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- EMDB-49341: Cryo-EM composite map of the NADPH-bound Pyrococcus furiosus SHI ... -

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Basic information

Entry
Database: EMDB / ID: EMD-49341
TitleCryo-EM composite map of the NADPH-bound Pyrococcus furiosus SHI complex
Map data
Sample
  • Complex: Quaternary complex of the NADPH-bound Pyrococcus furiosus SHI complex
    • Protein or peptide: Sulfhydrogenase 1 subunit delta
    • Protein or peptide: Sulfhydrogenase 1 subunit beta
    • Protein or peptide: Sulfhydrogenase 1 subunit gamma
    • Protein or peptide: Sulfhydrogenase 1 subunit alpha
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: formyl[bis(hydrocyanato-1kappaC)]ironnickel(Fe-Ni)
  • Ligand: MAGNESIUM ION
Keywordshydrogen production / [NiFe] hydrogenase / cryo-EM structure / electron transport
Function / homology
Function and homology information


sulfhydrogenase / sulfur reductase activity / hydrogen dehydrogenase (NADP+) / hydrogen dehydrogenase (NADP+) activity / [Ni-Fe] hydrogenase complex / pyrimidine nucleotide biosynthetic process / ferredoxin hydrogenase activity / 3 iron, 4 sulfur cluster binding / nickel cation binding / 2 iron, 2 sulfur cluster binding ...sulfhydrogenase / sulfur reductase activity / hydrogen dehydrogenase (NADP+) / hydrogen dehydrogenase (NADP+) activity / [Ni-Fe] hydrogenase complex / pyrimidine nucleotide biosynthetic process / ferredoxin hydrogenase activity / 3 iron, 4 sulfur cluster binding / nickel cation binding / 2 iron, 2 sulfur cluster binding / flavin adenine dinucleotide binding / 4 iron, 4 sulfur cluster binding / metal ion binding / cytoplasm
Similarity search - Function
: / : / : / 4Fe-4S dicluster domain / : / Dihydroorotate dehydrogenase, electron transfer subunit, Fe-S binding domain superfamily / Cytochrome-c3 hydrogenase, gamma subunit / Dihydroorotate dehydrogenase, electron transfer subunit, iron-sulphur cluster binding domain / : / Iron-sulfur cluster binding domain of dihydroorotate dehydrogenase B ...: / : / : / 4Fe-4S dicluster domain / : / Dihydroorotate dehydrogenase, electron transfer subunit, Fe-S binding domain superfamily / Cytochrome-c3 hydrogenase, gamma subunit / Dihydroorotate dehydrogenase, electron transfer subunit, iron-sulphur cluster binding domain / : / Iron-sulfur cluster binding domain of dihydroorotate dehydrogenase B / : / Flavoprotein pyridine nucleotide cytochrome reductase-like, FAD-binding domain / Oxidoreductase FAD-binding domain / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Alpha-helical ferredoxin / Nickel-dependent hydrogenases large subunit signature 2. / Nickel-dependent hydrogenase, large subunit, nickel binding site / Nickel-dependent hydrogenase, large subunit / Nickel-dependent hydrogenase / NADH:ubiquinone oxidoreductase-like, 20kDa subunit / NADH ubiquinone oxidoreductase, 20 Kd subunit / [NiFe]-hydrogenase, large subunit / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain
Similarity search - Domain/homology
Sulfhydrogenase 1 subunit delta / Sulfhydrogenase 1 subunit alpha / Sulfhydrogenase 1 subunit gamma / Sulfhydrogenase 1 subunit beta
Similarity search - Component
Biological speciesPyrococcus furiosus (archaea)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.06 Å
AuthorsXiao X / Li H
Funding support United States, 1 items
OrganizationGrant numberCountry
Department of Energy (DOE, United States) United States
CitationJournal: Structure / Year: 2025
Title: Structural insights into the biotechnologically relevant reversible NADPH-oxidizing NiFe-hydrogenase from P.furiosus.
Authors: Xiansha Xiao / Gerrit J Schut / Xiang Feng / Patrick M McTernan / Dominik K Haja / William N Lanzilotta / Michael W W Adams / Huilin Li /
Abstract: The cytoplasmic hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the group III hydrogenase family. SHI oxidizes NADPH rather than NADH to reduce protons and ...The cytoplasmic hydrogenase I (SHI) from the hyperthermophilic archaeon Pyrococcus furiosus belongs to the group III hydrogenase family. SHI oxidizes NADPH rather than NADH to reduce protons and evolve hydrogen gas, and because of this property, coupled with its high thermal stability, the enzyme holds great potential for economical hydrogen production. Despite decades of efforts, the SHI structure has remained unknown. Here, we report the cryoelectron microscopic (cryo-EM) structures of the heterotetrameric SHI holoenzyme (αδβγ). SHI is a symmetric dimer of two individually functional heterotetramers. SHI-αδ resembles the standard [NiFe] hydrogenase, and SHI-βγ function as the NADPH oxidoreductase. SHI-β contains three [4Fe-4S] clusters that relay electrons from NADPH in SHI-γ to the catalytic [NiFe] cluster in SHI-αδ for H production. These structures will guide the adaptation of this unique enzyme for biotechnological applications.
History
DepositionFeb 20, 2025-
Header (metadata) releaseJul 9, 2025-
Map releaseJul 9, 2025-
UpdateJul 9, 2025-
Current statusJul 9, 2025Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_49341.png

