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- EMDB-25775: Structure of STEAP2 in complex with ligands -

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Basic information

Entry
Database: EMDB / ID: EMD-25775
TitleStructure of STEAP2 in complex with ligands
Map data
Sample
  • Complex: Homo-trimeric complex of STEAP2
    • Protein or peptide: Metalloreductase STEAP2
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • Ligand: CHOLESTEROL
Keywordsmembrane-embedded oxidoreductase / membrane protein / OXIDOREDUCTASE
Function / homology
Function and homology information


copper ion import across plasma membrane / Oxidoreductases; Oxidizing metal ions; With NAD+ or NADP+ as acceptor / ferric-chelate reductase (NADPH) activity / cupric reductase (NADH) activity / iron ion import across plasma membrane / regulated exocytosis / Transferrin endocytosis and recycling / copper ion import / Golgi to plasma membrane transport / trans-Golgi network transport vesicle ...copper ion import across plasma membrane / Oxidoreductases; Oxidizing metal ions; With NAD+ or NADP+ as acceptor / ferric-chelate reductase (NADPH) activity / cupric reductase (NADH) activity / iron ion import across plasma membrane / regulated exocytosis / Transferrin endocytosis and recycling / copper ion import / Golgi to plasma membrane transport / trans-Golgi network transport vesicle / response to hormone / endocytosis / early endosome / endosome membrane / endosome / Golgi membrane / metal ion binding / plasma membrane / cytosol
Similarity search - Function
Ferric reductase transmembrane component-like domain / Ferric reductase like transmembrane component / Pyrroline-5-carboxylate reductase, catalytic, N-terminal / NADP oxidoreductase coenzyme F420-dependent / NAD(P)-binding domain superfamily
Similarity search - Domain/homology
Metalloreductase STEAP2
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.2 Å
AuthorsWang L / Chen KH
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/Eunice Kennedy Shriver National Institute of Child Health & Human Development (NIH/NICHD) United States
CitationJournal: Elife / Year: 2023
Title: Mechanism of stepwise electron transfer in six-transmembrane epithelial antigen of the prostate (STEAP) 1 and 2.
Authors: Kehan Chen / Lie Wang / Jiemin Shen / Ah-Lim Tsai / Ming Zhou / Gang Wu /
Abstract: Six transmembrane epithelial antigen of the prostate (STEAP) 1-4 are membrane-embedded hemoproteins that chelate a heme prosthetic group in a transmembrane domain (TMD). STEAP2-4, but not STEAP1, ...Six transmembrane epithelial antigen of the prostate (STEAP) 1-4 are membrane-embedded hemoproteins that chelate a heme prosthetic group in a transmembrane domain (TMD). STEAP2-4, but not STEAP1, have an intracellular oxidoreductase domain (OxRD) and can mediate cross-membrane electron transfer from NADPH via FAD and heme. However, it is unknown whether STEAP1 can establish a physiologically relevant electron transfer chain. Here, we show that STEAP1 can be reduced by reduced FAD or soluble cytochrome reductase that serves as a surrogate OxRD, providing the first evidence that STEAP1 can support a cross-membrane electron transfer chain. It is not clear whether FAD, which relays electrons from NADPH in OxRD to heme in TMD, remains constantly bound to the STEAPs. We found that FAD reduced by STEAP2 can be utilized by STEAP1, suggesting that FAD is diffusible rather than staying bound to STEAP2. We determined the structure of human STEAP2 in complex with NADP and FAD to an overall resolution of 3.2 Å by cryo-electron microscopy and found that the two cofactors bind STEAP2 similarly as in STEAP4, suggesting that a diffusible FAD is a general feature of the electron transfer mechanism in the STEAPs. We also demonstrated that STEAP2 reduces ferric nitrilotriacetic acid (Fe-NTA) significantly slower than STEAP1 and proposed that the slower reduction is due to the poor Fe-NTA binding to the highly flexible extracellular region in STEAP2. These results establish a solid foundation for understanding the function and mechanisms of the STEAPs.
History
DepositionDec 20, 2021-
Header (metadata) releaseJan 25, 2023-
Map releaseJan 25, 2023-
UpdateFeb 7, 2024-
Current statusFeb 7, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25775.png

