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- EMDB-5404: Molecular Architecture of Chemoreceptor Array in E. coli -

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Basic information

Entry
Database: EMDB / ID: EMD-5404
TitleMolecular Architecture of Chemoreceptor Array in E. coli
Map dataThis in situ structure of chemoreceptor array was determined by using cryo-electron tomography of E. coli minicell
Sample
  • Sample: Molecular Architecture of Chemoreceptor Arrays
  • Organelle or cellular component: Bacterial chemoreceptor arrays
KeywordsBacterial Chemotaxis Chemoreceptor array Signaling Transduction Minicell cryo-electron tomography
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / negative staining / Resolution: 32.0 Å
AuthorsLiu J / Hu B / Morado DR / Jani S / Manson MD / Margolin W
CitationJournal: Proc Natl Acad Sci U S A / Year: 2012
Title: Molecular architecture of chemoreceptor arrays revealed by cryoelectron tomography of Escherichia coli minicells.
Authors: Jun Liu / Bo Hu / Dustin R Morado / Sneha Jani / Michael D Manson / William Margolin /
Abstract: The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of ...The chemoreceptors of Escherichia coli localize to the cell poles and form a highly ordered array in concert with the CheA kinase and the CheW coupling factor. However, a high-resolution structure of the array has been lacking, and the molecular basis of array assembly has thus remained elusive. Here, we use cryoelectron tomography of flagellated E. coli minicells to derive a 3D map of the intact array. Docking of high-resolution structures into the 3D map provides a model of the core signaling complex, in which a CheA/CheW dimer bridges two adjacent receptor trimers via multiple hydrophobic interactions. A further, hitherto unknown, hydrophobic interaction between CheW and the homologous P5 domain of CheA in an adjacent core complex connects the complexes into an extended array. This architecture provides a structural basis for array formation and could explain the high sensitivity and cooperativity of chemotaxis signaling in E. coli.
History
DepositionFeb 20, 2012-
Header (metadata) releaseApr 23, 2012-
Map releaseApr 26, 2012-
UpdateFeb 12, 2014-
Current statusFeb 12, 2014Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.6
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.6
  • Imaged by UCSF Chimera
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Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5404_1.jpg

Downloads & links

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Map

FileDownload / File: emd_5404.map.gz / Format: CCP4 / Size: 7.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThis in situ structure of chemoreceptor array was determined by using cryo-electron tomography of E. coli minicell
Voxel sizeX=Y=Z: 5.7 Å
Density
Contour LevelBy AUTHOR: 1.6 / Movie #1: 1.6
Minimum - Maximum-10.09930325 - 11.679125790000001
Average (Standard dev.)0.0 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-64-64-64
Dimensions128128128
Spacing128128128
CellA=B=C: 729.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z5.75.75.7
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z729.600729.600729.600
α/β/γ90.00090.00090.000
start NX/NY/NZ-62-62-62
NX/NY/NZ125125125
MAP C/R/S123
start NC/NR/NS-64-64-64
NC/NR/NS128128128
D min/max/mean-10.09911.6790.000

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Supplemental data

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Sample components

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Entire : Molecular Architecture of Chemoreceptor Arrays

EntireName: Molecular Architecture of Chemoreceptor Arrays
Components
  • Sample: Molecular Architecture of Chemoreceptor Arrays
  • Organelle or cellular component: Bacterial chemoreceptor arrays

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Supramolecule #1000: Molecular Architecture of Chemoreceptor Arrays

SupramoleculeName: Molecular Architecture of Chemoreceptor Arrays / type: sample / ID: 1000 / Details: The sample is E. coli minicell. / Number unique components: 3

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Supramolecule #1: Bacterial chemoreceptor arrays

SupramoleculeName: Bacterial chemoreceptor arrays / type: organelle_or_cellular_component / ID: 1 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Escherichia coli (E. coli) / synonym: Escherichia coli

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Experimental details

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Structure determination

Methodnegative staining, cryo EM
Processingsubtomogram averaging
Aggregation stateparticle

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Sample preparation

BufferDetails: tryptone broth
StainingType: NEGATIVE
Details: Bacterial cultures were mixed with 15-nm colloidal gold (used as fiducial makers in image alignment) and then deposited onto freshly glow-discharged, holey carbon grids for 1 minute. The ...Details: Bacterial cultures were mixed with 15-nm colloidal gold (used as fiducial makers in image alignment) and then deposited onto freshly glow-discharged, holey carbon grids for 1 minute. The grids were blotted with filter paper and rapidly frozen in liquid ethane, using a gravity-driven plunger apparatus.
GridDetails: 200 mesh holey carbon grids, glow discharged.
VitrificationCryogen name: ETHANE / Chamber humidity: 80 % / Chamber temperature: 90 K / Instrument: HOMEMADE PLUNGER
Details: Grids were blotted for a few seconds before plunging into liquid ethane.
Method: Blot for few seconds before plunging.

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Electron microscopy

MicroscopeFEI POLARA 300
Alignment procedureLegacy - Astigmatism: Objective lens astigmatism was corrected at 200,000 times magnification
DateDec 1, 2010
Image recordingCategory: CCD / Film or detector model: GENERIC TVIPS (4k x 4k) / Number real images: 1024 / Average electron dose: 100 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 31000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.0 mm / Nominal defocus max: 6.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 31000
Sample stageSpecimen holder: LN cooled / Specimen holder model: OTHER / Tilt series - Axis1 - Min angle: -65 ° / Tilt series - Axis1 - Max angle: 65 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsThe particles were selected manually by visual inspection. The approximate local orientation of each small patch was estimated based on its location relative to the center of the minicell, therefore providing two of the three Euler angles. The sub-volume analysis of 2-D arrays was carried out by using Dr. Hanspeter Winkler's package.
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: IMOD,RAPTOR,PROTOMO / Number subtomograms used: 12483
Final 3D classificationNumber classes: 8

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Atomic model buiding 1

Initial modelPDB ID:

1qu7

SoftwareName: Chimera
DetailsProtocol: Rigid body
RefinementSpace: REAL / Protocol: RIGID BODY FIT

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