+Open data
-Basic information
Entry | Database: EMDB / ID: EMD-10733 | |||||||||
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Title | Structure of full-length CD20 in complex with Obinutuzumab Fab | |||||||||
Map data | Soft masked used for focused refinement | |||||||||
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 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 | |||||||||
Biological species | Homo sapiens (human) | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 4.33 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_10733.map.gz | 83.6 MB | EMDB map data format | |
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Header (meta data) | emd-10733-v30.xml emd-10733.xml | 19.4 KB 19.4 KB | Display Display | EMDB header |
Images | emd_10733.png | 52.3 KB | ||
Masks | emd_10733_msk_1.map | 163.6 MB | Mask map | |
Archive directory | http://ftp.pdbj.org/pub/emdb/structures/EMD-10733 ftp://ftp.pdbj.org/pub/emdb/structures/EMD-10733 | HTTPS FTP |
-Validation report
Summary document | emd_10733_validation.pdf.gz | 271.3 KB | Display | EMDB validaton report |
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Full document | emd_10733_full_validation.pdf.gz | 270.5 KB | Display | |
Data in XML | emd_10733_validation.xml.gz | 6.8 KB | Display | |
Arichive directory | https://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10733 ftp://ftp.pdbj.org/pub/emdb/validation_reports/EMD-10733 | HTTPS FTP |
-Related structure data
Related structure data | 6y97MC 6y90C 6y92C 6y9aC M: atomic model generated by this map C: citing same article (ref.) |
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Similar structure data |
-Links
EMDB pages | EMDB (EBI/PDBe) / EMDataResource |
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-Map
File | Download / File: emd_10733.map.gz / Format: CCP4 / Size: 163.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Annotation | Soft masked used for focused refinement | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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
-Mask #1
File | emd_10733_msk_1.map | ||||||||||||
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Projections & Slices |
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Density Histograms |
-Sample components
-Entire : Full-length human antigen CD20 in complex with Obinutuzumab Fab
Entire | Name: Full-length human antigen CD20 in complex with Obinutuzumab Fab |
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Components |
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-Supramolecule #1: Full-length human antigen CD20 in complex with Obinutuzumab Fab
Supramolecule | Name: Full-length human antigen CD20 in complex with Obinutuzumab Fab type: cell / ID: 1 / Parent: 0 / Macromolecule list: all |
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Source (natural) | Organism: Homo sapiens (human) / Tissue: B-lymphocyte |
-Supramolecule #2: Full-length human CD20
Supramolecule | Name: Full-length human CD20 / type: organelle_or_cellular_component / ID: 2 / Parent: 1 / Macromolecule list: #1 |
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Source (natural) | Organism: Homo sapiens (human) / Location in cell: Plasma membrane |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK-293F / Recombinant plasmid: pcDNA3.1 |
-Supramolecule #3: Obinutuzumab Fab
Supramolecule | Name: Obinutuzumab Fab / type: organelle_or_cellular_component / ID: 3 / Parent: 1 / Macromolecule list: #2-#3 |
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Source (natural) | Organism: Homo sapiens (human) / Location in cell: Extra cellular |
Recombinant expression | Organism: Homo sapiens (human) / Recombinant cell: HEK-293F / Recombinant plasmid: IgG1 Expression vector |
-Macromolecule #1: B-lymphocyte antigen CD20
Macromolecule | Name: B-lymphocyte antigen CD20 / type: protein_or_peptide / ID: 1 / Details: Wt CD20 / Number of copies: 2 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) / Cell: B-Lymphocyte |
Molecular weight | Theoretical: 18.735373 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 E |
-Macromolecule #2: Obinutuzumab Fab heavy chain
Macromolecule | Name: Obinutuzumab Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 12.855385 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: VQLVQSGAEV KKPGSSVKVS CKASGYAFSY SWINWVRQAP GQGLEWMGRI FPGDGDTDYN GKFKGRVTIT ADKSTSTAYM ELSSLRSED TAVYYCARNV FDGYWLVYWG QGTLVTV |
-Macromolecule #3: Obinutuzumab Fab light chain
Macromolecule | Name: Obinutuzumab Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO |
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Source (natural) | Organism: Homo sapiens (human) |
Molecular weight | Theoretical: 11.962462 KDa |
Recombinant expression | Organism: Homo sapiens (human) |
Sequence | String: DIVMTQTPLS LPVTPGEPAS ISCRSSKSLL HSNGITYLYW YLQKPGQSPQ LLIYQMSNLV SGVPDRFSGS GSGTDFTLKI SRVEAEDVG VYYCAQNLEL PYTFGGGTKV E |
-Experimental details
-Structure determination
Method | cryo EM |
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Processing | single particle reconstruction |
Aggregation state | particle |
-Sample preparation
Concentration | 1.2 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: AIR | |||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV / Details: Grids were blot for 3.5 Sec.. |
-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: 10545 / Average exposure time: 8.0 sec. / Average electron dose: 42.84 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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Refinement | Space: REAL / Protocol: RIGID BODY FIT | ||||||
Output model | PDB-6y97: |