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- EMDB-6320: Structure of bacterial chemotaxis signaling CheA2-hexamer core co... -

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Basic information

Entry
Database: EMDB / ID: EMD-6320
TitleStructure of bacterial chemotaxis signaling CheA2-hexamer core complex by cryo-electron tomography and subvolume averaging
Map dataStructure of bacterial chemotaxis signaling CheA2-hexamer core complex by cryo-electron tomography and subvolume averaging
Sample
  • Sample: tarCF CheA CheW
  • Protein or peptide: Chemotaxis protein CheA
  • Protein or peptide: Chemotaxis protein CheW
  • Protein or peptide: Methyl-accepting chemotaxis protein II
KeywordsBacterial chemotaxis / Signal transduction / cryo-Electron Tomography / Molecular dynamics simulation / all-atom
Function / homology
Function and homology information


negative regulation of protein modification process / detection of chemical stimulus / methyl accepting chemotaxis protein complex / protein histidine kinase binding / positive regulation of post-translational protein modification / bacterial-type flagellum-dependent swimming motility / cell tip / regulation of bacterial-type flagellum-dependent cell motility / aerotaxis / protein histidine kinase activity ...negative regulation of protein modification process / detection of chemical stimulus / methyl accepting chemotaxis protein complex / protein histidine kinase binding / positive regulation of post-translational protein modification / bacterial-type flagellum-dependent swimming motility / cell tip / regulation of bacterial-type flagellum-dependent cell motility / aerotaxis / protein histidine kinase activity / regulation of chemotaxis / thermotaxis / signal complex assembly / histidine kinase / phosphorelay sensor kinase activity / phosphorelay signal transduction system / establishment of localization in cell / cellular response to amino acid stimulus / protein homooligomerization / chemotaxis / transmembrane signaling receptor activity / protein domain specific binding / phosphorylation / signal transduction / protein homodimerization activity / ATP binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Chemotaxis protein CheW / CheY binding / CheY binding / Chemotaxis protein CheA, P2 response regulator-binding domain superfamily / Chemotaxis protein CheA, P2 response regulator-binding / P2 response regulator binding domain / Methyl-accepting chemotaxis protein, four helix bundle domain superfamily / Homologues of the ligand binding domain of Tar / Chemotaxis methyl-accepting receptor, methyl-accepting site / Bacterial chemotaxis sensory transducers signature. ...Chemotaxis protein CheW / CheY binding / CheY binding / Chemotaxis protein CheA, P2 response regulator-binding domain superfamily / Chemotaxis protein CheA, P2 response regulator-binding / P2 response regulator binding domain / Methyl-accepting chemotaxis protein, four helix bundle domain superfamily / Homologues of the ligand binding domain of Tar / Chemotaxis methyl-accepting receptor, methyl-accepting site / Bacterial chemotaxis sensory transducers signature. / Chemotaxis methyl-accepting receptor Tar-related, ligand-binding / Tar ligand binding domain homologue / Histidine kinase CheA-like, homodimeric domain / CheY-binding domain of CheA / Histidine kinase CheA-like, homodimeric domain superfamily / Signal transducing histidine kinase, homodimeric domain / Signal transducing histidine kinase, homodimeric domain / CheW-like domain profile. / CheW-like domain / CheW-like domain superfamily / CheW-like domain / Two component signalling adaptor domain / Histidine Phosphotransfer domain / Chemotaxis methyl-accepting receptor / Hpt domain / Histidine-containing phosphotransfer (HPt) domain profile. / Signal transduction histidine kinase, phosphotransfer (Hpt) domain / HPT domain superfamily / Target SNARE coiled-coil homology domain / Methyl-accepting chemotaxis protein (MCP) signalling domain / Methyl-accepting chemotaxis protein (MCP) signalling domain / Bacterial chemotaxis sensory transducers domain profile. / Methyl-accepting chemotaxis-like domains (chemotaxis sensory transducer). / HAMP domain / HAMP (Histidine kinases, Adenylyl cyclases, Methyl binding proteins, Phosphatases) domain / HAMP domain profile. / HAMP domain / Signal transduction histidine kinase, dimerisation/phosphoacceptor domain superfamily / Signal transduction histidine kinase-related protein, C-terminal / Histidine kinase domain / Histidine kinase domain profile. / Histidine kinase-, DNA gyrase B-, and HSP90-like ATPase / Histidine kinase-like ATPases / Histidine kinase/HSP90-like ATPase / Histidine kinase/HSP90-like ATPase superfamily
Similarity search - Domain/homology
Methyl-accepting chemotaxis protein II / Chemotaxis protein CheA / Chemotaxis protein CheW / Chemotaxis protein CheA / Chemotaxis protein CheW / Methyl-accepting chemotaxis protein 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodsubtomogram averaging / cryo EM / Resolution: 17.5 Å
AuthorsCassidy CK / Himes BA / Alvarez FJ / Ma J / Zhou G / Perilla JR / Schulten K / Zhang P
CitationJournal: Elife / Year: 2015
Title: CryoEM and computer simulations reveal a novel kinase conformational switch in bacterial chemotaxis signaling.
Authors: C Keith Cassidy / Benjamin A Himes / Frances J Alvarez / Jun Ma / Gongpu Zhao / Juan R Perilla / Klaus Schulten / Peijun Zhang /
Abstract: Chemotactic responses in bacteria require large, highly ordered arrays of sensory proteins to mediate the signal transduction that ultimately controls cell motility. A mechanistic understanding of ...Chemotactic responses in bacteria require large, highly ordered arrays of sensory proteins to mediate the signal transduction that ultimately controls cell motility. A mechanistic understanding of the molecular events underlying signaling, however, has been hampered by the lack of a high-resolution structural description of the extended array. Here, we report a novel reconstitution of the array, involving the receptor signaling domain, histidine kinase CheA, and adaptor protein CheW, as well as a density map of the core-signaling unit at 11.3 Å resolution, obtained by cryo-electron tomography and sub-tomogram averaging. Extracting key structural constraints from our density map, we computationally construct and refine an atomic model of the core array structure, exposing novel interfaces between the component proteins. Using all-atom molecular dynamics simulations, we further reveal a distinctive conformational change in CheA. Mutagenesis and chemical cross-linking experiments confirm the importance of the conformational dynamics of CheA for chemotactic function.
History
DepositionApr 17, 2015-
Header (metadata) releaseMay 20, 2015-
Map releaseDec 9, 2015-
UpdateDec 9, 2015-
Current statusDec 9, 2015Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 1.5
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by height
  • Surface level: 1.5
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_6320.png

