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- EMDB-10734: Structure of full-length CD20 in complex with Obinutuzumab Fab -

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Open data


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Basic information

Entry
Database: EMDB / ID: EMD-10734
TitleStructure of full-length CD20 in complex with Obinutuzumab Fab
Map dataFull-length human CD20 in complex with Obinutuzumab
Sample
  • Complex: Full-length human antigen CD20 in complex with Obinutuzumab
    • Complex: Full-length human CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Obinutuzumab Fab
      • Protein or peptide: Obinutuzumab Fab heavy chain
      • Protein or peptide: Obinutuzumab Fab Light chain
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: 4.2 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-
UpdateSep 16, 2020-
Current statusSep 16, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6y9a
  • Surface level: 0.3
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6y9a
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10734.png

Downloads & links

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Map

FileDownload / File: emd_10734.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationFull-length human CD20 in complex with Obinutuzumab
Voxel sizeX=Y=Z: 0.814 Å
Density
Contour LevelBy AUTHOR: 0.22 / Movie #1: 0.3
Minimum - Maximum-0.93773156 - 1.336014
Average (Standard dev.)0.0015358375 (±0.040006608)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions350350350
Spacing350350350
CellA=B=C: 284.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8140.8140.814
M x/y/z350350350
origin x/y/z0.0000.0000.000
length x/y/z284.900284.900284.900
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS350350350
D min/max/mean-0.9381.3360.002

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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 Obinutuzumab

EntireName: Full-length human antigen CD20 in complex with Obinutuzumab
Components
  • Complex: Full-length human antigen CD20 in complex with Obinutuzumab
    • Complex: Full-length human CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Obinutuzumab Fab
      • Protein or peptide: Obinutuzumab Fab heavy chain
      • Protein or peptide: Obinutuzumab Fab Light chain

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

SupramoleculeName: Full-length human antigen CD20 in complex with Obinutuzumab
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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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: Obinutuzumab Fab

SupramoleculeName: Obinutuzumab Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Homo sapiens (human)
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 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human) / Cell: B-Lymphocyte
Molecular weightTheoretical: 18.735373 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 E

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Macromolecule #2: Obinutuzumab Fab heavy chain

MacromoleculeName: Obinutuzumab Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.40726 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: VQLVQSGAEV KKPGSSVKVS CKASGYAFSY SWINWVRQAP GQGLEWMGRI FPGDGDTDYN GKFKGRVTIT ADKSTSTAYM ELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV ...String:
VQLVQSGAEV KKPGSSVKVS CKASGYAFSY SWINWVRQAP GQGLEWMGRI FPGDGDTDYN GKFKGRVTIT ADKSTSTAYM ELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTVSSA STKGPSVFPL APSSKSTSGG TAALGCLVKD YFPEPVTVSW N SGALTSGV HTFPAVLQSS GLYSLSSVVT VPSSSLGTQT YICNVNHKPS NTKVDKKVEP K

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Macromolecule #3: Obinutuzumab Fab Light chain

MacromoleculeName: Obinutuzumab Fab Light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.964822 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVG VYYCAQNLEL PYTFGGGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL Q SGNSQESV ...String:
DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVG VYYCAQNLEL PYTFGGGTKV EIKRTVAAPS VFIFPPSDEQ LKSGTASVVC LLNNFYPREA KVQWKVDNAL Q SGNSQESV TEQDSKDSTY SLSSTLTLSK ADYEKHKVYA CEVTHQGLSS PVTKSFNRGE C

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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: AIR
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Grids were blot for 3.5 Sec..

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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: 10545 / Average electron dose: 42.84 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: 1316678
CTF correctionSoftware - Name: Gctf (ver. 1.06) / Details: Gctf
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

3pp4

Final reconstructionNumber classes used: 3 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 2.14.2) / Number images used: 63547
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

3pp4

chain_id: H, residue_range: 2-220

3pp4

chain_id: L, residue_range: 1-219

6y90

residue_range: 45-213
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

PDB-6y9a:
Structure of full-length CD20 in complex with Obinutuzumab Fab

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