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Yorodumi- EMDB-15218: Medium resolution cryo-EM density map of Tn4430 TnpA transposase ... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-15218 | |||||||||
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Title | Medium resolution cryo-EM density map of Tn4430 TnpA transposase from Tn3 family in apo state | |||||||||
Map data | ||||||||||
Sample |
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Keywords | Tranposition / complex / hyperactive mutant / paired-end complex / DNA BINDING PROTEIN | |||||||||
Function / homology | Tn3 transposase DDE domain / Domain of unknown function DUF4158 / : / Tn3 transposase DDE domain / Domain of unknown function (DUF4158) / transposase activity / DNA transposition / DNA binding / Transposase for transposon Tn4430 Function and homology information | |||||||||
Biological species | Bacillus thuringiensis (bacteria) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.4 Å | |||||||||
Authors | Shkumatov AV / Liu Y / Efremov RG | |||||||||
Funding support | Belgium, 1 items
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Citation | Journal: Nucleic Acids Res / Year: 2023 Title: AFM-based force spectroscopy unravels stepwise formation of the DNA transposition complex in the widespread Tn3 family mobile genetic elements. Authors: Maricruz Fernandez / Alexander V Shkumatov / Yun Liu / Claire Stulemeijer / Sylvie Derclaye / Rouslan G Efremov / Bernard Hallet / David Alsteens / Abstract: Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent ...Transposon Tn4430 belongs to a widespread family of bacterial transposons, the Tn3 family, which plays a prevalent role in the dissemination of antibiotic resistance among pathogens. Despite recent data on the structural architecture of the transposition complex, the molecular mechanisms underlying the replicative transposition of these elements are still poorly understood. Here, we use force-distance curve-based atomic force microscopy to probe the binding of the TnpA transposase of Tn4430 to DNA molecules containing one or two transposon ends and to extract the thermodynamic and kinetic parameters of transposition complex assembly. Comparing wild-type TnpA with previously isolated deregulated TnpA mutants supports a stepwise pathway for transposition complex formation and activation during which TnpA first binds as a dimer to a single transposon end and then undergoes a structural transition that enables it to bind the second end cooperatively and to become activated for transposition catalysis, the latter step occurring at a much faster rate for the TnpA mutants. Our study thus provides an unprecedented approach to probe the dynamic of a complex DNA processing machinery at the single-particle level. | |||||||||
History |
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-Structure visualization
Supplemental images |
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-Downloads & links
-EMDB archive
Map data | emd_15218.map.gz | 117.5 MB | EMDB map data format | |
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Header (meta data) | emd-15218-v30.xml emd-15218.xml | 17.9 KB 17.9 KB | Display Display | EMDB header |
FSC (resolution estimation) | emd_15218_fsc.xml | 11.1 KB | Display | FSC data file |
Images | emd_15218.png | 98 KB | ||
Masks | emd_15218_msk_1.map | 125 MB | Mask map | |
Filedesc metadata | emd-15218.cif.gz | 5.9 KB | ||
Others | emd_15218_half_map_1.map.gz emd_15218_half_map_2.map.gz | 115.8 MB 115.8 MB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-15218 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-15218 | HTTPS FTP |
-Related structure data
Related structure data | C: citing same article (ref.) |
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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_15218.map.gz / Format: CCP4 / Size: 125 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: 0.786 Å | ||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
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-Supplemental data
-Mask #1
File | emd_15218_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #1
File | emd_15218_half_map_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Half map: #2
File | emd_15218_half_map_2.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : TnpA transposase in apo state.
Entire | Name: TnpA transposase in apo state. |
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Components |
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-Supramolecule #1: TnpA transposase in apo state.
