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- PDB-7utd: The 2.19-angstrom CryoEM structure of the [NiFe]-hydrogenase Huc ... -

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Basic information

Entry
Database: PDB / ID: 7utd
TitleThe 2.19-angstrom CryoEM structure of the [NiFe]-hydrogenase Huc from Mycobacterium smegmatis - Complex minus stalk
Components
  • (Hydrogenase-2, ...) x 2
  • Type 2 [NiFe]-hydrogenase Huc membrane adapter subunit
KeywordsOXIDOREDUCTASE / [NiFe] Hydrogenase / Membrane associated / Complex / Quinone Transport / ELECTRON TRANSPORT / OXIDOREDUCTASE (EC 1.12.99.6)
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
Protein of unknown function DUF1641 / Protein of unknown function (DUF1641) / [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 ...Protein of unknown function DUF1641 / Protein of unknown function (DUF1641) / [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
NICKEL (III) ION / FE3-S4 CLUSTER / CARBONMONOXIDE-(DICYANO) IRON / MENAQUINONE-9 / DUF1641 domain-containing protein / Hydrogenase-2, large subunit / NADH ubiquinone oxidoreductase 20 kDa subunit
Similarity search - Component
Biological speciesMycolicibacterium smegmatis MC2 155 (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.19 Å
AuthorsGrinter, R. / Venugopal, H. / Kropp, A. / Greening, C.
Funding support Australia, 2items
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 26, 2022Deposition site: RCSB / Processing site: RCSB
Revision 1.0Jan 4, 2023Provider: repository / Type: Initial release
Revision 1.1Jan 25, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.2Mar 8, 2023Group: Database references / Category: citation / citation_author
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_ASTM / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_DOI / _citation.title / _citation.year
Revision 1.3Mar 22, 2023Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.4Mar 29, 2023Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first / _citation.page_last
Revision 1.5Apr 5, 2023Group: Database references / Category: pdbx_database_related

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Hydrogenase-2, large subunit
B: Hydrogenase-2, small subunit
C: Hydrogenase-2, large subunit
D: Hydrogenase-2, small subunit
E: Hydrogenase-2, large subunit
F: Hydrogenase-2, small subunit
G: Hydrogenase-2, large subunit
H: Hydrogenase-2, small subunit
I: Hydrogenase-2, large subunit
J: Hydrogenase-2, small subunit
K: Hydrogenase-2, large subunit
L: Hydrogenase-2, small subunit
M: Hydrogenase-2, large subunit
N: Hydrogenase-2, small subunit
O: Hydrogenase-2, large subunit
P: Hydrogenase-2, small subunit
Q: Type 2 [NiFe]-hydrogenase Huc membrane adapter subunit
R: Type 2 [NiFe]-hydrogenase Huc membrane adapter subunit
S: Type 2 [NiFe]-hydrogenase Huc membrane adapter subunit
T: Type 2 [NiFe]-hydrogenase Huc membrane adapter subunit
hetero molecules


Theoretical massNumber of molelcules
Total (without water)815,81276
Polymers800,68020
Non-polymers15,13256
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: native gel electrophoresis, electron microscopy, gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Hydrogenase-2, ... , 2 types, 16 molecules ACEGIKMOBDFHJLNP

#1: Protein
Hydrogenase-2, large subunit /


Mass: 57217.078 Da / Num. of mol.: 8 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155 / References: UniProt: A0QUM7, hydrogenase (acceptor)
#2: Protein
Hydrogenase-2, small subunit / / NADH ubiquinone oxidoreductase 20 kDa subunit


Mass: 39659.770 Da / Num. of mol.: 8 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: ATCC 700084 / mc(2)155 / References: UniProt: I7G634, hydrogenase (acceptor)

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Protein , 1 types, 4 molecules QRST

#3: Protein
Type 2 [NiFe]-hydrogenase Huc membrane adapter subunit


Mass: 6416.264 Da / Num. of mol.: 4 / Source method: isolated from a natural source
Source: (natural) Mycolicibacterium smegmatis MC2 155 (bacteria)
Strain: MC2 155 / References: UniProt: A0QUM5

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Non-polymers , 5 types, 56 molecules

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


Mass: 58.693 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Ni / Feature type: SUBJECT OF INVESTIGATION
#5: Chemical
ChemComp-FCO / CARBONMONOXIDE-(DICYANO) IRON


Mass: 135.890 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C3FeN2O / Feature type: SUBJECT OF INVESTIGATION
#6: Chemical
ChemComp-MG / MAGNESIUM ION


Mass: 24.305 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: Mg
#7: Chemical
ChemComp-MQ9 / MENAQUINONE-9 / Vitamin K2


Mass: 785.233 Da / Num. of mol.: 8 / Source method: obtained synthetically / Formula: C56H80O2 / Feature type: SUBJECT OF INVESTIGATION
#8: Chemical...
ChemComp-F3S / FE3-S4 CLUSTER / Iron–sulfur cluster


Mass: 295.795 Da / Num. of mol.: 24 / Source method: obtained synthetically / Formula: Fe3S4 / Feature type: SUBJECT OF INVESTIGATION

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Details

Has ligand of interestY

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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: Complex of the type 2 [NiFe]-hydrogenase Huc from Mycobacterium smegmatis
Type: COMPLEX / Entity ID: #1-#3 / Source: NATURAL
Molecular weightValue: 0.833 MDa / Experimental value: NO
Source (natural)Organism: Mycolicibacterium smegmatis (bacteria) / Strain: MC2 155
Buffer solutionpH: 7.9 / Details: pH 7.9
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris1
2200 mMSodium chlorideNaClSodium chloride1
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid type: Quantifoil
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 295 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 1500 nm / Nominal defocus min: 500 nm / Cs: 2.7 mm
Specimen holderCryogen: HELIUM
Image recordingAverage exposure time: 6 sec. / Electron dose: 66 e/Å2 / Film or detector model: GATAN K3 (6k x 4k) / Num. of real images: 3113

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Processing

EM software
IDNameVersionCategory
4CTFFIND4.1CTF correction
7Coot0.92model fitting
8UCSF ChimeraX1.3model fitting
10cryoSPARC3.3.1initial Euler assignment
11cryoSPARC3.3.1final Euler assignment
12cryoSPARC3.3.1classification
13cryoSPARC3.3.13D reconstruction
14PHENIX1.9.2model refinement
15Coot0.92model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 825900
SymmetryPoint symmetry: C4 (4 fold cyclic)
3D reconstructionResolution: 2.19 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 139367 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: BACKBONE TRACE / Space: REAL / Target criteria: Correlation coefficient

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