Downloads & links

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Map

FileDownload / File: emd_49341.map.gz / Format: CCP4 / Size: 216 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
0.83 Å/pix.
x 384 pix.
= 317.952 Å		0.83 Å/pix.
x 384 pix.
= 317.952 Å		0.83 Å/pix.
x 384 pix.
= 317.952 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.828 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.7
Minimum - Maximum0.0 - 19.719360000000002
Average (Standard dev.)0.04365759 (±0.5813625)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions384384384
Spacing384384384
CellA=B=C: 317.952 Å
α=β=γ: 90.0 °

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Supplemental data

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Sample components

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Entire : Quaternary complex of the NADPH-bound Pyrococcus furiosus SHI complex

EntireName: Quaternary complex of the NADPH-bound Pyrococcus furiosus SHI complex
Components
  • Complex: Quaternary complex of the NADPH-bound Pyrococcus furiosus SHI complex
    • Protein or peptide: Sulfhydrogenase 1 subunit delta
    • Protein or peptide: Sulfhydrogenase 1 subunit beta
    • Protein or peptide: Sulfhydrogenase 1 subunit gamma
    • Protein or peptide: Sulfhydrogenase 1 subunit alpha
  • Ligand: IRON/SULFUR CLUSTER
  • Ligand: FE2/S2 (INORGANIC) CLUSTER
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: formyl[bis(hydrocyanato-1kappaC)]ironnickel(Fe-Ni)
  • Ligand: MAGNESIUM ION

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Supramolecule #1: Quaternary complex of the NADPH-bound Pyrococcus furiosus SHI complex

SupramoleculeName: Quaternary complex of the NADPH-bound Pyrococcus furiosus SHI complex
type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1-#4
Source (natural)Organism: Pyrococcus furiosus (archaea)

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Macromolecule #1: Sulfhydrogenase 1 subunit delta

MacromoleculeName: Sulfhydrogenase 1 subunit delta / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: hydrogen dehydrogenase (NADP+)
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightTheoretical: 29.269037 KDa
SequenceString: MGKVRIGFYA LTSCYGCQLQ LAMMDELLQL IPNAEIVCWF MIDRDSIEDE KVDIAFIEGS VSTEEEVELV KKIRENAKIV VAVGACAVQ GGVQSWSEKP LEELWKKVYG DAKVKFQPKK AEPVSKYIKV DYNIYGCPPE KKDFLYALGT FLIGSWPEDI D YPVCLECR ...String:
MGKVRIGFYA LTSCYGCQLQ LAMMDELLQL IPNAEIVCWF MIDRDSIEDE KVDIAFIEGS VSTEEEVELV KKIRENAKIV VAVGACAVQ GGVQSWSEKP LEELWKKVYG DAKVKFQPKK AEPVSKYIKV DYNIYGCPPE KKDFLYALGT FLIGSWPEDI D YPVCLECR LNGHPCILLE KGEPCLGPVT RAGCNARCPG FGVACIGCRG AIGYDVAWFD SLAKVFKEKG MTKEEIIERM KM FNGHDER VEKMVEKIFS GGEQ

