+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23722 | |||||||||
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Title | ADP-AlF3 bound TnsC structure in closed form | |||||||||
Map data | local filtered map | |||||||||
Sample |
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Biological species | Scytonema hofmannii (bacteria) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.8 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_23722.map.gz | 1.7 MB | EMDB map data format | |
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Header (meta data) | emd-23722-v30.xml emd-23722.xml | 26.4 KB 26.4 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_23722_fsc.xml | 9.1 KB | Display | FSC data file |
Images | emd_23722.png | 135.2 KB | ||
Masks | emd_23722_msk_1.map | 64 MB | Mask map | |
Others | emd_23722_additional_1.map.gz emd_23722_additional_2.map.gz emd_23722_half_map_1.map.gz emd_23722_half_map_2.map.gz | 49.7 MB 6 MB 49.7 MB 49.7 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23722 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23722 | HTTPS FTP |
-Related structure data
Related structure data | 7m9bMC 7m99C 7m9aC 7m9cC 7n6iC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_23722.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | local filtered map | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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_23722_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: full map
File | emd_23722_additional_1.map | ||||||||||||
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Annotation | full map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Additional map: sharpened map
File | emd_23722_additional_2.map | ||||||||||||
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Annotation | sharpened map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 1
File | emd_23722_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_23722_half_map_2.map | ||||||||||||
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Annotation | half map 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Head to head dimer of TnsC hexamers bound to DNA
Entire | Name: Head to head dimer of TnsC hexamers bound to DNA |
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Components |
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-Supramolecule #1: Head to head dimer of TnsC hexamers bound to DNA
Supramolecule | Name: Head to head dimer of TnsC hexamers bound to DNA / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 / Details: Reconstituted with ADP-AlF3, in closed state |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Molecular weight | Theoretical: 400 KDa |
-Macromolecule #1: TnsC
Macromolecule | Name: TnsC / type: protein_or_peptide / ID: 1 / Number of copies: 12 / Enantiomer: LEVO |
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Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Molecular weight | Theoretical: 31.444617 KDa |
Recombinant expression | Organism: Escherichia coli BL21 (bacteria) |
Sequence | String: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV ...String: MTEAQAIAKQ LGGVKPDDEW LQAEIARLKG KSIVPLQQVK TLHDWLDGKR KARKSCRVVG ESRTGKTVAC DAYRYRHKPQ QEAGRPPTV PVVYIRPHQK CGPKDLFKKI TEYLKYRVTK GTVSDFRDRT IEVLKGCGVE MLIIDEADRL KPETFADVRD I AEDLGIAV VLVGTDRLDA VIKRDEQVLE RFRAHLRFGK LSGEDFKNTV EMWEQMVLKL PVSSNLKSKE MLRILTSATE GY IGRLDEI LREAAIRSLS RGLKKIDKAV LQEVAKEYK |
-Macromolecule #2: DNA (27-MER)
Macromolecule | Name: DNA (27-MER) / type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.411627 KDa |
Sequence | String: (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA)(DA) (DA)(DA)(DA)(DA)(DA)(DA)(DA) |
-Macromolecule #3: DNA (27-MER)
Macromolecule | Name: DNA (27-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 8.168248 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT) |
-Macromolecule #4: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 4 / Number of copies: 10 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 10 mg/mL | |||||||||||||||||||||||||||
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Buffer | pH: 7.4 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Support film - Film thickness: 50.0 nm / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR | |||||||||||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TALOS ARCTICA |
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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 FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 63000 |
Specialist optics | Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV |
Sample stage | Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 45.0 e/Å2 |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |