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- EMDB-10735: Cryo-EM structure of trimeric human STEAP1 bound to three Fab120.... -

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Basic information

Entry
Database: EMDB / ID: EMD-10735
TitleCryo-EM structure of trimeric human STEAP1 bound to three Fab120.545 fragments
Map dataUnsharpened cryo-EM map of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545. The constant region of the Fabs is masked out.
Sample
  • Complex: Trimeric human six-transmembrane epithelial antigen of the prostate (STEAP1) bound to three Fab-fragments of antibody mAb120.545
    • Complex: Fab-fragment 1 of antibody mAb120.545
      • Protein or peptide: Fab120.545 light chain
      • Protein or peptide: Fab120.545 heavy chain
    • Complex: Homo-trimeric STEAP1, chain a, b and c.
      • Protein or peptide: Metalloreductase STEAP1
    • Complex: Fab Fragment 2 of antibody mAb120.545
    • Complex: Fab-fragment 3 of antibody mAb120.545
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE
Function / homology
Function and homology information


cell-cell junction / endosome membrane / endosome / heme binding / membrane / metal ion binding / plasma membrane
Similarity search - Function
Ferric reductase transmembrane component-like domain / Ferric reductase like transmembrane component
Similarity search - Domain/homology
Biological speciesHomo sapiens (human) / house mouse (house mouse) / Mus musculus (house mouse)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.97 Å
AuthorsOosterheert W / Gros P
Funding support Netherlands, 1 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research (NWO)731.015.201 Netherlands
CitationJournal: 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
DepositionMar 6, 2020-
Header (metadata) releaseMay 20, 2020-
Map releaseMay 20, 2020-
UpdateJul 22, 2020-
Current statusJul 22, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6y9b
  • Surface level: 0.022
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_10735.png

Downloads & links

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Map

FileDownload / File: emd_10735.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationUnsharpened cryo-EM map of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545. The constant region of the Fabs is masked out.
Voxel sizeX=Y=Z: 1.0285 Å
Density
Contour LevelBy AUTHOR: 0.022 / Movie #1: 0.022
Minimum - Maximum-0.037630104 - 0.0994739
Average (Standard dev.)0.00015654201 (±0.002010027)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 308.55 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.02851.02851.0285
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z308.550308.550308.550
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.0380.0990.000

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Supplemental data

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Mask #1

Fileemd_10735_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened, unmasked cryo-EM map of homotrimeric STEAP1 bound...

Fileemd_10735_additional_1.map
AnnotationUnsharpened, unmasked cryo-EM map of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Sharpened cryo-EM map of homotrimeric STEAP1 bound to...

Fileemd_10735_additional_2.map
AnnotationSharpened cryo-EM map of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545. The constant region of the Fabs is masked out. The map is autosharpened (B = -69 Angstrom) in Relion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 1 of homotrimeric STEAP1 bound to...

Fileemd_10735_half_map_1.map
AnnotationHalf map 1 of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map 2 of homotrimeric STEAP1 bound to...

Fileemd_10735_half_map_2.map
AnnotationHalf map 2 of homotrimeric STEAP1 bound to three Fab-fragments of antibody mAb120.545.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Trimeric human six-transmembrane epithelial antigen of the prosta...

EntireName: Trimeric human six-transmembrane epithelial antigen of the prostate (STEAP1) bound to three Fab-fragments of antibody mAb120.545
Components
  • Complex: Trimeric human six-transmembrane epithelial antigen of the prostate (STEAP1) bound to three Fab-fragments of antibody mAb120.545
    • Complex: Fab-fragment 1 of antibody mAb120.545
      • Protein or peptide: Fab120.545 light chain
      • Protein or peptide: Fab120.545 heavy chain
    • Complex: Homo-trimeric STEAP1, chain a, b and c.
      • Protein or peptide: Metalloreductase STEAP1
    • Complex: Fab Fragment 2 of antibody mAb120.545
    • Complex: Fab-fragment 3 of antibody mAb120.545
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE

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Supramolecule #1: Trimeric human six-transmembrane epithelial antigen of the prosta...