UniProtKB: Sulfhydrogenase 1 subunit delta

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Macromolecule #2: Sulfhydrogenase 1 subunit beta

MacromoleculeName: Sulfhydrogenase 1 subunit beta / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: sulfhydrogenase
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightTheoretical: 43.464688 KDa
SequenceString: MRYVKLPKEN TYEFLERLKD WGKLYAPVKI SDKFYDFREI DDVRKIEFHY NRTIMPPKKF FFKPREKLFE FDISKPEYRE VIEEVEPFI IFGVHACDIY GLKILDTVYL DEFPDKYYKV RREKGIIIGI SCMPDEYCFC NLRETDFADD GFDLFFHELP D GWLVRVGT ...String:
MRYVKLPKEN TYEFLERLKD WGKLYAPVKI SDKFYDFREI DDVRKIEFHY NRTIMPPKKF FFKPREKLFE FDISKPEYRE VIEEVEPFI IFGVHACDIY GLKILDTVYL DEFPDKYYKV RREKGIIIGI SCMPDEYCFC NLRETDFADD GFDLFFHELP D GWLVRVGT PTGHRLVDKN IKLFEEVTDK DICAFRDFEK RRQQAFKYHE DWGNLRYLLE LEMEHPMWDE EADKCLACGI CN TTCPTCR CYEVQDIVNL DGVTGYRERR WDSCQFRSHG LVAGGHNFRP TKKDRFRNRY LCKNAYNEKL GLSYCVGCGR CTA FCPANI SFVGNLRRIL GLEENKCPPT VSEEIPKRGF AYSSNIRGDG V

UniProtKB: Sulfhydrogenase 1 subunit beta

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Macromolecule #3: Sulfhydrogenase 1 subunit gamma

MacromoleculeName: Sulfhydrogenase 1 subunit gamma / type: protein_or_peptide / ID: 3 / Number of copies: 2 / Enantiomer: LEVO / EC number: sulfhydrogenase
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightTheoretical: 33.093527 KDa
SequenceString: MMLPKEIMMP NDNPYALHRV KVLKVYSLTE TEKLFLFRFE DPELAEKWTF KPGQFVQLTI PGVGEVPISI CSSPMRKGFF ELCIRKAGR VTTVVHRLKP GDTVLVRGPY GNGFPVDEWE GMDLLLIAAG LGTAPLRSVF LYAMDNRWKY GNITFINTAR Y GKDLLFYK ...String:
MMLPKEIMMP NDNPYALHRV KVLKVYSLTE TEKLFLFRFE DPELAEKWTF KPGQFVQLTI PGVGEVPISI CSSPMRKGFF ELCIRKAGR VTTVVHRLKP GDTVLVRGPY GNGFPVDEWE GMDLLLIAAG LGTAPLRSVF LYAMDNRWKY GNITFINTAR Y GKDLLFYK ELEAMKDLAE AENVKIIQSV TRDPNWPGLK GRPQQFIVEA NTNPKNTAVA ICGPPRMYKS VFEALINYGY RP ENIFVTL ERRMKCGIGK CGHCNVGTST SWKYICKDGP VFTYFDIVST PGLLD

UniProtKB: Sulfhydrogenase 1 subunit gamma

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Macromolecule #4: Sulfhydrogenase 1 subunit alpha

