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- EMDB-25708: GPC2 HEP CT3 complex -

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Basic information

Entry
Database: EMDB / ID: EMD-25708
TitleGPC2 HEP CT3 complex
Map data
Sample
  • Complex: GPC2 HET CT3 complex
    • Protein or peptide: Glypican-2
    • Protein or peptide: CT3
Function / homology
Function and homology information


regulation of protein localization to membrane / Defective B3GALT6 causes EDSP2 and SEMDJL1 / Defective B4GALT7 causes EDS, progeroid type / Defective B3GAT3 causes JDSSDHD / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / A tetrasaccharide linker sequence is required for GAG synthesis / HS-GAG biosynthesis / HS-GAG degradation / smoothened signaling pathway ...regulation of protein localization to membrane / Defective B3GALT6 causes EDSP2 and SEMDJL1 / Defective B4GALT7 causes EDS, progeroid type / Defective B3GAT3 causes JDSSDHD / Defective EXT2 causes exostoses 2 / Defective EXT1 causes exostoses 1, TRPS2 and CHDS / A tetrasaccharide linker sequence is required for GAG synthesis / HS-GAG biosynthesis / HS-GAG degradation / smoothened signaling pathway / regulation of signal transduction / Retinoid metabolism and transport / lysosomal lumen / positive regulation of neuron projection development / neuron differentiation / Golgi lumen / cell migration / collagen-containing extracellular matrix / Attachment and Entry / synapse / cell surface / endoplasmic reticulum / extracellular space / plasma membrane
Similarity search - Function
Glypican-2 / Glypican, conserved site / Glypicans signature. / Glypican / Glypican
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / Mus musculus (house mouse)
Methodsingle particle reconstruction / negative staining / Resolution: 21.0 Å
AuthorsZhu J / Cachau R / De Val Alda N / Li N / Ho M
Funding support United States, 2 items
OrganizationGrant numberCountry
National Institutes of Health/National Cancer Institute (NIH/NCI)Z01 BC010891 United States
National Institutes of Health/National Cancer Institute (NIH/NCI)Cancer Moonshot Program United States
CitationJournal: Cell Rep Med / Year: 2021
Title: CAR T cells targeting tumor-associated exons of glypican 2 regress neuroblastoma in mice.
Authors: Nan Li / Madeline B Torres / Madeline R Spetz / Ruixue Wang / Luyi Peng / Meijie Tian / Christopher M Dower / Rosa Nguyen / Ming Sun / Chin-Hsien Tai / Natalia de Val / Raul Cachau / Xiaolin ...Authors: Nan Li / Madeline B Torres / Madeline R Spetz / Ruixue Wang / Luyi Peng / Meijie Tian / Christopher M Dower / Rosa Nguyen / Ming Sun / Chin-Hsien Tai / Natalia de Val / Raul Cachau / Xiaolin Wu / Stephen M Hewitt / Rosandra N Kaplan / Javed Khan / Brad St Croix / Carol J Thiele / Mitchell Ho /
Abstract: Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. ...Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.
History
DepositionDec 13, 2021-
Header (metadata) releaseDec 22, 2021-
Map releaseDec 22, 2021-
UpdateDec 22, 2021-
Current statusDec 22, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7t62
  • Surface level: 0.026
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_25708.png

Downloads & links

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Map

FileDownload / File: emd_25708.map.gz / Format: CCP4 / Size: 30.5 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 2.19 Å
Density
Contour LevelBy AUTHOR: 0.026 / Movie #1: 0.026
Minimum - Maximum-0.0573553 - 0.13237461
Average (Standard dev.)4.621865e-06 (±0.003369515)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions200200200
Spacing200200200
CellA=B=C: 438.0 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z2.192.192.19
M x/y/z200200200
origin x/y/z0.0000.0000.000
length x/y/z438.000438.000438.000
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS200200200
D min/max/mean-0.0570.1320.000

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Supplemental data

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Sample components

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Entire : GPC2 HET CT3 complex

EntireName: GPC2 HET CT3 complex
Components
  • Complex: GPC2 HET CT3 complex
    • Protein or peptide: Glypican-2
    • Protein or peptide: CT3

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Supramolecule #1: GPC2 HET CT3 complex

SupramoleculeName: GPC2 HET CT3 complex / type: complex / Chimera: Yes / ID: 1 / Parent: 0 / Macromolecule list: all

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Macromolecule #1: Glypican-2

