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Basic information

Entry
Database: PDB / ID: 3ja6
TitleCryo-electron Tomography and All-atom Molecular Dynamics Simulations Reveal a Novel Kinase Conformational Switch in Bacterial Chemotaxis Signaling
Components
  • (Methyl-accepting chemotaxis protein 2) x 2
  • Chemotaxis protein CheA
  • Chemotaxis protein CheW
KeywordsSIGNALING PROTEIN / bacterial chemotaxis / core-signaling unit / adaptor protein / histidine kinase / chemoreceptor
Function / homology
Function and homology information


histidine kinase / phosphorelay sensor kinase activity / chemotaxis / transmembrane signaling receptor activity / protein domain specific binding / signal transduction / ATP binding / plasma membrane / cytosol / cytoplasm
Similarity search - Function
Chemotaxis protein CheW / Chemotaxis protein CheA, P2 response regulator-binding domain superfamily / Chemotaxis protein CheA, P2 response regulator-binding / P2 response regulator binding domain / 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. ...Chemotaxis protein CheW / Chemotaxis protein CheA, P2 response regulator-binding domain superfamily / Chemotaxis protein CheA, P2 response regulator-binding / P2 response regulator binding domain / 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). / 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
Chemotaxis protein CheA / Chemotaxis protein CheW / Methyl-accepting chemotaxis protein 2
Similarity search - Component
Biological speciesEscherichia coli (E. coli)
MethodELECTRON MICROSCOPY / electron tomography / cryo EM / Resolution: 12.7 Å
AuthorsCassidy, C.K. / Himes, B.A. / Alvarez, F.J. / Ma, J. / Zhao, G. / Perilla, J.R. / 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 21, 2015Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 9, 2015Provider: repository / Type: Initial release
Revision 1.1Feb 21, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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  • Deposited structure unit
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  • Simplified surface model + fitted atomic model
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Chemotaxis protein CheW
B: Chemotaxis protein CheW
C: Chemotaxis protein CheA
D: Chemotaxis protein CheW
E: Chemotaxis protein CheA
F: Chemotaxis protein CheW
G: Methyl-accepting chemotaxis protein 2
H: Methyl-accepting chemotaxis protein 2
I: Methyl-accepting chemotaxis protein 2
J: Methyl-accepting chemotaxis protein 2
K: Methyl-accepting chemotaxis protein 2
L: Methyl-accepting chemotaxis protein 2
M: Methyl-accepting chemotaxis protein 2
N: Methyl-accepting chemotaxis protein 2
O: Methyl-accepting chemotaxis protein 2
P: Methyl-accepting chemotaxis protein 2
Q: Methyl-accepting chemotaxis protein 2
R: Methyl-accepting chemotaxis protein 2


Theoretical massNumber of molelcules
Total (without water)547,59818
Polymers547,59818
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein
Chemotaxis protein CheW /


Mass: 15718.318 Da / Num. of mol.: 4 / Fragment: SEE REMARK 999
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: PPA770 / Production host: Escherichia coli (E. coli) / Strain (production host): RP3098 / References: UniProt: Q56311*PLUS
#2: Protein Chemotaxis protein CheA /


Mass: 42489.328 Da / Num. of mol.: 2
Fragment: dimerization domain, kinase domain, and regulatory domain (SEE REMARK 999)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: pKJ9 / Production host: Escherichia coli (E. coli) / Strain (production host): RP3098 / References: UniProt: Q56310*PLUS
#3: Protein
Methyl-accepting chemotaxis protein 2


Mass: 33398.828 Da / Num. of mol.: 6 / Fragment: cytoplasmic domain (SEE REMARK 999)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: pHTCF / Production host: Escherichia coli (E. coli) / Strain (production host): RP3098 / References: UniProt: Q9X0M7*PLUS
#4: Protein
Methyl-accepting chemotaxis protein 2


Mass: 33225.594 Da / Num. of mol.: 6 / Fragment: cytoplasmic domain (SEE REMARK 999)
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Escherichia coli (E. coli) / Plasmid: pHTCF / Production host: Escherichia coli (E. coli) / Strain (production host): RP3098 / References: UniProt: Q9X0M7*PLUS
Sequence detailsTHE IMAGED PROTEINS WERE FROM ESCHERICHIA COLI, BUT THE MODELED SEQUENCES ARE FROM THERMOTOGA MARITIMA MSB8.

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: electron tomography

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Sample preparation

Component
IDNameTypeDetailsParent-ID
1CheA2-trimer core complexCOMPLEXTrimer of (CheA dimer, dimer of tarCF trimers of dimers, 2 CheW subunits)0
2Chemotaxis protein CheA1
3Chemotaxis protein CheW1
4Methyl-accepting chemotaxis protein II1
Molecular weightValue: 1.66 MDa / Experimental value: NO
Buffer solutionName: 75 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2 / pH: 7.4 / Details: 75 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: Perforated R2/2 Quantifoil grids precoated with 10 nm fiducial gold beads on the backside of the grid
VitrificationInstrument: HOMEMADE PLUNGER / Cryogen name: ETHANE
Details: Single-sided blotting (to avoid disruption of the monolayer) before plunging into liquid ethane
Method: Single-sided blotting to avoid disruption of the monolayer

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Electron microscopy imaging

Experimental equipment
Model: Tecnai Polara / Image courtesy: FEI Company
MicroscopyModel: FEI POLARA 300 / Date: Jan 7, 2009
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: SPOT SCAN
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 39000 X / Calibrated magnification: 49834 X / Nominal defocus max: 8000 nm / Nominal defocus min: 4000 nm / Cs: 2 mm
Specimen holderSpecimen holder model: OTHER / Specimen holder type: Polara cartridge / Tilt angle max: 70 ° / Tilt angle min: -70 °
Image recordingElectron dose: 60 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k)
Image scansNum. digital images: 60

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Processing

CTF correctionDetails: TomoCTF (strip-based periodogram)
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionMethod: SIRTSirte / Resolution: 12.7 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 4000 / Nominal pixel size: 3.01 Å / Actual pixel size: 3.01 Å / Details: Gold-standard refinement / Refinement type: HALF-MAPS REFINED INDEPENDENTLY / Symmetry type: 2D CRYSTAL
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL / Details: REFINEMENT PROTOCOL--FLEXIBLE
Atomic model buildingPDB-ID: 1QU7

1qu7


Accession code: 1QU7 / Source name: PDB / Type: experimental model
Refinement stepCycle: LAST
ProteinNucleic acidLigandSolventTotal
Num. atoms38314 0 0 0 38314

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