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Yorodumi- EMDB-23757: Cryo-electron microscopy structure of TnsC(1-503)A225V bound to DNA -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-23757 | |||||||||
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Title | Cryo-electron microscopy structure of TnsC(1-503)A225V bound to DNA | |||||||||
Map data | ||||||||||
Sample |
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Keywords | AAA+ ATPase / DNA binding / Transposition / 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) / synthetic construct (others) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.56 Å | |||||||||
Authors | Shen Y / Ortega J | |||||||||
Funding support | Canada, 1 items
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Citation | Journal: Nat Struct Mol Biol / Year: 2022 Title: Structural basis for DNA targeting by the Tn7 transposon. Authors: Yao Shen / Josue Gomez-Blanco / Michael T Petassi / Joseph E Peters / Joaquin Ortega / Alba Guarné / Abstract: Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ ...Tn7 transposable elements are unique for their highly specific, and sometimes programmable, target-site selection mechanisms and precise insertions. All the elements in the Tn7 family utilize an AAA+ adaptor (TnsC) to coordinate target-site selection with transpososome assembly and to prevent insertions at sites already containing a Tn7 element. Owing to its multiple functions, TnsC is considered the linchpin in the Tn7 element. Here we present the high-resolution cryo-EM structure of TnsC bound to DNA using a gain-of-function variant of the protein and a DNA substrate that together recapitulate the recruitment to a specific DNA target site. TnsC forms an asymmetric ring on target DNA that segregates target-site selection and interaction with the paired-end complex to opposite faces of the ring. Unlike most AAA+ ATPases, TnsC uses a DNA distortion to find the target site but does not remodel DNA to activate transposition. By recognizing pre-distorted substrates, TnsC creates a built-in regulatory mechanism where ATP hydrolysis abolishes ring formation proximal to an existing element. This work unveils how Tn7 and Tn7-like elements determine the strict spacing between the target and integration sites. | |||||||||
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_23757.map.gz | 94.5 MB | EMDB map data format | |
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Header (meta data) | emd-23757-v30.xml emd-23757.xml | 14.7 KB 14.7 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_23757_fsc.xml | 10.7 KB | Display | FSC data file |
Images | emd_23757.png | 155.8 KB | ||
Filedesc metadata | emd-23757.cif.gz | 6 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-23757 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-23757 | HTTPS FTP |
-Validation report
Summary document | emd_23757_validation.pdf.gz | 625.4 KB | Display | EMDB validaton report |
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Full document | emd_23757_full_validation.pdf.gz | 625 KB | Display | |
Data in XML | emd_23757_validation.xml.gz | 11.6 KB | Display | |
Data in CIF | emd_23757_validation.cif.gz | 15.3 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23757 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-23757 | HTTPS FTP |
-Related structure data
Related structure data | 7mcsMC 7mbwC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_23757.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Voxel size | X=Y=Z: 0.855 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Complex of TnsC bound to DNA in the presence of AMPPnP.
Entire | Name: Complex of TnsC bound to DNA in the presence of AMPPnP. |
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Components |
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-Supramolecule #1: Complex of TnsC bound to DNA in the presence of AMPPnP.
Supramolecule | Name: Complex of TnsC bound to DNA in the presence of AMPPnP. type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
Molecular weight | Theoretical: 30 KDa |
-Supramolecule #2: TnsC(1-503)A225V bound to DNA
Supramolecule | Name: TnsC(1-503)A225V bound to DNA / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#3 |
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Source (natural) | Organism: Escherichia coli (E. coli) |
-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: 59.34698 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 QIANAHVLGL 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: DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3')
Macromolecule | Name: DNA (5'-D(P*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*C)-3') type: dna / ID: 2 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 4.572937 KDa |
Sequence | String: (DC)(DG)(DC)(DG)(DC)(DG)(DC)(DG)(DC)(DG) (DC)(DG)(DC)(DG)(DC) |
-Macromolecule #3: DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3')
Macromolecule | Name: DNA (5'-D(P*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*GP*CP*G)-3') type: dna / ID: 3 / Number of copies: 1 / Classification: DNA |
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Source (natural) | Organism: synthetic construct (others) |
Molecular weight | Theoretical: 4.612961 KDa |
Sequence | String: (DG)(DC)(DG)(DC)(DG)(DC)(DG)(DC)(DG)(DC) (DG)(DC)(DG)(DC)(DG) |
-Macromolecule #4: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER
Macromolecule | Name: PHOSPHOAMINOPHOSPHONIC ACID-ADENYLATE ESTER / type: ligand / ID: 4 / Number of copies: 6 / Formula: ANP |
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Molecular weight | Theoretical: 506.196 Da |
Chemical component information | ChemComp-ANP: |
-Macromolecule #5: MAGNESIUM ION
Macromolecule | Name: MAGNESIUM ION / type: ligand / ID: 5 / Number of copies: 6 / Formula: MG |
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Molecular weight | Theoretical: 24.305 Da |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 8 |
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Grid | Model: C-flat-2/2 / Material: COPPER / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 15 sec. |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film or detector model: GATAN K3 (6k x 4k) / Average electron dose: 78.0 e/Å2 |
Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.75 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000 |
Sample stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Refinement | Space: REAL |
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Output model | PDB-7mcs: |