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- PDB-6qq5: Cryo-EM structure of dimeric quinol dependent nitric oxide reduct... -

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Basic information

Database: PDB / ID: 6qq5
TitleCryo-EM structure of dimeric quinol dependent nitric oxide reductase (qNOR) 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 / integral component of membrane
Cytochrome c oxidase subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome c oxidase-like, subunit I domain / Cytochrome C Oxidase; Chain A / Up-down Bundle / Mainly Alpha
Nitric oxide reductase subunit B
Biological speciesAlcaligenes xylosoxydans xylosoxydans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.9 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.
Validation Report
SummaryFull reportAbout validation report
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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Deposited unit
A: Nitric oxide reductase subunit B
B: Nitric oxide reductase subunit B
hetero molecules

Theoretical massNumber of molelcules
Total (without water)168,90210

TypeNameSymmetry operationNumber
identity operation1_555x,y,z1
Buried area10060 Å2
ΔGint-202 kcal/mol
Surface area52190 Å2


#1: Protein Nitric oxide reductase subunit B

Mass: 83121.984 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alcaligenes xylosoxydans xylosoxydans (bacteria)
Gene: norB_1, ERS451415_02175 / Plasmid: pET-26b (+) / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41
References: UniProt: A0A0D6H8R3, nitric oxide reductase (cytochrome c)
#2: Chemical

Mass: 616.487 Da / Num. of mol.: 4 / Source method: obtained synthetically / 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 / Calcium

Mass: 40.078 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: Ca

Experimental details


EM experimentAggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction

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
SpecimenConc.: 3 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

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: 75000 X / Nominal defocus max: -3500 nm / Nominal defocus min: -1500 nm / Cs: 2.7 mm
Image recordingElectron dose: 65 e/Å2 / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of real images: 3213
Image scansMovie frames/image: 40


EM software
1RELION3particle selection
2EPUimage acquisition
4CTFFIND4.1CTF correction
7Coot0.8.9model fitting
12RELION33D reconstruction
13PHENIX1.14model refinement
Particle selectionNum. of particles selected: 707246
SymmetryPoint symmetry: C2 (2 fold cyclic)
3D reconstructionResolution: 3.9 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 56134 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 3AYF
Pdb chain-ID: A

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