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- PDB-8vgo: CryoEM structure of CD20 in complex with engineered conformationa... -

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Basic information

Entry
Database: PDB / ID: 8vgo
TitleCryoEM structure of CD20 in complex with engineered conformationally rigid Rituximab.4DS Fab
Components
  • B-lymphocyte antigen CD20
  • Rituximab.4DS Fab heavy chain
  • Rituximab.4DS Fab light chain
KeywordsMEMBRANE PROTEIN/IMMUNE SYSTEM / antibody 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 / immunoglobulin binding / B cell activation / humoral immune response / B cell proliferation / 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 / immunoglobulin binding / B cell activation / humoral immune response / B cell proliferation / plasma membrane raft / B cell differentiation / B cell receptor signaling pathway / protein tetramerization / 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
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)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.6 Å
AuthorsKung, J.E. / Jao, C.C. / Arthur, C.P. / Sudhamsu, J.
Funding support1items
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, 2023Deposition site: RCSB / Processing site: RCSB
Revision 1.0Nov 6, 2024Provider: repository / Type: Initial release
Revision 1.1Nov 27, 2024Group: Data collection / Category: em_admin / Item: _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Rituximab.4DS Fab heavy chain
B: Rituximab.4DS Fab light chain
G: B-lymphocyte antigen CD20
H: Rituximab.4DS Fab heavy chain
I: B-lymphocyte antigen CD20
L: Rituximab.4DS Fab light chain


Theoretical massNumber of molelcules
Total (without water)159,5466
Polymers159,5466
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Antibody Rituximab.4DS Fab heavy chain


Mass: 25345.355 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#2: Antibody Rituximab.4DS Fab light chain


Mass: 23106.811 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Production host: Cricetulus griseus (Chinese hamster)
#3: 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 / Fragment: residues 41-297
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
Has protein modificationY

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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

Component
IDNameTypeEntity IDParent-IDSource
1CD20 in complex with Rituximab.4DS FabCOMPLEXall0MULTIPLE SOURCES
2CD20COMPLEX#31RECOMBINANT
3Rituximab.4DS FabCOMPLEX#1-#21RECOMBINANT
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Homo sapiens (human)9606
43Homo sapiens (human)9606
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
32Trichoplusia ni (cabbage looper)7111
43Cricetulus griseus (Chinese hamster)10029
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
125 mMTris1
2150 mMsodium chlorideNaCl1
30.02 %GDN1
40.002 %CHS1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportDetails: grid was treated overnight with 4 mM monothiolalkane(C11)PEG6-OH (11-mercaptoundecyl) hexaethyleneglycol then rinsed in ethanol prior to sample application
Grid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R0.6/1
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE

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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 FIELD / Nominal magnification: 165000 X / Nominal defocus max: 1800 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm
Image recordingElectron dose: 44 e/Å2 / Detector mode: COUNTING / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 4

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Processing

EM software
IDNameVersionCategory
1cryoSPARCparticle selection
2EPUimage acquisition
4cryoSPARCCTF correction
7UCSF ChimeraXmodel fitting
8ISOLDEmodel fitting
10PHENIX1.21rc1_5049model refinement
11cryoSPARCinitial Euler assignment
12cryoSPARCfinal Euler assignment
14cryoSPARC3D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 2.6 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 375469 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6VJA

6vja


Accession code: 6VJA / Source name: PDB / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0039368
ELECTRON MICROSCOPYf_angle_d0.67412730
ELECTRON MICROSCOPYf_dihedral_angle_d13.6763330
ELECTRON MICROSCOPYf_chiral_restr0.0441442
ELECTRON MICROSCOPYf_plane_restr0.0081594

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