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- PDB-9j4c: Cryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1... -

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Basic information

Entry
Database: PDB / ID: 9j4c
TitleCryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer
Components
  • Heavy chain of aPlexinA1-19-43 Fab
  • Light chain of aPlexinA1-19-43 Fab
  • Plexin-A1
KeywordsPROTEIN BINDING/IMMUNE SYSTEM / Signalling / Fab / Complex / PROTEIN BINDING / PROTEIN BINDING-IMMUNE SYSTEM complex
Function / homology
Function and homology information


olfactory nerve formation / neuron projection guidance / dichotomous subdivision of terminal units involved in salivary gland branching / gonadotrophin-releasing hormone neuronal migration to the hypothalamus / Other semaphorin interactions / T cell activation via T cell receptor contact with antigen bound to MHC molecule on antigen presenting cell / semaphorin receptor complex / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / semaphorin receptor activity / CRMPs in Sema3A signaling ...olfactory nerve formation / neuron projection guidance / dichotomous subdivision of terminal units involved in salivary gland branching / gonadotrophin-releasing hormone neuronal migration to the hypothalamus / Other semaphorin interactions / T cell activation via T cell receptor contact with antigen bound to MHC molecule on antigen presenting cell / semaphorin receptor complex / SEMA3A-Plexin repulsion signaling by inhibiting Integrin adhesion / semaphorin receptor activity / CRMPs in Sema3A signaling / regulation of smooth muscle cell migration / RHOD GTPase cycle / RND1 GTPase cycle / neuron projection extension / semaphorin-plexin signaling pathway / Sema3A PAK dependent Axon repulsion / synapse assembly / regulation of cell migration / glutamatergic synapse / extracellular exosome / nucleoplasm / plasma membrane / cytosol
Similarity search - Function
Plexin-A1, Sema domain / Plexin, TIG domain 2 / TIG domain found in plexin / Plexin A/B, PSI domain / Plexin, TIG domain 1 / TIG domain / Plexin, cytoplasmic RasGAP domain / Plexin, cytoplasmic RhoGTPase-binding domain / Plexin cytoplasmic RasGAP domain / Plexin cytoplasmic RhoGTPase-binding domain ...Plexin-A1, Sema domain / Plexin, TIG domain 2 / TIG domain found in plexin / Plexin A/B, PSI domain / Plexin, TIG domain 1 / TIG domain / Plexin, cytoplasmic RasGAP domain / Plexin, cytoplasmic RhoGTPase-binding domain / Plexin cytoplasmic RasGAP domain / Plexin cytoplasmic RhoGTPase-binding domain / Plexin family / Plexin repeat / Plexin repeat / Sema domain / semaphorin domain / Sema domain / Sema domain superfamily / Sema domain profile. / IPT/TIG domain / ig-like, plexins, transcription factors / Rho GTPase activation protein / IPT domain / PSI domain / domain found in Plexins, Semaphorins and Integrins / Immunoglobulin E-set / WD40/YVTN repeat-like-containing domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Biological speciesHomo sapiens (human)
Mus musculus (house mouse)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsTian, H. / Fung, C.P.
Funding support Hong Kong, 1items
OrganizationGrant numberCountry
Other governmentPRP/065/19FX Hong Kong
CitationJournal: J Biol Chem / Year: 2025
Title: A bispecific antibody designed to act as a NRP2/PLXNA1 agonist mimics anticancer activity of SEMA3F.
Authors: Honglei Tian / Chun Po Fung / Luke Burman / Yeeting E Chong / Changdong Liu / Yanyan Geng / Lam Yang / Man Wai Chow / Yingyi Zhang / Kwok Wa Hugo Ho / Guang Zhu / Zhenguo Wu / Xiang-Lei Yang ...Authors: Honglei Tian / Chun Po Fung / Luke Burman / Yeeting E Chong / Changdong Liu / Yanyan Geng / Lam Yang / Man Wai Chow / Yingyi Zhang / Kwok Wa Hugo Ho / Guang Zhu / Zhenguo Wu / Xiang-Lei Yang / Zhiwen Xu / Leslie A Nangle /
Abstract: Neuropilin-2 (NRP2) is a pleiotropic receptor with diverse roles across biological systems. Recent work detailed its role as an immunomodulatory receptor target that is currently being explored in ...Neuropilin-2 (NRP2) is a pleiotropic receptor with diverse roles across biological systems. Recent work detailed its role as an immunomodulatory receptor target that is currently being explored in clinical development for interstitial lung diseases, establishing it as a viable therapeutic target. To mediate its diverse effects, NRP2 interacts with endogenous ligands, including semaphorins (SEMAs) and vascular endothelial growth factors, signaling via ligand-induced heterodimerization with various receptor families. One of these ligands, SEMA3F exhibits well-documented tumor-suppressive activities mediated through NRP2 and plexinA1 (PLXNA1). Despite its observed benefits, SEMA3F is not therapeutically viable due to the multifaceted nature of its functions through non-NRP2-mediated interactions, leading to concerns around potential toxicity. Here, we describe development of bispecific antibodies (bsAbs) that dimerize PLXNA1 and NRP2, selectively mimicking the beneficial aspects of SEMA3F signaling as a basis for a novel anticancer therapy. Using a single B cell-based mAb discovery platform, anti-PLXNA1 mAbs with diverse lineages were generated and combined with anti-NRP2 mAbs to produce over 200 PLXNA1-NRP2 bsAbs. Antibodies were screened in cell-based assays (receptor dimerization, phospho-AKT, oncogene expression, and cell proliferation), yielding one bsAb capable of mimicking NRP2-mediated SEMA3F activities in all assays. Structural studies revealed that this bsAb binds to PLXNA1/NRP2 at sites distinct from the SEMA3F-binding site, but in a manner that allows proper spacing for receptor complex formation and flexibility of conformational changes for signaling. This study demonstrates the potential of these receptors as targets for agonistic bsAbs development and provides the groundwork for further exploration in tumor models.
History
DepositionAug 9, 2024Deposition site: PDBJ / Processing site: PDBJ
Revision 1.0Jan 21, 2026Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0Jan 21, 2026Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Plexin-A1
L: Light chain of aPlexinA1-19-43 Fab
H: Heavy chain of aPlexinA1-19-43 Fab