Downloads & links

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Map

FileDownload / File: emd_6320.map.gz / Format: CCP4 / Size: 4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of bacterial chemotaxis signaling CheA2-hexamer core complex by cryo-electron tomography and subvolume averaging
Voxel sizeX=Y=Z: 3.01 Å
Density
Contour LevelBy AUTHOR: 1.5 / Movie #1: 1.5
Minimum - Maximum-6.19167137 - 9.26665974
Average (Standard dev.)-0.00508755 (±1.45572317)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions13013064
Spacing13013064
CellA: 391.3 Å / B: 391.3 Å / C: 192.64 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z3.013.013.01
M x/y/z13013064
origin x/y/z0.0000.0000.000
length x/y/z391.300391.300192.640
α/β/γ90.00090.00090.000
start NX/NY/NZ-8211244
NX/NY/NZ115138149
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS13013064
D min/max/mean-6.1929.267-0.005

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Supplemental data

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Sample components

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Entire : tarCF CheA CheW

EntireName: tarCF CheA CheW
Components
  • Sample: tarCF CheA CheW
  • Protein or peptide: Chemotaxis protein CheA
  • Protein or peptide: Chemotaxis protein CheW
  • Protein or peptide: Methyl-accepting chemotaxis protein II

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Supramolecule #1000: tarCF CheA CheW

SupramoleculeName: tarCF CheA CheW / type: sample / ID: 1000
Oligomeric state: Hexamer of (CheA dimer, dimer of tarCF trimers of dimers, 2 CheW subunits)
Number unique components: 3
Molecular weightTheoretical: 3.32 MDa

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Macromolecule #1: Chemotaxis protein CheA

MacromoleculeName: Chemotaxis protein CheA / type: protein_or_peptide / ID: 1 / Name.synonym: Bacterial Chemotaxis Histidine Kinase CheA / Number of copies: 12 / Oligomeric state: Dimer / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: Inner Membrane
Molecular weightTheoretical: 71.384 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: RP3098 / Recombinant plasmid: pKJ9
SequenceUniProtKB: Chemotaxis protein CheA

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Macromolecule #2: Chemotaxis protein CheW

MacromoleculeName: Chemotaxis protein CheW / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Oligomeric state: monomeric / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: Inner Membrane
Molecular weightTheoretical: 18.083 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: RP3098 / Recombinant plasmid: PPA770
SequenceUniProtKB: Chemotaxis protein CheW

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Macromolecule #3: Methyl-accepting chemotaxis protein II

MacromoleculeName: Methyl-accepting chemotaxis protein II / type: protein_or_peptide / ID: 3 / Name.synonym: tarCF
Details: Cytoplasmic fragment of wild-type aspartate receptor
Number of copies: 6 / Oligomeric state: trimer of dimers / Recombinant expression: Yes
Source (natural)Organism: Escherichia coli (E. coli) / Location in cell: Inner Membrane
Molecular weightTheoretical: 31.209 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli) / Recombinant strain: RP3098 / Recombinant plasmid: pHTCF
SequenceUniProtKB: Methyl-accepting chemotaxis protein II

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation state2D array

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Sample preparation

BufferpH: 7.4 / Details: 75 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2
GridDetails: Perforated R2/2 Quantifoil grids precoated with 10 nm fiducial gold beads on the backside of the grid
VitrificationCryogen name: ETHANE / Instrument: HOMEMADE PLUNGER
Method: Single-sided blotting to avoid disruption of the monolayer
DetailsPseudo-crystalline 2D monolayer reconstituted on a lipid monolayer

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Electron microscopy

MicroscopeFEI POLARA 300
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49834 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.0 mm / Nominal defocus max: 8.0 µm / Nominal defocus min: 4.0 µm / Nominal magnification: 39000
Sample stageSpecimen holder model: OTHER / Tilt series - Axis1 - Min angle: -70 ° / Tilt series - Axis1 - Max angle: 70 °
DateJan 7, 2009
Image recordingCategory: CCD / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Number real images: 60 / Average electron dose: 60 e/Å2 / Bits/pixel: 16
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

Crystal parametersPlane group: P 1
CTF correctionDetails: TomoCTF (strip-based periodogram)
Final reconstructionAlgorithm: OTHER / Resolution.type: BY AUTHOR / Resolution: 17.5 Å / Resolution method: OTHER / Software - Name: IMOD / Number subtomograms used: 400
DetailsSubtomograms were initially selected using template matching. Positions were refined using alignment to class averages. Classification was also used to select symmetry centers and to remove high variance outliers. Cross-correlation with missing-wedge compensation was used for alignment, with SVD and HAC used for statistical analysis.
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

1qu7

RefinementSpace: REAL / Protocol: FLEXIBLE FIT

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