Supramolecule | Name: TnpA transposase in apo state. / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all Details: Medium resolution map with some preferential orientation. |
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Source (natural) | Organism: Bacillus thuringiensis (bacteria) |
-Macromolecule #1: Transposase for transposon Tn4430
Macromolecule | Name: Transposase for transposon Tn4430 / type: protein_or_peptide / ID: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Bacillus thuringiensis (bacteria) |
Recombinant expression | Organism: Escherichia coli (E. coli) |
Sequence | String: MGVKQLLSEA QRNELMDLSR LTEWDLVTFH TFSKHDLHLI LKHRRGYNRL GFALQLVLIR YPGWSLTEYK DIPQYVVAYV ASQLQIPPE EFLVYAKRGN TLWEHLGEIR TEYGYQNFSS EYKETLLQFL VQQAMDNNNT LYLIEITIST LRKMKVILPA M YVIEDIVW ...String: MGVKQLLSEA QRNELMDLSR LTEWDLVTFH TFSKHDLHLI LKHRRGYNRL GFALQLVLIR YPGWSLTEYK DIPQYVVAYV ASQLQIPPE EFLVYAKRGN TLWEHLGEIR TEYGYQNFSS EYKETLLQFL VQQAMDNNNT LYLIEITIST LRKMKVILPA M YVIEDIVW EAKQQADQKV YSILHDGLVQ EQKDQLDALL LPTINGKSPL AWLKDVPAQP SPESFLKVID RLQFVQKIGL TI DTTKINT NRLRQLARLG SKYEPYAFRR FNEVKRYSML VSFLLEITQD LIDYAIEIHD RLMMNLQTKG KKEQDEIQQA NGK KLNEKI LQFITVCGTL IEAKETGKDA FAALDEVMSW NEMVESVEEA KQLSRPLNYD YLDLLNTRYS YVRRYAPTLL RSLH FRATK SGEPVLQALD TIHELNETGK RKVPHGAPLH FVSNRWQKHV YDDDGNINRH YYELAALTEL RNHIRSGDIF VSGSR HHKA FDDYLIPYDE WNEVSNIPNG LTAPLKAEDY ITDRINRLNE HLEWLSKNSE KLEGVDISQG KLHVERLDRG TPEEAK AFS KLLHSMLPRI KLTDLLIEVA SWTGFHDQFI HASTNQSPDQ EEQNIVLATL MAMGTNIGLT KMAEATPGIS YRQMANA SQ WRMYDDAMVR AQSILVNFQK EQKLSSYWGD GTTSSSDGMR LSIAVRSLHA DSNPHYGTGK GGTIYRFVSD QLSAYHVK V ITTNARDALH VLDGLLHHET DLKIEEHYTD TAGYTDQVFA LTHLLGFRFA PRIRDLADTK LFSIPGGEEY ENVQALLKG KINVKLIKEN YEDIRRLAYS VQTGKVSSAL IMGKLGSYAR QNKLATALGE MGRIEKTLFT LDYISNKAVR RRVQKGLNKG EAINALARI IFFGQRGEFR ERALQDQLQR ASALNIIINA ISVWNTVYME KAVEELKARG EFREDLMPYA WPLGWEHINF L GEYKFEGL HDTGQMNLRP LRIKEPFYSP IRSFLEQKLI SEEDLNSAVD HHHHHH UniProtKB: Transposase for transposon Tn4430 |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 0.15 mg/mL |
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Buffer | pH: 7.5 / Details: 50 mM HEPES (pH 7.5), 100 mM NaCl, 30 mM L-Arg HCL |
Grid | Model: Quantifoil R2/1 / Material: COPPER / Mesh: 300 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 60 sec. / Pretreatment - Atmosphere: AIR |
Vitrification | Cryogen name: ETHANE / Chamber humidity: 90 % / Instrument: GATAN CRYOPLUNGE 3 |
-Electron microscopy
Microscope | JEOL CRYO ARM 300 |
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Electron beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm |
Specialist optics | Energy filter - Name: In-column Omega Filter / Energy filter - Slit width: 20 eV |
Sample stage | Specimen holder model: JEOL CRYOSPECPORTER / Cooling holder cryogen: NITROGEN |
Image recording | Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 4872 / Average exposure time: 10.0 sec. / Average electron dose: 58.0 e/Å2 |