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- PDB-6t6v: Glu-494-Ala inactive monomer of a quinol dependent Nitric Oxide R... -

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Basic information

Entry
Database: PDB / ID: 6t6v
TitleGlu-494-Ala inactive monomer of a quinol dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans
ComponentsNitric oxide reductase subunit B
KeywordsOXIDOREDUCTASE / Monomer / Proton Transfer / Nitric Oxide
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 subunit I / Cytochrome c oxidase-like, subunit I superfamily / Cytochrome C and Quinol oxidase polypeptide I
Similarity search - Domain/homology
PROTOPORPHYRIN IX CONTAINING FE / Nitric oxide reductase subunit B
Similarity search - Component
Biological speciesAlcaligenes xylosoxydans xylosoxydans (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å
AuthorsGopalasingam, C.C. / Johnson, R.M. / 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: IUCrJ / Year: 2020
Title: The active form of quinol-dependent nitric oxide reductase from is a dimer.
Authors: M Arif M Jamali / Chai C Gopalasingam / Rachel M Johnson / Takehiko Tosha / Kazumasa Muramoto / Stephen P Muench / Svetlana V Antonyuk / Yoshitsugu Shiro / Samar S Hasnain /
Abstract: is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of ... is carried by nearly a billion humans, causing developmental impairment and over 100 000 deaths a year. A quinol-dependent nitric oxide reductase (qNOR) plays a critical role in the survival of the bacterium in the human host. X-ray crystallographic analyses of qNOR, including that from (qNOR) reported here at 3.15 Å resolution, show monomeric assemblies, despite the more active dimeric sample being used for crystallization. Cryo-electron microscopic analysis of the same chromatographic fraction of qNOR, however, revealed a dimeric assembly at 3.06 Å resolution. It is shown that zinc (which is used in crystallization) binding near the dimer-stabilizing TMII region contributes to the disruption of the dimer. A similar destabilization is observed in the monomeric (∼85 kDa) cryo-EM structure of a mutant (Glu494Ala) qNOR from the opportunistic pathogen () , which primarily migrates as a monomer. The monomer-dimer transition of qNORs seen in the cryo-EM and crystallographic structures has wider implications for structural studies of multimeric membrane proteins. X-ray crystallographic and cryo-EM structural analyses have been performed on the same chromatographic fraction of qNOR to high resolution. This represents one of the first examples in which the two approaches have been used to reveal a monomeric assembly and a dimeric assembly in vitrified cryo-EM grids. A number of factors have been identified that may trigger the destabilization of helices that are necessary to preserve the integrity of the dimer. These include zinc binding near the entry of the putative proton-transfer channel and the preservation of the conformational integrity of the active site. The mutation near the active site results in disruption of the active site, causing an additional destabilization of helices (TMIX and TMX) that flank the proton-transfer channel helices, creating an inert monomeric enzyme.
History
DepositionOct 19, 2019Deposition site: PDBE / Processing site: PDBE
Revision 1.0Apr 1, 2020Provider: repository / Type: Initial release
Revision 1.1May 27, 2020Group: 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 / _citation_author.name
Revision 1.2May 22, 2024Group: Data collection / Database references / Refinement description
Category: chem_comp_atom / chem_comp_bond ...chem_comp_atom / chem_comp_bond / database_2 / em_3d_fitting_list / pdbx_initial_refinement_model
Item: _database_2.pdbx_DOI / _database_2.pdbx_database_accession ..._database_2.pdbx_DOI / _database_2.pdbx_database_accession / _em_3d_fitting_list.accession_code / _em_3d_fitting_list.initial_refinement_model_id / _em_3d_fitting_list.source_name / _em_3d_fitting_list.type

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Structure visualization

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Assembly

Deposited unit
A: Nitric oxide reductase subunit B
hetero molecules


Theoretical massNumber of molelcules
Total (without water)86,0394
Polymers84,7661
Non-polymers1,2733
Water00
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: microscopy
TypeNameSymmetry operationNumber
identity operation1_5551
Buried area2710 Å2
ΔGint-60 kcal/mol
Surface area31310 Å2
MethodPISA

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Components

#1: Protein Nitric oxide reductase subunit B


Mass: 84765.961 Da / Num. of mol.: 1 / Mutation: E494A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Alcaligenes xylosoxydans xylosoxydans (bacteria)
Gene: norB_1, ERS451415_02175 / Plasmid: pET26b(+) / Production host: Escherichia coli BL21(DE3) (bacteria) / Strain (production host): BL21(DE3) / Variant (production host): C41
References: UniProt: A0A0D6H8R3, nitric oxide reductase (cytochrome c)
#2: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4
#3: Chemical ChemComp-CA / CALCIUM ION


Mass: 40.078 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: Ca
Has ligand of interestN

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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: Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.095 MDa / Experimental value: YES
Source (natural)Organism: Achromobacter xylosoxidans (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
150 mMTris-HCL1
2150 mMNaCl1
30.05 % (v/v)n-decyl-D-thiomaltosideDTM1
SpecimenConc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: COPPER / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 98 % / Chamber temperature: 277 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: 48000 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1500 nm / Cs: 2.7 mm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 50 e/Å2 / Detector mode: COUNTING / Film or detector model: GATAN K2 SUMMIT (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 2239
Image scansMovie frames/image: 40 / Used frames/image: 1-40

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Processing

EM software
IDNameVersionCategory
2EPU2.3image acquisition
4CTFFIND4.1CTF correction
7UCSF Chimera1.13.1model fitting
9PHENIX1.15model refinement
12RELION3classification
13RELION33D reconstruction
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 684000
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 4.5 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 144424 / Algorithm: BACK PROJECTION / Num. of class averages: 2 / Symmetry type: POINT
Atomic model buildingProtocol: FLEXIBLE FIT / Space: REAL
Atomic model buildingPDB-ID: 6QQ6

6qq6


Pdb chain-ID: A / Accession code: 6QQ6 / Source name: PDB / Type: experimental model

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