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- EMDB-38155: Fab domain of Immunoglobulin (IgG) reconstruction using conventio... -

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Basic information

Entry
Database: EMDB / ID: EMD-38155
TitleFab domain of Immunoglobulin (IgG) reconstruction using conventional single-particle cryo-EM
Map dataMap was reconstructed by cryoSPARC.
Sample
  • Complex: Fab domain of Rituximab
Keywordsimmunoglobulin / rituximab / Fab / cryoEM / volta phase plate / IMMUNE SYSTEM
Biological speciesMus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 5.69 Å
AuthorsLin HH / Wang CH / Wu YM / Tu IP / Chang WH
Funding support Taiwan, 6 items
OrganizationGrant numberCountry
Academia Sinica (Taiwan)AS-SUMMIT-107 Taiwan
Academia Sinica (Taiwan)AS-SUMMIT-108 Taiwan
Academia Sinica (Taiwan)AS-SUMMIT-109 Taiwan
Academia Sinica (Taiwan)AS-GCS-108-08 Taiwan
Academia Sinica (Taiwan)AS-IA-110-M05 Taiwan
Academia Sinica (Taiwan)AS-GCS-112-M03 Taiwan
CitationJournal: Sci Rep / Year: 2024
Title: Use of phase plate cryo-EM reveals conformation diversity of therapeutic IgG with 50 kDa Fab fragment resolved below 6 Å.
Authors: Hsin-Hung Lin / Chun-Hsiung Wang / Shih-Hsin Huang / Sung-Yao Lin / Takayuki Kato / Keiichi Namba / Naoki Hosogi / Chihong Song / Kazuyoshi Murata / Ching-Hsuan Yen / Tsui-Ling Hsu / Chi- ...Authors: Hsin-Hung Lin / Chun-Hsiung Wang / Shih-Hsin Huang / Sung-Yao Lin / Takayuki Kato / Keiichi Namba / Naoki Hosogi / Chihong Song / Kazuyoshi Murata / Ching-Hsuan Yen / Tsui-Ling Hsu / Chi-Huey Wong / Yi-Min Wu / I-Ping Tu / Wei-Hau Chang /
Abstract: While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small ...While cryogenic electron microscopy (cryo-EM) is fruitfully used for harvesting high-resolution structures of sizable macromolecules, its application to small or flexible proteins composed of small domains like immunoglobulin (IgG) remain challenging. Here, we applied single particle cryo-EM to Rituximab, a therapeutic IgG mediating anti-tumor toxicity, to explore its solution conformations. We found Rituximab molecules exhibited aggregates in cryo-EM specimens contrary to its solution behavior, and utilized a non-ionic detergent to successfully disperse them as isolated particles amenable to single particle analysis. As the detergent adversely reduced the protein-to-solvent contrast, we employed phase plate contrast to mitigate the impaired protein visibility. Assisted by phase plate imaging, we obtained a canonical three-arm IgG structure with other structures displaying variable arm densities co-existing in solution, affirming high flexibility of arm-connecting linkers. Furthermore, we showed phase plate imaging enables reliable structure determination of Fab to sub-nanometer resolution from ab initio, yielding a characteristic two-lobe structure that could be unambiguously docked with crystal structure. Our findings revealed conformation diversity of IgG and demonstrated phase plate was viable for cryo-EM analysis of small proteins without symmetry. This work helps extend cryo-EM boundaries, providing a valuable imaging and structural analysis framework for macromolecules with similar challenging features.
History
DepositionNov 28, 2023-
Header (metadata) releaseNov 27, 2024-
Map releaseNov 27, 2024-
UpdateDec 4, 2024-
Current statusDec 4, 2024Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_38155.png

Downloads & links

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Map

FileDownload / File: emd_38155.map.gz / Format: CCP4 / Size: 22.2 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMap was reconstructed by cryoSPARC.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 180 pix.
= 149.4 Å		0.83 Å/pix.
x 180 pix.
= 149.4 Å		0.83 Å/pix.
x 180 pix.
= 149.4 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.83 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.05
Minimum - Maximum-0.0 - 0.500458
Average (Standard dev.)0.0020885263 (±0.019216057)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions180180180
Spacing180180180
CellA=B=C: 149.4 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_38155_msk_1.map
Projections & Slices
AxesZYX

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Histogram

Histogram (log scale)

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Half map: Half map B.

Fileemd_38155_half_map_1.map
AnnotationHalf map B.
Projections & Slices
AxesZYX

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Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A.

Fileemd_38155_half_map_2.map
AnnotationHalf map A.
Projections & Slices
AxesZYX

Projections

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Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Fab domain of Rituximab

EntireName: Fab domain of Rituximab
Components
  • Complex: Fab domain of Rituximab

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Supramolecule #1: Fab domain of Rituximab

SupramoleculeName: Fab domain of Rituximab / type: complex / ID: 1 / Parent: 0
Details: The segmentation of Fab domain from Rituximab IgG was treated by using the PierceTM Fab preparation kit (Thermo Fisher Scientific Inc., Waltham, MA, USA)
Source (natural)Organism: Mus musculus (house mouse)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.2 mg/mL
BufferpH: 6.5
VitrificationCryogen name: ETHANE / Chamber humidity: 98 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: OTHER / Number real images: 50 / Average electron dose: 1.01 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.3000000000000003 µm / Nominal defocus min: 1.6 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsSuper Resolution mode
Particle selectionNumber selected: 12650536
Startup modelType of model: OTHER / Details: ab-initio map was generated by cryoSPARC.
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 5.69 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 228492
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cryoSPARC (ver. 4.1.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.1)
FSC plot (resolution estimation)

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