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- EMDB-10731: Structure of full-length CD20 in complex with Rituximab Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-10731
TitleStructure of full-length CD20 in complex with Rituximab Fab
Map data
SampleFull-length human antigen CD20 in complex with Rituximab Fab:
Full-length human CD20 / Rituximab Fab / B-lymphocyte antigen CD20 / (Rituximab Fab ...) x 2 / (ligand) x 3
Function / homology
Function and homology information


calcium ion import into cytosol / store-operated calcium entry / B cell proliferation / epidermal growth factor receptor binding / B cell activation / humoral immune response / immunoglobulin binding / plasma membrane raft / MHC class II protein complex binding / B cell differentiation ...calcium ion import into cytosol / store-operated calcium entry / B cell proliferation / epidermal growth factor receptor binding / B cell activation / humoral immune response / immunoglobulin binding / plasma membrane raft / MHC class II protein complex binding / B cell differentiation / response to bacterium / protein tetramerization / B cell receptor signaling pathway / external side of plasma membrane / cell surface / integral component of plasma membrane / extracellular space / extracellular exosome / nucleoplasm / identical protein binding / plasma membrane
B-lymphocyte antigen CD20 / Membrane-spanning 4-domains subfamily A / CD20-like family
B-lymphocyte antigen CD20
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.69 Å
AuthorsKumar A / Fronzes R / Reyes N
Funding support France, 1 items
OrganizationGrant numberCountry
European Research Council (ERC)309657 France
CitationJournal: Science / Year: 2020
Title: Binding mechanisms of therapeutic antibodies to human CD20.
Authors: Anand Kumar / Cyril Planchais / Rémi Fronzes / Hugo Mouquet / Nicolas Reyes /
Abstract: Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I ...Monoclonal antibodies (mAbs) targeting human antigen CD20 (cluster of differentiation 20) constitute important immunotherapies for the treatment of B cell malignancies and autoimmune diseases. Type I and II therapeutic mAbs differ in B cell binding properties and cytotoxic effects, reflecting differential interaction mechanisms with CD20. Here we present 3.7- to 4.7-angstrom cryo-electron microscopy structures of full-length CD20 in complexes with prototypical type I rituximab and ofatumumab and type II obinutuzumab. The structures and binding thermodynamics demonstrate that upon binding to CD20, type II mAbs form terminal complexes that preclude recruitment of additional mAbs and complement components, whereas type I complexes act as molecular seeds to increase mAb local concentration for efficient complement activation. Among type I mAbs, ofatumumab complexes display optimal geometry for complement recruitment. The uncovered mechanisms should aid rational design of next-generation immunotherapies targeting CD20.
Validation ReportSummary, Full report, XML, About validation report
History
DepositionMar 6, 2020-
Header (metadata) releaseAug 26, 2020-
Map releaseAug 26, 2020-
UpdateAug 26, 2020-
Current statusAug 26, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.34
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.34
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6y90
  • Surface level: 0.34
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10731.png

Downloads & links

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Map

FileDownload / File: emd_10731.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.81 Å/pix.
x 300 pix.
= 244.2 Å		0.81 Å/pix.
x 300 pix.
= 244.2 Å		0.81 Å/pix.
x 300 pix.
= 244.2 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.814 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.34 / Movie #1: 0.34
Minimum - Maximum-1.3570058 - 1.8360746
Average (Standard dev.)0.0036067262 (±0.056543805)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 244.2 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8140.8140.814
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z244.200244.200244.200
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-1.3571.8360.004

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Supplemental data

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Sample components

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Entire Full-length human antigen CD20 in complex with Rituximab Fab

EntireName: Full-length human antigen CD20 in complex with Rituximab Fab
Number of components: 9

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Component #1: protein, Full-length human antigen CD20 in complex with Rituximab Fab

ProteinName: Full-length human antigen CD20 in complex with Rituximab Fab
Recombinant expression: No

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Component #2: protein, Full-length human CD20

ProteinName: Full-length human CD20 / Recombinant expression: No
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human) / Vector: pcDNA3.1+ / Cell of expression system: HEK-293F

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Component #3: protein, Rituximab Fab

ProteinName: Rituximab Fab / Recombinant expression: No
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Homo sapiens (human) / Vector: IgG1 / Cell of expression system: HEK-293F

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Component #4: protein, B-lymphocyte antigen CD20

ProteinName: B-lymphocyte antigen CD20 / Details: Full-length wild type Human CD20 / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 19.164797 kDa
SourceSpecies: Homo sapiens (human)
Source (engineered)Expression System: Homo sapiens (human)
Source (natural)Cell: B-Lymphocyte

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Component #5: protein, Rituximab Fab Heavy Chain

ProteinName: Rituximab Fab Heavy Chain / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 23.733541 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Homo sapiens (human)

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Component #6: protein, Rituximab Fab Light Chain

ProteinName: Rituximab Fab Light Chain / Details: Rituximab Light Chain / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 23.078623 kDa
SourceSpecies: Mus musculus (house mouse)
Source (engineered)Expression System: Homo sapiens (human)

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Component #7: ligand, CHOLESTEROL HEMISUCCINATE

LigandName: CHOLESTEROL HEMISUCCINATE / Number of Copies: 8 / Recombinant expression: No
MassTheoretical: 0.486726 kDa

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Component #8: ligand, 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

LigandName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / Number of Copies: 2 / Recombinant expression: No
MassTheoretical: 0.790145 kDa

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Component #9: ligand, PENTADECANE

LigandName: PENTADECANE / Number of Copies: 10 / Recombinant expression: No
MassTheoretical: 0.212415 kDa

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Experimental details

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Sample preparation

SpecimenSpecimen state: Particle / Method: cryo EM
Sample solutionSpecimen conc.: 1.5 mg/mL / pH: 7.4
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 277.15 K / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
ImagingMicroscope: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 41.3 e/Å2 / Illumination mode: FLOOD BEAM
LensCs: 2.7 mm / Imaging mode: BRIGHT FIELD
Specimen HolderModel: OTHER
CameraDetector: GATAN K2 SUMMIT (4k x 4k)

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Image acquisition

Image acquisitionNumber of digital images: 9263

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Image processing

ProcessingMethod: single particle reconstruction / Applied symmetry: C2 (2 fold cyclic) / Number of projections: 65203
3D reconstructionSoftware: cryoSPARC / Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF

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Atomic model buiding

Modeling #1Refinement space: REAL
Details: The CD20-Rituximab fab model was built by placing CD20 peptide (residues 167-186) and Rituximab fab from PDB 2OSL into the EM map. The initial model was fitted manually and extended to a ...Details: The CD20-Rituximab fab model was built by placing CD20 peptide (residues 167-186) and Rituximab fab from PDB 2OSL into the EM map. The initial model was fitted manually and extended to a full CD20 model encompassing residues 45-216.
Input PDB model: 2OSL, 2OSL

2osl

2osl


Chain ID: H, L
Output model

6y90

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