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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 / homologyFerric reductase like transmembrane component / NADP oxidoreductase coenzyme F420-dependent / NAD(P)-binding domain superfamily / Pyrroline-5-carboxylate reductase, catalytic, N-terminal / Ferric reductase transmembrane component-like domain / ferric-chelate reductase (NADPH) activity / cupric reductase activity / With NAD or NADP as acceptor / iron ion import across cell outer membrane / copper ion import ...Ferric reductase like transmembrane component / NADP oxidoreductase coenzyme F420-dependent / NAD(P)-binding domain superfamily / Pyrroline-5-carboxylate reductase, catalytic, N-terminal / Ferric reductase transmembrane component-like domain / ferric-chelate reductase (NADPH) activity / cupric reductase activity / With NAD or NADP as acceptor / iron ion import across cell outer membrane / copper ion import / fat cell differentiation / early endosome membrane / iron ion homeostasis / endosome / Golgi membrane / integral component of plasma membrane / Golgi apparatus / extracellular exosome / plasma membrane / metal ion binding / Metalloreductase STEAP4
Function and homology information
Specimen sourceHomo sapiens (human)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / 3.8 Å resolution
AuthorsOosterheert, W. / van Bezouwen, L.S. / Rodenburg, R.N.P. / Forster, F. / Mattevi, A. / Gros, P.
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
Validation Report
SummaryFull reportAbout validation report
DateDeposition: Aug 17, 2018 / Release: Oct 24, 2018
RevisionDateData content typeGroupCategoryItemProviderType
1.0Oct 24, 2018Structure modelrepositoryInitial release
1.1Oct 31, 2018Structure modelData collection / Database referencescitation / citation_author_citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title

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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
Polyers156,1083
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)
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TypeNameSymmetry operationNumber
identity operation1_5551
Buried area (Å2)27820
ΔGint (kcal/M)-210
Surface area (Å2)57180
MethodPISA

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Components

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Protein/peptide , 1 types, 3 molecules CAB

#1: Protein/peptide Metalloreductase STEAP4 / 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: (gene. exp.) Homo sapiens (human) / Gene: STEAP4, STAMP2, TNFAIP9 / Plasmid name: 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, With NAD or NADP as acceptor

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Non-polymers , 5 types, 15 molecules

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


Mass: 743.405 Da / Num. of mol.: 3 / Formula: C21H28N7O17P3 / Nicotinamide adenine dinucleotide phosphate
#3: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 3 / Formula: C34H32FeN4O4 / Heme
#4: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 3 / Formula: C27H33N9O15P2 / Flavin adenine dinucleotide / Comment: FAD *YM
#5: Chemical ChemComp-NAG / N-ACETYL-D-GLUCOSAMINE


Mass: 221.208 Da / Num. of mol.: 3 / Formula: C8H15NO6 / N-Acetylglucosamine
#6: Chemical ChemComp-44E / (2R)-3-(phosphonooxy)propane-1,2-diyl dihexanoate


Mass: 368.360 Da / Num. of mol.: 3 / Formula: C15H29O8P

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: PARTICLE / 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)Cell: HEK293 GNTI- / Organism: Homo sapiens (human) / Plasmid: pUPE 3423
Buffer solutionDetails: 25 mM MES pH 5.5 200 mM NaCl 0.08% digitonin (w/v) / pH: 5.5
Buffer component
IDConc.NameFormulaBuffer ID
1200 mMsodium chlorideNaCl1
225 mM2-(N-morpholino)ethanesulfonic acidC6H13NO4S1
30.08 % (w/v)DIGITONINC56H92O291
SpecimenConc.: 4 mg/ml
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.
Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 200 / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 293 kelvins / Details: blot time 4seconds blot force 0

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Electron microscopy imaging

Experimental equipment
Model: Talos Arctica / Image courtesy: FEI Company
MicroscopyMicroscope model: 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 / Nominal defocus max: 3000 nm / Nominal defocus min: 800 nm / Cs: 2.7 mm / C2 aperture diameter: 50 mm / 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) / Number of grids imaged: 1 / Number 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

Classification: refinement / Contact author: Paul D. Adams / Contact author email: pdadams[at]lbl.gov / Location: https://www.phenix-online.org/ / Type: program / Version: 1.13rc1_2958

Name
phenix.real_space_refine
PHENIX
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 selectionDetails: Autopicked in Relion. / Number of particles selected: 421438
SymmetryPoint symmetry: C3
3D reconstructionResolution: 3.8 Å / Resolution method: FSC 0.143 CUT-OFF / Number of particles: 209075 / Details: performed by RELION. / Number of class averages: 1 / Symmetry type: POINT
RefineStereochemistry 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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