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- EMDB-0199: human STEAP4 bound to NADP, FAD, heme and Fe(III)-NTA. -

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Basic information

Entry
Database: EMDB / ID: EMD-0199
Titlehuman STEAP4 bound to NADP, FAD, heme and Fe(III)-NTA.
Map dataNone
Sample
  • Complex: Human Six-Transmembrane Epithelial Antigen of the Prostate 4
    • Protein or peptide: Metalloreductase STEAP4
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDEFlavin adenine dinucleotide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate
Function / homology
Function and homology information


ferric-chelate reductase (NADPH) / ferric-chelate reductase (NADPH) activity / cupric reductase activity / iron import into cell / copper ion import / fat cell differentiation / protein homotrimerization / FAD binding / early endosome membrane / electron transfer activity ...ferric-chelate reductase (NADPH) / ferric-chelate reductase (NADPH) activity / cupric reductase activity / iron import into cell / copper ion import / fat cell differentiation / protein homotrimerization / FAD binding / early endosome membrane / electron transfer activity / endosome / Golgi membrane / heme binding / extracellular exosome / nucleoplasm / membrane / metal ion binding / plasma membrane
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 STEAP4
Similarity search - Component
Biological speciesHomo sapiens (human)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsOosterheert W / van Bezouwen LS / Rodenburg RNP / Forster F / Mattevi A / Gros P
Funding support Netherlands, Italy, 3 items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research731.015.201 Netherlands
Italian Association for Cancer ResearchIG19808 Italy
European Union653706 Netherlands
CitationJournal: Nat Commun / Year: 2018
Title: Cryo-EM structures of human STEAP4 reveal mechanism of iron(III) reduction.
Authors: Wout Oosterheert / Laura S van Bezouwen / Remco N P Rodenburg / Joke Granneman / Friedrich Förster / Andrea Mattevi / Piet Gros /
Abstract: Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe and Cu ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several ...Enzymes of the six-transmembrane epithelial antigen of the prostate (STEAP) family reduce Fe and Cu ions to facilitate metal-ion uptake by mammalian cells. STEAPs are highly upregulated in several types of cancer, making them potential therapeutic targets. However, the structural basis for STEAP-catalyzed electron transfer through an array of cofactors to metals at the membrane luminal side remains elusive. Here, we report cryo-electron microscopy structures of human STEAP4 in absence and presence of Fe-NTA. Domain-swapped, trimeric STEAP4 orients NADPH bound to a cytosolic domain onto axially aligned flavin-adenine dinucleotide (FAD) and a single b-type heme that cross the transmembrane-domain to enable electron transfer. Substrate binding within a positively charged ring indicates that iron gets reduced while in complex with its chelator. These molecular principles of iron reduction provide a basis for exploring STEAPs as therapeutic targets.
History
DepositionAug 17, 2018-
Header (metadata) releaseOct 24, 2018-
Map releaseOct 24, 2018-
UpdateJul 29, 2020-
Current statusJul 29, 2020Processing site: PDBe / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.053
  • Imaged by UCSF Chimera
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  • Surface view colored by cylindrical radius
  • Surface level: 0.053
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-6hcy
  • Surface level: 0.053
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_0199.png

Downloads & links

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Map

FileDownload / File: emd_0199.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationNone
Voxel sizeX=Y=Z: 0.8127 Å
Density
Contour LevelBy AUTHOR: 0.053 / Movie #1: 0.053
Minimum - Maximum-0.13904265 - 0.22599746
Average (Standard dev.)0.0004582962 (±0.0058541913)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 243.81 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.81270.81270.8127
M x/y/z300300300
origin x/y/z0.0000.0000.000
length x/y/z243.810243.810243.810
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS300300300
D min/max/mean-0.1390.2260.000

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Supplemental data

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Mask #1

Fileemd_0199_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened, unmasked density map of human STEAP4 in...

Fileemd_0199_additional_1.map
AnnotationUnsharpened, unmasked density map of human STEAP4 in the presence of NADP, FAD, heme and Fe(III)-NTA (cofactor/substrate-bound state). Generated by 3D auto-refine in Relion2.1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Unsharpened, density map of human STEAP4 in cofactor/substrate-bound...

Fileemd_0199_additional_2.map
AnnotationUnsharpened, density map of human STEAP4 in cofactor/substrate-bound state, with its power spectrum adjusted to be the same as for the lower resolution cofactor-bound map, generated by Relion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Difference map between STEAP in the absence and...

Fileemd_0199_additional_3.map
AnnotationDifference map between STEAP in the absence and presence of substrate Fe(III)-NTA. Generated through map subtraction in Relion.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: The first unfiltered-half map of the final refinement...

Fileemd_0199_half_map_1.map
AnnotationThe first unfiltered-half map of the final refinement of human STEAP4 in the presence of NADP, FAD, heme and Fe(III)-NTA (cofactor/substrate-bound state). Generated by 3D auto-refine in Relion2.1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: The second unfiltered-half map of the final refinement...