SupramoleculeName: Trimeric human six-transmembrane epithelial antigen of the prostate (STEAP1) bound to three Fab-fragments of antibody mAb120.545
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#3
Details: STEAP1 was expressed in HEK293 GNT1- cells and purified in digitonin. Fab120.545 was a kind gift from Genmab BV and was produced upon our request.
Source (natural)Organism: Homo sapiens (human) / Location in cell: plasma membrane
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 GNT1- / Recombinant plasmid: pUPE 2959
Molecular weightTheoretical: 134 KDa

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Supramolecule #2: Fab-fragment 1 of antibody mAb120.545

SupramoleculeName: Fab-fragment 1 of antibody mAb120.545 / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: house mouse (house mouse) / Location in cell: secreted
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Supramolecule #3: Homo-trimeric STEAP1, chain a, b and c.

SupramoleculeName: Homo-trimeric STEAP1, chain a, b and c. / type: complex / ID: 3 / Parent: 1 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human) / Location in cell: plasma membrane
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293 GNT1- / Recombinant plasmid: pUPE 2959

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Supramolecule #4: Fab Fragment 2 of antibody mAb120.545

SupramoleculeName: Fab Fragment 2 of antibody mAb120.545 / type: complex / ID: 4 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse) / Location in cell: secreted
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Supramolecule #5: Fab-fragment 3 of antibody mAb120.545

SupramoleculeName: Fab-fragment 3 of antibody mAb120.545 / type: complex / ID: 5 / Parent: 1 / Macromolecule list: #2-#3
Source (natural)Organism: Mus musculus (house mouse) / Location in cell: secreted
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant cell: HEK293

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Macromolecule #1: Metalloreductase STEAP1

MacromoleculeName: Metalloreductase STEAP1 / type: protein_or_peptide / ID: 1 / Number of copies: 3 / Enantiomer: LEVO
EC number: Oxidoreductases; Oxidizing metal ions; With NAD+ or NADP+ as acceptor
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.183652 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MGSESRKDIT NQEELWKMKP RRNLEEDDYL HKDTGETSML KRPVLLHLHQ TAHADEFDCP SELQHTQELF PQWHLPIKIA AIIASLTFL YTLLREVIHP LATSHQQYFY KIPILVINKV LPMVSITLLA LVYLPGVIAA IVQLHNGTKY KKFPHWLDKW M LTRKQFGL ...String:
MGSESRKDIT NQEELWKMKP RRNLEEDDYL HKDTGETSML KRPVLLHLHQ TAHADEFDCP SELQHTQELF PQWHLPIKIA AIIASLTFL YTLLREVIHP LATSHQQYFY KIPILVINKV LPMVSITLLA LVYLPGVIAA IVQLHNGTKY KKFPHWLDKW M LTRKQFGL LSFFFAVLHA IYSLSYPMRR SYRYKLLNWA YQQVQQNKED AWIEHDVWRM EIYVSLGIVG LAILALLAVT SI PSVSDSL TWREFHYIQS KLGIVSLLLG TIHALIFAWN KWIDIKQFVW YTPPTFMIAV FLPIVVLIFK SILFLPCLRK KIL KIRHGW EDVTKINKTE ICSQLAAAGA AWSHPQFEKG AAWSHPQFEK GAAWSHPQFE KGAA

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Macromolecule #2: Fab120.545 light chain

MacromoleculeName: Fab120.545 light chain / type: protein_or_peptide / ID: 2 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 24.344135 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DIVMSQSPSS LAVSVGEKVT MSCKSSQSLL YRSNQKNYLA WYQQKPGQSP KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVKAEDL AVYYCQQYYN YPRTFGGGTK LEIKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA L QSGNSQES ...String:
DIVMSQSPSS LAVSVGEKVT MSCKSSQSLL YRSNQKNYLA WYQQKPGQSP KLLIYWASTR ESGVPDRFTG SGSGTDFTLT ISSVKAEDL AVYYCQQYYN YPRTFGGGTK LEIKRTVAAP SVFIFPPSDE QLKSGTASVV CLLNNFYPRE AKVQWKVDNA L QSGNSQES VTEQDSKDST YSLSSTLTLS KADYEKHKVY ACEVTHQGLS SPVTKSFNRG EC

