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- EMDB-26801: The 1.67 Angstrom CryoEM structure of the [NiFe]-hydrogenase Huc ... -

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Basic information

Entry
Database: EMDB / ID: EMD-26801
TitleThe 1.67 Angstrom CryoEM structure of the [NiFe]-hydrogenase Huc from Mycobacterium smegmatis - catalytic dimer (Huc2S2L)
Map data
Sample
  • Complex: Complex of the type 2 [NiFe]-hydrogenase Huc from Mycobacterium smegmatis
    • Protein or peptide: Hydrogenase-2, large subunit
    • Protein or peptide: Hydrogenase-2, small subunit
  • Ligand: NICKEL (III) ION
  • Ligand: CARBONMONOXIDE-(DICYANO) IRON
  • Ligand: HYDROXIDE ION
  • Ligand: MAGNESIUM ION
  • Ligand: MENADIONE
  • Ligand: FE3-S4 CLUSTER
  • Ligand: water
Function / homology
Function and homology information


hydrogenase (acceptor) / ferredoxin hydrogenase complex / hydrogenase (acceptor) activity / ferredoxin hydrogenase activity / 3 iron, 4 sulfur cluster binding / nickel cation binding / 4 iron, 4 sulfur cluster binding / metal ion binding
Similarity search - Function
[NiFe]-hydrogenase, small subunit / Cytochrome-c3 hydrogenase, C-terminal / [NiFe]-hydrogenase, small subunit, C-terminal domain superfamily / NiFe/NiFeSe hydrogenase small subunit C-terminal / Nickel-dependent hydrogenases large subunit signature 1. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / Nickel-dependent hydrogenase, large subunit, nickel binding site / Nickel-dependent hydrogenase, large subunit / Nickel-dependent hydrogenase / NADH:ubiquinone oxidoreductase-like, 20kDa subunit ...[NiFe]-hydrogenase, small subunit / Cytochrome-c3 hydrogenase, C-terminal / [NiFe]-hydrogenase, small subunit, C-terminal domain superfamily / NiFe/NiFeSe hydrogenase small subunit C-terminal / Nickel-dependent hydrogenases large subunit signature 1. / [NiFe]-hydrogenase, small subunit, N-terminal domain superfamily / 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
Similarity search - Domain/homology
Hydrogenase-2, large subunit / NADH ubiquinone oxidoreductase 20 kDa subunit
Similarity search - Component
Biological speciesMycolicibacterium smegmatis (bacteria) / Mycolicibacterium smegmatis MC2 155 (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 1.67 Å
AuthorsGrinter R / Venugopal H / Kropp A / Greening C
Funding support Australia, 2 items
OrganizationGrant numberCountry
Australian Research Council (ARC)DP200103074 Australia
National Health and Medical Research Council (NHMRC, Australia)APP1197376 Australia
CitationJournal: Nature / Year: 2023
Title: Structural basis for bacterial energy extraction from atmospheric hydrogen.
Authors: Rhys Grinter / Ashleigh Kropp / Hari Venugopal / Moritz Senger / Jack Badley / Princess R Cabotaje / Ruyu Jia / Zehui Duan / Ping Huang / Sven T Stripp / Christopher K Barlow / Matthew ...Authors: Rhys Grinter / Ashleigh Kropp / Hari Venugopal / Moritz Senger / Jack Badley / Princess R Cabotaje / Ruyu Jia / Zehui Duan / Ping Huang / Sven T Stripp / Christopher K Barlow / Matthew Belousoff / Hannah S Shafaat / Gregory M Cook / Ralf B Schittenhelm / Kylie A Vincent / Syma Khalid / Gustav Berggren / Chris Greening /
Abstract: Diverse aerobic bacteria use atmospheric H as an energy source for growth and survival. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and ...Diverse aerobic bacteria use atmospheric H as an energy source for growth and survival. This globally significant process regulates the composition of the atmosphere, enhances soil biodiversity and drives primary production in extreme environments. Atmospheric H oxidation is attributed to uncharacterized members of the [NiFe] hydrogenase superfamily. However, it remains unresolved how these enzymes overcome the extraordinary catalytic challenge of oxidizing picomolar levels of H amid ambient levels of the catalytic poison O and how the derived electrons are transferred to the respiratory chain. Here we determined the cryo-electron microscopy structure of the Mycobacterium smegmatis hydrogenase Huc and investigated its mechanism. Huc is a highly efficient oxygen-insensitive enzyme that couples oxidation of atmospheric H to the hydrogenation of the respiratory electron carrier menaquinone. Huc uses narrow hydrophobic gas channels to selectively bind atmospheric H at the expense of O, and 3 [3Fe-4S] clusters modulate the properties of the enzyme so that atmospheric H oxidation is energetically feasible. The Huc catalytic subunits form an octameric 833 kDa complex around a membrane-associated stalk, which transports and reduces menaquinone 94 Å from the membrane. These findings provide a mechanistic basis for the biogeochemically and ecologically important process of atmospheric H oxidation, uncover a mode of energy coupling dependent on long-range quinone transport, and pave the way for the development of catalysts that oxidize H in ambient air.
History
DepositionApr 28, 2022-
Header (metadata) releaseJan 4, 2023-
Map releaseJan 4, 2023-
UpdateMar 29, 2023-
Current statusMar 29, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_26801.png

