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- PDB-6vja: Structure of CD20 in complex with rituximab Fab -

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Basic information

Entry
Database: PDB / ID: 6vja
TitleStructure of CD20 in complex with rituximab Fab
Components
  • B-lymphocyte antigen CD20
  • Rituximab Fab heavy chain
  • Rituximab Fab light chain
KeywordsIMMUNE SYSTEM / MS4A1 / CD20 / human / Rituximab / therapeutic / antibody
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 / response to bacterium / protein tetramerization / B cell receptor signaling pathway / 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
CHOLESTEROL HEMISUCCINATE / B-lymphocyte antigen CD20
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRohou, A. / Croll, T.I.
CitationJournal: Science / Year: 2020
Title: Structure of CD20 in complex with the therapeutic monoclonal antibody rituximab.
Authors: Lionel Rougé / Nancy Chiang / Micah Steffek / Christine Kugel / Tristan I Croll / Christine Tam / Alberto Estevez / Christopher P Arthur / Christopher M Koth / Claudio Ciferri / Edward ...Authors: Lionel Rougé / Nancy Chiang / Micah Steffek / Christine Kugel / Tristan I Croll / Christine Tam / Alberto Estevez / Christopher P Arthur / Christopher M Koth / Claudio Ciferri / Edward Kraft / Jian Payandeh / Gerald Nakamura / James T Koerber / Alexis Rohou /
Abstract: Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function ...Cluster of differentiation 20 (CD20) is a B cell membrane protein that is targeted by monoclonal antibodies for the treatment of malignancies and autoimmune disorders but whose structure and function are unknown. Rituximab (RTX) has been in clinical use for two decades, but how it activates complement to kill B cells remains poorly understood. We obtained a structure of CD20 in complex with RTX, revealing CD20 as a compact double-barrel dimer bound by two RTX antigen-binding fragments (Fabs), each of which engages a composite epitope and an extensive homotypic Fab:Fab interface. Our data suggest that RTX cross-links CD20 into circular assemblies and lead to a structural model for complement recruitment. Our results further highlight the potential relevance of homotypic Fab:Fab interactions in targeting oligomeric cell-surface markers.
History
DepositionJan 15, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Feb 26, 2020Provider: repository / Type: Initial release
Revision 1.1Mar 4, 2020Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.pdbx_database_id_PubMed / _citation.title / _citation.year / _citation_author.identifier_ORCID / _citation_author.name
Revision 1.2Mar 25, 2020Group: Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation_author.identifier_ORCID

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-21212
  • Imaged by UCSF Chimera
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Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
C: B-lymphocyte antigen CD20
D: B-lymphocyte antigen CD20
H: Rituximab Fab heavy chain
I: Rituximab Fab heavy chain
L: Rituximab Fab light chain
M: Rituximab Fab light chain
hetero molecules


Theoretical massNumber of molelcules
Total (without water)159,18612
Polymers156,2666
Non-polymers2,9206
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#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: 31320.789 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: MS4A1, CD20 / Production host: Trichoplusia ni (cabbage looper) / References: UniProt: P11836
#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.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / Strain (production host): 34B8
#3: Antibody Rituximab Fab light chain


Mass: 23078.623 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Escherichia coli (E. coli) / Strain (production host): 34B8
#4: Chemical
ChemComp-Y01 / CHOLESTEROL HEMISUCCINATE


Mass: 486.726 Da / Num. of mol.: 6 / Source method: obtained synthetically / Formula: C31H50O4
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

ComponentName: CD20 in complex with rituximab Fab / Type: COMPLEX / Entity ID: #1-#3 / Source: MULTIPLE SOURCES
Molecular weightValue: 0.135 MDa / Experimental value: YES
Source (natural)Organism: Homo sapiens (human)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2150 mMNaClSodium chloride1
30.01 %GDN1
SpecimenConc.: 2 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, UltrAuFoil, R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K / Details: 5s blot time

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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 FIELDBright-field microscopy / Nominal magnification: 165000 X / Calibrated magnification: 58906 X / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 10 sec. / Electron dose: 53 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 QUANTUM (4k x 4k) / Num. of grids imaged: 3 / Num. of real images: 24743
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV
Image scansSampling size: 5 µm / Movie frames/image: 40 / Used frames/image: 1-40

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Processing

SoftwareName: PHENIX / Version: 1.17_3643: / Classification: refinement
EM software
IDNameCategoryFitting-ID
1cisTEMparticle selection
2SerialEMimage acquisition
4cisTEMCTF correction
7Cootmodel fitting1
9cisTEMinitial Euler assignment
10cisTEMfinal Euler assignment
11cisTEMclassification
12cisTEM3D reconstruction
26PHENIXmodel refinement1
45UCSF Chimeramodel fitting2
46Cootmodel fitting2
47PHENIXmodel refinement2
48ISOLDEmodel fitting2
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 4114800
Details: 15,630 movies were selected because of the quality of their CTF fits. 4,114,800 coordinates were selected, leading to 466,632 particles which clustered into high-quality 2D class averages
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 149322 / Algorithm: FOURIER SPACE / Details: No data beyond 1/5 A-1 were used in refinement / Num. of class averages: 1 / Symmetry type: POINT
Atomic model building
IDProtocolSpaceDetails
1AB INITIO MODELREALAtomic model for CD20 was built ab initio and manually in COOT. It was then re-built using ISOLDE, and finally refined in Phenix.
2FLEXIBLE FITREALThe X-ray crystallographic atomic model for Rituximab Fab was rigid-body fit into the map, modified using Cootand ISOLDE, and refined with Phenix
Atomic model buildingPDB-ID: 2OSL

2osl

Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0139582
ELECTRON MICROSCOPYf_angle_d1.21513058
ELECTRON MICROSCOPYf_dihedral_angle_d16.013418
ELECTRON MICROSCOPYf_chiral_restr0.0641490
ELECTRON MICROSCOPYf_plane_restr0.0061608

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