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- EMDB-8575: Negative stain 3D reconstruction of hexameric ComM in the presenc... -

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Basic information

Entry
Database: EMDB / ID: EMD-8575
TitleNegative stain 3D reconstruction of hexameric ComM in the presence of ATP and ssDNA
Map dataHexameric ComM in the presence of ATP and ssDNA
Sample
  • Complex: Hexameric ComM protein in the presence of ATP and ssDNA
Function / homology
Function and homology information


establishment of competence for transformation / DNA duplex unwinding / DNA binding / ATP binding
Similarity search - Function
Mg chelatase-related protein / Mg chelatase-related protein, C-terminal domain / Magnesium chelatase, subunit ChlI C-terminal / Magnesium chelatase ChlI-like, catalytic domain / Magnesium chelatase, subunit ChlI / MCM domain / Ribosomal protein S5 domain 2-type fold, subgroup / Ribosomal protein S5 domain 2-type fold / ATPases associated with a variety of cellular activities / AAA+ ATPase domain / P-loop containing nucleoside triphosphate hydrolase
Similarity search - Domain/homology
Competence protein ComM
Similarity search - Component
Biological speciesHaemophilus influenzae (bacteria)
Methodsingle particle reconstruction / negative staining / Resolution: 13.8 Å
AuthorsNero TM / Dalia TN / Wang JC-Y / Bochman ML / Dalia AB
CitationJournal: 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
DepositionJan 26, 2017-
Header (metadata) releaseFeb 22, 2017-
Map releaseJan 24, 2018-
UpdateJul 18, 2018-
Current statusJul 18, 2018Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8575.png

Downloads & links

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Map

FileDownload / File: emd_8575.map.gz / Format: CCP4 / Size: 3.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationHexameric ComM in the presence of ATP and ssDNA
Voxel sizeX=Y=Z: 1.8 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.039537575 - 0.041225474
Average (Standard dev.)0.0010368424 (±0.006935026)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions100100100
Spacing100100100
CellA=B=C: 180.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.81.81.8
M x/y/z100100100
origin x/y/z0.0000.0000.000
length x/y/z180.000180.000180.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-152-37
NX/NY/NZ998271
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS100100100
D min/max/mean-0.0400.0410.001

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Supplemental data

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Sample components

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Entire : Hexameric ComM protein in the presence of ATP and ssDNA

EntireName: Hexameric ComM protein in the presence of ATP and ssDNA
Components
  • Complex: Hexameric ComM protein in the presence of ATP and ssDNA

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Supramolecule #1: Hexameric ComM protein in the presence of ATP and ssDNA

SupramoleculeName: Hexameric ComM protein in the presence of ATP and ssDNA
type: complex / ID: 1 / Parent: 0
Source (natural)Organism: Haemophilus influenzae (bacteria)
Recombinant expressionOrganism: Escherichia coli K-12 (bacteria) / Recombinant strain: Rosetta 2(DE3) pLysS
Molecular weightExperimental: 342 KDa

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

BufferpH: 7.5
Details: 25 mM Na-HEPES (pH 7.5), 5% glycerol, 300 mM NaCl, 5 mM MgCl2, and 0.05% Tween-20
StainingType: NEGATIVE / Material: Uranyl Formate / Details: 0.75% (w/v) Uranyl Formate
GridModel: EMS CF300-Cu / Material: COPPER / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeJEOL 3200FS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 60000
Sample stageSpecimen holder model: JEOL
Image recordingFilm 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

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Image processing

CTF correctionSoftware - Name: CTFFIND4 (ver. 4)
Startup modelType of model: OTHER
Details: De novo built using EMAN2 from Relion 2D class averages
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final 3D classificationSoftware - Name: RELION (ver. 1.4)
Final angle assignmentType: PROJECTION MATCHING / Software - Name: RELION (ver. 1.4)
Final reconstructionApplied 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

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