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- EMDB-43216: CryoEM structure of CD20 in complex with engineered conformationa... -

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Basic information

Entry
Database: EMDB / ID: EMD-43216
TitleCryoEM structure of CD20 in complex with engineered conformationally rigid Rituximab.4DS Fab
Map data
Sample
  • Complex: CD20 in complex with Rituximab.4DS Fab
    • Complex: CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Rituximab.4DS Fab
      • Protein or peptide: Rituximab.4DS Fab heavy chain
      • Protein or peptide: Rituximab.4DS Fab light chain
Keywordsantibody fragment / fab / protein engineering / membrane protein / MEMBRANE PROTEIN-IMMUNE SYSTEM complex
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 / humoral immune response / B cell proliferation / immunoglobulin binding / plasma membrane raft / 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 / humoral immune response / B cell proliferation / immunoglobulin binding / plasma membrane raft / B cell differentiation / B cell receptor signaling pathway / response to bacterium / 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
Membrane-spanning 4-domains subfamily A / CD20-like family / CD20-like family
Similarity search - Domain/homology
B-lymphocyte antigen CD20
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsKung JE / Jao CC / Arthur CP / Sudhamsu J
Funding support1 items
OrganizationGrant numberCountry
Not funded
CitationJournal: bioRxiv / Year: 2024
Title: Disulfi de constrained Fabs overcome target size limitation for high-resolution single-particle cryo-EM.
Authors: Jennifer E Kung / Matthew C Johnson / Christine C Jao / Christopher P Arthur / Dimitry Tegunov / Alexis Rohou / Jawahar Sudhamsu /
Abstract: High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure ...High-resolution structures of proteins are critical to understanding molecular mechanisms of biological processes and in the discovery of therapeutic molecules. Cryo-EM has revolutionized structure determination of large proteins and their complexes, but a vast majority of proteins that underlie human diseases are small (< 50 kDa) and usually beyond its reach due to low signal-to-noise images and difficulties in particle alignment. Current strategies to overcome this problem increase the overall size of small protein targets using scaffold proteins that bind to the target, but are limited by inherent flexibility and not being bound to their targets in a rigid manner, resulting in the target being poorly resolved compared to the scaffolds. Here we present an iteratively engineered molecular design for transforming Fabs (antibody fragments), into conformationally rigid scaffolds (Rigid-Fabs) that, when bound to small proteins (~20 kDa), can enable high-resolution structure determination using cryo-EM. This design introduces multiple disulfide bonds at strategic locations, generates a well-folded Fab constrained into a rigid conformation and can be applied to Fabs from various species, isotypes and chimeric Fabs. We present examples of the Rigid Fab design enabling high-resolution (2.3-2.5 Å) structures of small proteins, Ang2 (26 kDa) and KRAS (21 kDa) by cryo-EM. The strategies for designing disulfide constrained Rigid Fabs in our work thus establish a general approach to overcome the target size limitation of single particle cryo-EM.
History
DepositionDec 27, 2023-
Header (metadata) releaseNov 6, 2024-
Map releaseNov 6, 2024-
UpdateNov 27, 2024-
Current statusNov 27, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43216.png

Downloads & links

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Map

FileDownload / File: emd_43216.map.gz / Format: CCP4 / Size: 244.1 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
0.94 Å/pix.
x 400 pix.
= 374.28 Å		0.94 Å/pix.
x 400 pix.
= 374.28 Å		0.94 Å/pix.
x 400 pix.
= 374.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9357 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 11.699999999999999
Minimum - Maximum-31.753183 - 81.899240000000006
Average (Standard dev.)-0.0000986716 (±1.0036033)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions400400400
Spacing400400400
CellA=B=C: 374.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #2

Fileemd_43216_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_43216_half_map_2.map
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.4DS Fab

EntireName: CD20 in complex with Rituximab.4DS Fab
Components
  • Complex: CD20 in complex with Rituximab.4DS Fab
    • Complex: CD20
      • Protein or peptide: B-lymphocyte antigen CD20
    • Complex: Rituximab.4DS Fab
      • Protein or peptide: Rituximab.4DS Fab heavy chain
      • Protein or peptide: Rituximab.4DS Fab light chain

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Supramolecule #1: CD20 in complex with Rituximab.4DS Fab

SupramoleculeName: CD20 in complex with Rituximab.4DS Fab / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all

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Supramolecule #2: CD20

SupramoleculeName: CD20 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #3
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: Rituximab.4DS Fab

SupramoleculeName: Rituximab.4DS Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Rituximab.4DS Fab heavy chain

MacromoleculeName: Rituximab.4DS Fab heavy chain / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 25.345355 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QVQLQQPGAE CVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY NQKFKGKATL TADKSSSTAY MQLSSLTSE DSAVYYCARS TYYGGDWYFN VWGAGTCVTV SAASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFCECPV T VSWNSGAL ...String:
QVQLQQPGAE CVKPGASVKM SCKASGYTFT SYNMHWVKQT PGRGLEWIGA IYPGNGDTSY NQKFKGKATL TADKSSSTAY MQLSSLTSE DSAVYYCARS TYYGGDWYFN VWGAGTCVTV SAASTKGPSV FPLAPSSKST SGGTAALGCL VKDYFCECPV T VSWNSGAL TSGVHTFPAV LQSSGLYSLS SVVTVPSSSL GTQTYICNVN HKPSNTKVDK KVEPKSCDKT HTHHHHHHP

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Macromolecule #2: Rituximab.4DS Fab light chain

MacromoleculeName: Rituximab.4DS Fab light chain / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 23.106811 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKCG SSPKPWIYAT SNLASGVPVR FSGSGSGTSY SLTISRVECE DAATYYCQQ WTSNPPTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTCQDSK ...String:
QIVLSQSPAI LSASPGEKVT MTCRASSSVS YIHWFQQKCG SSPKPWIYAT SNLASGVPVR FSGSGSGTSY SLTISRVECE DAATYYCQQ WTSNPPTFGG GTKLEIKRTV AAPSVFIFPP SDEQLKSGTA SVVCLLNNFY PREAKVQWKV DNALQSGNSQ E SVTCQDSK DCTYSLSSTL TLSKADYEKH KVYACEVTHQ GLSSPVTKSF NRGEC

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Macromolecule #3: B-lymphocyte antigen CD20

MacromoleculeName: B-lymphocyte antigen CD20 / type: protein_or_peptide / ID: 3 / 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

UniProtKB: B-lymphocyte antigen CD20

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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

BufferpH: 7.5
Component:
ConcentrationNameFormula
25.0 mMTris
150.0 mMsodium chlorideNaCl
0.02 %GDN
0.002 %CHS
GridModel: Quantifoil R0.6/1 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY
Details: grid was treated overnight with 4 mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol then rinsed in ethanol prior to sample application
VitrificationCryogen name: ETHANE / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Detector mode: COUNTING / Number grids imaged: 4 / Average electron dose: 44.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 70.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 375469
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: OTHER / Software - Name: cryoSPARC

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Atomic model buiding 1

Initial modelPDB ID:

6vja


Chain - Source name: PDB / Chain - Initial model type: experimental model
RefinementSpace: REAL / Protocol: FLEXIBLE FIT
Output model

PDB-8vgo:
CryoEM structure of CD20 in complex with engineered conformationally rigid Rituximab.4DS Fab

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