+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-25454 | |||||||||
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Title | TnsBctd-TnsC complex | |||||||||
Map data | Relion sharpened map | |||||||||
Sample |
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Keywords | CAST / transposase / AAA+ ATPase / AAA+ / CRISPR / Cas / DNA BINDING PROTEIN-DNA complex | |||||||||
Biological species | [Scytonema hofmanni] UTEX 2349 (bacteria) / synthetic construct (others) | |||||||||
Method | helical reconstruction / cryo EM / Resolution: 3.54 Å | |||||||||
Authors | Park J / Tsai AWT | |||||||||
Funding support | United States, 1 items
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Citation | Journal: Proc Natl Acad Sci U S A / Year: 2022 Title: Mechanistic details of CRISPR-associated transposon recruitment and integration revealed by cryo-EM. Authors: Jung-Un Park / Amy Wei-Lun Tsai / Tiffany H Chen / Joseph E Peters / Elizabeth H Kellogg / Abstract: CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the ...CRISPR-associated transposons (CASTs) are Tn7-like elements that are capable of RNA-guided DNA integration. Although structural data are known for nearly all core transposition components, the transposase component, TnsB, remains uncharacterized. Using cryo-electron microscopy (cryo-EM) structure determination, we reveal the conformation of TnsB during transposon integration for the type V-K CAST system from (ShCAST). Our structure of TnsB is a tetramer, revealing strong mechanistic relationships with the overall architecture of RNaseH transposases/integrases in general, and in particular the MuA transposase from bacteriophage Mu. However, key structural differences in the C-terminal domains indicate that TnsB's tetrameric architecture is stabilized by a different set of protein-protein interactions compared with MuA. We describe the base-specific interactions along the TnsB binding site, which explain how different CAST elements can function on cognate mobile elements independent of one another. We observe that melting of the 5' nontransferred strand of the transposon end is a structural feature stabilized by TnsB and furthermore is crucial for donor-DNA integration. Although not observed in the TnsB strand-transfer complex, the C-terminal end of TnsB serves a crucial role in transposase recruitment to the target site. The C-terminal end of TnsB adopts a short, structured 15-residue "hook" that decorates TnsC filaments. Unlike full-length TnsB, C-terminal fragments do not appear to stimulate filament disassembly using two different assays, suggesting that additional interactions between TnsB and TnsC are required for redistributing TnsC to appropriate targets. The structural information presented here will help guide future work in modifying these important systems as programmable gene integration tools. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_25454.map.gz | 7.1 MB | EMDB map data format | |
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Header (meta data) | emd-25454-v30.xml emd-25454.xml | 22.8 KB 22.8 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_25454_fsc.xml | 9.1 KB | Display | FSC data file |
Images | emd_25454.png | 101 KB | ||
Masks | emd_25454_msk_1.map | 64 MB | Mask map | |
Filedesc metadata | emd-25454.cif.gz | 6.4 KB | ||
Others | emd_25454_additional_1.map.gz emd_25454_half_map_1.map.gz emd_25454_half_map_2.map.gz | 48.6 MB 49.3 MB 49.3 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-25454 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-25454 | HTTPS FTP |
-Validation report
Summary document | emd_25454_validation.pdf.gz | 807 KB | Display | EMDB validaton report |
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Full document | emd_25454_full_validation.pdf.gz | 806.6 KB | Display | |
Data in XML | emd_25454_validation.xml.gz | 15.8 KB | Display | |
Data in CIF | emd_25454_validation.cif.gz | 20.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25454 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-25454 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_25454.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
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Annotation | Relion sharpened map | ||||||||||||||||||||
Voxel size | X=Y=Z: 1.33 Å | ||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_25454_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Additional map: full map
File | emd_25454_additional_1.map | ||||||||||||
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Annotation | full map | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: halfmap 1
File | emd_25454_half_map_1.map | ||||||||||||
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Annotation | halfmap 1 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Half map: halfmap 2
File | emd_25454_half_map_2.map | ||||||||||||
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Annotation | halfmap 2 | ||||||||||||
Projections & Slices |
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Density Histograms |
-Sample components
-Entire : AMPPNP-bound TnsBctd-TnsC filament from ShCAST element
Entire | Name: AMPPNP-bound TnsBctd-TnsC filament from ShCAST element |
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Components |
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-Supramolecule #1: AMPPNP-bound TnsBctd-TnsC filament from ShCAST element
Supramolecule | Name: AMPPNP-bound TnsBctd-TnsC filament from ShCAST element type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: [Scytonema hofmanni] UTEX 2349 (bacteria) |
Molecular weight | Theoretical: 400 KDa |
-Macromolecule #1: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP...
Macromolecule | Name: DNA (5'-D(P*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*TP*T)-3') type: dna / ID: 1 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 5.734706 KDa |
Sequence | String: (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) (DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT)(DT) |
-Macromolecule #2: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP...
Macromolecule | Name: DNA (5'-D(P*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*AP*A)-3') type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 6.845593 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) |
-Macromolecule #3: TnsC filament
Macromolecule | Name: TnsC filament / type: protein_or_peptide / ID: 3 / Number of copies: 10 / Enantiomer: LEVO |
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Source (natural) | Organism: [Scytonema hofmanni] UTEX 2349 (bacteria) |
Molecular weight | Theoretical: 31.444617 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
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 #4: TnsBctd
Macromolecule | Name: TnsBctd / type: protein_or_peptide / ID: 4 / Number of copies: 10 / Enantiomer: LEVO |
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Source (natural) | Organism: [Scytonema hofmanni] UTEX 2349 (bacteria) |
Molecular weight | Theoretical: 1.97711 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: IEVWDYEQLR EEYGF |
-Macromolecule #5: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 5 / Number of copies: 10 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ChemComp-ANP: |
-Macromolecule #6: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 10 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | helical reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1 mg/mL | |||||||||||||||||||||
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Buffer | pH: 7.5 Component:
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Grid | Model: UltrAuFoil R1.2/1.3 / Support film - Material: GOLD / Support film - topology: HOLEY | |||||||||||||||||||||
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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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 50.0 e/Å2 |
Electron beam | Acceleration voltage: 200 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 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |