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Open data
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Basic information
Entry | Database: EMDB / ID: EMD-23726 | |||||||||
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Title | ATP-bound TnsC-TniQ complex from ShCAST system | |||||||||
![]() | local resolution filtered map | |||||||||
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Biological species | ![]() | |||||||||
Method | ![]() ![]() | |||||||||
![]() | Park J / Tsai AWL / Mehrotra E / Kellogg EH | |||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure viewer | EM map: ![]() ![]() ![]() |
Supplemental images |
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Downloads & links
-EMDB archive
Map data | ![]() | 1.9 MB | ![]() | |
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Header (meta data) | ![]() ![]() | 25.8 KB 25.8 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 9.1 KB | Display | ![]() |
Images | ![]() | 70.5 KB | ||
Masks | ![]() | 64 MB | ![]() | |
Others | ![]() ![]() ![]() ![]() | 49.7 MB 5.6 MB 49.7 MB 49.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 7n6iMC ![]() 7m99C ![]() 7m9aC ![]() 7m9bC ![]() 7m9cC C: citing same article ( M: atomic model generated by this map |
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Similar structure data |
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Links
EMDB pages | ![]() ![]() |
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Map
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Annotation | local resolution 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
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-Additional map: Full map
File | emd_23726_additional_1.map | ||||||||||||
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Annotation | Full map | ||||||||||||
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Density Histograms |
-Additional map: sharpened map using uniform B-factor
File | emd_23726_additional_2.map | ||||||||||||
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Annotation | sharpened map using uniform B-factor | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: half map 1
File | emd_23726_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_23726_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 : Helical filament of TnsC monomers bound to DNA and TniQ
Entire | Name: Helical filament of TnsC monomers bound to DNA and TniQ |
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Components |
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-Supramolecule #1: Helical filament of TnsC monomers bound to DNA and TniQ
Supramolecule | Name: Helical filament of TnsC monomers bound to DNA and TniQ type: complex / ID: 1 / Parent: 0 / Macromolecule list: all / Details: Reconstituted with ATP |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() ![]() |
Molecular weight | Theoretical: 400 KDa |
-Macromolecule #1: TnsC
Macromolecule | Name: TnsC / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() ![]() |
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 |
-Macromolecule #2: TniQ
Macromolecule | Name: TniQ / type: protein_or_peptide / ID: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: ![]() |
Recombinant expression | Organism: ![]() ![]() ![]() |
Sequence | String: MIEAPDVKPW LFLIKPYEGE SLSHFLGRFR RANHLSASGL GTLAGIGAIV ARWERFHFNP RPSQQELEAI ASVVEVAAQR LAQMLPPAGV GMQHEPIRLC GACYAESPCH RIEWQYKSVW KCDRHQLKIL AKCPNCQAPF KMPALWEDGC CHRCRMPFAE MAKLQKV |
-Experimental details
-Structure determination
Method | ![]() |
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Aggregation state | particle |
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Sample preparation
Concentration | 1 mg/mL | |||||||||||||||||||||
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Buffer | pH: 7.4 Component:
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Grid | Model: UltrAuFoil R2/2 / 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 / 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 7 seconds before plunging. | |||||||||||||||||||||
Details | Excess of TniQ was added to ATP-bound TnsC filaments |
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Electron microscopy
Microscope | FEI TALOS ARCTICA |
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Electron beam | Acceleration voltage: 200 kV / Electron source: ![]() |
Electron optics | C2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD![]() |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
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: 4172 / Average exposure time: 3.0 sec. / Average electron dose: 50.0 e/Å2 |
Experimental equipment | ![]() Model: Talos Arctica / Image courtesy: FEI Company |
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Image processing
-Atomic model buiding 1
Refinement | Protocol: RIGID BODY FIT |
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Output model | ![]() PDB-7n6i: |