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- PDB-3ja6: Cryo-electron Tomography and All-atom Molecular Dynamics Simulati... -
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Basic information
Entry | Database: PDB / ID: 3ja6 | ||||||
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Title | Cryo-electron Tomography and All-atom Molecular Dynamics Simulations Reveal a Novel Kinase Conformational Switch in Bacterial Chemotaxis Signaling | ||||||
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![]() | SIGNALING PROTEIN / bacterial chemotaxis / core-signaling unit / adaptor protein / histidine kinase / chemoreceptor | ||||||
Function / homology | ![]() histidine kinase / phosphorelay sensor kinase activity / transmembrane signaling receptor activity / chemotaxis / lysozyme activity / protein domain specific binding / signal transduction / ATP binding / plasma membrane / cytoplasm / cytosol Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / electron tomography / cryo EM / Resolution: 12.7 Å | ||||||
![]() | Cassidy, C.K. / Himes, B.A. / Alvarez, F.J. / Ma, J. / Zhao, G. / Perilla, J.R. / Schulten, K. / Zhang, P. | ||||||
![]() | ![]() 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. | ||||||
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 1.5 MB | Display | ![]() |
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PDB format | ![]() | 1.2 MB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 754.4 KB | Display | ![]() |
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Full document | ![]() | 782.9 KB | Display | |
Data in XML | ![]() | 111.3 KB | Display | |
Data in CIF | ![]() | 185.2 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 6319MC ![]() 3234C ![]() 6320C M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 15718.318 Da / Num. of mol.: 4 / Fragment: SEE REMARK 999 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() #2: Protein | 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.) ![]() ![]() ![]() ![]() #3: Protein | 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.) ![]() ![]() ![]() ![]() #4: Protein | 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.) ![]() ![]() ![]() ![]() Sequence details | THE IMAGED PROTEINS WERE FROM ESCHERICHI | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: electron tomography |
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Sample preparation
Component |
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Molecular weight | Value: 1.66 MDa / Experimental value: NO | |||||||||||||||||||||||||
Buffer solution | Name: 75 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2 / pH: 7.4 / Details: 75 mM Tris-HCl, 100 mM KCl, 5 mM MgCl2 | |||||||||||||||||||||||||
Specimen | Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | |||||||||||||||||||||||||
Specimen support | Details: Perforated R2/2 Quantifoil grids precoated with 10 nm fiducial gold beads on the backside of the grid | |||||||||||||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI POLARA 300 / Date: Jan 7, 2009 |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 39000 X / Calibrated magnification: 49834 X / Nominal defocus max: 8000 nm / Nominal defocus min: 4000 nm / Cs: 2 mm |
Specimen holder | Specimen holder model: OTHER / Specimen holder type: Polara cartridge / Tilt angle max: 70 ° / Tilt angle min: -70 ° |
Image recording | Electron dose: 60 e/Å2 / Film or detector model: GATAN ULTRASCAN 4000 (4k x 4k) |
Image scans | Num. digital images: 60 |
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Processing
CTF correction | Details: TomoCTF (strip-based periodogram) | ||||||||||||
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Symmetry | Point symmetry: C2 (2 fold cyclic) | ||||||||||||
3D reconstruction | Method: SIRT / 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 building | Protocol: FLEXIBLE FIT / Space: REAL / Details: REFINEMENT PROTOCOL--FLEXIBLE | ||||||||||||
Atomic model building | PDB-ID: 1QU7 Accession code: 1QU7 / Source name: PDB / Type: experimental model | ||||||||||||
Refinement step | Cycle: LAST
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