details | This sample comprised monodisperse filaments reconstituted using dialysis. |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Digitization - Dimensions - Width: 5760 pixel / Digitization - Dimensions - Height: 4092 pixel / Number grids imaged: 1 / Number real images: 2910 / Average exposure time: 3.0 sec. / Average electron dose: 45.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 63000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |