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- EMDB-7372: Single Molecule 3D Image of a Hole-Hole Homodimer Species Generat... -

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Basic information

Entry
Database: EMDB / ID: EMD-7372
TitleSingle Molecule 3D Image of a Hole-Hole Homodimer Species Generated in the Production of a Bispecific Recombinant IgG1 (No. 020)
Map dataSingle Molecule 3D Image of a Hole-Hole Homodimer Species Generated in the Production of a Bispecific Recombinant IgG1 (No. 020)
Sample
  • Organelle or cellular component: IgG1 hole-hole homodimer
Biological speciesunidentified (others)
Methodelectron tomography / negative staining / Resolution: 13.93 Å
AuthorsLei D / Liu J / Liu H / Lei M / Ren G
Funding support United States, 4 items
OrganizationGrant numberCountry
National Science Foundation (NSF, United States)DMR-1344290 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM104427 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL115153 United States
Department of Energy (DOE, United States)DE-AC02-05CH11231 United States
CitationJournal: 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.
History
DepositionJan 23, 2018-
Header (metadata) releaseFeb 28, 2018-
Map releaseJan 30, 2019-
UpdateDec 25, 2019-
Current statusDec 25, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by radius
  • Surface level: 0.2
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_7372.png

Downloads & links

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Map

FileDownload / File: emd_7372.map.gz / Format: CCP4 / Size: 8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSingle Molecule 3D Image of a Hole-Hole Homodimer Species Generated in the Production of a Bispecific Recombinant IgG1 (No. 020)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
2.96 Å/pix.
x 128 pix.
= 378.88 Å		2.96 Å/pix.
x 128 pix.
= 378.88 Å		2.96 Å/pix.
x 128 pix.
= 378.88 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 2.96 Å
Density

Histogram

Histogram (log scale)
Contour LevelMovie #1: 0.2
Minimum - Maximum-0.33066773 - 1.9942639
Average (Standard dev.)0.0010865752 (±0.055288576)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin0430
Dimensions128128128
Spacing128128128
CellA=B=C: 378.88 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.962.962.96
M x/y/z128128128
origin x/y/z0.0000.0000.000
length x/y/z378.880378.880378.880
α/β/γ90.00090.00090.000
start NX/NY/NZ000
NX/NY/NZ281156
MAP C/R/S123
start NC/NR/NS4300
NC/NR/NS128128128
D min/max/mean-0.3311.9940.001

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Supplemental data

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Sample components

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Entire : IgG1 hole-hole homodimer

EntireName: IgG1 hole-hole homodimer
Components
  • Organelle or cellular component: IgG1 hole-hole homodimer

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Supramolecule #1: IgG1 hole-hole homodimer

SupramoleculeName: IgG1 hole-hole homodimer / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: unidentified (others)
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster) / Recombinant cell: ovary

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Experimental details

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Structure determination

Methodnegative staining
Processingelectron tomography
Aggregation stateparticle

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Sample preparation

BufferpH: 7 / Details: Dulbecco's phosphate buffered saline (DPBS)
StainingType: NEGATIVE / Material: Uranyl Formate / Details: The grid was stained with 1% (w/v) uranyl formate
GridMaterial: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 0.039 kPa
SectioningOther: NO SECTIONING

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Electron microscopy

MicroscopeZEISS LIBRA120PLUS
Specialist opticsEnergy filter - Name: In-column Omega Filter
Image recordingFilm or detector model: GATAN ULTRASCAN 4000 (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Average electron dose: 71.1 e/Å2
Electron beamAcceleration voltage: 120 kV / Electron source: LAB6
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.2 mm / Nominal magnification: 80000

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Image processing

DetailsX-ray speckles in images were removed before CTF correction.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 13.93 Å / Resolution method: FSC 0.5 CUT-OFF
Details: The 3D reconstruction was performed by using Individual-Particle Electron Tomography (IPET). The obtained 3D map was Gaussian low-pass filtered to 2 nm. Image of the 3D map was taken in ...Details: The 3D reconstruction was performed by using Individual-Particle Electron Tomography (IPET). The obtained 3D map was Gaussian low-pass filtered to 2 nm. Image of the 3D map was taken in Chimera with the 'hide dust' function activated.
Number images used: 61
FSC plot (resolution estimation)

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