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- PDB-6qq6: Cryo-EM structure of dimeric quinol dependent nitric oxide reduct... -

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Basic information

Entry
Database: PDB / ID: 6qq6
TitleCryo-EM structure of dimeric quinol dependent nitric oxide reductase (qNOR) Val495Ala mutant from Alcaligenes xylosoxidans
ComponentsNitric oxide reductase subunit B
KeywordsOXIDOREDUCTASE / Proton Transfer / Membrane Protein / Homodimer
Function / homology
Function and homology information


nitric oxide reductase (cytochrome c) / nitric oxide reductase activity / cytochrome-c oxidase activity / aerobic respiration / heme binding / membrane
Similarity search - Function
Cytochrome C Oxidase; Chain A / Cytochrome c oxidase-like, subunit I domain / Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I / Up-down Bundle / Mainly Alpha
Similarity search - Domain/homology
: / PROTOPORPHYRIN IX CONTAINING FE / Chem-LOP / Nitric oxide reductase subunit B
Similarity search - Component
Biological speciesAlcaligenes xylosoxydans xylosoxydans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.3 Å
AuthorsGopalasingam, C.C. / Johnson, R.M. / Chiduza, G.N. / Tosha, T. / Yamamoto, M. / Shiro, Y. / Antonyuk, S.V. / Muench, S.P. / Hasnain, S.S.
Funding support United Kingdom, 4items
OrganizationGrant numberCountry
Biotechnology and Biological Sciences Research CouncilBB/L006960/1 United Kingdom
Biotechnology and Biological Sciences Research CouncilBB/N013972/1 United Kingdom
Wellcome Trust109158/B/15/Z United Kingdom
Wellcome Trust108466/Z/15/Z United Kingdom
CitationJournal: Sci Adv / Year: 2019
Title: Dimeric structures of quinol-dependent nitric oxide reductases (qNORs) revealed by cryo-electron microscopy.
Authors: Chai C Gopalasingam / Rachel M Johnson / George N Chiduza / Takehiko Tosha / Masaki Yamamoto / Yoshitsugu Shiro / Svetlana V Antonyuk / Stephen P Muench / S Samar Hasnain /
Abstract: Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone ...Quinol-dependent nitric oxide reductases (qNORs) are membrane-integrated, iron-containing enzymes of the denitrification pathway, which catalyze the reduction of nitric oxide (NO) to the major ozone destroying gas nitrous oxide (NO). Cryo-electron microscopy structures of active qNOR from and an activity-enhancing mutant have been determined to be at local resolutions of 3.7 and 3.2 Å, respectively. They unexpectedly reveal a dimeric conformation (also confirmed for qNOR from ) and define the active-site configuration, with a clear water channel from the cytoplasm. Structure-based mutagenesis has identified key residues involved in proton transport and substrate delivery to the active site of qNORs. The proton supply direction differs from cytochrome c-dependent NOR (cNOR), where water molecules from the cytoplasm serve as a proton source similar to those from cytochrome c oxidase.
History
DepositionFeb 17, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Sep 11, 2019Provider: repository / Type: Initial release
Revision 1.1Sep 18, 2019Group: Data collection / Database references / Category: citation / citation_author
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID
Revision 1.2Dec 18, 2019Group: Other / Category: atom_sites
Item: _atom_sites.fract_transf_matrix[1][1] / _atom_sites.fract_transf_matrix[2][2] / _atom_sites.fract_transf_matrix[3][3]

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Structure visualization

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  • EMDB-4619
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Assembly

Deposited unit
A: Nitric oxide reductase subunit B
B: Nitric oxide reductase subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)171,85315
Polymers166,1882
Non-polymers5,66513
Water32418
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area14770 Å2
ΔGint-199 kcal/mol
Surface area47820 Å2
MethodPISA

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Components

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Protein / Sugars , 2 types, 4 molecules AB

#1: Protein Nitric oxide reductase subunit B


Mass: 83093.938 Da / Num. of mol.: 2 / Mutation: V495A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alcaligenes xylosoxydans xylosoxydans (bacteria)
Gene: norB_1, ERS451415_02175 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: A0A0D6H8R3, nitric oxide reductase (cytochrome c)
#6: Sugar ChemComp-LMT / DODECYL-BETA-D-MALTOSIDE


Type: D-saccharide / Mass: 510.615 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C24H46O11 / Comment: detergent*YM

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Non-polymers , 5 types, 29 molecules

#2: Chemical
ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME / Heme B


Mass: 616.487 Da / Num. of mol.: 4
Source method: isolated from a genetically manipulated source
Formula: C34H32FeN4O4
#3: Chemical ChemComp-FE / FE (III) ION / Iron


Mass: 55.845 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Fe
#4: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca
#5: Chemical ChemComp-LOP / (1R)-2-{[(R)-(2-AMINOETHOXY)(HYDROXY)PHOSPHORYL]OXY}-1-[(DODECANOYLOXY)METHYL]ETHYL (9Z)-OCTADEC-9-ENOATE / LAURYL OLEYL PHOSPHATIDYL ETHANOLAMINE


Mass: 661.890 Da / Num. of mol.: 3 / Source method: obtained synthetically / Formula: C35H68NO8P / Comment: phospholipid*YM
#7: Water ChemComp-HOH / water / Water


Mass: 18.015 Da / Num. of mol.: 18 / Source method: isolated from a natural source / Formula: H2O

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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: Quinol Dependent Nitric Oxide Reductase / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.17 MDa / Experimental value: NO
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41 (DE3)
Buffer solutionpH: 7.5
SpecimenConc.: 5 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 277 K
Details: Blot for 6 seconds with blot force of 6 prior to plunge freezing

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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 magnification: 47710 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1200 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 49 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 1803
Image scansMovie frames/image: 40

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Processing

EM software
IDNameVersionCategory
1RELION3particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7Coot0.8.9model fitting
12RELION33D reconstruction
13PHENIX1.14model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 614931
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.3 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 227359 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6QQ5

6qq5

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