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- PDB-7p5h: TmHydABC- D2 map -

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Basic information

Entry
Database: PDB / ID: 7p5h
TitleTmHydABC- D2 map
Components(Fe-hydrogenase, subunit ...) x 3
KeywordsOXIDOREDUCTASE / Hydrogenase / bifurcation / confurcaction / cryoEM / electron transfer / complex
Function / homology
Function and homology information


hydrogenase (NAD+, ferredoxin) / NADH dehydrogenase (ubiquinone) activity / ATP synthesis coupled electron transport / 2 iron, 2 sulfur cluster binding / FMN binding / 4 iron, 4 sulfur cluster binding / oxidoreductase activity / membrane / metal ion binding / cytoplasm
Similarity search - Function
NADP-reducing hydrogenase subunit HndA / Iron hydrogenase, small subunit / Iron hydrogenase small subunit / Iron hydrogenase small subunit / Iron hydrogenase, large subunit, C-terminal / Iron hydrogenase / Iron only hydrogenase large subunit, C-terminal domain / 4Fe-4S dicluster domain / Respiratory-chain NADH dehydrogenase 75 Kd subunit signature 1. / 2Fe-2S iron-sulfur cluster binding domain ...NADP-reducing hydrogenase subunit HndA / Iron hydrogenase, small subunit / Iron hydrogenase small subunit / Iron hydrogenase small subunit / Iron hydrogenase, large subunit, C-terminal / Iron hydrogenase / Iron only hydrogenase large subunit, C-terminal domain / 4Fe-4S dicluster domain / Respiratory-chain NADH dehydrogenase 75 Kd subunit signature 1. / 2Fe-2S iron-sulfur cluster binding domain / NADH:ubiquinone oxidoreductase, 75kDa subunit, conserved site / NADH-ubiquinone oxidoreductase-G iron-sulfur binding region / NADH-ubiquinone oxidoreductase-G iron-sulfur binding region / Respiratory-chain NADH dehydrogenase 24 Kd subunit signature. / NuoE domain / NADH-quinone oxidoreductase subunit E-like / Thioredoxin-like [2Fe-2S] ferredoxin / NADH-quinone oxidoreductase subunit E, N-terminal / NADH:ubiquinone oxidoreductase, 51kDa subunit, conserved site / Respiratory-chain NADH dehydrogenase 51 Kd subunit signature 2. / NADH:ubiquinone oxidoreductase, subunit G, iron-sulphur binding / His(Cys)3-ligated-type [4Fe-4S] domain profile. / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, iron-sulphur binding domain / NADH-ubiquinone oxidoreductase 51kDa subunit, iron-sulphur binding domain superfamily / NADH-ubiquinone oxidoreductase 51kDa subunit, FMN-binding domain superfamily / Respiratory-chain NADH dehydrogenase 51 Kd subunit / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / NADH-ubiquinone oxidoreductase-F iron-sulfur binding region / 4Fe-4S dicluster domain / 2Fe-2S ferredoxin-type iron-sulfur binding domain profile. / 2Fe-2S ferredoxin-type iron-sulfur binding domain / 2Fe-2S ferredoxin-like superfamily / 4Fe-4S ferredoxin, iron-sulphur binding, conserved site / 4Fe-4S ferredoxin-type iron-sulfur binding region signature. / 4Fe-4S ferredoxin-type iron-sulfur binding domain profile. / 4Fe-4S ferredoxin-type, iron-sulphur binding domain / Thioredoxin-like superfamily
Similarity search - Domain/homology
FE2/S2 (INORGANIC) CLUSTER / FLAVIN MONONUCLEOTIDE / IRON/SULFUR CLUSTER / Bifurcating [FeFe] hydrogenase beta subunit / Bifurcating [FeFe] hydrogenase alpha subunit / Bifurcating [FeFe] hydrogenase gamma subunit
Similarity search - Component
Biological speciesThermotoga maritima (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.3 Å
AuthorsFurlan, C. / Chongdar, N. / Gupta, P. / Lubitz, W. / Ogata, H. / Blaza, J.N. / Birrell, J.A.
Funding support United Kingdom, Germany, Japan, 3items
OrganizationGrant numberCountry
UK Research and Innovation (UKRI)MR/T040742/1 United Kingdom
German Research Foundation (DFG)BI 2198/1-1 Germany
Japan Society for the Promotion of Science (JSPS)16K21748 Japan
CitationJournal: Elife / Year: 2022
Title: Structural insight on the mechanism of an electron-bifurcating [FeFe] hydrogenase.
Authors: Chris Furlan / Nipa Chongdar / Pooja Gupta / Wolfgang Lubitz / Hideaki Ogata / James N Blaza / James A Birrell /
Abstract: Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential ...Electron bifurcation is a fundamental energy conservation mechanism in nature in which two electrons from an intermediate-potential electron donor are split so that one is sent along a high-potential pathway to a high-potential acceptor and the other is sent along a low-potential pathway to a low-potential acceptor. This process allows endergonic reactions to be driven by exergonic ones and is an alternative, less recognized, mechanism of energy coupling to the well-known chemiosmotic principle. The electron-bifurcating [FeFe] hydrogenase from (HydABC) requires both NADH and ferredoxin to reduce protons generating hydrogen. The mechanism of electron bifurcation in HydABC remains enigmatic in spite of intense research efforts over the last few years. Structural information may provide the basis for a better understanding of spectroscopic and functional information. Here, we present a 2.3 Å electron cryo-microscopy structure of HydABC. The structure shows a heterododecamer composed of two independent 'halves' each made of two strongly interacting HydABC heterotrimers connected via a [4Fe-4S] cluster. A central electron transfer pathway connects the active sites for NADH oxidation and for proton reduction. We identified two conformations of a flexible iron-sulfur cluster domain: a 'closed bridge' and an 'open bridge' conformation, where a Zn site may act as a 'hinge' allowing domain movement. Based on these structural revelations, we propose a possible mechanism of electron bifurcation in HydABC where the flavin mononucleotide serves a dual role as both the electron bifurcation center and as the NAD reduction/NADH oxidation site.
History
DepositionJul 14, 2021Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 14, 2022Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: Fe-hydrogenase, subunit alpha
B: Fe-hydrogenase, subunit beta
C: Fe-hydrogenase, subunit gamma
D: Fe-hydrogenase, subunit alpha
E: Fe-hydrogenase, subunit beta
F: Fe-hydrogenase, subunit gamma
a: Fe-hydrogenase, subunit alpha
b: Fe-hydrogenase, subunit beta
c: Fe-hydrogenase, subunit gamma
d: Fe-hydrogenase, subunit alpha
e: Fe-hydrogenase, subunit beta
f: Fe-hydrogenase, subunit gamma
hetero molecules


