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- PDB-6hd1: human STEAP4 bound to NADPH, FAD and heme. -

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Basic information

Entry
Database: PDB / ID: 6hd1
Titlehuman STEAP4 bound to NADPH, FAD and heme.
ComponentsMetalloreductase STEAP4
KeywordsMEMBRANE PROTEIN / Enzyme / Metalloreductase / Electron Transfer / Cofactor-binding
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
(2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate / FLAVIN-ADENINE DINUCLEOTIDE / PROTOPORPHYRIN IX CONTAINING FE / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / Metalloreductase STEAP4
Similarity search - Component
Biological speciesHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.8 Å
AuthorsOosterheert, W. / van Bezouwen, L.S. / Rodenburg, R.N.P. / Forster, F. / Mattevi, A. / Gros, P.
Funding support Netherlands, Italy, 3items
OrganizationGrant numberCountry
Netherlands Organisation for Scientific Research731.015.201 Netherlands
European Union653706 Netherlands
Italian Association for Cancer ResearchIG19808 Italy
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, 2018Deposition site: PDBE / Processing site: PDBE
Revision 1.0Oct 24, 2018Provider: repository / Type: Initial release
Revision 1.1Oct 31, 2018Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Nov 6, 2019Group: Data collection / Refinement description / Category: software / Item: _software.name
Revision 1.3Dec 11, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]
Revision 1.4Jul 29, 2020Group: Data collection / Derived calculations / Structure summary
Category: chem_comp / entity ...chem_comp / entity / pdbx_chem_comp_identifier / pdbx_entity_nonpoly / struct_conn / struct_conn_type / struct_site / struct_site_gen
Item: _chem_comp.name / _chem_comp.type ..._chem_comp.name / _chem_comp.type / _entity.pdbx_description / _pdbx_entity_nonpoly.name / _struct_conn.conn_type_id / _struct_conn.id / _struct_conn.pdbx_dist_value / _struct_conn.pdbx_leaving_atom_flag / _struct_conn.pdbx_role / _struct_conn.ptnr1_auth_asym_id / _struct_conn.ptnr1_auth_comp_id / _struct_conn.ptnr1_auth_seq_id / _struct_conn.ptnr1_label_asym_id / _struct_conn.ptnr1_label_atom_id / _struct_conn.ptnr1_label_comp_id / _struct_conn.ptnr1_label_seq_id / _struct_conn.ptnr2_auth_asym_id / _struct_conn.ptnr2_auth_comp_id / _struct_conn.ptnr2_auth_seq_id / _struct_conn.ptnr2_label_asym_id / _struct_conn.ptnr2_label_atom_id / _struct_conn.ptnr2_label_comp_id / _struct_conn_type.id
Description: Carbohydrate remediation / Provider: repository / Type: Remediation

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Structure visualization

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Assembly

Deposited unit
C: Metalloreductase STEAP4
A: Metalloreductase STEAP4
B: Metalloreductase STEAP4
hetero molecules


Theoretical massNumber of molelcules
Total (without water)164,31318
Polymers156,1083
Non-polymers8,20515
Water0
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: light scattering, Trimeric arrangement confirmed by multi-angle laser light scattering (MALLS)
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area27820 Å2
ΔGint-210 kcal/mol
Surface area57180 Å2
MethodPISA

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Components

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Protein / Sugars , 2 types, 6 molecules CAB

#1: Protein Metalloreductase STEAP4 / Six-transmembrane epithelial antigen of prostate 4 / SixTransMembrane protein of prostate 2 / Tumor ...Six-transmembrane epithelial antigen of prostate 4 / SixTransMembrane protein of prostate 2 / Tumor necrosis factor / alpha-induced protein 9


