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- EMDB-17732: Structure of the human mitochondrial iron-sulfur cluster biosynth... -

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Entry
Database: EMDB / ID: EMD-17732
TitleStructure of the human mitochondrial iron-sulfur cluster biosynthesis complex during persulfide transfer (persulfide on ISCU2)
Map data
Sample
  • Complex: Iron-bound human NFS1-ISD11-ACP1-ISCU2-FXN (Fe-NIAUX) complex during persulfide transfer
    • Protein or peptide: Cysteine desulfurase
    • Protein or peptide: LYR motif-containing protein 4
    • Protein or peptide: Acyl carrier protein
    • Protein or peptide: Iron-sulfur cluster assembly enzyme ISCU
    • Protein or peptide: Frataxin mature form
  • Ligand: PYRIDOXAL-5'-PHOSPHATEPyridoxal phosphate
  • Ligand: S-[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] dodecanethioate
  • Ligand: FE (II) ION
  • Ligand: water
Keywordscysteine desulfurase / FeS biosynthesis / FeS biogenesis / mitochondria / Friedreich's ataxia / persulfide / frataxin / TRANSFERASE
Function / homology
Function and homology information


regulation of ferrochelatase activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / Molybdenum cofactor biosynthesis / proprioception / [4Fe-4S] cluster assembly / iron incorporation into metallo-sulfur cluster / positive regulation of lyase activity / positive regulation of succinate dehydrogenase activity / L-cysteine desulfurase complex ...regulation of ferrochelatase activity / negative regulation of iron ion import across plasma membrane / molybdopterin cofactor metabolic process / Molybdenum cofactor biosynthesis / proprioception / [4Fe-4S] cluster assembly / iron incorporation into metallo-sulfur cluster / positive regulation of lyase activity / positive regulation of succinate dehydrogenase activity / L-cysteine desulfurase complex / Mitochondrial iron-sulfur cluster biogenesis / mitochondrial ISCU complex / positive regulation of aconitate hydratase activity / positive regulation of mitochondrial electron transport, NADH to ubiquinone / iron chaperone activity / cysteine desulfurase / cysteine desulfurase activity / negative regulation of organ growth / Mo-molybdopterin cofactor biosynthetic process / iron-sulfur cluster assembly complex / Mitochondrial protein import / [2Fe-2S] cluster assembly / adult walking behavior / oxidative phosphorylation / response to iron ion / acyl binding / embryo development ending in birth or egg hatching / lipid A biosynthetic process / lipid biosynthetic process / iron-sulfur cluster assembly / heme biosynthetic process / acyl carrier activity / negative regulation of multicellular organism growth / organ growth / muscle cell cellular homeostasis / positive regulation of catalytic activity / ferroxidase / iron-sulfur cluster binding / negative regulation of release of cytochrome c from mitochondria / phosphopantetheine binding / ferroxidase activity / protein autoprocessing / mitochondrion organization / ferric iron binding / ferrous iron binding / 2 iron, 2 sulfur cluster binding / cellular response to hydrogen peroxide / fatty acid biosynthetic process / pyridoxal phosphate binding / Maturation of replicase proteins / iron ion transport / positive regulation of cell growth / intracellular iron ion homeostasis / molecular adaptor activity / nuclear body / mitochondrial matrix / response to xenobiotic stimulus / iron ion binding / centrosome / lipid binding / positive regulation of cell population proliferation / negative regulation of apoptotic process / protein homodimerization activity / mitochondrion / zinc ion binding / nucleoplasm / metal ion binding / nucleus / cytosol / cytoplasm
Similarity search - Function
LYRM4, LYR domain / Frataxin / ISC system FeS cluster assembly, IscU scaffold / Cysteine desulfurase IscS / Frataxin/CyaY / Frataxin conserved site / Frataxin-like domain / Frataxin family signature. / Frataxin family profile. / Frataxin-like domain ...LYRM4, LYR domain / Frataxin / ISC system FeS cluster assembly, IscU scaffold / Cysteine desulfurase IscS / Frataxin/CyaY / Frataxin conserved site / Frataxin-like domain / Frataxin family signature. / Frataxin family profile. / Frataxin-like domain / NIF system FeS cluster assembly, NifU, N-terminal / NifU-like N terminal domain / Frataxin/CyaY superfamily / Cysteine desulfurase / Aminotransferase class-V, pyridoxal-phosphate binding site / Aminotransferases class-V pyridoxal-phosphate attachment site. / Aminotransferase class V domain / Aminotransferase class-V / Complex 1 LYR protein domain / Complex 1 protein (LYR family) / Acyl carrier protein (ACP) / Phosphopantetheine attachment site / Phosphopantetheine attachment site. / Phosphopantetheine attachment site / ACP-like superfamily / Carrier protein (CP) domain profile. / Phosphopantetheine binding ACP domain / Pyridoxal phosphate-dependent transferase, small domain / Pyridoxal phosphate-dependent transferase, major domain / Pyridoxal phosphate-dependent transferase
Similarity search - Domain/homology
Acyl carrier protein / Frataxin, mitochondrial / Iron-sulfur cluster assembly enzyme ISCU / LYR motif-containing protein 4 / Cysteine desulfurase
Similarity search - Component
Biological speciesHomo sapiens (human) / Escherichia coli BL21(DE3) (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.49 Å
AuthorsSteinhilper R / Murphy BJ
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Commun / Year: 2024
Title: Mechanism and structural dynamics of sulfur transfer during de novo [2Fe-2S] cluster assembly on ISCU2.
Authors: Vinzent Schulz / Ralf Steinhilper / Jonathan Oltmanns / Sven-A Freibert / Nils Krapoth / Uwe Linne / Sonja Welsch / Maren H Hoock / Volker Schünemann / Bonnie J Murphy / Roland Lill /
Abstract: Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11- ...Maturation of iron-sulfur proteins in eukaryotes is initiated in mitochondria by the core iron-sulfur cluster assembly (ISC) complex, consisting of the cysteine desulfurase sub-complex NFS1-ISD11-ACP1, the scaffold protein ISCU2, the electron donor ferredoxin FDX2, and frataxin, a protein dysfunctional in Friedreich's ataxia. The core ISC complex synthesizes [2Fe-2S] clusters de novo from Fe and a persulfide (SSH) bound at conserved cluster assembly site residues. Here, we elucidate the poorly understood Fe-dependent mechanism of persulfide transfer from cysteine desulfurase NFS1 to ISCU2. High-resolution cryo-EM structures obtained from anaerobically prepared samples provide snapshots that both visualize different stages of persulfide transfer from Cys381 to Cys138 and clarify the molecular role of frataxin in optimally positioning assembly site residues for fast sulfur transfer. Biochemical analyses assign ISCU2 residues essential for sulfur transfer, and reveal that Cys138 rapidly receives the persulfide without a detectable intermediate. Mössbauer spectroscopy assessing the Fe coordination of various sulfur transfer intermediates shows a dynamic equilibrium between pre- and post-sulfur-transfer states shifted by frataxin. Collectively, our study defines crucial mechanistic stages of physiological [2Fe-2S] cluster assembly and clarifies frataxin's molecular role in this fundamental process.
History
DepositionJun 26, 2023-
Header (metadata) releaseMay 1, 2024-
Map releaseMay 1, 2024-
UpdateMay 1, 2024-
Current statusMay 1, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_17732.png

