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- PDB-9fe1: Cryo-EM structure of the ternary DARPin NY_1/HLA-A0201/NY-ESO1 co... -

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Basic information

Entry
Database: PDB / ID: 9fe1
TitleCryo-EM structure of the ternary DARPin NY_1/HLA-A0201/NY-ESO1 complex.
Components
  • Beta-2-microglobulin
  • Cancer/testis antigen 1
  • DARPin NY_1
  • MHC class I antigen
KeywordsIMMUNE SYSTEM / DARPin targeting MHC molecules in complex with tumor-associated peptide antigens
Function / homology
Function and homology information


tRNA threonylcarbamoyladenosine metabolic process / negative regulation of receptor binding / early endosome lumen / Nef mediated downregulation of MHC class I complex cell surface expression / DAP12 interactions / transferrin transport / cellular response to iron ion / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / peptide antigen assembly with MHC class II protein complex ...tRNA threonylcarbamoyladenosine metabolic process / negative regulation of receptor binding / early endosome lumen / Nef mediated downregulation of MHC class I complex cell surface expression / DAP12 interactions / transferrin transport / cellular response to iron ion / Endosomal/Vacuolar pathway / Antigen Presentation: Folding, assembly and peptide loading of class I MHC / peptide antigen assembly with MHC class II protein complex / cellular response to iron(III) ion / MHC class II protein complex / negative regulation of forebrain neuron differentiation / antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent / ER to Golgi transport vesicle membrane / peptide antigen assembly with MHC class I protein complex / regulation of iron ion transport / regulation of erythrocyte differentiation / HFE-transferrin receptor complex / response to molecule of bacterial origin / MHC class I peptide loading complex / T cell mediated cytotoxicity / positive regulation of T cell cytokine production / antigen processing and presentation of endogenous peptide antigen via MHC class I / antigen processing and presentation of exogenous peptide antigen via MHC class II / positive regulation of immune response / MHC class I protein complex / positive regulation of T cell activation / peptide antigen binding / positive regulation of receptor-mediated endocytosis / negative regulation of neurogenesis / cellular response to nicotine / positive regulation of T cell mediated cytotoxicity / multicellular organismal-level iron ion homeostasis / specific granule lumen / phagocytic vesicle membrane / recycling endosome membrane / Interferon gamma signaling / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / negative regulation of epithelial cell proliferation / MHC class II protein complex binding / Modulation by Mtb of host immune system / late endosome membrane / sensory perception of smell / positive regulation of cellular senescence / tertiary granule lumen / DAP12 signaling / T cell differentiation in thymus / negative regulation of neuron projection development / ER-Phagosome pathway / protein refolding / early endosome membrane / protein homotetramerization / amyloid fibril formation / intracellular iron ion homeostasis / learning or memory / endoplasmic reticulum lumen / Amyloid fiber formation / Golgi membrane / lysosomal membrane / external side of plasma membrane / focal adhesion / Neutrophil degranulation / SARS-CoV-2 activates/modulates innate and adaptive immune responses / structural molecule activity / endoplasmic reticulum / Golgi apparatus / protein homodimerization activity / extracellular space / extracellular exosome / extracellular region / identical protein binding / membrane / plasma membrane / cytoplasm / cytosol
Similarity search - Function
CTAG/Pcc1 family / Transcription factor Pcc1 / MHC class I alpha chain, alpha1 alpha2 domains / Class I Histocompatibility antigen, domains alpha 1 and 2 / Beta-2-Microglobulin / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / MHC classes I/II-like antigen recognition protein / : ...CTAG/Pcc1 family / Transcription factor Pcc1 / MHC class I alpha chain, alpha1 alpha2 domains / Class I Histocompatibility antigen, domains alpha 1 and 2 / Beta-2-Microglobulin / : / MHC class I-like antigen recognition-like / MHC class I-like antigen recognition-like superfamily / MHC classes I/II-like antigen recognition protein / : / Immunoglobulin/major histocompatibility complex, conserved site / Immunoglobulins and major histocompatibility complex proteins signature. / Immunoglobulin C-Type / Immunoglobulin C1-set / Immunoglobulin C1-set domain / Ig-like domain profile. / Immunoglobulin-like domain / Immunoglobulin-like domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
Beta-2-microglobulin / Cancer/testis antigen 1 / MHC class I antigen
Similarity search - Component
Biological speciesHomo sapiens (human)
synthetic construct (others)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsSchulte, T. / Wallden, K. / Carroni, M. / Sandalova, T. / Walser, M. / Mueller, S. / Venetz, N. / Achour, A.
Funding support Sweden, 3items
OrganizationGrant numberCountry
Knut and Alice Wallenberg Foundation Sweden
Cancerfonden Sweden
Swedish Research Council Sweden
CitationJournal: To Be Published
Title: Development of highly specific and potent HLA/peptide-targeting DARPin T cell engagers
Authors: Venetz, N. / Schulte, T. / Mueller, S. / Wallden, K. / Fischer, S. / Kadri, N. / Paladino, M. / Pina, N. / Radom, F. / Villemagne, D. / Bruckmaier, S. / Cornelius, A. / Hospodarsch, T. / ...Authors: Venetz, N. / Schulte, T. / Mueller, S. / Wallden, K. / Fischer, S. / Kadri, N. / Paladino, M. / Pina, N. / Radom, F. / Villemagne, D. / Bruckmaier, S. / Cornelius, A. / Hospodarsch, T. / Sun, R. / Chambers, B.J. / Carroni, M. / Levitsky, V. / Sandalova, T. / Walser, M. / Achour, A.
History
DepositionMay 17, 2024Deposition site: PDBE / Processing site: PDBE
Revision 1.0May 28, 2025Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: EM metadata / Data content type: EM metadata / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Additional map / Part number: 1 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Additional map / Part number: 2 / Data content type: Additional map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: FSC / Data content type: FSC / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Half map / Part number: 1 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Half map / Part number: 2 / Data content type: Half map / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Image / Data content type: Image / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Mask / Part number: 1 / Data content type: Mask / Provider: repository / Type: Initial release
Revision 1.0May 28, 2025Data content type: Primary map / Data content type: Primary map / Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: MHC class I antigen
B: Beta-2-microglobulin
C: Cancer/testis antigen 1
D: DARPin NY_1


Theoretical massNumber of molelcules
Total (without water)65,1074
Polymers65,1074
Non-polymers00
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable, gel filtration, Complex isolated from Superdex 200 GL 10/300 SEC in 25 mM HEPES, 150 mM NaCl, pH 7.4; eluted at retention volume corresponding to the expected MW.
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

#1: Protein MHC class I antigen


Mass: 33541.047 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: HLA-A / Production host: Escherichia coli (E. coli) / References: UniProt: Q8WLS4
#2: Protein Beta-2-microglobulin


Mass: 11879.356 Da / Num. of mol.: 1 / Fragment: UNP residues 21-119
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: B2M, CDABP0092, HDCMA22P / Production host: Escherichia coli (E. coli) / References: UniProt: P61769
#3: Protein/peptide Cancer/testis antigen 1 / Autoimmunogenic cancer/testis antigen NY-ESO-1 / Cancer/testis antigen 6.1 / CT6.1 / L antigen ...Autoimmunogenic cancer/testis antigen NY-ESO-1 / Cancer/testis antigen 6.1 / CT6.1 / L antigen family member 2 / LAGE-2


Mass: 1090.335 Da / Num. of mol.: 1 / Source method: obtained synthetically / Source: (synth.) Homo sapiens (human) / References: UniProt: P78358
#4: Protein DARPin NY_1


Mass: 18596.217 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) synthetic construct (others) / Production host: Escherichia coli (E. coli)
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

