|Entry||Database: EMDB / ID: EMD-7356|
|Title||Single Molecule 3D Image of a Hole-Hole Homodimer Species Generated in the Production of a Bispecific Recombinant IgG1 (No. 004)|
|Sample||IgG1 hole-hole homodimer|
|Biological species||unidentified (others)|
|Method||electron tomography / negative staining / Resolution: 12.81 Å|
|Authors||Lei D / Liu J / Liu H / Lei M / Ren G|
|Funding support|| United States, 4 items |
|Citation||Journal: Sci Rep / Year: 2019|
Title: Single-Molecule 3D Images of "Hole-Hole" IgG1 Homodimers by Individual-Particle Electron Tomography.
Authors: Dongsheng Lei / Jianfang Liu / Hongbin Liu / Thomas E Cleveland / John P Marino / Ming Lei / Gang Ren /
Abstract: The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more ...The engineering of immunoglobulin-G molecules (IgGs) is of wide interest for improving therapeutics, for example by modulating the activity or multiplexing the specificity of IgGs to recognize more than one antigen. Optimization of engineered IgG requires knowledge of three-dimensional (3D) structure of synthetic IgG. However, due to flexible nature of the molecules, their structural characterization is challenging. Here, we use our reported individual-particle electron tomography (IPET) method with optimized negative-staining (OpNS) for direct 3D reconstruction of individual IgG hole-hole homodimer molecules. The hole-hole homodimer is an undesired variant generated during the production of a bispecific antibody using the knob-into-hole heterodimer technology. A total of 64 IPET 3D density maps at ~15 Å resolutions were reconstructed from 64 individual molecules, revealing 64 unique conformations. In addition to the known Y-shaped conformation, we also observed an unusual X-shaped conformation. The 3D structure of the X-shaped conformation contributes to our understanding of the structural details of the interaction between two heavy chains in the Fc domain. The IPET approach, as an orthogonal technique to characterize the 3D structure of therapeutic antibodies, provides insight into the 3D structural variety and dynamics of heterogeneous IgG molecules.
|Validation Report||Summary, Full report, XML, About validation report|
|Structure viewer||EM map: |
Downloads & links
|File||Download / File: emd_7356.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)|
|Projections & slices|
Images are generated by Spider.
|Voxel size||X=Y=Z: 2.96 Å|
|Symmetry||Space group: 1|
CCP4 map header:
-Entire IgG1 hole-hole homodimer
|Entire||Name: IgG1 hole-hole homodimer / Number of components: 1|
-Component #1: protein, IgG1 hole-hole homodimer
|Protein||Name: IgG1 hole-hole homodimer / Recombinant expression: No|
|Source||Species: unidentified (others)|
|Source (engineered)||Expression System: Cricetulus griseus (Chinese hamster) / Cell of expression system: ovary|
|Specimen||Specimen state: Particle / Method: negative staining|
|Sample solution||Buffer solution: Dulbecco's phosphate buffered saline (DPBS)|
|Staining||The grid was stained with 1% (w/v) uranyl formate|
|Vitrification||Cryogen name: NONE|
-Electron microscopy imaging
|Imaging||Microscope: ZEISS LIBRA120PLUS|
|Electron gun||Electron source: LAB6 / Accelerating voltage: 120 kV / Electron dose: 100.03 e/Å2 / Illumination mode: FLOOD BEAM|
|Lens||Magnification: 80000.0 X (nominal) / Cs: 2.2 mm / Imaging mode: BRIGHT FIELD / Energy filter: In-column Omega Filter|
|Specimen Holder||Model: OTHER|
|Camera||Detector: GATAN ULTRASCAN 4000 (4k x 4k)|
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