Downloads & links

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Map

FileDownload / File: emd_25775.map.gz / Format: CCP4 / Size: 52.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 1.069 Å
Density
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum-1.8521452 - 3.0089824
Average (Standard dev.)0.006155498 (±0.081661396)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions240240240
Spacing240240240
CellA=B=C: 256.56 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: #1

Fileemd_25775_half_map_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_25775_half_map_2.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
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Histogram

Histogram (log scale)

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Sample components

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Entire : Homo-trimeric complex of STEAP2

EntireName: Homo-trimeric complex of STEAP2
Components
  • Complex: Homo-trimeric complex of STEAP2
    • Protein or peptide: Metalloreductase STEAP2
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine
  • Ligand: CHOLESTEROL

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Supramolecule #1: Homo-trimeric complex of STEAP2

SupramoleculeName: Homo-trimeric complex of STEAP2 / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 56 KDa

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Macromolecule #1: Metalloreductase STEAP2

MacromoleculeName: Metalloreductase STEAP2 / 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: 56.115305 KDa
Recombinant expressionOrganism: Trichoplusia ni (cabbage looper)
SequenceString: MESISMMGSP KSLSETFLPN GINGIKDARK VTVGVIGSGD FAKSLTIRLI RCGYHVVIGS RNPKFASEFF PHVVDVTHHE DALTKTNII FVAIHREHYT SLWDLRHLLV GKILIDVSNN MRINQYPESN AEYLASLFPD SLIVKGFNVV SAWALQLGPK D ASRQVYIC ...String:
MESISMMGSP KSLSETFLPN GINGIKDARK VTVGVIGSGD FAKSLTIRLI RCGYHVVIGS RNPKFASEFF PHVVDVTHHE DALTKTNII FVAIHREHYT SLWDLRHLLV GKILIDVSNN MRINQYPESN AEYLASLFPD SLIVKGFNVV SAWALQLGPK D ASRQVYIC SNNIQARQQV IELARQLNFI PIDLGSLSSA REIENLPLRL FTLWRGPVVV AISLATFFFL YSFVRDVIHP YA RNQQSDF YKIPIEIVNK TLPIVAITLL SLVYLAGLLA AAYQLYYGTK YRRFPPWLET WLQCRKQLGL LSFFFAMVHV AYS LCLPMR RSERYLFLNM AYQQVHANIE NSWNEEEVWR IEMYISFGIM SLGLLSLLAV TSIPSVSNAL NWREFSFIQS TLGY VALLI STFHVLIYGW KRAFEEEYYR FYTPPNFVLA LVLPSIVILG KIILFLPCIS RKLKRIKKGW EKSQFLEEGM GGTIP HVSP ERVTVM

UniProtKB: Metalloreductase STEAP2

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Macromolecule #2: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 3 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Macromolecule #3: PROTOPORPHYRIN IX CONTAINING FE

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

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #4: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 4 / Number of copies: 3 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Macromolecule #5: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

MacromoleculeName: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / type: ligand / ID: 5 / Number of copies: 3 / Formula: LBN
Molecular weightTheoretical: 760.076 Da
Chemical component information

ChemComp-LBN:
1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine / phospholipid*YM

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Macromolecule #6: CHOLESTEROL

MacromoleculeName: CHOLESTEROL / type: ligand / ID: 6 / Number of copies: 6 / Formula: CLR
Molecular weightTheoretical: 386.654 Da
Chemical component information

ChemComp-CLR:
CHOLESTEROL

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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 mg/mL
BufferpH: 7.5
VitrificationCryogen name: OTHER

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 1.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: TUNGSTEN HAIRPIN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.2 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 305849
Initial angle assignmentType: NOT APPLICABLE
Final angle assignmentType: NOT APPLICABLE

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