Theoretical massNumber of molelcules
Total (without water)659,47852
Polymers648,24812
Non-polymers11,23040
Water11,530640
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area53370 Å2
ΔGint-842 kcal/mol
Surface area177810 Å2
MethodPISA

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Components

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Fe-hydrogenase, subunit ... , 3 types, 12 molecules ADadBEbeCFcf

#1: Protein
Fe-hydrogenase, subunit alpha


Mass: 72351.391 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) (bacteria)
Strain: ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8
Gene: TM_1426, HydA
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: G4FFG1, hydrogenase (NAD+, ferredoxin)
#2: Protein
Fe-hydrogenase, subunit beta


Mass: 68769.406 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) (bacteria)
Strain: ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8
Gene: TM_1425, HydB
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: G4FFG0, hydrogenase (NAD+, ferredoxin)
#3: Protein
Fe-hydrogenase, subunit gamma


Mass: 20941.172 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Details: M initiation codon AS is a linker WSHPQFEK strep tag SGGGGG is a linker ENLYFQ is tev sequence SA is a linker
Source: (gene. exp.) Thermotoga maritima (strain ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8) (bacteria)
Strain: ATCC 43589 / DSM 3109 / JCM 10099 / NBRC 100826 / MSB8
Gene: TM_1424, HydC
Production host: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
References: UniProt: Q9S5X7, hydrogenase (NAD+, ferredoxin)

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Non-polymers , 5 types, 680 molecules

#4: Chemical
ChemComp-SF4 / IRON/SULFUR CLUSTER


Mass: 351.640 Da / Num. of mol.: 20 / Source method: obtained synthetically / Formula: Fe4S4
#5: Chemical
ChemComp-FES / FE2/S2 (INORGANIC) CLUSTER


Mass: 175.820 Da / Num. of mol.: 12 / Source method: obtained synthetically / Formula: Fe2S2
#6: Chemical
ChemComp-FMN / FLAVIN MONONUCLEOTIDE / RIBOFLAVIN MONOPHOSPHATE


Mass: 456.344 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: C17H21N4O9P / Feature type: SUBJECT OF INVESTIGATION
#7: Chemical
ChemComp-ZN / ZINC ION


Mass: 65.409 Da / Num. of mol.: 4 / Source method: obtained synthetically / Formula: Zn / Feature type: SUBJECT OF INVESTIGATION
#8: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 640 / Source method: isolated from a natural source / Formula: H2O

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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: Tetrameric complex of HydABC protomers / Type: COMPLEX / Entity ID: #1-#3 / Source: RECOMBINANT
Molecular weightValue: 0.64 MDa / Experimental value: NO
Source (natural)Organism: Thermotoga maritima MSB8 (bacteria)
Source (recombinant)Organism: Escherichia coli 'BL21-Gold(DE3)pLysS AG' (bacteria)
Buffer solutionpH: 8
Buffer component
IDConc.NameBuffer-ID
110 mMTris hydrochloride1
2150 mMNaCl1
SpecimenConc.: 1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES / Details: Purified by gel filtration
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277.15 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 FIELD / Nominal magnification: 165000 X / Calibrated magnification: 60700 X / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm / Cs: 2.7 mm / C2 aperture diameter: 70 µm / Alignment procedure: COMA FREE
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 6 sec. / Electron dose: 57 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 4790

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Processing

SoftwareName: PHENIX / Version: 1.19.2_4158: / Classification: refinement
EM software
IDNameVersionCategory
7UCSF Chimeramodel fitting
9RELION3.1initial Euler assignment
10RELION3.1final Euler assignment
11RELION3.1classification
12RELION3.13D reconstruction
13PHENIXmodel refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 885306
SymmetryPoint symmetry: D2 (2x2 fold dihedral)
3D reconstructionResolution: 2.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 278793 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.00440612
ELECTRON MICROSCOPYf_angle_d0.61754892
ELECTRON MICROSCOPYf_dihedral_angle_d6.4775592
ELECTRON MICROSCOPYf_chiral_restr0.0436164
ELECTRON MICROSCOPYf_plane_restr0.0056992

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