Downloads & links

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Map

FileDownload / File: emd_17732.map.gz / Format: CCP4 / Size: 4.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesX (Sec.)Y (Row.)Z (Col.)
1.03 Å/pix.
x 146 pix.
= 150.38 Å		1.03 Å/pix.
x 86 pix.
= 88.58 Å		1.03 Å/pix.
x 100 pix.
= 103. Å

Surface

Projections

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Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 1.03 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-3.5320296 - 6.521454
Average (Standard dev.)0.0027551448 (±0.40663755)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderZYX
Origin634533
Dimensions86100146
Spacing14686100
CellA: 150.37999 Å / B: 88.579994 Å / C: 103.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_17732_msk_1.map
Projections & Slices
AxesZYX

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Half map: #2

Fileemd_17732_half_map_1.map
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Half map: #1

Fileemd_17732_half_map_2.map
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Sample components

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Entire : Iron-bound human NFS1-ISD11-ACP1-ISCU2-FXN (Fe-NIAUX) complex dur...

EntireName: Iron-bound human NFS1-ISD11-ACP1-ISCU2-FXN (Fe-NIAUX) complex during persulfide transfer
Components
  • Complex: Iron-bound human NFS1-ISD11-ACP1-ISCU2-FXN (Fe-NIAUX) complex during persulfide transfer
    • Protein or peptide: Cysteine desulfurase
    • Protein or peptide: LYR motif-containing protein 4
    • Protein or peptide: Acyl carrier protein
    • Protein or peptide: Iron-sulfur cluster assembly enzyme ISCU
    • Protein or peptide: Frataxin mature form
  • Ligand: PYRIDOXAL-5'-PHOSPHATEPyridoxal phosphate
  • Ligand: S-[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] dodecanethioate
  • Ligand: FE (II) ION
  • Ligand: water

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Supramolecule #1: Iron-bound human NFS1-ISD11-ACP1-ISCU2-FXN (Fe-NIAUX) complex dur...