Downloads & links

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Map

FileDownload / File: emd_26801.map.gz / Format: CCP4 / Size: 103 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.82 Å
Density
Contour LevelBy AUTHOR: 1.157
Minimum - Maximum-6.412333 - 19.718851
Average (Standard dev.)0.0045369444 (±0.21863388)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions300300300
Spacing300300300
CellA=B=C: 246.0 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_26801_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #2

Fileemd_26801_half_map_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: #1

Fileemd_26801_half_map_2.map
Projections & Slices
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Histogram (log scale)

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Sample components

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Entire : Complex of the type 2 [NiFe]-hydrogenase Huc from Mycobacterium s...

EntireName: Complex of the type 2 [NiFe]-hydrogenase Huc from Mycobacterium smegmatis
Components
  • Complex: Complex of the type 2 [NiFe]-hydrogenase Huc from Mycobacterium smegmatis
    • Protein or peptide: Hydrogenase-2, large subunit
    • Protein or peptide: Hydrogenase-2, small subunit
  • Ligand: NICKEL (III) ION
  • Ligand: CARBONMONOXIDE-(DICYANO) IRON
  • Ligand: HYDROXIDE ION
  • Ligand: MAGNESIUM ION
  • Ligand: MENADIONE
  • Ligand: FE3-S4 CLUSTER
  • Ligand: water

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Supramolecule #1: Complex of the type 2 [NiFe]-hydrogenase Huc from Mycobacterium s...

SupramoleculeName: Complex of the type 2 [NiFe]-hydrogenase Huc from Mycobacterium smegmatis
type: complex / ID: 1 / Chimera: Yes / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria) / Strain: MC2 155
Molecular weightTheoretical: 833 KDa

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Macromolecule #1: Hydrogenase-2, large subunit

MacromoleculeName: Hydrogenase-2, large subunit / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO / EC number: hydrogenase (acceptor)
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: ATCC 700084 / mc(2)155
Molecular weightTheoretical: 57.217078 KDa
SequenceString: LDLFVSPLGR VEGDLDVRVT INDGVVTSAW TEAAMFRGFE IILRGKDPQA GLIVCPRICG ICGGSHLYKS AYALDTAWRT HMPPNATLI RNICQACETL QSIPRYFYAL FAIDLTNKNY AKSKLYDEAV RRFAPYVGTS YQPGVVLSAK PVEVYAIFGG Q WP(DHI)SSFMV ...String:
LDLFVSPLGR VEGDLDVRVT INDGVVTSAW TEAAMFRGFE IILRGKDPQA GLIVCPRICG ICGGSHLYKS AYALDTAWRT HMPPNATLI RNICQACETL QSIPRYFYAL FAIDLTNKNY AKSKLYDEAV RRFAPYVGTS YQPGVVLSAK PVEVYAIFGG Q WP(DHI)SSFMV PGGVMSAPTL SDVTRAIAIL EHWNDNWLEK QWLGCSVDRW LENKTWNDVL AWVDENESQY NSDCGFFI R YCLDVGLDKY GQGVGNYLAT GTYFEPSLYE NPTIEGRNAA LIGRSGVFAD GRYFEFDQAN VTEDVTHSFY EGNRPLHPF EGETIPVNPE DGRRQGKYSW AKSPRYAVPG LGNVPLETGP LARRMAASAP DAETHQDDDP LFADIYNAIG PSVMVRQLAR MHEGPKYYK WVRQWLDDLE LKESFYTKPV EYAEGKGFGS TEAARGALSD WIVIEDSKIK NYQVVTPTAW NIGPRDASEV L GPIEQALV GSPIVDAEDP VELGHVARSF DSCLVCTVH

