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- EMDB-61131: Cryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1... -

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Basic information

Entry
Database: EMDB / ID: EMD-61131
TitleCryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer
Map dataCryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer. Sharpened main map.
Sample
  • Complex: Dimer complex of Human Plexin-A1 27-710 with aPlexinA1-19-43 Fab binding
    • Complex: Dimer of Human Plexin-A1 27-710
      • Protein or peptide: Plexin-A1
    • Complex: aPlexinA1-19-43 Fab
      • Protein or peptide: Light chain of aPlexinA1-19-43 Fab
      • Protein or peptide: Heavy chain of aPlexinA1-19-43 Fab
KeywordsSignalling / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.33 Å
AuthorsTian H / Fung CP
Funding support Hong Kong, 1 items
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, 2024-
Header (metadata) releaseJan 21, 2026-
Map releaseJan 21, 2026-
UpdateJan 21, 2026-
Current statusJan 21, 2026Processing site: PDBj / Status: Released

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Structure visualization

Supplemental images

emd_61131.png

Downloads & links

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Map

FileDownload / File: emd_61131.map.gz / Format: CCP4 / Size: 83.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer. Sharpened main map.
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.03 Å/pix.
x 280 pix.
= 287.28 Å		1.03 Å/pix.
x 280 pix.
= 287.28 Å		1.03 Å/pix.
x 280 pix.
= 287.28 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.026 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.173
Minimum - Maximum-1.4498564 - 2.5345814
Average (Standard dev.)0.00083649304 (±0.055359047)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 287.28 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: Cryo-EM structure of aPlexinA1-19-43 Fab in complex with...

Fileemd_61131_additional_1.map
AnnotationCryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer. Raw Map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of aPlexinA1-19-43 Fab in complex with...

Fileemd_61131_half_map_1.map
AnnotationCryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer. Half Map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Cryo-EM structure of aPlexinA1-19-43 Fab in complex with...

Fileemd_61131_half_map_2.map
AnnotationCryo-EM structure of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer. Half Map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimer complex of Human Plexin-A1 27-710 with aPlexinA1-19-43 Fab ...

EntireName: Dimer complex of Human Plexin-A1 27-710 with aPlexinA1-19-43 Fab binding
Components
  • Complex: Dimer complex of Human Plexin-A1 27-710 with aPlexinA1-19-43 Fab binding
    • Complex: Dimer of Human Plexin-A1 27-710
      • Protein or peptide: Plexin-A1
    • Complex: aPlexinA1-19-43 Fab
      • Protein or peptide: Light chain of aPlexinA1-19-43 Fab
      • Protein or peptide: Heavy chain of aPlexinA1-19-43 Fab

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Supramolecule #1: Dimer complex of Human Plexin-A1 27-710 with aPlexinA1-19-43 Fab ...

SupramoleculeName: Dimer complex of Human Plexin-A1 27-710 with aPlexinA1-19-43 Fab binding
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Details: Fab 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.

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Supramolecule #2: Dimer of Human Plexin-A1 27-710

SupramoleculeName: Dimer of Human Plexin-A1 27-710 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)

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Supramolecule #3: aPlexinA1-19-43 Fab

SupramoleculeName: aPlexinA1-19-43 Fab / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #2-#3
Details: Heavy chain and light chain of aPlexinA1-19-43 Fab expressed and purified from CHO cells
Source (natural)Organism: Mus musculus (house mouse)

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Macromolecule #1: Plexin-A1

MacromoleculeName: Plexin-A1 / type: protein_or_peptide / ID: 1 / Details: Human Plexin A1 27 to 710 with His Tag / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 76.710734 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EAGLPRAGGG SQPPFRTFSA SDWGLTHLVV HEQTGEVYVG AVNRIYKLSG NLTLLRAHVT GPVEDNEKCY PPPSVQSCPH GLGSTDNVN KLLLLDYAAN RLLACGSASQ GICQFLRLDD LFKLGEPHHR KEHYLSSVQE AGSMAGVLIA GPPGQGQAKL F VGTPIDGK ...String:
EAGLPRAGGG SQPPFRTFSA SDWGLTHLVV HEQTGEVYVG AVNRIYKLSG NLTLLRAHVT GPVEDNEKCY PPPSVQSCPH GLGSTDNVN KLLLLDYAAN RLLACGSASQ GICQFLRLDD LFKLGEPHHR KEHYLSSVQE AGSMAGVLIA GPPGQGQAKL F VGTPIDGK SEYFPTLSSR RLMANEEDAD MFGFVYQDEF VSSQLKIPSD TLSKFPAFDI YYVYSFRSEQ FVYYLTLQLD TQ LTSPDAA GEHFFTSKIV RLCVDDPKFY SYVEFPIGCE QAGVEYRLVQ DAYLSRPGRA LAHQLGLAED EDVLFTVFAQ GQK NRVKPP KESALCLFTL RAIKEKIKER IQSCYRGEGK LSLPWLLNKE LGCINSPLQI DDDFCGQDFN QPLGGTVTIE GTPL FVDKD DGLTAVAAYD YRGRTVVFAG TRSGRIRKIL VDLSNPGGRP ALAYESVVAQ EGSPILRDLV LSPNHQYLYA MTEKQ VTRV PVESCVQYTS CELCLGSRDP HCGWCVLHSI CSRRDACERA DEPQRFAADL LQCVQLTVQP RNVSVTMSQV PLVLQA WNV PDLSAGVNCS FEDFTESESV LEDGRIHCRS PSAREVAPIT RGQGDQRVVK LYLKSKETGK KFASVDFVFY NCSVHQS CL SCVNGSFPCH WCKYRHVCTH NVADCAFLEG RVNVSEDCPQ ILHHHHHH