Theoretical massNumber of molelcules
Total (without water)125,4273
Polymers125,4273
Non-polymers00
Water00
1
A: Plexin-A1
L: Light chain of aPlexinA1-19-43 Fab
H: Heavy chain of aPlexinA1-19-43 Fab

A: Plexin-A1
L: Light chain of aPlexinA1-19-43 Fab
H: Heavy chain of aPlexinA1-19-43 Fab


Theoretical massNumber of molelcules
Total (without water)250,8536
Polymers250,8536
Non-polymers00
Water0
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
point symmetry operation1
2


  • Idetical with deposited unit
  • point asymmetric unit
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
3


  • Idetical with deposited unit in distinct coordinate
  • point asymmetric unit, std point frame
TypeNameSymmetry operationNumber
transform to point frame1
SymmetryPoint symmetry: (Schoenflies symbol: C2 (2 fold cyclic))

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Components

#1: Protein Plexin-A1 / Semaphorin receptor NOV


Mass: 76710.734 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Details: Human Plexin A1 27 to 710 with His Tag / Source: (gene. exp.) Homo sapiens (human) / Gene: PLXNA1, NOV, PLXN1 / Cell line (production host): CHO / Production host: Cricetulus griseus (Chinese hamster) / References: UniProt: Q9UIW2
#2: Antibody Light chain of aPlexinA1-19-43 Fab


Mass: 23413.873 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): CHO / Production host: Cricetulus griseus (Chinese hamster)
#3: Antibody Heavy chain of aPlexinA1-19-43 Fab


