+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15344 | |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Title | Type V-K CAST TnsB bound to LTR-SR | |||||||||||||||
Map data | ||||||||||||||||
Sample |
| |||||||||||||||
Biological species | Scytonema hofmannii (bacteria) | |||||||||||||||
Method | single particle reconstruction / cryo EM / Resolution: 11.2 Å | |||||||||||||||
Authors | Tenjo-Castano F / Sofos N / Lopez-Mendez B / Stutzke LS / Fuglsang A / Stella S / Montoya G | |||||||||||||||
Funding support | Denmark, 4 items
| |||||||||||||||
Citation | Journal: Nat Commun / Year: 2022 Title: Structure of the TnsB transposase-DNA complex of type V-K CRISPR-associated transposon. Authors: Francisco Tenjo-Castaño / Nicholas Sofos / Blanca López-Méndez / Luisa S Stutzke / Anders Fuglsang / Stefano Stella / Guillermo Montoya / Abstract: CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema ...CRISPR-associated transposons (CASTs) are mobile genetic elements that co-opted CRISPR-Cas systems for RNA-guided transposition. Here we present the 2.4 Å cryo-EM structure of the Scytonema hofmannii (sh) TnsB transposase from Type V-K CAST, bound to the strand transfer DNA. The strand transfer complex displays an intertwined pseudo-symmetrical architecture. Two protomers involved in strand transfer display a catalytically competent active site composed by DDE residues, while other two, which play a key structural role, show active sites where the catalytic residues are not properly positioned for phosphodiester hydrolysis. Transposon end recognition is accomplished by the NTD1/2 helical domains. A singular in trans association of NTD1 domains of the catalytically competent subunits with the inactive DDE domains reinforces the assembly. Collectively, the structural features suggest that catalysis is coupled to protein-DNA assembly to secure proper DNA integration. DNA binding residue mutants reveal that lack of specificity decreases activity, but it could increase transposition in some cases. Our structure sheds light on the strand transfer reaction of DDE transposases and offers new insights into CAST transposition. | |||||||||||||||
History |
|
-Structure visualization
Supplemental images |
---|
-Downloads & links
-EMDB archive
Map data | emd_15344.map.gz | 118.5 MB | EMDB map data format | |
---|---|---|---|---|
Header (meta data) | emd-15344-v30.xml emd-15344.xml | 19.5 KB 19.5 KB | Display Display | EMDB header |
Images | emd_15344.png | 25.2 KB | ||
Others | emd_15344_half_map_1.map.gz emd_15344_half_map_2.map.gz | 226.8 MB 226.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15344 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15344 | HTTPS FTP |
-Validation report
Summary document | emd_15344_validation.pdf.gz | 708.8 KB | Display | EMDB validaton report |
---|---|---|---|---|
Full document | emd_15344_full_validation.pdf.gz | 708.3 KB | Display | |
Data in XML | emd_15344_validation.xml.gz | 16 KB | Display | |
Data in CIF | emd_15344_validation.cif.gz | 18.9 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15344 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-15344 | HTTPS FTP |
-Related structure data
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
---|
-Map
File | Download / File: emd_15344.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Voxel size | X=Y=Z: 0.98 Å | ||||||||||||||||||||
Density |
| ||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Half map: #1
File | emd_15344_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: #2
File | emd_15344_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Sample components
-Entire : Type V-K CAST TnsB bound to LTR-SR
Entire | Name: Type V-K CAST TnsB bound to LTR-SR |
---|---|
Components |
|
-Supramolecule #1: Type V-K CAST TnsB bound to LTR-SR
Supramolecule | Name: Type V-K CAST TnsB bound to LTR-SR / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all |
---|---|
Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Molecular weight | Experimental: 359 KDa |
-Macromolecule #1: TnsB
Macromolecule | Name: TnsB / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQDG LVGLTQTSRA DKGKHRIGEF WEN FITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV LAPILEKQQK ...String: MNSQQNPDLA VHPLAIPMEG LLGESATTLE KNVIATQLSE EAQVKLEVIQ SLLEPCDRTT YGQKLREAAE KLNVSLRTVQ RLVKNWEQDG LVGLTQTSRA DKGKHRIGEF WEN FITKTY KEGNKGSKRM TPKQVALRVE AKARELKDSK PPNYKTVLRV LAPILEKQQK AKSIRSPGWR GTTLSVKTRE GKDLSVDYSN HVWQCDHTRV DVLLVDQHGE ILSRPWLT T VIDTYSRCIM GINLGFDAPS SGVVALALRH AILPKRYGSE YKLHCEWGTY GKPEHFYTDG GKDFRSNHLS QIGAQLGFVC HLRDRPSEGG VVERPFKTLN DQLFSTLPGY TGSN VQERP EDAEKDARLT LRELEQLLVR YIVDRYNQSI DARMGDQTRF ERWEAGLPTV PVPIPERDLD ICLMKQSRRT VQRGGCLQFQ NLMYRGEYLA GYAGETVNLR FDPRDITTI LVYRQENNQE VFLTRAHAQG LETEQLALDE AEAASRRLRT AGKTISNQSL LQEVVDRDAL VATKKSRKER QKLEQTVLRS AAVDESNRES LPSQIVEPDE VESTETVHSQ YEDIEVWDYE QLREEYGFGS EFELENLYFQ |
-Macromolecule #2: RightElement
Macromolecule | Name: RightElement / type: dna / ID: 2 / Classification: DNA |
---|---|
Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Sequence | String: TGCTACGTCT CTACGTGTAC AGTGACTAAT TATATGTCGT TGTGACAAAT TATTGTCATC AGTAAAATCC TTATACAGTA TAGATTATAG CGCTTTGGCA GTTTTAGCAT AACCTCTTTG CAGTGACAAA ATAGATGTCG TTGTCCGTGA TTGTGACAAA TTAGCTGTCG ...String: TGCTACGTCT CTACGTGTAC AGTGACTAAT TATATGTCGT TGTGACAAAT TATTGTCATC AGTAAAATCC TTATACAGTA TAGATTATAG CGCTTTGGCA GTTTTAGCAT AACCTCTTTG CAGTGACAAA ATAGATGTCG TTGTCCGTGA TTGTGACAAA TTAGCTGTCG CTTTGCAAGA TAGGAAAAAG CTTTTGTGTA TTTTCATAAT GACAAATTGA CTGTCGCAGG AGGTAA |
-Macromolecule #3: LeftElement
Macromolecule | Name: LeftElement / type: dna / ID: 3 / Classification: DNA |
---|---|
Source (natural) | Organism: Scytonema hofmannii (bacteria) |
Sequence | String: ATTATATTGA TGACATTTAA TTTGTCATCA ATTAATTAAG CAACGCTGAT GGGTCACGAC GACAATTAAA TAGTCACAAT GACATTAATC TGTCACCGAC GACAGATAAT TTGTCACTGT ACACTACGCC TTTTGTGG |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 Component:
| |||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | TFS GLACIOS |
---|---|
Image recording | Film or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 738 / Average exposure time: 40.0 sec. / Average electron dose: 40.0 e/Å2 |
Electron beam | Acceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.9 µm / Nominal magnification: 150000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL / Protocol: AB INITIO MODEL |
---|