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Yorodumi- PDB-6y9b: Cryo-EM structure of trimeric human STEAP1 bound to three Fab120.... -
+Open data
-Basic information
Entry | Database: PDB / ID: 6y9b | ||||||
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Title | Cryo-EM structure of trimeric human STEAP1 bound to three Fab120.545 fragments | ||||||
Components |
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Keywords | MEMBRANE PROTEIN / Integral membrane protein Heme-binding protein Oxidoreductase Antibody-antigen complex | ||||||
Function / homology | Function and homology information cell-cell junction / endosome / endosome membrane / heme binding / membrane / metal ion binding / plasma membrane Similarity search - Function | ||||||
Biological species | Homo sapiens (human) Mus musculus (house mouse) | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.97 Å | ||||||
Authors | Oosterheert, W. / Gros, P. | ||||||
Funding support | Netherlands, 1items
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Citation | Journal: J Biol Chem / Year: 2020 Title: Cryo-electron microscopy structure and potential enzymatic function of human six-transmembrane epithelial antigen of the prostate 1 (STEAP1). Authors: Wout Oosterheert / Piet Gros / Abstract: Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising ...Six-transmembrane epithelial antigen of the prostate 1 (STEAP1) is an integral membrane protein that is highly up-regulated on the cell surface of several human cancers, making it a promising therapeutic target to manage these diseases. It shares sequence homology with three enzymes (STEAP2-STEAP4) that catalyze the NADPH-dependent reduction of iron(III). However, STEAP1 lacks an intracellular NADPH-binding domain and does not exhibit cellular ferric reductase activity. Thus, both the molecular function of STEAP1 and its role in cancer progression remain elusive. Here, we present a ∼3.0-Å cryo-EM structure of trimeric human STEAP1 bound to three antigen-binding fragments (Fabs) of the clinically used antibody mAb120.545. The structure revealed that STEAP1 adopts a reductase-like conformation and interacts with the Fabs through its extracellular helices. Enzymatic assays in human cells revealed that STEAP1 promotes iron(III) reduction when fused to the intracellular NADPH-binding domain of its family member STEAP4, suggesting that STEAP1 functions as a ferric reductase in STEAP heterotrimers. Our work provides a foundation for deciphering the molecular mechanisms of STEAP1 and may be useful in the design of new therapeutic strategies to target STEAP1 in cancer. | ||||||
History |
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-Structure visualization
Movie |
Movie viewer |
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Structure viewer | Molecule: MolmilJmol/JSmol |
-Downloads & links
-Download
PDBx/mmCIF format | 6y9b.cif.gz | 281.7 KB | Display | PDBx/mmCIF format |
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PDB format | pdb6y9b.ent.gz | 226.9 KB | Display | PDB format |
PDBx/mmJSON format | 6y9b.json.gz | Tree view | PDBx/mmJSON format | |
Others | Other downloads |
-Validation report
Summary document | 6y9b_validation.pdf.gz | 1.4 MB | Display | wwPDB validaton report |
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Full document | 6y9b_full_validation.pdf.gz | 1.4 MB | Display | |
Data in XML | 6y9b_validation.xml.gz | 53.9 KB | Display | |
Data in CIF | 6y9b_validation.cif.gz | 78.5 KB | Display | |
Arichive directory | https://data.pdbj.org/pub/pdb/validation_reports/y9/6y9b ftp://data.pdbj.org/pub/pdb/validation_reports/y9/6y9b | HTTPS FTP |
-Related structure data
Related structure data | 10735MC M: map data used to model this data C: citing same article (ref.) |
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Similar structure data |
-Links
-Assembly
Deposited unit |
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1 |
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-Components
#1: Protein | Mass: 44183.652 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Source: (gene. exp.) Homo sapiens (human) / Gene: STEAP1, PRSS24, STEAP / Plasmid: pUPE 2959 Details (production host): Plasmid contains C-terminal Strep3-tag Cell line (production host): HEK293 GNT1- / Organ (production host): kidney / Production host: Homo sapiens (human) / Tissue (production host): embryonic kidney References: UniProt: Q9UHE8, Oxidoreductases; Oxidizing metal ions; With NAD+ or NADP+ as acceptor #2: Antibody | Mass: 24344.135 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Details: Antibody fragment generated through mouse immunization Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293 / Production host: Homo sapiens (human) #3: Antibody | Mass: 26149.922 Da / Num. of mol.: 3 Source method: isolated from a genetically manipulated source Details: Antibody fragment generated through mouse immunization Source: (gene. exp.) Mus musculus (house mouse) / Cell line (production host): HEK293 / Production host: Homo sapiens (human) #4: Chemical | #5: Chemical | Has ligand of interest | Y | |
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-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
-Sample preparation
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Molecular weight |
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Buffer solution | pH: 7.8 Details: Protein sample was subjected to size-exclusion chromatography in 20 mM Tris pH 7.8, 200 mM NaCl, 0.08% (w/v) digitonin. 1 mM FAD (final concentration) was added to the sample before vitrification. | ||||||||||||||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES Details: STEAP1-Fab120.545 complex purified in digitonin. The sample was monodisperse. | ||||||||||||||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE-PROPANE / Humidity: 100 % / Chamber temperature: 293 K / Details: blot 4 seconds, blotforce 0 |
-Electron microscopy imaging
Experimental equipment | Model: Talos Arctica / Image courtesy: FEI Company |
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Microscopy | Model: FEI TALOS ARCTICA Details: Data was collected on a 200 kV Talos Arctica, located at Utrecht University, the Netherlands. |
Electron gun | Electron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Calibrated defocus min: 400 nm / Calibrated defocus max: 3500 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN |
Image recording | Average exposure time: 6.5 sec. / Electron dose: 49.5 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 5325 |
EM imaging optics | Energyfilter name: GIF Quantum LS / Energyfilter slit width: 20 eV |
Image scans | Movie frames/image: 26 / Used frames/image: 1-26 |
-Processing
Software | Name: PHENIX / Version: dev_3689: / Classification: refinement | ||||||||||||||||||||||||||||||||||||
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EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 616302 / Details: Picking was performed in Relion. | ||||||||||||||||||||||||||||||||||||
Symmetry | Point symmetry: C3 (3 fold cyclic) | ||||||||||||||||||||||||||||||||||||
3D reconstruction | Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 169426 / Details: Final reconstruction was generated in Relion. / Symmetry type: POINT | ||||||||||||||||||||||||||||||||||||
Atomic model building | B value: 100.9 / Protocol: RIGID BODY FIT / Space: REAL | ||||||||||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6HCY Pdb chain-ID: A / Pdb chain residue range: 196-454 | ||||||||||||||||||||||||||||||||||||
Refine LS restraints |
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