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- EMDB-3234: Representative tomogram as used in: Structure of bacterial chemot... -

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Basic information

Entry
Database: EMDB / ID: EMD-3234
TitleRepresentative tomogram as used in: Structure of bacterial chemotaxis signaling CheA2-trimer core complex by cryo-electron tomography and subvolume averaging
Map dataThe tomogram is is binned by 4 while those used in the corresponding subTomogram work were used at full sampling. The protein array is best viewed by rotating around the X-axis 3.3 degrees, and the Y-axis 0.3 degrees as Imod Slicer angles.
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 / positive regulation of post-translational protein modification / protein histidine kinase binding / bacterial-type flagellum-dependent swimming motility / regulation of bacterial-type flagellum-dependent cell motility / aerotaxis / cell tip / protein histidine kinase activity ...negative regulation of protein modification process / detection of chemical stimulus / methyl accepting chemotaxis protein complex / positive regulation of post-translational protein modification / protein histidine kinase binding / bacterial-type flagellum-dependent swimming motility / regulation of bacterial-type flagellum-dependent cell motility / aerotaxis / cell tip / 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 / signal transduction / protein homodimerization activity / ATP binding / identical protein binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Chemotaxis protein CheW / CheY binding / CheY binding / 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 / : ...Chemotaxis protein CheW / CheY binding / CheY binding / 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 / 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-related protein, C-terminal / Signal transduction histidine kinase, dimerisation/phosphoacceptor domain superfamily / 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
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
Methodelectron tomography / cryo EM
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
DepositionNov 6, 2015-
Header (metadata) releaseNov 25, 2015-
Map releaseNov 25, 2015-
UpdateDec 16, 2015-
Current statusDec 16, 2015Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Solid view (volume rendering)
  • Imaged by UCSF Chimera
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  • Simplified surface model
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

EMD-3234.tif

emd_3234.tif

Downloads & links

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Map

FileDownload / File: emd_3234.map.gz / Format: CCP4 / Size: 75.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationThe tomogram is is binned by 4 while those used in the corresponding subTomogram work were used at full sampling. The protein array is best viewed by rotating around the X-axis 3.3 degrees, and the Y-axis 0.3 degrees as Imod Slicer angles.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
12.04 Å/pix.
x 100 pix.
= 1204. Å		12.04 Å/pix.
x 450 pix.
= 5418. Å		12.04 Å/pix.
x 450 pix.
= 5418. Å

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 12.04 Å
Density

Histogram

Histogram (log scale)
Minimum - Maximum-3.36340594 - 3.49809051
Average (Standard dev.)0.0 (±0.47615752)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin07517
Dimensions450450100
Spacing450450100
CellA: 5418.0 Å / B: 5418.0 Å / C: 1204.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z12.0412.0412.04
M x/y/z450450100
origin x/y/z0.0000.0000.000
length x/y/z5418.0005418.0001204.000
α/β/γ90.00090.00090.000
start NX/NY/NZ-190-190-189
NX/NY/NZ380380380
MAP C/R/S123
start NC/NR/NS75017
NC/NR/NS450450100
D min/max/mean-3.3633.4980.000

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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: Trimer of (CheA dimer, dimer of tarCF trimers of dimers, 2 CheW subunits)
Number unique components: 3
Molecular weightTheoretical: 1.66 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: 6 / 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
Processingelectron tomography
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
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
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsCalibrated magnification: 49834 / Illumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELD / 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 ° / Tilt series - Axis1 - Angle increment: 3 °
Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company

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Image processing

DetailsCTF correction (phase only) performed on projections rotated to have tilt axis coincide with Y-axis. Projections aligned using area matching with geometry refinement in Protomo. Reconstruction with SIRT in IMOD.
Final reconstructionAlgorithm: OTHER / Resolution method: OTHER / Software - Name: IMOD / Number images used: 60
CTF correctionDetails: TomoCTF (strip-based periodogram)
Crystal parametersPlane group: P 1

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