SupramoleculeName: Iron-bound human NFS1-ISD11-ACP1-ISCU2-FXN (Fe-NIAUX) complex during persulfide transfer
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#5
Source (natural)Organism: Homo sapiens (human)

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Macromolecule #1: Cysteine desulfurase

MacromoleculeName: Cysteine desulfurase / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO / EC number: cysteine desulfurase
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 44.937449 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: MSLRPLYMDV QATTPLDPRV LDAMLPYLIN YYGNPHSRTH AYGWESEAAM ERARQQVASL IGADPREIIF TSGATESNNI AIKGVARFY RSRKKHLITT QTEHKCVLDS CRSLEAEGFQ VTYLPVQKSG IIDLKELEAA IQPDTSLVSV MTVNNEIGVK Q PIAEIGRI ...String:
MSLRPLYMDV QATTPLDPRV LDAMLPYLIN YYGNPHSRTH AYGWESEAAM ERARQQVASL IGADPREIIF TSGATESNNI AIKGVARFY RSRKKHLITT QTEHKCVLDS CRSLEAEGFQ VTYLPVQKSG IIDLKELEAA IQPDTSLVSV MTVNNEIGVK Q PIAEIGRI CSSRKVYFHT DAAQAVGKIP LDVNDMKIDL MSISGHKIYG PKGVGAIYIR RRPRVRVEAL QSGGGQERGM RS GTVPTPL VVGLGAACEV AQQEMEYDHK RISKLSERLI QNIMKSLPDV VMNGDPKHHY PGCINLSFAY VEGESLLMAL KDV ALSSGS ACTSASLEPS YVLRAIGTDE DLAHSSIRFG IGRFTTEEEV DYTVEKCIQH VKRLREMSPL WEMVQDGIDL KSIK WTQH

UniProtKB: Cysteine desulfurase

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Macromolecule #2: LYR motif-containing protein 4

MacromoleculeName: LYR motif-containing protein 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 13.502373 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MGSSHHHHHH GSPTTENLYF QGHNMAASSR AQVLALYRAM LRESKRFSAY NYRTYAVRRI RDAFRENKNV KDPVEIQTLV NKAKRDLGV IRRQVHIGQL YSTDKLIIEN RDMPRT

UniProtKB: LYR motif-containing protein 4

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Macromolecule #3: Acyl carrier protein

MacromoleculeName: Acyl carrier protein / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Escherichia coli BL21(DE3) (bacteria)
Molecular weightTheoretical: 8.64546 KDa
SequenceString:
MSTIEERVKK IIGEQLGVKQ EEVTNNASFV EDLGADSLDT VELVMALEEE FDTEIPDEEA EKITTVQAAI DYINGHQA

UniProtKB: Acyl carrier protein

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Macromolecule #4: Iron-sulfur cluster assembly enzyme ISCU

MacromoleculeName: Iron-sulfur cluster assembly enzyme ISCU / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 15.716184 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
MAYHKKVVDH YENPRNVGSL DKTSKNVGTG LVGAPACGDV MKLQIQVDEK GKIVDARFKT FGCGSAIASS SLATEWVKGK TVEEALTIK NTDIAKELCL PPVKLH(CSS)SML AEDAIKAALA DYKLKQEPKK GEAEKKLEHH HHHH

UniProtKB: Iron-sulfur cluster assembly enzyme ISCU

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Macromolecule #5: Frataxin mature form

MacromoleculeName: Frataxin mature form / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Homo sapiens (human)
Molecular weightTheoretical: 14.554985 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString:
SNASGTLGHP GSLDETTYER LAEETLDSLA EFFEDLADKP YTFEDYDVSF GSGVLTVKLG GDLGTYVINK QTPNKQIWLS SPSSGPKRY DWTGKNWVYS HDGVSLHELL AAELTKALKT KLDLSSLAYS GKDA

UniProtKB: Frataxin, mitochondrial

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Macromolecule #6: PYRIDOXAL-5'-PHOSPHATE

MacromoleculeName: PYRIDOXAL-5'-PHOSPHATE / type: ligand / ID: 6 / Number of copies: 1 / Formula: PLP
Molecular weightTheoretical: 247.142 Da
Chemical component information

ChemComp-PLP:
PYRIDOXAL-5'-PHOSPHATE / Pyridoxal phosphate

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Macromolecule #7: S-[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-b...

MacromoleculeName: S-[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] dodecanethioate
type: ligand / ID: 7 / Number of copies: 1 / Formula: 8Q1
Molecular weightTheoretical: 540.651 Da
Chemical component information

ChemComp-8Q1:
S-[2-({N-[(2R)-2-hydroxy-3,3-dimethyl-4-(phosphonooxy)butanoyl]-beta-alanyl}amino)ethyl] dodecanethioate

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Macromolecule #8: FE (II) ION

MacromoleculeName: FE (II) ION / type: ligand / ID: 8 / Number of copies: 1 / Formula: FE2
Molecular weightTheoretical: 55.845 Da

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 143 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER / Water

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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

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeTFS KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 2.5 µm / Nominal defocus min: 0.8 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: GATAN K3 BIOQUANTUM (6k x 4k) / Average electron dose: 62.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Startup modelType of model: INSILICO MODEL
Initial angle assignmentType: MAXIMUM LIKELIHOOD
Final angle assignmentType: MAXIMUM LIKELIHOOD
Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 2.49 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 507565
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelPDB ID:

6nzu


Chain - Source name: PDB / Chain - Initial model type: experimental model
Output model

PDB-8pk8:
Structure of the human mitochondrial iron-sulfur cluster biosynthesis complex during persulfide transfer (persulfide on ISCU2)

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