MacromoleculeName: Glypican-2 / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 61.002066 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: GPGPGSEAKV TRSCAETRQV LGARGYSLNL IPPALISGEH LRVCPQEYTC CSSETEQRLI RETEATFRGL VEDSGSFLVH TLAARHRKF DEFFLEMLSV AQHSLTQLFS HSYGRLYAQH ALIFNGLFSR LRDFYGESGE GLDDTLADFW AQLLERVFPL L HPQYSFPP ...String:
GPGPGSEAKV TRSCAETRQV LGARGYSLNL IPPALISGEH LRVCPQEYTC CSSETEQRLI RETEATFRGL VEDSGSFLVH TLAARHRKF DEFFLEMLSV AQHSLTQLFS HSYGRLYAQH ALIFNGLFSR LRDFYGESGE GLDDTLADFW AQLLERVFPL L HPQYSFPP DYLLCLSRLA SSTDGSLQPF GDSPRRLRLQ ITRTLVAARA FVQGLETGRN VVSEALKVPV SEGCSQALMR LI GCPLCRG VPSLMPCQGF CLNVVRGCLS SRGLEPDWGN YLDGLLILAD KLQGPFSFEL TAESIGVKIS EGLMYLQENS AKV SAQVFQ ECGPPDPVPA RNRRAPPPRE EAGRLWSMVT EEERPTTAAG TNLHRLVWEL RERLARMRGF WARLSLTVCG DSRM AADAS LEAAPCWTGA GRGRYLPPVV GGSPAEQVNN PELKVDASGP DVPTRRRRLQ LRAATARMKT AALGHDLDGQ DADED ASGS GGGQQYADDW MAGAVAPPAR PPRPPYPPRR DGSGGKGGGG SARYNQGRSR SGGASIGFHT QTILILSLSA LALLGP R

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Macromolecule #2: CT3

MacromoleculeName: CT3 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 47.359551 KDa
Recombinant expressionOrganism: Mus musculus (house mouse)
SequenceString: EVQLQQSGPE LVKPGASVKM SCKASRFTFT DYNIHWVKQS PGKTLEWIGY INPNNGDIFY KQKFNGKATL TINKSSNTAY MELRSLTSE DSAVYYCVRS SNIRYTFDRF FDVWGTGTTV TVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP V TVTWNSGS ...String:
EVQLQQSGPE LVKPGASVKM SCKASRFTFT DYNIHWVKQS PGKTLEWIGY INPNNGDIFY KQKFNGKATL TINKSSNTAY MELRSLTSE DSAVYYCVRS SNIRYTFDRF FDVWGTGTTV TVSSAKTTPP SVYPLAPGSA AQTNSMVTLG CLVKGYFPEP V TVTWNSGS LSSGVHTFPA VLESDLYTLS SSVTVPSSPR PSETVTCNVA HPASSTKVDK KIENVLTQSP AIMSASLGEK VT MSCRASS SVNYIYWYQQ KSDASPKLWI YYTSNLAPGV PARFSGSGSG NSYSLTISSM EGEDAATYYC QQFSSSPSTF GTG TKLELK RADAAPTVSI FPPSSEQLTS GGASVVCFLN NFYPKDINVK WKIDGSERQN GVLNSWTDQD SKDSTYSMSS TLTL TKDEY ERHNSYTCEA THKTSTSPIV KSFNRNEC

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Experimental details

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Structure determination

Methodnegative staining
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.5
StainingType: NEGATIVE / Material: uranyl formate
Details: A 3 uL aliquot containing ~0.01 mg/mL of the samples was applied for 20 s onto a carbon-coated 200 Cu mesh grid (Electron Microscopy Sciences, Protochips, Inc.) that had been glow discharged ...Details: A 3 uL aliquot containing ~0.01 mg/mL of the samples was applied for 20 s onto a carbon-coated 200 Cu mesh grid (Electron Microscopy Sciences, Protochips, Inc.) that had been glow discharged at 30 mA for 30 s (Pelco easiGlow, Ted Pella, Inc.), then negatively stained with 0.7% (w/v) uranyl formate for 40 sec.
GridModel: Quantifoil / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: CONTINUOUS / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Atmosphere: AIR

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Electron microscopy

MicroscopeFEI TECNAI 20
Electron beamAcceleration voltage: 200 kV / Electron source: LAB6
Electron opticsIllumination mode: SPOT SCAN / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 5.0 µm / Nominal defocus min: 2.0 µm / Nominal magnification: 100000
Sample stageCooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI EAGLE (2k x 2k) / Digitization - Dimensions - Width: 2048 pixel / Digitization - Dimensions - Height: 2048 pixel / Number real images: 200 / Average electron dose: 40.0 e/Å2

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Image processing

Particle selectionNumber selected: 21000
Startup modelType of model: PDB ENTRY
PDB model - PDB ID:

6wjl

Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final 3D classificationNumber classes: 1 / Software - Name: RELION (ver. 3.08)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.08)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 21.0 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.08) / Number images used: 21000
FSC plot (resolution estimation)

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