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

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Basic information

Entry
Database: PDB / ID: 6y90
TitleStructure of full-length CD20 in complex with Rituximab Fab
Components
  • (Rituximab Fab ...) x 2
  • B-lymphocyte antigen CD20
KeywordsMEMBRANE PROTEIN / cancer immunotherapy / therapeutic antibody
Function / homology
Function and homology information


store-operated calcium entry / calcium ion import into cytosol / positive regulation of calcium ion import across plasma membrane / epidermal growth factor receptor binding / B cell activation / immunoglobulin binding / plasma membrane raft / B cell proliferation / humoral immune response / B cell differentiation ...store-operated calcium entry / calcium ion import into cytosol / positive regulation of calcium ion import across plasma membrane / epidermal growth factor receptor binding / B cell activation / immunoglobulin binding / plasma membrane raft / B cell proliferation / humoral immune response / B cell differentiation / B cell receptor signaling pathway / protein tetramerization / 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
Membrane-spanning 4-domains subfamily A / CD20-like family / CD20-like family / Immunoglobulins / Immunoglobulin-like / Sandwich / Mainly Beta
Similarity search - Domain/homology
PENTADECANE / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / CHOLESTEROL HEMISUCCINATE / B-lymphocyte antigen CD20
Similarity search - Component
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.69 Å
AuthorsKumar, A. / Reyes, N.
Funding support France, 1items
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, 2020Deposition site: PDBE / Processing site: PDBE
Revision 1.0Aug 26, 2020Provider: repository / Type: Initial release

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Structure visualization

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  • Deposited structure unit
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  • Superimposition on EM map
  • EMDB-10731
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
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Assembly

Deposited unit
A: B-lymphocyte antigen CD20
B: B-lymphocyte antigen CD20
C: Rituximab Fab Heavy Chain
D: Rituximab Fab Light Chain
H: Rituximab Fab Heavy Chain
L: Rituximab Fab Light Chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)139,55226
Polymers131,9546
Non-polymers7,59820
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration, microscopy, Negative staining
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area22170 Å2
ΔGint-113 kcal/mol
Surface area56260 Å2
MethodPISA

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Components

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Protein , 1 types, 2 molecules AB

#1: Protein B-lymphocyte antigen CD20 / B-lymphocyte surface antigen B1 / Bp35 / Leukocyte surface antigen Leu-16 / Membrane-spanning 4- ...B-lymphocyte surface antigen B1 / Bp35 / Leukocyte surface antigen Leu-16 / Membrane-spanning 4-domains subfamily A member 1


Mass: 19164.797 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Full-length wild type Human CD20 / Source: (gene. exp.) Homo sapiens (human) / Cell: B-Lymphocyte / Gene: MS4A1, CD20 / Plasmid: pcDNA3.1+ / Cell line (production host): HEK-293F / Production host: Homo sapiens (human) / References: UniProt: P11836

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Antibody , 2 types, 4 molecules CHDL

#2: Antibody Rituximab Fab Heavy Chain


Mass: 23733.541 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Plasmid: IgG1 expression vector / Cell line (production host): HEK-293F / Production host: Homo sapiens (human)
#3: Antibody Rituximab Fab Light Chain


Mass: 23078.623 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Details: Rituximab Light Chain / Source: (gene. exp.) Mus musculus (house mouse) / Plasmid: IgG1 expression vector / Cell line (production host): HEK-293F / Production host: Homo sapiens (human)

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Non-polymers , 3 types, 20 molecules

#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C31H50O4
#5: Chemical ChemComp-PC1 / 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / 3-SN-PHOSPHATIDYLCHOLINE


Mass: 790.145 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C44H88NO8P / Comment: phospholipid*YM
#6: Chemical
ChemComp-MYS / PENTADECANE


Mass: 212.415 Da / Num. of mol.: 10 / Source method: obtained synthetically / Formula: C15H32

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Details

Has ligand of interestN

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

Component
IDNameTypeEntity IDParent-IDSource
1Full-length human antigen CD20 in complex with Rituximab FabCOMPLEX#1-#30MULTIPLE SOURCES
2Full-length human CD20COMPLEX#11RECOMBINANT
3Rituximab FabCOMPLEX#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11
22NO
33NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
12Homo sapiens (human)9606
23Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-IDCellPlasmid
12Homo sapiens (human)9606HEK-293FpcDNA3.1+
23Homo sapiens (human)9606HEK-293FIgG1
Buffer solutionpH: 7.4
Buffer component
IDConc.NameFormulaBuffer-ID
150 mM(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)HEPES1
2100 mMSodium ChlorideNaCl1
30.0084 PercentGDN101GDN1
40.00168 PercentCholesterol hemisuccinateCHS1
SpecimenConc.: 1.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELD / Calibrated defocus min: 800 nm / Calibrated defocus max: 2000 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 8 sec. / Electron dose: 41.3 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 9263
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.18.2_3874: / Classification: refinement
EM software
IDNameVersionCategoryDetails
2SerialEMimage acquisition
4Gctf1.06CTF correction
7UCSF Chimera1.13.1model fittingRigid Body fit
8Coot0.8.9.2model fittingmodel manual fitting and extension
10PHENIX1.17.1model refinement
11cryoSPARC2.14.2initial Euler assignment
12cryoSPARC2.14.2final Euler assignment
13cryoSPARC2.14.2classification
14cryoSPARC2.14.23D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3103137
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.69 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 65203 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / 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.
Atomic model building
IDPDB-IDPdb chain-ID 3D fitting-IDPdb chain residue range
12OSL

2osl

H11-224
22OSL

2osl

L11-213
RefinementCross valid method: NONE
Stereochemistry target values: GeoStd + Monomer Library + CDL v1.2
Displacement parametersBiso mean: 72.58 Å2
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00310018
ELECTRON MICROSCOPYf_angle_d0.62213590
ELECTRON MICROSCOPYf_dihedral_angle_d15.9742080
ELECTRON MICROSCOPYf_chiral_restr0.0411532
ELECTRON MICROSCOPYf_plane_restr0.0031650

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