UniProtKB: Plexin-A1

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Macromolecule #2: Light chain of aPlexinA1-19-43 Fab

MacromoleculeName: Light chain of aPlexinA1-19-43 Fab / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 23.413873 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: QIVLTQSPAI LSASPGEKVT MSCSVSSSIT YMHWYQQKPG TSPKRWIYDT SKLASGVPAR FSGSGSGTSF SLTISNMEAE DAATYYCHQ RSSYPYSFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK ...String:
QIVLTQSPAI LSASPGEKVT MSCSVSSSIT YMHWYQQKPG TSPKRWIYDT SKLASGVPAR FSGSGSGTSF SLTISNMEAE DAATYYCHQ RSSYPYSFGG GTKLEIKRAD AAPTVSIFPP SSEQLTSGGA SVVCFLNNFY PKDINVKWKI DGSERQNGVL N SWTDQDSK DSTYSMSSTL TLTKDEYERH NSYTCEATHK TSTSPIVKSF NRNEC

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Macromolecule #3: Heavy chain of aPlexinA1-19-43 Fab

MacromoleculeName: Heavy chain of aPlexinA1-19-43 Fab / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 25.302014 KDa
Recombinant expressionOrganism: Cricetulus griseus (Chinese hamster)
SequenceString: EVQLQQSGPE LVKPGASVKI SCKASGYKFT ENYMDWVKQS HGESLEWIGD ISPDNGDTSY NQKFRDKATL TVDKSSSTAY MELRSLTSE DSAVYYCAQI IYYDYVGYAL DYWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL ...String:
EVQLQQSGPE LVKPGASVKI SCKASGYKFT ENYMDWVKQS HGESLEWIGD ISPDNGDTSY NQKFRDKATL TVDKSSSTAY MELRSLTSE DSAVYYCAQI IYYDYVGYAL DYWGQGTSVT VSSAKTTPPS VYPLAPGSAA QTNSMVTLGC LVKGYFPEPV T VTWNSGSL SSGVHTFPAV LQSDLYTLSS SVTVPSSTWP SETVTCNVAH PASSTKVDKK IVPRDCGGSH HHHHH

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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

Concentration0.1 mg/mL
BufferpH: 7.4 / Details: 1x PBS 7.4
GridModel: Quantifoil R2/2 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 291.15 K / Instrument: FEI VITROBOT MARK IV
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).
DetailsThis sample is a 1:1 mixture of aPlexinA1-19-43 Fab in complex with PlexinA1 dimer. The sample was mono disperse.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: FEI FALCON III (4k x 4k) / Number real images: 1989 / Average electron dose: 50.0 e/Å2
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)
Electron beamAcceleration voltage: 300 kV / Electron source: OTHER
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.2 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber 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
CTF correctionSoftware - Name: cryoSPARC (ver. 4.1.2) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionNumber classes used: 1 / Resolution.type: BY AUTHOR / Resolution: 3.33 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 4.1.2) / Number images used: 253734
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 4.1.2)
Final 3D classificationNumber classes: 6 / Software - Name: cryoSPARC (ver. 4.1.2)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

5l59

chain_id: A, residue_range: 27-710, source_name: PDB, initial_model_type: experimental modelInital model was based on PDB entry 5L59, and truncated to the appropriate lenght of 27-710
chain_id: H, source_name: AlphaFold, initial_model_type: in silico modelIntial model of Fab was generated in silico using alphafold servers.
chain_id: L, source_name: AlphaFold, initial_model_type: in silico modelIntial model of Fab was generated in silico using alphafold servers.
DetailsInitial local fitting was done using Chimera X, followed by manual fitting using Coot. Finally it was refined using Phenix real space refinement
RefinementSpace: REAL / Protocol: AB INITIO MODEL
Output model

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

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