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- EMDB-21212: Structure of CD20 in complex with rituximab Fab -

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Basic information

Entry
Database: EMDB / ID: EMD-21212
TitleStructure of CD20 in complex with rituximab Fab
Map dataSharpened map, used for figure making and model building.
Sample
  • Complex: CD20 in complex with rituximab Fab
    • Protein or peptide: B-lymphocyte antigen CD20
    • Protein or peptide: Rituximab Fab heavy chain
    • Protein or peptide: Rituximab Fab light chain
  • Ligand: CHOLESTEROL HEMISUCCINATE
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
B-lymphocyte antigen CD20
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsRohou A
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, 2020-
Header (metadata) releaseFeb 19, 2020-
Map releaseFeb 26, 2020-
UpdateDec 2, 2020-
Current statusDec 2, 2020Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.244
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.244
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-6vja
  • Surface level: 0.244
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_21212.png

Downloads & links

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Map

FileDownload / File: emd_21212.map.gz / Format: CCP4 / Size: 113.6 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSharpened map, used for figure making and model building.
Voxel sizeX=Y=Z: 1 Å
Density
Contour LevelBy AUTHOR: 0.244 / Movie #1: 0.244
Minimum - Maximum-0.6907387 - 1.2992085
Average (Standard dev.)-0.002559343 (±0.037191927)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions310310310
Spacing310310310
CellA=B=C: 310.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z111
M x/y/z310310310
origin x/y/z0.0000.0000.000
length x/y/z310.000310.000310.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS310310310
D min/max/mean-0.6911.299-0.003

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Supplemental data

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Mask #1

Fileemd_21212_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: FSC map 1

Fileemd_21212_half_map_1.map
AnnotationFSC map 1
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: FSC map 2

Fileemd_21212_half_map_2.map
AnnotationFSC map 2
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : CD20 in complex with rituximab Fab

EntireName: CD20 in complex with rituximab Fab
Components
  • Complex: CD20 in complex with rituximab Fab
    • Protein or peptide: B-lymphocyte antigen CD20
    • Protein or peptide: Rituximab Fab heavy chain
    • Protein or peptide: Rituximab Fab light chain
  • Ligand: CHOLESTEROL HEMISUCCINATE

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Supramolecule #1: CD20 in complex with rituximab Fab

SupramoleculeName: CD20 in complex with rituximab Fab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Source (natural)Organism: Homo sapiens (human)
Molecular weightExperimental: 135 KDa

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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)
Molecular weightTheoretical: 31.320789 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MGSTQSFFMR ESKTLGAVQI MNGLFHIALG GLLMIPAGIY APICVTVWYP LWGGIMYIIS GSLLAATEKN SRKCLVKGKM IMNSLSLFA AISGMILSIM DILNIKISHF LKMESLNFIR AHTPYINIYN CEPANPSEKN SPSTQYCYSI QSLFLGILSV M LIFAFFQE ...String:
MGSTQSFFMR ESKTLGAVQI MNGLFHIALG GLLMIPAGIY APICVTVWYP LWGGIMYIIS GSLLAATEKN SRKCLVKGKM IMNSLSLFA AISGMILSIM DILNIKISHF LKMESLNFIR AHTPYINIYN CEPANPSEKN SPSTQYCYSI QSLFLGILSV M LIFAFFQE LVIAGIVENE WKRTCSRPKS NIVLLSAEEK KEQTIEIKEE VVGLTETSSQ PKNEEDIEII PIQEEEEEET ET NFPEPPQ DQESSPIEND SSPGNSENLY FQGHHHHHHH H

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Macromolecule #2: Rituximab Fab heavy chain

MacromoleculeName: Rituximab Fab heavy chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.733541 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #3: Rituximab Fab light chain

MacromoleculeName: Rituximab Fab light chain / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.078623 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: 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

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Macromolecule #4: CHOLESTEROL HEMISUCCINATE

MacromoleculeName: CHOLESTEROL HEMISUCCINATE / type: ligand / ID: 4 / Number of copies: 6 / Formula: Y01
Molecular weightTheoretical: 486.726 Da
Chemical component information

ChemComp-Y01:
CHOLESTEROL HEMISUCCINATE

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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

Concentration2 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMTris
150.0 mMNaClSodium chloride
0.01 %GDN
GridModel: Quantifoil, UltrAuFoil, R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV / Details: 5s blot time.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Calibrated magnification: 58906 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal magnification: 165000
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K2 QUANTUM (4k x 4k) / Detector mode: COUNTING / Digitization - Sampling interval: 5.0 µm / Digitization - Frames/image: 1-40 / Number grids imaged: 3 / Number real images: 24743 / Average exposure time: 10.0 sec. / Average electron dose: 53.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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
CTF correctionSoftware - Name: cisTEM
Initial angle assignmentType: RANDOM ASSIGNMENT / Software - Name: cisTEM
Final 3D classificationNumber classes: 6 / Software - Name: cisTEM
Final angle assignmentType: OTHER / Software - Name: cisTEM
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cisTEM / Details: No data beyond 1/5 A-1 were used in refinement / Number images used: 149322
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

2osl

DetailsAtomic model for CD20 was built ab initio and manually in COOT. It was then re-built using ISOLDE, and finally refined in Phenix.
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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Atomic model buiding 2

DetailsThe X-ray crystallographic atomic model for Rituximab Fab was rigid-body fit into the map, modified using Cootand ISOLDE, and refined with Phenix
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

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

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