Mass: 52036.000 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Homo sapiens (human) / Gene: STEAP4, STAMP2, TNFAIP9 / Plasmid: pUPE / Details (production host): pUPE 3423 / Cell (production host): HEK293 GNTI- / Cell line (production host): HEK293 GNTI- / Organ (production host): KIDNEY / Production host: Homo sapiens (human) / Tissue (production host): KIDNEY
References: UniProt: Q687X5, Oxidoreductases; Oxidizing metal ions; With NAD+ or NADP+ as acceptor
#5: Sugar ChemComp-NAG / 2-acetamido-2-deoxy-beta-D-glucopyranose / N-Acetylglucosamine


Type: D-saccharide, beta linking / Mass: 221.208 Da / Num. of mol.: 3
Source method: isolated from a genetically manipulated source
Formula: C8H15NO6
IdentifierTypeProgram
DGlcpNAcbCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
N-acetyl-b-D-glucopyranosamineCOMMON NAMEGMML 1.0
b-D-GlcpNAcIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GlcNAcSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Non-polymers , 4 types, 12 molecules

#2: Chemical ChemComp-NAP / NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE / Nicotinamide adenine dinucleotide phosphate


Mass: 743.405 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C21H28N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#4: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE / Flavin adenine dinucleotide


Mass: 785.550 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#6: Chemical ChemComp-44E / (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate


Mass: 368.360 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C15H29O8P / Comment: phospholipid*YM

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: homotrimer of human STEAP4. / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.152 MDa / Experimental value: NO
Source (natural)Organism: Homo sapiens (human)
Source (recombinant)Organism: Homo sapiens (human) / Cell: HEK293 GNTI- / Plasmid: pUPE 3423
Buffer solutionpH: 5.5 / Details: 25 mM MES pH 5.5 200 mM NaCl 0.08% digitonin (w/v)
Buffer component
IDConc.NameFormulaBuffer-ID
1200 mMsodium chlorideNaClSodium chloride1
225 mM2-(N-morpholino)ethanesulfonic acidC6H13NO4S1
30.08 % (w/v)DIGITONINC56H92O291
SpecimenConc.: 4 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: human STEAP4 was purified from the HEK293 GNTI- cell membrane through Strep-affinity chromatography and size-exclusion chromatography (SEC). After SEC in digitonin, the sample was ...Details: human STEAP4 was purified from the HEK293 GNTI- cell membrane through Strep-affinity chromatography and size-exclusion chromatography (SEC). After SEC in digitonin, the sample was monodisperse. Cofactors NADPH and FAD were added before grid freezing.
Specimen supportGrid material: GOLD / Grid mesh size: 200 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 K / Details: blot time 4seconds blot force 0

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyModel: FEI TALOS ARCTICA
Details: 200 kV Talos Arctica at Utrecht University, the Netherlands.
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 200 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal magnification: 130000 X / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN
Image recordingAverage exposure time: 6 sec. / Electron dose: 45.5 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: 2321
EM imaging opticsEnergyfilter slit width: 20 eV
Image scansWidth: 3838 / Height: 3710 / Movie frames/image: 24 / Used frames/image: 1-24

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Processing

Software
NameVersionClassificationNB
PHENIX1.13rc1_2958refinement
PHENIX1.13rc1_2958refinement
EM software
IDNameVersionCategory
1RELION2.1b1particle selection
2EPUimage acquisition
4Gctf1.06CTF correction
10RELION2.1b1initial Euler assignment
11RELION2.1b1final Euler assignment
12RELION2.1b1classification
13RELION2.1b13D reconstruction
CTF correctionDetails: performed in GCTF. / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 421438 / Details: Autopicked in Relion.
SymmetryPoint symmetry: C3 (3 fold cyclic)
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 209075 / Details: performed by RELION. / Num. of class averages: 1 / Symmetry type: POINT
RefinementStereochemistry target values: GeoStd + Monomer Library
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.009499118895811328
ELECTRON MICROSCOPYf_angle_d0.998348232615507
ELECTRON MICROSCOPYf_chiral_restr0.06087392454561749
ELECTRON MICROSCOPYf_plane_restr0.007551135118321848
ELECTRON MICROSCOPYf_dihedral_angle_d13.60393588256456

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