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Macromolecule #2: Hydrogenase-2, small subunit

MacromoleculeName: Hydrogenase-2, small subunit / type: protein_or_peptide / ID: 2 / Number of copies: 2 / Enantiomer: LEVO / EC number: hydrogenase (acceptor)
Source (natural)Organism: Mycolicibacterium smegmatis MC2 155 (bacteria) / Strain: ATCC 700084 / mc(2)155
Molecular weightTheoretical: 35.071988 KDa
Recombinant expressionOrganism: Mycolicibacterium smegmatis MC2 155 (bacteria)
SequenceString: ASVLWFQGGA CSGNTMSFLN ADEPNVVDLI VDFGLDLLWH PSLGLELGNN AQKVFWDCAK GERPLDIFVF EGTVIEAPNG TGQMDMFAG RPMKDWVTDL AGAAQIVVAI GDCACFGGIP AMEPNPSGST GLQFHKREKG GFLGPDFRSK MGLPVINVPG C PAHPDWIT ...String:
ASVLWFQGGA CSGNTMSFLN ADEPNVVDLI VDFGLDLLWH PSLGLELGNN AQKVFWDCAK GERPLDIFVF EGTVIEAPNG TGQMDMFAG RPMKDWVTDL AGAAQIVVAI GDCACFGGIP AMEPNPSGST GLQFHKREKG GFLGPDFRSK MGLPVINVPG C PAHPDWIT QILVALATGR AGDITLDDLH RPETFFKTFT QTGCTRVQFF EYKQSTLSFG EGTRTGCLFY EFGCRGPMTH SP CNRILWN RQSSKTRAGM PCLGCTEPEF PHFDLAPGTV FKTQKVSGMI PKEVPEGTDH LTYMGLAAAA RIAAPQWSKE DMF VV

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Macromolecule #3: NICKEL (III) ION

MacromoleculeName: NICKEL (III) ION / type: ligand / ID: 3 / Number of copies: 2 / Formula: 3NI
Molecular weightTheoretical: 58.693 Da
Chemical component information

ChemComp-3NI:
NICKEL (III) ION / Nickel

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Macromolecule #4: CARBONMONOXIDE-(DICYANO) IRON

MacromoleculeName: CARBONMONOXIDE-(DICYANO) IRON / type: ligand / ID: 4 / Number of copies: 2 / Formula: FCO
Molecular weightTheoretical: 135.89 Da
Chemical component information

ChemComp-FCO:
CARBONMONOXIDE-(DICYANO) IRON

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Macromolecule #5: HYDROXIDE ION

MacromoleculeName: HYDROXIDE ION / type: ligand / ID: 5 / Number of copies: 2 / Formula: OH
Molecular weightTheoretical: 17.007 Da
Chemical component information

ChemComp-OH:
HYDROXIDE ION / Hydroxide

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Macromolecule #6: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 6 / Number of copies: 2 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #7: MENADIONE

MacromoleculeName: MENADIONE / type: ligand / ID: 7 / Number of copies: 2 / Formula: VK3
Molecular weightTheoretical: 172.18 Da
Chemical component information

ChemComp-VK3:
MENADIONE / Menadione

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Macromolecule #8: FE3-S4 CLUSTER

MacromoleculeName: FE3-S4 CLUSTER / type: ligand / ID: 8 / Number of copies: 6 / Formula: F3S
Molecular weightTheoretical: 295.795 Da
Chemical component information

ChemComp-F3S:
FE3-S4 CLUSTER / Iron–sulfur cluster

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Macromolecule #9: water

MacromoleculeName: water / type: ligand / ID: 9 / Number of copies: 603 / 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

Concentration5.0 mg/mL
BufferpH: 7.9
Component:
ConcentrationNameFormula
50.0 mMTris
200.0 mMSodium chlorideNaClSodium chloride

Details: pH 7.9
GridModel: Quantifoil / Material: GOLD / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.5 µm / Nominal magnification: 105000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: HELIUM
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 2 / Number real images: 3133 / Average exposure time: 6.0 sec. / Average electron dose: 66.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 9348375
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.2)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC (ver. 3.3.1)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 3.3.1)
Final reconstructionApplied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 1.67 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 422614
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE / Target criteria: Correlation coefficient
Output model

PDB-7uur:
The 1.67 Angstrom CryoEM structure of the [NiFe]-hydrogenase Huc from Mycobacterium smegmatis - catalytic dimer (Huc2S2L)

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