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Yorodumi- EMDB-40221: CryoEM structure of the TnsC(1-503)-TnsD(1-318)-DNA complex in a ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-40221 | |||||||||
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Title | CryoEM structure of the TnsC(1-503)-TnsD(1-318)-DNA complex in a 7:2:1 stoichiometry from E. coli Tn7 | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Transposon / AAA+ ATPase / Oligomer / Complex / DNA BINDING PROTEIN / DNA BINDING PROTEIN-DNA complex | |||||||||
Function / homology | Function and homology information transposition / DNA recombination / ATP hydrolysis activity / DNA binding / ATP binding Similarity search - Function | |||||||||
Biological species | Escherichia coli (E. coli) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.51 Å | |||||||||
Authors | Shen Y / Guarne A | |||||||||
Funding support | Canada, 1 items
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Citation | Journal: Mol Cell / Year: 2024 Title: Assembly of the Tn7 targeting complex by a regulated stepwise process. Authors: Yao Shen / Shreya S Krishnan / Michael T Petassi / Mark A Hancock / Joseph E Peters / Alba Guarné / Abstract: The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection ...The Tn7 family of transposons is notable for its highly regulated integration mechanisms, including programmable RNA-guided transposition. The targeting pathways rely on dedicated target selection proteins from the TniQ family and the AAA+ adaptor TnsC to recruit and activate the transposase at specific target sites. Here, we report the cryoelectron microscopy (cryo-EM) structures of TnsC bound to the TniQ domain of TnsD from prototypical Tn7 and unveil key regulatory steps stemming from unique behaviors of ATP- versus ADP-bound TnsC. We show that TnsD recruits ADP-bound dimers of TnsC and acts as an exchange factor to release one protomer with exchange to ATP. This loading process explains how TnsC assembles a heptameric ring unidirectionally from the target site. This unique loading process results in functionally distinct TnsC protomers within the ring, providing a checkpoint for target immunity and explaining how insertions at programmed sites precisely occur in a specific orientation across Tn7 elements. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_40221.map.gz | 227.7 MB | EMDB map data format | |
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Header (meta data) | emd-40221-v30.xml emd-40221.xml | 23.7 KB 23.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_40221_fsc.xml | 13.3 KB | Display | FSC data file |
Images | emd_40221.png | 162.9 KB | ||
Filedesc metadata | emd-40221.cif.gz | 6.8 KB | ||
Others | emd_40221_additional_1.map.gz emd_40221_additional_2.map.gz emd_40221_half_map_1.map.gz emd_40221_half_map_2.map.gz | 230 MB 230 MB 226.6 MB 226.6 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-40221 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-40221 | HTTPS FTP |
-Validation report
Summary document | emd_40221_validation.pdf.gz | 866.2 KB | Display | EMDB validaton report |
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Full document | emd_40221_full_validation.pdf.gz | 865.8 KB | Display | |
Data in XML | emd_40221_validation.xml.gz | 22.6 KB | Display | |
Data in CIF | emd_40221_validation.cif.gz | 28.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40221 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-40221 | HTTPS FTP |
-Related structure data
Related structure data | 8glwMC 8gluC 8glxC 8vcjC 8vctC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data | Similarity search - Function & homologyF&H Search |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_40221.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.855 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Additional map: #2
File | emd_40221_additional_1.map | ||||||||||||
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Density Histograms |
-Additional map: #1
File | emd_40221_additional_2.map | ||||||||||||
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Density Histograms |
-Half map: #2
File | emd_40221_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_40221_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactor...
Entire | Name: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactors ATP and ADP |
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Components |
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-Supramolecule #1: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactor...
