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Yorodumi- EMDB-8575: Negative stain 3D reconstruction of hexameric ComM in the presenc... -
+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-8575 | |||||||||
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Title | Negative stain 3D reconstruction of hexameric ComM in the presence of ATP and ssDNA | |||||||||
Map data | Hexameric ComM in the presence of ATP and ssDNA | |||||||||
Sample |
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Function / homology | Function and homology information establishment of competence for transformation / DNA duplex unwinding / DNA binding / ATP binding Similarity search - Function | |||||||||
Biological species | Haemophilus influenzae (bacteria) | |||||||||
Method | single particle reconstruction / negative staining / Resolution: 13.8 Å | |||||||||
Authors | Nero TM / Dalia TN / Wang JC-Y / Bochman ML / Dalia AB | |||||||||
Citation | Journal: Nucleic Acids Res / Year: 2018 Title: ComM is a hexameric helicase that promotes branch migration during natural transformation in diverse Gram-negative species. Authors: Thomas M Nero / Triana N Dalia / Joseph Che-Yen Wang / David T Kysela / Matthew L Bochman / Ankur B Dalia / Abstract: Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly ...Acquisition of foreign DNA by natural transformation is an important mechanism of adaptation and evolution in diverse microbial species. Here, we characterize the mechanism of ComM, a broadly conserved AAA+ protein previously implicated in homologous recombination of transforming DNA (tDNA) in naturally competent Gram-negative bacterial species. In vivo, we found that ComM was required for efficient comigration of linked genetic markers in Vibrio cholerae and Acinetobacter baylyi, which is consistent with a role in branch migration. Also, ComM was particularly important for integration of tDNA with increased sequence heterology, suggesting that its activity promotes the acquisition of novel DNA sequences. In vitro, we showed that purified ComM binds ssDNA, oligomerizes into a hexameric ring, and has bidirectional helicase and branch migration activity. Based on these data, we propose a model for tDNA integration during natural transformation. This study provides mechanistic insight into the enigmatic steps involved in tDNA integration and uncovers the function of a protein required for this conserved mechanism of horizontal gene transfer. | |||||||||
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_8575.map.gz | 2.4 MB | EMDB map data format | |
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Header (meta data) | emd-8575-v30.xml emd-8575.xml | 10.9 KB 10.9 KB | Display Display | EMDB header |
Images | emd_8575.png | 73.9 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-8575 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-8575 | HTTPS FTP |
-Related structure data
Similar structure data |
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-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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Related items in Molecule of the Month |
-Map
File | Download / File: emd_8575.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Hexameric ComM in the presence of ATP and ssDNA | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 1.8 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 : Hexameric ComM protein in the presence of ATP and ssDNA
Entire | Name: Hexameric ComM protein in the presence of ATP and ssDNA |
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Components |
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-Supramolecule #1: Hexameric ComM protein in the presence of ATP and ssDNA
Supramolecule | Name: Hexameric ComM protein in the presence of ATP and ssDNA type: complex / ID: 1 / Parent: 0 |
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Source (natural) | Organism: Haemophilus influenzae (bacteria) |
Recombinant expression | Organism: Escherichia coli K-12 (bacteria) / Recombinant strain: Rosetta 2(DE3) pLysS |
Molecular weight | Experimental: 342 KDa |
-Experimental details
-Structure determination
Method | negative staining |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Buffer | pH: 7.5 Details: 25 mM Na-HEPES (pH 7.5), 5% glycerol, 300 mM NaCl, 5 mM MgCl2, and 0.05% Tween-20 |
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Staining | Type: NEGATIVE / Material: Uranyl Formate / Details: 0.75% (w/v) Uranyl Formate |
Grid | Model: EMS CF300-Cu / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR |
-Electron microscopy
Microscope | JEOL 3200FS |
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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 magnification: 60000 |
Sample stage | Specimen holder model: JEOL |
Image recording | Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 1 / Average exposure time: 0.3 sec. / Average electron dose: 14.0 e/Å2 |
-Image processing
CTF correction | Software - Name: CTFFIND4 (ver. 4) |
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Startup model | Type of model: OTHER Details: De novo built using EMAN2 from Relion 2D class averages |
Initial angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4) |
Final 3D classification | Software - Name: RELION (ver. 1.4) |
Final angle assignment | Type: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4) |
Final reconstruction | Applied symmetry - Point group: C6 (6 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 13.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 1.4) / Details: Relion 1.4 was used / Number images used: 32958 |