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- EMDB-50336: Cryo-EM structure of the ternary DARPin NY_1/HLA-A0201/NY-ESO1 co... -

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Basic information

Entry
Database: EMDB / ID: EMD-50336
TitleCryo-EM structure of the ternary DARPin NY_1/HLA-A0201/NY-ESO1 complex.
Map dataPhenix auto-sharpened, with model
Sample
  • Complex: Ternary complex formed between the DARPin NY_1 and HLA-A0201/hb2m/NY-ESO1_157-165(9V)
    • Protein or peptide: MHC class I antigen
    • Protein or peptide: Beta-2-microglobulin
    • Protein or peptide: Cancer/testis antigen 1
    • Protein or peptide: DARPin NY_1
KeywordsDARPin targeting MHC molecules in complex with tumor-associated peptide antigens / IMMUNE SYSTEM
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)
Methodsingle 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, 3 items
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 / Sun R / Chambers BJ / Carroni ...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 BJ / Carroni M / Levitsky V / Sandalova T / Walser M / Achour A
History
DepositionMay 17, 2024-
Header (metadata) releaseMay 28, 2025-
Map releaseMay 28, 2025-
UpdateMay 28, 2025-
Current statusMay 28, 2025Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_50336.png

Downloads & links

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Map

FileDownload / File: emd_50336.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationPhenix auto-sharpened, with model
Projections & slices

Image control

Size
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AxesX (Sec.)Y (Row.)Z (Col.)
0.98 Å/pix.
x 256 pix.
= 249.6 Å		0.98 Å/pix.
x 256 pix.
= 249.6 Å		0.98 Å/pix.
x 256 pix.
= 249.6 Å

Surface

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Images are generated by Spider.

Voxel sizeX=Y=Z: 0.975 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 12.0
Minimum - Maximum-26.410246000000001 - 59.019302000000003
Average (Standard dev.)-0.000000000000989 (±1.0)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 249.6 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_50336_msk_1.map
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Additional map: Phenix density-modified, using model

Fileemd_50336_additional_1.map
AnnotationPhenix density-modified, using model
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Additional map: Phenix auto-sharpened, without model

Fileemd_50336_additional_2.map
AnnotationPhenix auto-sharpened, without model
Projections & Slices
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Half map: half-map A

Fileemd_50336_half_map_1.map
Annotationhalf-map A
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Half map: #1

Fileemd_50336_half_map_2.map
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Sample components

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Entire : Ternary complex formed between the DARPin NY_1 and HLA-A0201/hb2m...

EntireName: Ternary complex formed between the DARPin NY_1 and HLA-A0201/hb2m/NY-ESO1_157-165(9V)
Components
  • Complex: Ternary complex formed between the DARPin NY_1 and HLA-A0201/hb2m/NY-ESO1_157-165(9V)
    • Protein or peptide: MHC class I antigen
    • Protein or peptide: Beta-2-microglobulin
    • Protein or peptide: Cancer/testis antigen 1
    • Protein or peptide: DARPin NY_1

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Supramolecule #1: Ternary complex formed between the DARPin NY_1 and HLA-A0201/hb2m...

SupramoleculeName: Ternary complex formed between the DARPin NY_1 and HLA-A0201/hb2m/NY-ESO1_157-165(9V)
type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
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.
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 60 KDa

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Macromolecule #1: MHC class I antigen

MacromoleculeName: MHC class I antigen / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 33.541047 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString: MGSHSMRYFF TSVSRPGRGE PRFIAVGYVD DTQFVRFDSD AASQRMEPRA PWIEQEGPEY WDGETRKVKA HSQTHRVDLG TLRGYYNQS EAGSHTVQRM YGCDVGSDWR FLRGYHQYAY DGKDYIALKE DLRSWTAADM AAQTTKHKWE AAHVAEQLRA Y LEGTCVEW ...String:
MGSHSMRYFF TSVSRPGRGE PRFIAVGYVD DTQFVRFDSD AASQRMEPRA PWIEQEGPEY WDGETRKVKA HSQTHRVDLG TLRGYYNQS EAGSHTVQRM YGCDVGSDWR FLRGYHQYAY DGKDYIALKE DLRSWTAADM AAQTTKHKWE AAHVAEQLRA Y LEGTCVEW LRRYLENGKE TLQRTDAPKT HMTHHAVSDH EATLRCWALS FYPAEITLTW QRDGEDQTQD TELVETRPAG DG TFQKWAA VVVPSGQEQR YTCHVQHEGL PKPLTLRWEP GSGGSAWSHP QFEK

UniProtKB: MHC class I antigen

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Macromolecule #2: Beta-2-microglobulin

MacromoleculeName: Beta-2-microglobulin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 11.879356 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MIQRTPKIQV YSRHPAENGK SNFLNCYVSG FHPSDIEVDL LKNGERIEKV EHSDLSFSKD WSFYLLYYTE FTPTEKDEYA CRVNHVTLS QPKIVKWDRD M

UniProtKB: Beta-2-microglobulin

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Macromolecule #3: Cancer/testis antigen 1

MacromoleculeName: Cancer/testis antigen 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 1.090335 KDa
SequenceString:
SLLMWITQV

UniProtKB: Cancer/testis antigen 1

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Macromolecule #4: DARPin NY_1

MacromoleculeName: DARPin NY_1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: synthetic construct (others)
Molecular weightTheoretical: 18.596217 KDa
Recombinant expressionOrganism: Escherichia coli (E. coli)
SequenceString:
MRGSHHHHHH ENLYFQGSDL GKKLLQAARA GQLDEVRELL KAGADVNAKD LIGVTPLHLA AFSGHLEIVE VLLKASADVN AKDVSGRTP LHVAAKHGHL EIVEVLLKAG ADVNAKDLIG FTPLHLAAQF GHLEIVEVLL KAGADVNAQD KSGKTPADLA A RAGHQDIA EVLQKAA

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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

Concentration0.5 mg/mL
BufferpH: 7.4 / Details: 25 mM HEPES, 150 mM NaCl, pH 7.4
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Number grids imaged: 1 / Number real images: 10855 / Average exposure time: 2.3 sec. / Average electron dose: 57.5 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.6 µm / Nominal magnification: 130000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 7726585
CTF correctionSoftware - Name: cryoSPARC (ver. 3.3.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC (ver. 3.3.1) / Number images used: 204743
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final 3D classificationNumber classes: 1 / Software - Name: cryoSPARC (ver. 3.3.1)
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial model
PDB IDChainDetails

1s9w

source_name: PDB, initial_model_type: experimental model
source_name: Other, initial_model_type: experimental modelNY_1 crystal structure, PDB code given in publication
DetailsThe 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
RefinementSpace: REAL
Output model

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

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