Supramolecule | Name: Complex of TnsC(1-503) bound to TnsD(1-318) and DNA with cofactors ATP and ADP type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 550 KDa |
-Macromolecule #1: Transposon Tn7 transposition protein TnsC
Macromolecule | Name: Transposon Tn7 transposition protein TnsC / type: protein_or_peptide / ID: 1 / Number of copies: 7 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 59.318926 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGATRIQAVY RDTGVEAYRD NPFIEALPPL QESVNSAASL KSSLQLTSSD LQKSRVIRAH TICRIPDDYF QPLGTHLLLS ERISVMIRG GYVGRNPKTG DLQKHLQNGY ERVQTGELET FRFEEARSTA QSLLLIGCSG SGKTTSLHRI LATYPQVIYH R ELNVEQVV ...String: MGATRIQAVY RDTGVEAYRD NPFIEALPPL QESVNSAASL KSSLQLTSSD LQKSRVIRAH TICRIPDDYF QPLGTHLLLS ERISVMIRG GYVGRNPKTG DLQKHLQNGY ERVQTGELET FRFEEARSTA QSLLLIGCSG SGKTTSLHRI LATYPQVIYH R ELNVEQVV YLKIDCSHNG SLKEICLNFF RALDRALGSN YERRYGLKRH GIETMLALMS QIANAHALGL LVIDEIQHLS RS RSGGSQE MLNFFVTMVN IIGVPVMLIG TPKAREIFEA DLRSARRGAG FGAIFWDPIQ QTQRGKPNQE WIAFTDNLWQ LQL LQRKDA LLSDEVRDVW YELSQGVMDI VVKLFVLAQL RALALGNERI TAGLLRQVYQ DELKPVHPML EALRSGIPER IARY SDLVV PEIDKRLIQL QLDIAAIQEQ TPEEKALQEL DTEDQRHLYL MLKEDYDSSL LIPTIKKAFS QNPTMTRQKL LPLVL QWLM EGETVVSELE KPSKSKKVSP NSSSVDKLAA ALEHHHHHH UniProtKB: Transposon Tn7 transposition protein TnsC |
-Macromolecule #2: Transposon Tn7 transposition protein TnsD
Macromolecule | Name: Transposon Tn7 transposition protein TnsD / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 36.63602 KDa |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MRNFPVPYSN ELIYSTIARA GVYQGIVSPK QLLDEVYGNR KVVATLGLPS HLGVIARHLH QTGRYAVQQL IYEHTLFPLY APFVGKERR DEAIRLMEYQ AQGAVHLMLG VAASRVKSDN RFRYCPDCVA LQLNRYGEAF WQRDWYLPAL PYCPKHGALV F FDRAVDDH ...String: MRNFPVPYSN ELIYSTIARA GVYQGIVSPK QLLDEVYGNR KVVATLGLPS HLGVIARHLH QTGRYAVQQL IYEHTLFPLY APFVGKERR DEAIRLMEYQ AQGAVHLMLG VAASRVKSDN RFRYCPDCVA LQLNRYGEAF WQRDWYLPAL PYCPKHGALV F FDRAVDDH RHQFWALGHT ELLSDYPKDS LSQLTALAAY IAPLLDAPRA QELSPSLEQW TLFYQRLAQD LGLTKSKHIR HD LVAERVR QTFSDEALEK LDLKLAENKD TCWLKSIFRK HRKAFSYLQH SIVWQALLPK LTVIEALQQA SALTEHSITT R UniProtKB: Transposon Tn7 transposition protein TnsD |
-Macromolecule #3: DNA (50-MER)
Macromolecule | Name: DNA (50-MER) / type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 15.438931 KDa |
Sequence | String: (DA)(DT)(DA)(DC)(DT)(DG)(DT)(DG)(DG)(DA) (DC)(DC)(DA)(DG)(DA)(DA)(DC)(DC)(DC)(DT) (DG)(DA)(DT)(DA)(DA)(DA)(DT)(DG)(DC) (DA)(DA)(DC)(DG)(DC)(DT)(DC)(DA)(DT)(DA) (DG) (DC)(DG)(DG)(DG)(DC)(DA)(DG)(DA) (DC)(DG) |
-Macromolecule #4: DNA (50-MER)
Macromolecule | Name: DNA (50-MER) / type: dna / ID: 4 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 15.366818 KDa |
Sequence | String: (DC)(DG)(DT)(DC)(DT)(DG)(DC)(DC)(DC)(DG) (DC)(DT)(DA)(DT)(DG)(DA)(DG)(DC)(DG)(DT) (DT)(DG)(DC)(DA)(DT)(DT)(DT)(DA)(DT) (DC)(DA)(DG)(DG)(DG)(DT)(DT)(DC)(DT)(DG) (DG) (DT)(DC)(DC)(DA)(DC)(DA)(DG)(DT) (DA)(DT) |
-Macromolecule #5: ADENOSINE-5'-DIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 5 / Formula: ADP |
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Molecular weight | Theoretical: 427.201 Da |
Chemical component information | ChemComp-ADP: |
-Macromolecule #6: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 7 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Macromolecule #7: ADENOSINE-5'-TRIPHOSPHATE
Macromolecule | Name: ADENOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 7 / Number of copies: 2 / Formula: ATP |
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Molecular weight | Theoretical: 507.181 Da |
Chemical component information | ChemComp-ATP: |
-Macromolecule #8: ZINC ION
Macromolecule | Name: ZINC ION / type: ligand / ID: 8 / Number of copies: 2 / Formula: ZN |
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Molecular weight | Theoretical: 65.409 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.5 mg/mL |
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Buffer | pH: 8 Details: 20 mM Tris pH 8.0, 150 mM NaCl, 1.4 mM beta-mercaptoethanol, 5 mM MgCl2 |
Grid | Model: C-flat-2/1 / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 79.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.75 µm / Nominal defocus min: 1.25 µm |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model | PDB ID: Chain - Source name: PDB / Chain - Initial model type: experimental model / Details: TnsC and TnsD were modelled using PDB 8GLU. |
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Refinement | Space: REAL / Protocol: OTHER |
Output model | PDB-8glw: |