Fileemd_0199_half_map_2.map
AnnotationThe second unfiltered-half map of the final refinement of human STEAP4 in the presence of NADP, FAD, heme and Fe(III)-NTA (cofactor/substrate-bound state). Generated by 3D auto-refine in Relion2.1.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Human Six-Transmembrane Epithelial Antigen of the Prostate 4

EntireName: Human Six-Transmembrane Epithelial Antigen of the Prostate 4
Components
  • Complex: Human Six-Transmembrane Epithelial Antigen of the Prostate 4
    • Protein or peptide: Metalloreductase STEAP4
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDEFlavin adenine dinucleotide
  • Ligand: 2-acetamido-2-deoxy-beta-D-glucopyranose
  • Ligand: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate

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Supramolecule #1: Human Six-Transmembrane Epithelial Antigen of the Prostate 4

SupramoleculeName: Human Six-Transmembrane Epithelial Antigen of the Prostate 4
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Homo sapiens (human)
Recombinant expressionOrganism: Homo sapiens (human) / Recombinant strain: HEK293 GNTI- / Recombinant cell: HEK293 GNTI- / Recombinant plasmid: pUPE 3423
Molecular weightTheoretical: 152 KDa

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Macromolecule #1: Metalloreductase STEAP4

MacromoleculeName: Metalloreductase STEAP4 / 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: 52.036 KDa
Recombinant expressionOrganism: Homo sapiens (human)
SequenceString: MEKTCIDALP LTMNSSEKQE TVCIFGTGDF GRSLGLKMLQ CGYSVVFGSR NPQKTTLLPS GAEVLSYSEA AKKSGIIIIA IHREHYDFL TELTEVLNGK ILVDISNNLK INQYPESNAE YLAHLVPGAH VVKAFNTISA WALQSGALDA SRQVFVCGND S KAKQRVMD ...String:
MEKTCIDALP LTMNSSEKQE TVCIFGTGDF GRSLGLKMLQ CGYSVVFGSR NPQKTTLLPS GAEVLSYSEA AKKSGIIIIA IHREHYDFL TELTEVLNGK ILVDISNNLK INQYPESNAE YLAHLVPGAH VVKAFNTISA WALQSGALDA SRQVFVCGND S KAKQRVMD IVRNLGLTPM DQGSLMAAKE IEKYPLQLFP MWRFPFYLSA VLCVFLFFYC VIRDVIYPYV YEKKDNTFRM AI SIPNRIF PITALTLLAL VYLPGVIAAI LQLYRGTKYR RFPDWLDHWM LCRKQLGLVA LGFAFLHVLY TLVIPIRYYV RWR LGNLTV TQAILKKENP FSTSSAWLSD SYVALGILGF FLFVLLGITS LPSVSNAVNW REFRFVQSKL GYLTLILCTA HTLV YGGKR FLSPSNLRWY LPAAYVLGLI IPCTVLVIKF VLIMPCVDNT LTRIRQGWER NSKH

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Macromolecule #2: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 2 / Number of copies: 3 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Nicotinamide adenine dinucleotide phosphate

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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 / Heme B

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Macromolecule #4: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 4 / Number of copies: 3 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM / Flavin adenine dinucleotide

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Macromolecule #5: 2-acetamido-2-deoxy-beta-D-glucopyranose

MacromoleculeName: 2-acetamido-2-deoxy-beta-D-glucopyranose / type: ligand / ID: 5 / Number of copies: 3 / Formula: NAG
Molecular weightTheoretical: 221.208 Da
Chemical component information

ChemComp-NAG:
2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine

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Macromolecule #6: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate

MacromoleculeName: (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / type: ligand / ID: 6 / Number of copies: 3 / Formula: 44E
Molecular weightTheoretical: 368.36 Da
Chemical component information

ChemComp-44E:
(2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / 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

Concentration4 mg/mL
BufferpH: 5.5
Component:
ConcentrationFormulaName
200.0 mMNaClSodium chloridesodium chloride
25.0 mMC6H13NO4S2-(N-morpholino)ethanesulfonic acid
0.08 % (w/v)C56H92O29DIGITONIN

Details: 25 mM MES pH 5.5 200 mM NaCl 0.08% (w/v) digitonin
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 200 / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV / Details: Blotted for 4 seconds Blotforce 0.
DetailsThe sample was purified from the HEK293 GNTI- cell membrane using STREP-affinity chromatography and size-exclusion chromatography (SEC). The sample was monodisperse after SEC in digitonin.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 2.0 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 165000
Specialist opticsEnergy filter - Slit width: 20 eV
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
DetailsGrid was prescreened on a 200 kV Talos Arctica Microscope. Microscope alignment was performed by Wim Hagen at EMBL Heidelberg.
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Digitization - Frames/image: 1-40 / Number grids imaged: 1 / Number real images: 8211 / Average exposure time: 12.0 sec. / Average electron dose: 47.4 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1098075
CTF correctionSoftware - Name: Gctf (ver. 1.18) / Details: GCTF within the Relion GUI.
Startup modelType of model: INSILICO MODEL
In silico model: As initial model, the map of STEAP4 in the cofactor-bound state was used.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final 3D classificationNumber classes: 5 / Avg.num./class: 55684 / Software - Name: RELION (ver. 2.1)
Details: Several rounds of 3D classification were performed in Relion 2.1.
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 2.1)
Final reconstructionNumber classes used: 2 / Applied symmetry - Point group: C3 (3 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 2.1) / Details: Standard Refinement ant Post processing in Relion. / Number images used: 255144
DetailsTitan Krios at EMBL Heidelberg.
FSC plot (resolution estimation)

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Atomic model buiding 1

DetailsNo Fitting was performed.
Output model

PDB-6hcy:
human STEAP4 bound to NADP, FAD, heme and Fe(III)-NTA.

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