Mass: 25302.014 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): CHO / Production host: Cricetulus griseus (Chinese hamster)
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
IDNameTypeDetailsEntity IDParent-IDSource
1Dimer complex of Human Plexin-A1 27-710 with aPlexinA1-19-43 Fab bindingCOMPLEXFab fragments generated from cloning the variable region of PlexinA1 antibody generated from mouse, into Fab vector. Fab fragments and PlexinA1 27-710 are expressed and purified from CHO cells.all0RECOMBINANT
2Dimer of Human Plexin-A1 27-710COMPLEX#11RECOMBINANT
3aPlexinA1-19-43 FabCOMPLEXHeavy chain and light chain of aPlexinA1-19-43 Fab expressed and purified from CHO cells#2-#31RECOMBINANT
Molecular weight
IDEntity assembly-IDExperimental value
11NO
21NO
31NO
Source (natural)
IDEntity assembly-IDOrganismNcbi tax-ID
32Homo sapiens (human)9606
43Mus musculus (house mouse)10090
Source (recombinant)
IDEntity assembly-IDOrganismNcbi tax-ID
21Cricetulus griseus (Chinese hamster)10029
32Cricetulus griseus (Chinese hamster)10029
43Cricetulus griseus (Chinese hamster)10029
Buffer solutionpH: 7.4 / Details: 1x PBS 7.4
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: This sample is a 1:1 mixture of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer. The sample was mono disperse.
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R2/2
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 291.15 K
Details: To prepare cryo-grids, 3 ul samples were applied to glow discharged Quantifoil Au grids (R2/2, 300 mesh), which were subsequently blotted with filter paper (Ted Pella) for 3 seconds at 18oC ...Details: To prepare cryo-grids, 3 ul samples were applied to glow discharged Quantifoil Au grids (R2/2, 300 mesh), which were subsequently blotted with filter paper (Ted Pella) for 3 seconds at 18oC and 100% humidity. The grids were immediately plunge frozen in liquid ethane using a FEI Vitrobot IV (ThermoFisher)

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: OTHER / Accelerating voltage: 300 kV / Illumination mode: OTHER
Electron lensMode: OTHER / Nominal defocus max: 2500 nm / Nominal defocus min: 1200 nm
Image recordingElectron dose: 50 e/Å2 / Film or detector model: FEI FALCON III (4k x 4k) / Num. of real images: 1989
Details: A total of 2,084 movies were acquired and imported into Cryo-SPARC using the following parameters: Raw pixel size 1.026 A, Accelerating Voltage 300 kV, Spherical Aberration 2.7 mm, and Total ...Details: A total of 2,084 movies were acquired and imported into Cryo-SPARC using the following parameters: Raw pixel size 1.026 A, Accelerating Voltage 300 kV, Spherical Aberration 2.7 mm, and Total exposure dose 50 e/A^2. Motion correction and CTF estimation were performed using Full-frame Motion Correction and Patch CTF. After that, 1,989 micrographs were selected from the 2,084 micrographs based on average intensity (-10055.05 to 2041.77)

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC4.1.2particle selection
4cryoSPARC4.1.2CTF correction
7UCSF ChimeraXmodel fittingInitial models were rough fitted using the Fit in Map function of UCSF ChimeraX
8Coot0.9.8.7model fittingAtomic modelling and refinement were done through Coot
10cryoSPARC4.1.2initial Euler assignment
11cryoSPARC4.1.2final Euler assignment
12cryoSPARC4.1.2classification
13cryoSPARC4.1.23D reconstruction
14PHENIX1.21model refinementReal-space refinement function of PHENIX was used to refine the structure
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 3741859
Details: After Inspect Particle Picks (NCC score > 0.620, 10421 < local power score < 17132) and Extract From Micrographs (Extraction box size 320 pix), a total of 3,741,859 particles were extracted
3D reconstructionResolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 253734 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: AB INITIO MODEL / Space: REAL
Details: Initial local fitting was done using Chimera X, followed by manual fitting using Coot. Finally it was refined using Phenix real space refinement
Atomic model building

3D fitting-ID: 1

IDPDB-IDPdb chain-IDAccession codeChain-IDChain residue rangeDetailsInitial refinement model-IDPdb chain residue rangeSource nameType
15L59

5l59

A5L59A27-710Inital model was based on PDB entry 5L59, and truncated to the appropriate lenght of 27-710127-710PDBexperimental model
2HIntial model of Fab was generated in silico using alphafold servers.AlphaFoldin silico model
3LIntial model of Fab was generated in silico using alphafold servers.AlphaFoldin silico model

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