MacromoleculeName: Sulfhydrogenase 1 subunit alpha / type: protein_or_peptide / ID: 4 / Number of copies: 2 / Enantiomer: LEVO / EC number: hydrogen dehydrogenase (NADP+)
Source (natural)Organism: Pyrococcus furiosus (archaea)
Molecular weightTheoretical: 48.381871 KDa
SequenceString: MKNLYLPITI DHIARVEGKG GVEIIIGDDG VKEVKLNIIE GPRFFEAITI GKKLEEALAI YPRICSFCSA AHKLTALEAA EKAVGFVPR EEIQALREVL YIGDMIESHA LHLYLLVLPD YRGYSSPLKM VNEYKREIEI ALKLKNLGTW MMDILGSRAI H QENAVLGG ...String:
MKNLYLPITI DHIARVEGKG GVEIIIGDDG VKEVKLNIIE GPRFFEAITI GKKLEEALAI YPRICSFCSA AHKLTALEAA EKAVGFVPR EEIQALREVL YIGDMIESHA LHLYLLVLPD YRGYSSPLKM VNEYKREIEI ALKLKNLGTW MMDILGSRAI H QENAVLGG FGKLPEKSVL EKMKAELREA LPLAEYTFEL FAKLEQYSEV EGPITHLAVK PRGDAYGIYG DYIKASDGEE FP SEKYRDY IKEFVVEHSF AKHSHYKGRP FMVGAISRVI NNADLLYGKA KELYEANKDL LKGTNPFANN LAQALEIVYF IER AIDLLD EALAKWPIKP RDEVEIKDGF GVSTTEAPRG ILVYALKVEN GRVSYADIIT PTAFNLAMME EHVRMMAEKH YNDD PERLK ILAEMVVRAY DPCISCSVHV VRL

UniProtKB: Sulfhydrogenase 1 subunit alpha

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Macromolecule #5: IRON/SULFUR CLUSTER

MacromoleculeName: IRON/SULFUR CLUSTER / type: ligand / ID: 5 / Number of copies: 14 / Formula: SF4
Molecular weightTheoretical: 351.64 Da
Chemical component information

ChemComp-FS1:
IRON/SULFUR CLUSTER

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Macromolecule #6: FE2/S2 (INORGANIC) CLUSTER

MacromoleculeName: FE2/S2 (INORGANIC) CLUSTER / type: ligand / ID: 6 / Number of copies: 2 / Formula: FES
Molecular weightTheoretical: 175.82 Da
Chemical component information

ChemComp-FES:
FE2/S2 (INORGANIC) CLUSTER

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Macromolecule #7: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 7 / Number of copies: 2 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

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

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Macromolecule #8: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE / type: ligand / ID: 8 / Number of copies: 2 / Formula: NAP
Molecular weightTheoretical: 743.405 Da
Chemical component information

ChemComp-NAP:
NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Macromolecule #9: formyl[bis(hydrocyanato-1kappaC)]ironnickel(Fe-Ni)

MacromoleculeName: formyl[bis(hydrocyanato-1kappaC)]ironnickel(Fe-Ni) / type: ligand / ID: 9 / Number of copies: 2 / Formula: NFU
Molecular weightTheoretical: 195.591 Da
Chemical component information

ChemComp-NFU:
formyl[bis(hydrocyanato-1kappaC)]ironnickel(Fe-Ni)

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Macromolecule #10: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 10 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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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

Concentration2 mg/mL
BufferpH: 7.5
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: PLASMA CLEANING / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 283.15 K / Instrument: FEI VITROBOT MARK II

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: GIF Bioquantum / Energy filter - Slit width: 20 eV
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number real images: 31397 / Average exposure time: 1.5 sec. / Average electron dose: 60.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 100.0 µm / Calibrated magnification: 105000 / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 1.8 µm / Nominal defocus min: 1.3 µm
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1215778
CTF correctionSoftware - Name: cryoSPARC / Type: PHASE FLIPPING AND AMPLITUDE CORRECTION
Startup modelType of model: NONE
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.06 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 313849
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: AlphaFold / Chain - Initial model type: in silico model
RefinementProtocol: AB INITIO MODEL
Output model

PDB-9nf0:
Structure of the NADPH-bound Pyrococcus furiosus SHI complex

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