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- EMDB-5542: Signaling in Chemoreceptor Arrays Through Mobility Control of Kin... -

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Basic information

Entry
Database: EMDB / ID: EMD-5542
TitleSignaling in Chemoreceptor Arrays Through Mobility Control of Kinase Domains - low kinase activity state - tsr mutant P221DCell signaling
Map dataSubvolume average of mutant receptor tsrP221D/CheA/CheW
Sample
  • Sample: chemoreceptor array with mutant receptor tsr P221D
  • Protein or peptide: tsr P221D
  • Protein or peptide: CheW
  • Protein or peptide: CheA
Keywordschemotaxis / E.coli / CheA
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 25.0 Å
AuthorsBriegel A / Ames P / Gumbart JC / Oikonomou CM / Parkinson JS / Jensen GJ
CitationJournal: Mol Microbiol / Year: 2013
Title: The mobility of two kinase domains in the Escherichia coli chemoreceptor array varies with signalling state.
Authors: Ariane Briegel / Peter Ames / James C Gumbart / Catherine M Oikonomou / John S Parkinson / Grant J Jensen /
Abstract: Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn ...Motile bacteria sense their physical and chemical environment through highly cooperative, ordered arrays of chemoreceptors. These signalling complexes phosphorylate a response regulator which in turn governs flagellar motor reversals, driving cells towards favourable environments. The structural changes that translate chemoeffector binding into the appropriate kinase output are not known. Here, we apply high-resolution electron cryotomography to visualize mutant chemoreceptor signalling arrays in well-defined kinase activity states. The arrays were well ordered in all signalling states, with no discernible differences in receptor conformation at 2-3 nm resolution. Differences were observed, however, in a keel-like density that we identify here as CheA kinase domains P1 and P2, the phosphorylation site domain and the binding domain for response regulator target proteins. Mutant receptor arrays with high kinase activities all exhibited small keels and high proteolysis susceptibility, indicative of mobile P1 and P2 domains. In contrast, arrays in kinase-off signalling states exhibited a range of keel sizes. These findings confirm that chemoreceptor arrays do not undergo large structural changes during signalling, and suggest instead that kinase activity is modulated at least in part by changes in the mobility of key domains.
History
DepositionDec 14, 2012-
Header (metadata) releaseDec 26, 2012-
Map releaseJul 17, 2013-
UpdateSep 11, 2013-
Current statusSep 11, 2013Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.275
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 0.275
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_5542_1.jpg

emd_5542_2.tif

Downloads & links

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Map

FileDownload / File: emd_5542.map.gz / Format: CCP4 / Size: 825.2 KB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubvolume average of mutant receptor tsrP221D/CheA/CheW
Voxel sizeX=Y=Z: 6.44 Å
Density
Contour LevelBy AUTHOR: 0.275 / Movie #1: 0.275
Minimum - Maximum0.0 - 0.36071506
Average (Standard dev.)0.2218239 (±0.08268128)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions606060
Spacing606060
CellA=B=C: 386.4 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z6.446.446.44
M x/y/z606060
origin x/y/z0.0000.0000.000
length x/y/z386.400386.400386.400
α/β/γ90.00090.00090.000
start NX/NY/NZ-5029166
NX/NY/NZ106122134
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS606060
D min/max/mean0.0000.3610.222

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Supplemental data

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Sample components

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Entire : chemoreceptor array with mutant receptor tsr P221D

EntireName: chemoreceptor array with mutant receptor tsr P221D
Components
  • Sample: chemoreceptor array with mutant receptor tsr P221D
  • Protein or peptide: tsr P221D
  • Protein or peptide: CheW
  • Protein or peptide: CheA

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Supramolecule #1000: chemoreceptor array with mutant receptor tsr P221D

SupramoleculeName: chemoreceptor array with mutant receptor tsr P221D / type: sample / ID: 1000 / Number unique components: 3

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Macromolecule #1: tsr P221D

MacromoleculeName: tsr P221D / type: protein_or_peptide / ID: 1 / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli)
Recombinant expressionOrganism: Escherichia coli (E. coli)

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Macromolecule #2: CheW

MacromoleculeName: CheW / type: protein_or_peptide / ID: 2 / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Escherichia coli (E. coli)

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Macromolecule #3: CheA

MacromoleculeName: CheA / type: protein_or_peptide / ID: 3 / Oligomeric state: dimer / Recombinant expression: No / Database: NCBI
Source (natural)Organism: Escherichia coli (E. coli)

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging

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Sample preparation

BufferDetails: Tryptone Broth (10 g/L Tryptone, 5 g/L NaCl). Cells were incubated with penicillin for 1 hour prior to freezing.
GridDetails: R 2/2 copper/rhodium grids, glow discharged
VitrificationCryogen name: ETHANE-PROPANE MIXTURE / Chamber humidity: 100 % / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.2 mm / Nominal defocus min: 10.0 µm / Nominal magnification: 34000
Specialist opticsEnergy filter - Name: FEI / Energy filter - Lower energy threshold: 0.0 eV / Energy filter - Upper energy threshold: 20.0 eV
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -66.93 ° / Tilt series - Axis1 - Max angle: 59.52 °
DateAug 18, 2011
Image recordingAverage electron dose: 150 e/Å2
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

CTF correctionDetails: IMOD
Final reconstructionResolution.type: BY AUTHOR / Resolution: 25.0 Å / Software - Name: IMOD, tomo3D
DetailsCTF-corrected. Average number of tilts used in the 3D reconstructions: 128. Average tomographic tilt angle increment: 1.

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