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

Basic information

Entry

Database: EMDB / ID: EMD-50336

• Title: Cryo-EM structure of the ternary DARPin NY_1/HLA-A0201/NY-ESO1 complex.
• Map data: Phenix 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

• Keywords: DARPin targeting MHC molecules in complex with tumor-associated peptide antigens / IMMUNE SYSTEM

Function / homology

Function:

tRNA threonylcarbamoyladenosine metabolic process / : / : / positive regulation of receptor binding / early endosome lumen / Nef mediated downregulation of MHC class I complex cell surface expression / negative regulation of receptor binding / DAP12 interactions / 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 / antigen processing and presentation of exogenous protein antigen via MHC class Ib, TAP-dependent / MHC class II protein complex / negative regulation of forebrain neuron differentiation / ER to Golgi transport vesicle membrane / peptide antigen assembly with MHC class I protein complex / regulation of erythrocyte differentiation / regulation of iron ion transport / MHC class I peptide loading complex / response to molecule of bacterial origin / HFE-transferrin receptor complex / T cell mediated cytotoxicity / antigen processing and presentation of endogenous peptide antigen via MHC class I / positive regulation of T cell cytokine production / antigen processing and presentation of exogenous peptide antigen via MHC class II / MHC class I protein complex / positive regulation of immune response / peptide antigen binding / negative regulation of neurogenesis / positive regulation of T cell mediated cytotoxicity / positive regulation of receptor-mediated endocytosis / multicellular organismal-level iron ion homeostasis / positive regulation of T cell activation / cellular response to nicotine / specific granule lumen / recycling endosome membrane / phagocytic vesicle membrane / positive regulation of cellular senescence / Immunoregulatory interactions between a Lymphoid and a non-Lymphoid cell / negative regulation of epithelial cell proliferation / Interferon gamma signaling / MHC class II protein complex binding / positive regulation of protein binding / Modulation by Mtb of host immune system / late endosome membrane / sensory perception of smell / tertiary granule lumen / DAP12 signaling / negative regulation of neuron projection development / iron ion transport / T cell differentiation in thymus / ER-Phagosome pathway / protein refolding / early endosome membrane / protein homotetramerization / amyloid fibril formation / intracellular iron ion homeostasis / learning or memory / Amyloid fiber formation / endoplasmic reticulum lumen / Golgi membrane / external side of plasma membrane / lysosomal 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 / cytosol / cytoplasm

Domain/homology:

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

Component:

Beta-2-microglobulin / Cancer/testis antigen 1 / MHC class I antigen

• Biological species: Homo sapiens (human) / synthetic construct (others)
• Method: single particle reconstruction / cryo EM / Resolution: 3.1 Å
• Authors: Schulte T / Wallden K / Carroni M / Sandalova T / Walser M / Mueller S / Venetz N / Achour A

Funding support

Sweden, 3 items

Organization	Grant number	Country
Knut and Alice Wallenberg Foundation		Sweden
Cancerfonden		Sweden
Swedish Research Council		Sweden

• Citation: Journal: 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 M / Levitsky V / Sandalova T / Walser M / Achour A

History

Deposition	May 17, 2024	-
Header (metadata) release	May 28, 2025	-
Map release	May 28, 2025	-
Update	May 28, 2025	-
Current status	May 28, 2025	Processing site: PDBe / Status: Released

Map

• File: Download / File: emd_50336.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
• Annotation: Phenix auto-sharpened, with model
• Voxel size: X=Y=Z: 0.975 Å

Density

Contour Level	By AUTHOR: 12.0
Minimum - Maximum	-26.410246000000001 - 59.019302000000003
Average (Standard dev.)	-0.000000000000989 (±1.0)

• Symmetry: Space group: 1

Details

EMDB XML:

Map geometry	Axis order Z Y X Origin 0 0 0 Dimensions 256 256 256 Spacing 256 256 256
Cell	A=B=C: 249.6 Å α=β=γ: 90.0 °

Supplemental data

Mask #1

• File: emd_50336_msk_1.map

Additional map: Phenix density-modified, using model

• File: emd_50336_additional_1.map
• Annotation: Phenix density-modified, using model

Additional map: Phenix auto-sharpened, without model

• File: emd_50336_additional_2.map
• Annotation: Phenix auto-sharpened, without model

Half map: half-map A

• File: emd_50336_half_map_1.map
• Annotation: half-map A

Half map: #1

• File: emd_50336_half_map_2.map

Sample components

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

• Entire: Name: 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

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

• Supramolecule: Name: 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 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 weight: Theoretical: 60 KDa

Macromolecule #1: MHC class I antigen

• Macromolecule: Name: MHC class I antigen / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 33.541047 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

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

Macromolecule #2: Beta-2-microglobulin

• Macromolecule: Name: Beta-2-microglobulin / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 11.879356 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MIQRTPKIQV YSRHPAENGK SNFLNCYVSG FHPSDIEVDL LKNGERIEKV EHSDLSFSKD WSFYLLYYTE FTPTEKDEYA CRVNHVTLS QPKIVKWDRD M

UniProtKB: Beta-2-microglobulin

Macromolecule #3: Cancer/testis antigen 1

• Macromolecule: Name: Cancer/testis antigen 1 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: Homo sapiens (human)
• Molecular weight: Theoretical: 1.090335 KDa

Sequence

String:

SLLMWITQV

UniProtKB: Cancer/testis antigen 1

Macromolecule #4: DARPin NY_1

• Macromolecule: Name: DARPin NY_1 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
• Source (natural): Organism: synthetic construct (others)
• Molecular weight: Theoretical: 18.596217 KDa
• Recombinant expression: Organism: Escherichia coli (E. coli)

Sequence

String:

MRGSHHHHHH ENLYFQGSDL GKKLLQAARA GQLDEVRELL KAGADVNAKD LIGVTPLHLA AFSGHLEIVE VLLKASADVN AKDVSGRTP LHVAAKHGHL EIVEVLLKAG ADVNAKDLIG FTPLHLAAQF GHLEIVEVLL KAGADVNAQD KSGKTPADLA A RAGHQDIA EVLQKAA

Experimental details

Structure determination

• Method: cryo EM
• Processing: single particle reconstruction
• Aggregation state: particle

Sample preparation

• Concentration: 0.5 mg/mL
• Buffer: pH: 7.4 / Details: 25 mM HEPES, 150 mM NaCl, pH 7.4
• Vitrification: Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK IV

Electron microscopy

• Microscope: FEI TITAN KRIOS
• Specialist optics: Energy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
• Image recording: Film 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/Ų
• Electron beam: Acceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
• Electron optics: C2 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 stage: Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
• Experimental equipment: Model: Titan Krios / Image courtesy: FEI Company

Image processing

• Particle selection: Number selected: 7726585
• CTF correction: Software - Name: cryoSPARC (ver. 3.3.1) / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
• Startup model: Type of model: NONE
• Final reconstruction: Applied 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 assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
• Final angle assignment: Type: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
• Final 3D classification: Number classes: 1 / Software - Name: cryoSPARC (ver. 3.3.1)

Atomic model buiding 1

Initial model

PDB ID	Chain	Details
1s9w	source_name: PDB, initial_model_type: experimental model
	source_name: Other, initial_model_type: experimental model	NY_1 crystal structure, PDB code given in publication

• 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
• Refinement: Space: REAL

Output model

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