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Macromolecule #3: Fab120.545 heavy chain

MacromoleculeName: Fab120.545 heavy chain / type: protein_or_peptide / ID: 3 / Number of copies: 3 / Enantiomer: LEVO
Source (natural)Organism: Mus musculus (house mouse)
Molecular weightTheoretical: 26.149922 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: DVQVQESGPG LVKPSQSLSL TCTVTGYSIT SDYAWNWIRQ FPGNKLEWMG YISNSGSTSY NPSLKSRISI TRDTSKNQFF LQLISVTTE DTATYYCARE RNYDYDDYYY AMDYWGQGTT LTVSAASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ...String:
DVQVQESGPG LVKPSQSLSL TCTVTGYSIT SDYAWNWIRQ FPGNKLEWMG YISNSGSTSY NPSLKSRISI TRDTSKNQFF LQLISVTTE DTATYYCARE RNYDYDDYYY AMDYWGQGTT LTVSAASTKG PSVFPLAPSS KSTSGGTAAL GCLVKDYFPE P VTVSWNSG ALTSGVHTFP AVLQSSGLYS LSSVVTVPSS SLGTQTYICN VNHKPSNTKV DKRVEPKSCD KTHTHHHHHH

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Macromolecule #4: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 4 / Number of copies: 3 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE / Heme B

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Macromolecule #5: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE

MacromoleculeName: 1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE / type: ligand / ID: 5 / Number of copies: 3 / Formula: XP4
Molecular weightTheoretical: 591.777 Da
Chemical component information

ChemComp-XP4:
1,2-DIMYRISTOYL-SN-GLYCERO-3-PHOSPHATE / DMPA, phospholipid*YM

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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

Concentration5.0 mg/mL
BufferpH: 7.8
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
20.0 mMTris
0.08 % (w/v)digitonin
1.0 mMFlavin-Adenine dinucleotide (FAD)

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.
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE-PROPANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: blot 4 seconds, blotforce 0.
DetailsSTEAP1-Fab120.545 complex purified in digitonin. The sample was monodisperse.

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Electron microscopy

MicroscopeFEI TALOS ARCTICA
Electron beamAcceleration voltage: 200 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Calibrated defocus max: 3.5 µm / Calibrated defocus min: 0.4 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.0 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Name: GIF Quantum LS / Energy filter - Slit width: 20 eV
Sample stageCooling holder cryogen: NITROGEN
DetailsData was collected on a 200 kV Talos Arctica, located at Utrecht University, the Netherlands.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Digitization - Frames/image: 1-26 / Number grids imaged: 1 / Number real images: 5325 / Average exposure time: 6.5 sec. / Average electron dose: 49.5 e/Å2
Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 616302 / Details: Picking was performed in Relion.
CTF correctionSoftware - Name: Gctf (ver. 1.18)
Startup modelType of model: EMDB MAP
EMDB ID:

0199


Details: First 3D classification was performed using the low-pass filtered EM map of STEAP4 with the interacellular domain removed. Classifications performed in Relion.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0) / Details: All processing performed in Relion.
Final 3D classificationSoftware - Name: RELION (ver. 3.1beta)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1beta) / Details: All processing performed in Relion.
Final reconstructionApplied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 2.97 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1beta) / Details: Final reconstruction was generated in Relion. / Number images used: 169426
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6hcy


Chain - Chain ID: A / Chain - Residue range: 196-454
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Overall B value: 100.9
Output model

PDB-6y9b:
Cryo-EM structure of trimeric human STEAP1 bound to three Fab120.545 fragments

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