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- EMDB-10732: Structure of full-length CD20 in complex with Ofatumumab Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-10732
TitleStructure of full-length CD20 in complex with Ofatumumab Fab
Map dataStructure of Human CD20 in complex with Ofatumumab
Sample
  • Complex: Full-length human antigen CD20 in complex with Ofatumumab Fab
    • Complex: Full-length human CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Ofatumumab Fab
      • Protein or peptide: Ofatumumab Fab light chain
      • Protein or peptide: Ofatumumab Fab heavy chain
  • Ligand: CHOLESTEROL HEMISUCCINATE
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)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.73 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: 0.61
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.61
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6y92
  • Surface level: 0.61
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10732.png

Downloads & links

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Map

FileDownload / File: emd_10732.map.gz / Format: CCP4 / Size: 75.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationStructure of Human CD20 in complex with Ofatumumab
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.61 / Movie #1: 0.61
Minimum - Maximum-1.9058784 - 2.7761757
Average (Standard dev.)0.0032388624 (±0.09052563)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions270270270
Spacing270270270
CellA=B=C: 270.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z270270270
origin x/y/z0.0000.0000.000
length x/y/z270.000270.000270.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS270270270
D min/max/mean-1.9062.7760.003

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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 Ofatumumab Fab

EntireName: Full-length human antigen CD20 in complex with Ofatumumab Fab
Components
  • Complex: Full-length human antigen CD20 in complex with Ofatumumab Fab
    • Complex: Full-length human CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Ofatumumab Fab
      • Protein or peptide: Ofatumumab Fab light chain
      • Protein or peptide: Ofatumumab Fab heavy chain
  • Ligand: CHOLESTEROL HEMISUCCINATE

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

SupramoleculeName: Full-length human antigen CD20 in complex with Ofatumumab 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: Ofatumumab Fab

SupramoleculeName: Ofatumumab Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human) / Location in cell: extra cellular
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK-293F / Recombinant plasmid: IgG1 expression vector

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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: 19.914656 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString:
TQSFFMRESK TLGAVQIMNG LFHIALGGLL MIPAGIYAPI CVTVWYPLWG GIMYIISGSL LAATEKNSRK CLVKGKMIMN SLSLFAAIS GMILSIMDIL NIKISHFLKM ESLNFIRAHT PYINIYNCEP ANPSEKNSPS TQYCYSIQSL FLGILSVMLI F AFFQELVI AGIVENEWKR

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Macromolecule #2: Ofatumumab Fab light chain

MacromoleculeName: Ofatumumab Fab light chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.174719 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQ QRSNWPITFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS ...String:
EIVLTQSPAT LSLSPGERAT LSCRASQSVS SYLAWYQQKP GQAPRLLIYD ASNRATGIPA RFSGSGSGTD FTLTISSLEP EDFAVYYCQ QRSNWPITFG QGTRLEIKRT VAAPSVFIFP PSDEQLKSGT ASVVCLLNNF YPREAKVQWK VDNALQSGNS Q ESVTEQDS KDSTYSLSST LTLSKADYEK HKVYACEVTH QGLSSPVTKS FNR

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Macromolecule #3: Ofatumumab Fab heavy chain

MacromoleculeName: Ofatumumab Fab heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.138934 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: EVQLVESGGG LVQPGRSLRL SCAASGFTFN DYAMHWVRQA PGKGLEWVST ISWNSGSIGY ADSVKGRFTI SRDNAKKSLY LQMNSLRAE DTALYYCAKD IQYGNYYYGM DVWGQGTTVT VSSASTKGPS VFPLAPGGTA ALGCLVKDYF PEPVTVSWNS G ALTSGVHT ...String:
EVQLVESGGG LVQPGRSLRL SCAASGFTFN DYAMHWVRQA PGKGLEWVST ISWNSGSIGY ADSVKGRFTI SRDNAKKSLY LQMNSLRAE DTALYYCAKD IQYGNYYYGM DVWGQGTTVT VSSASTKGPS VFPLAPGGTA ALGCLVKDYF PEPVTVSWNS G ALTSGVHT FPAVLQSSGL YSLSSVVTVP SSSLGTQTYI CNVNHKPSNT KVDKKVEP

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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

Concentration1.2 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

MicroscopeTFS GLACIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 1 / Number real images: 2540 / Average exposure time: 40.0 sec. / Average electron dose: 40.0 e/Å2
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated defocus max: 2.2 µm / Calibrated defocus min: 0.8 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm
Sample stageCooling holder cryogen: NITROGEN

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Image processing

Particle selectionNumber selected: 919699
CTF correctionSoftware - Name: Gctf (ver. 1.06)
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3giz

Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 4.73 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14.2) / Number images used: 99146
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: 3 / Software - Name: cryoSPARC (ver. 2.14.2)

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Atomic model buiding 1

Initial model
PDB IDChain

3giz

chain_id: H, residue_range: 1-216

3giz

chain_id: L, residue_range: 1-211
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6y92:
Structure of full-length CD20 in complex with Ofatumumab Fab

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