ComponentName: Ternary complex formed between the DARPin NY_1 and HLA-A0201/hb2m/NY-ESO1_157-165(9V)
Type: COMPLEX
Details: Size-exclusion chromatography (SEC) was used to purify refolded complexes of HLA-A0201 with human beta-2-microglobulin (hb2m) and the peptide NY-ESO1157-165(9V). Strep-Tactin Superflow high ...Details: Size-exclusion chromatography (SEC) was used to purify refolded complexes of HLA-A0201 with human beta-2-microglobulin (hb2m) and the peptide NY-ESO1157-165(9V). Strep-Tactin Superflow high capacity columns (1 mL, IBA lifescience) run in 20 mM HEPES, 300 mM NaCl pH 7.5 were used for further purification. After a column wash, the protein was eluted using the same buffer supplemented with 2.5 mM desthiobiotin. TEV-cleaved DARPin NY_1 was reverse-purified via IMAC and the monomer was isolated from Superdex 200 equilibrated in 20 mM HEPES, 150 mM NaCl pH 7.5. For cross-linking, HLA-A0201/NY-ESO1157-165(9V) was mixed in a 1:2 molar ratio with NY_1, concentrated to an absorbance at 280 nm (Abs280) of 2.3, and incubated for 45 min in 25 mM HEPES, 150 mM NaCl, supplemented with 1.4 mM BS(PEG)5 (BS5) (ThermoScientific). The cross-linker was quenched by addition of 25 mM Tris. Samples for grid screening were isolated from Superdex 200 GL 10/300 SEC in 25 mM HEPES, 150 mM NaCl, pH 7.4, and concentrated to Abs280 values of 2.
Entity ID: all / Source: RECOMBINANT
Molecular weightValue: 0.06 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Escherichia coli (E. coli)
Buffer solutionpH: 7.4 / Details: 25 mM HEPES, 150 mM NaCl, pH 7.4
SpecimenConc.: 0.5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K

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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: 130000 X / Nominal defocus max: 2500 nm / Nominal defocus min: 600 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 2.3 sec. / Electron dose: 57.5 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Num. of grids imaged: 1 / Num. of real images: 10855
EM imaging opticsEnergyfilter name: GIF Bioquantum / Energyfilter slit width: 20 eV

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Processing

EM software
IDNameVersionCategoryDetails
1cryoSPARC3.3.1particle selectionTemplate picker
2EPU2.8.1image acquisition
4cryoSPARC3.3.1CTF correction
7UCSF ChimeraX1.1.1model fitting
8ISOLDE1.1.0model fitting
9Coot0.9.5model fitting
11PHENIX1.19model refinement
12cryoSPARC3.3.1initial Euler assignment
13cryoSPARC3.3.1final Euler assignment
14cryoSPARC3.3.1classification
15cryoSPARC3.3.13D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 7726585
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 204743 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingSpace: REAL
Details: The crystal structures of NY_1 and HLA-A0201/NY-ESO1 (PDB 1S9W) were placed into the initial map in ChimeraX. The model was iteratively refined by model building in Coot and ChimeraX-Isolde, ...Details: The crystal structures of NY_1 and HLA-A0201/NY-ESO1 (PDB 1S9W) were placed into the initial map in ChimeraX. The model was iteratively refined by model building in Coot and ChimeraX-Isolde, as well as Phenix real space refinement with integrated amber force field with Ramachandran, secondary structure and reference structure restraints
Atomic model building

3D fitting-ID: 1 / Type: experimental model

IDPDB-IDAccession codeInitial refinement model-IDSource nameDetails
11S9W

1s9w

1S9W1PDB
2OtherNY_1 crystal structure, PDB code given in publication
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0054400
ELECTRON MICROSCOPYf_angle_d0.8515967
ELECTRON MICROSCOPYf_dihedral_angle_d15.2041600
ELECTRON MICROSCOPYf_chiral_restr0.051641
ELECTRON MICROSCOPYf_plane_restr0.012777

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