Journal: Mol Immunol / Year: 2017 Title: Human myeloma IgG4 reveals relatively rigid asymmetric Y-like structure with different conformational stability of C2 domains. Authors: Vladimir M Tischenko / Vladimir P Zav'yalov / Sergey N Ryazantsev / Abstract: Human IgG4 (hIgG4) has weak pro-inflammatory activity. The structural basis for this is still unclear. Here a 3D model of myeloma hIgG4 was created at ∼3nm resolution using electron microscopy (EM) ...Human IgG4 (hIgG4) has weak pro-inflammatory activity. The structural basis for this is still unclear. Here a 3D model of myeloma hIgG4 was created at ∼3nm resolution using electron microscopy (EM) with negative staining and single-particle 3D reconstruction. The hIgG4 model reveals relatively rigid asymmetric Y-like structure. The model shows that one Fab subunit is closer to the upper portion of the Fc subunit (C2 domain) than the other Fab. This is in agreement with X-ray crystallography and X-ray/neutron scattering, recently published by others. The same hIgG4 sample was studied with differential scanning calorimetry (DSC) and fluorescence. The thermodynamics and fluorescence observations indicate that one C2 domain displays less conformational stability than the other. This finding is consistent with the flipping of one C2 domain, observed in pembrolizumab (recombinant hIgG4) by X-ray crystallography. The specific feature of hIgG4 C2 domains together with relatively rigid asymmetric Y-like structure, in which one Fab subunit is closer to the upper portion of the Fc subunit (C2 domain) than the other Fab, can explain the unique biological properties of hIgG4, such as its weak pro-inflammatory activity.
History
Deposition
Nov 1, 2016
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Header (metadata) release
Feb 8, 2017
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Map release
Feb 14, 2018
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Update
Nov 25, 2020
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Current status
Nov 25, 2020
Processing site: RCSB / Status: Released
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Structure visualization
Movie
Surface view with section colored by density value
Type: NEGATIVE / Material: 1% uranyl acetate Details: Negative staining with uranyl acetate was carried out using the approach described by Valentine et al. with modification.
Grid
Model: Homemade / Material: COPPER / Mesh: 150 / Support film - Material: CARBON / Support film - topology: HOLEY
Details
Human myeloma IgG4
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Electron microscopy
Microscope
JEOL 100CX
Details
images were close to focus
Image recording
Film or detector model: KODAK 4489 FILM / Digitization - Scanner: NIKON SUPER COOLSCAN 9000 / Average exposure time: 1.5 sec. / Average electron dose: 100.0 e/Å2
Electron beam
Acceleration voltage: 80 kV / Electron source: TUNGSTEN HAIRPIN
Electron optics
Calibrated magnification: 80000 / Illumination mode: OTHER / Imaging mode: BRIGHT FIELD / Cs: 1.9 mm
Sample stage
Specimen holder model: JEOL
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Image processing
Details
Grids were analyzed in JEM100C (JEOL, Japan) transmission electron microscope at magnification x80000. EM plates with images were scanned using Nikon Cool SuperScan 9000 scanner at 0.158 nm/pix.
Particle selection
Number selected: 1500 Details: We boxed particles with 0.158 nm/pix and box size 196x196 pixels.
CTF correction
Details: Images were close to focus, no CTF correction implemented
Startup model
Type of model: OTHER Details: features ellipsoid (2:1) was used as initial model in the first round, than model obtained in the first step were used for 3D reconstruction
Final reconstruction
Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 32.0 Å / Resolution method: FSC 0.5 CUT-OFF / Software - Name: EMAN (ver. 2) / Number images used: 1000
Initial angle assignment
Type: ANGULAR RECONSTITUTION / Software - Name: EMAN (ver. 2)
Final angle assignment
Type: ANGULAR RECONSTITUTION / Software - Name: EMAN (ver. 2) / Details: EMAN2 system
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