+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-10731 | |||||||||
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Title | Structure of full-length CD20 in complex with Rituximab Fab | |||||||||
Map data | ||||||||||
Sample |
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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 proliferation / plasma membrane raft / B cell activation / 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 proliferation / plasma membrane raft / B cell activation / immunoglobulin binding / humoral immune response / B cell differentiation / response to bacterium / B cell receptor signaling pathway / protein tetramerization / 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 | |||||||||
Biological species | Homo sapiens (human) / Mus musculus (house mouse) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 3.69 Å | |||||||||
Authors | Kumar A / Fronzes R / Reyes N | |||||||||
Funding support | France, 1 items
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Citation | Journal: 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 |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | EM map: SurfViewMolmilJmol/JSmol |
Supplemental images |
-Downloads & links
-EMDB archive
Map data | emd_10731.map.gz | 52.7 MB | EMDB map data format | |
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Header (meta data) | emd-10731-v30.xml emd-10731.xml | 20.3 KB 20.3 KB | Display Display | EMDB header |
Images | emd_10731.png | 68.4 KB | ||
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10731 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10731 | HTTPS FTP |
-Validation report
Summary document | emd_10731_validation.pdf.gz | 261.3 KB | Display | EMDB validaton report |
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Full document | emd_10731_full_validation.pdf.gz | 260.4 KB | Display | |
Data in XML | emd_10731_validation.xml.gz | 6.5 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10731 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10731 | HTTPS FTP |
-Related structure data
Related structure data | 6y90MC 6y92C 6y97C 6y9aC C: citing same article (ref.) M: atomic model generated by this map |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_10731.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Projections & slices | Image control
Images are generated by Spider. | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Voxel size | X=Y=Z: 0.814 Å | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Density |
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Symmetry | Space group: 1 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Details | EMDB XML:
CCP4 map header:
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-Supplemental data
-Sample components
-Entire : Full-length human antigen CD20 in complex with Rituximab Fab
Entire | Name: Full-length human antigen CD20 in complex with Rituximab Fab |
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Components |
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-Supramolecule #1: Full-length human antigen CD20 in complex with Rituximab Fab
Supramolecule | Name: 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
Supramolecule | Name: Full-length human CD20 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK-293F / Recombinant plasmid: pcDNA3.1+ |
-Supramolecule #3: Rituximab Fab
Supramolecule | Name: Rituximab Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: Mus musculus (house mouse) |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK-293F / Recombinant plasmid: IgG1 |
-Macromolecule #1: B-lymphocyte antigen CD20
Macromolecule | Name: B-lymphocyte antigen CD20 / type: protein_or_peptide / ID: 1 / Details: Full-length wild type Human CD20 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) / Cell: B-Lymphocyte |
Molecular weight | Theoretical: 19.164797 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: FMRESKTLGA VQIMNGLFHI ALGGLLMIPA GIYAPICVTV WYPLWGGIMY IISGSLLAAT EKNSRKCLVK GKMIMNSLSL FAAISGMIL SIMDILNIKI SHFLKMESLN FIRAHTPYIN IYNCEPANPS EKNSPSTQYC YSIQSLFLGI LSVMLIFAFF Q ELVIAGIV ENEW |
-Macromolecule #2: Rituximab Fab Heavy Chain
Macromolecule | Name: Rituximab Fab Heavy Chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 23.733541 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 |
-Macromolecule #3: Rituximab Fab Light Chain
Macromolecule | Name: Rituximab Fab Light Chain / type: protein_or_peptide / ID: 3 / Details: Rituximab Light Chain / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Mus musculus (house mouse) |
Molecular weight | Theoretical: 23.078623 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: 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 |
-Macromolecule #4: CHOLESTEROL HEMISUCCINATE
Macromolecule | Name: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 8 / Formula: Y01 |
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Molecular weight | Theoretical: 486.726 Da |
Chemical component information | ChemComp-Y01: |
-Macromolecule #5: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE
Macromolecule | Name: 1,2-DIACYL-SN-GLYCERO-3-PHOSPHOCHOLINE / type: ligand / ID: 5 / Number of copies: 2 / Formula: PC1 |
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Molecular weight | Theoretical: 790.145 Da |
Chemical component information | ChemComp-PC1: |
-Macromolecule #6: PENTADECANE
Macromolecule | Name: PENTADECANE / type: ligand / ID: 6 / Number of copies: 10 / Formula: MYS |
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Molecular weight | Theoretical: 212.415 Da |
Chemical component information | ChemComp-MYS: |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.5 mg/mL | |||||||||||||||
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Buffer | pH: 7.4 Component:
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Grid | Model: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: OTHER | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV |
-Electron microscopy
Microscope | FEI TITAN KRIOS |
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Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN |
Electron optics | C2 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 stage | Cooling holder cryogen: NITROGEN |
Experimental equipment | Model: Titan Krios / Image courtesy: FEI Company |
+Image processing
-Atomic model buiding 1
Initial model |
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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. | ||||||
Refinement | Space: REAL / Protocol: AB INITIO MODEL | ||||||
Output model | PDB-6y90: |