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

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-10731
TitleStructure of full-length CD20 in complex with Rituximab Fab
Map data
Sample
  • Complex: Full-length human antigen CD20 in complex with Rituximab Fab
    • Complex: Full-length human CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Rituximab Fab
      • Protein or peptide: Rituximab Fab Heavy Chain
      • Protein or peptide: Rituximab Fab Light Chain
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: PENTADECANE
Function / homology
Function and homology information


store-operated calcium entry / positive regulation of calcium ion import across plasma membrane / calcium ion import into cytosol / epidermal growth factor receptor binding / B cell activation / B cell proliferation / plasma membrane raft / immunoglobulin binding / humoral immune response / B cell differentiation ...store-operated calcium entry / positive regulation of calcium ion import across plasma membrane / calcium ion import into cytosol / epidermal growth factor receptor binding / B cell activation / B cell proliferation / plasma membrane raft / immunoglobulin binding / humoral immune response / B cell differentiation / protein tetramerization / B cell receptor signaling pathway / response to bacterium / MHC class II protein complex binding / cell surface receptor signaling pathway / external side of plasma membrane / cell surface / extracellular space / extracellular exosome / nucleoplasm / identical protein binding / plasma membrane
Similarity search - Function
CD20-like family / Membrane-spanning 4-domains subfamily A / CD20-like family
Similarity search - Domain/homology
B-lymphocyte antigen CD20
Similarity search - Component
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.
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
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  • 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)
Voxel sizeX=Y=Z: 0.814 Å
Density
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
Components
  • Complex: Full-length human antigen CD20 in complex with Rituximab Fab
    • Complex: Full-length human CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Rituximab Fab
      • Protein or peptide: Rituximab Fab Heavy Chain
      • Protein or peptide: Rituximab Fab Light Chain
  • Ligand: CHOLESTEROL HEMISUCCINATE
  • Ligand: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
  • Ligand: PENTADECANE

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Supramolecule #1: Full-length human antigen CD20 in complex with Rituximab Fab

SupramoleculeName: Full-length human antigen CD20 in complex with Rituximab Fab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3

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Supramolecule #2: Full-length human CD20

SupramoleculeName: Full-length human CD20 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK-293F / Recombinant plasmid: pcDNA3.1+

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Supramolecule #3: Rituximab Fab

SupramoleculeName: Rituximab Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK-293F / Recombinant plasmid: IgG1

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Macromolecule #1: B-lymphocyte antigen CD20

MacromoleculeName: B-lymphocyte antigen CD20 / type: protein_or_peptide / ID: 1 / Details: Full-length wild type Human CD20 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Cell: B-Lymphocyte
Molecular weightTheoretical: 19.164797 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
FMRESKTLGA VQIMNGLFHI ALGGLLMIPA GIYAPICVTV WYPLWGGIMY IISGSLLAAT EKNSRKCLVK GKMIMNSLSL FAAISGMIL SIMDILNIKI SHFLKMESLN FIRAHTPYIN IYNCEPANPS EKNSPSTQYC YSIQSLFLGI LSVMLIFAFF Q ELVIAGIV ENEW

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Macromolecule #2: Rituximab Fab Heavy Chain

MacromoleculeName: Rituximab Fab Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.733541 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY NQKFKGKATL TADKSSSTAY MQLSSLTSE DSAVYYCARS TYYGGDWYFN VWGAGTTVTV SAASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT ...String:
QVQLQQPGAE LVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY NQKFKGKATL TADKSSSTAY MQLSSLTSE DSAVYYCARS TYYGGDWYFN VWGAGTTVTV SAASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFPEPVT V SWNSGALT SGVHTFPAVL QSSGLYSLSS VVTVPSSSLG TQTYICNVNH KPSNTKVDKK VEPKSC

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Macromolecule #3: Rituximab Fab Light Chain

MacromoleculeName: Rituximab Fab Light Chain / type: protein_or_peptide / ID: 3 / Details: Rituximab Light Chain / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.078623 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT SNLASGVPVR FSGSGSGTSY SLTISRVEAE DAATYYCQQ WTSNPPTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTEQDSK ...String:
QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKPG SSPKPWIYAT SNLASGVPVR FSGSGSGTSY SLTISRVEAE DAATYYCQQ WTSNPPTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTEQDSK DSTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 8 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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Macromolecule #5: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE

MacromoleculeName: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 5 / Number of copies: 2 / Formula: PC1
Molecular weightTheoretical: 790.145 Da
Chemical component information

ChemComp-PC1:
1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / phospholipid*YM

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Macromolecule #6: PENTADECANE

MacromoleculeName: PENTADECANE / type: ligand / ID: 6 / Number of copies: 10 / Formula: MYS
Molecular weightTheoretical: 212.415 Da
Chemical component information

ChemComp-MYS:
PENTADECANE

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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

Concentration1.5 mg/mL
BufferpH: 7.4
Component:
ConcentrationFormulaName
50.0 mMHEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)
100.0 mMNaClSodium Chloride
0.0084 PercentGDNGDN101
0.00168 PercentCHSCholesterol hemisuccinate
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 9263 / Average exposure time: 8.0 sec. / Average electron dose: 41.3 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 2.0 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 3103137
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

2osl

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14.2) / Number images used: 65203
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.14.2)
Final 3D classificationNumber classes: 4 / Software - Name: cryoSPARC (ver. 2.14.2)

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Atomic model buiding 1

Initial model
PDB IDChain

2osl

chain_id: H, residue_range: 1-224

2osl

chain_id: L, residue_range: 1-213
DetailsThe 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.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

PDB-6y90:
Structure of full-length CD20 in complex with Rituximab Fab

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