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- PDB-6t6v: Glu-494-Ala inactive monomer of a quinol dependent Nitric Oxide R... -
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Open data
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Basic information
Entry | Database: PDB / ID: 6t6v | |||||||||||||||
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Title | Glu-494-Ala inactive monomer of a quinol dependent Nitric Oxide Reductase (qNOR) from Alcaligenes xylosoxidans | |||||||||||||||
![]() | Nitric oxide reductase subunit B | |||||||||||||||
![]() | OXIDOREDUCTASE / Monomer / Proton Transfer / Nitric Oxide | |||||||||||||||
Function / homology | ![]() nitric oxide reductase (cytochrome c) / nitric oxide reductase activity / cytochrome-c oxidase activity / aerobic respiration / heme binding / membrane Similarity search - Function | |||||||||||||||
Biological species | ![]() | |||||||||||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 4.5 Å | |||||||||||||||
![]() | Gopalasingam, C.C. / Johnson, R.M. / Antonyuk, S.V. / Muench, S.P. / Hasnain, S.S. | |||||||||||||||
Funding support | ![]()
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![]() | ![]() 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 |
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Structure visualization
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Structure viewer | Molecule: ![]() ![]() |
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PDBx/mmCIF format | ![]() | 142.6 KB | Display | ![]() |
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PDB format | ![]() | 110 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Summary document | ![]() | 1012.6 KB | Display | ![]() |
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Full document | ![]() | 1 MB | Display | |
Data in XML | ![]() | 30.6 KB | Display | |
Data in CIF | ![]() | 44.6 KB | Display | |
Arichive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 10387MC ![]() 0822C ![]() 6l1xC ![]() 6l3hC M: map data used to model this data C: citing same article ( |
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Similar structure data |
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 84765.961 Da / Num. of mol.: 1 / Mutation: E494A Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() Gene: norB_1, ERS451415_02175 / Plasmid: pET26b(+) / Production host: ![]() ![]() References: UniProt: A0A0D6H8R3, nitric oxide reductase (cytochrome c) | ||||
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#2: Chemical | #3: Chemical | ChemComp-CA / | Has ligand of interest | N | |
-Experimental details
-Experiment
Experiment | Method: ELECTRON MICROSCOPY |
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EM experiment | Aggregation state: PARTICLE / 3D reconstruction method: single particle reconstruction |
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Sample preparation
Component | Name: Glu-494-Ala mutant of quinol dependent Nitric Oxide Reductase Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||
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Molecular weight | Value: 0.095 MDa / Experimental value: YES | ||||||||||||||||||||
Source (natural) | Organism: ![]() | ||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 3 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||
Specimen support | Grid material: COPPER / Grid type: Quantifoil R1.2/1.3 | ||||||||||||||||||||
Vitrification | Instrument: 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 |
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Microscopy | Model: FEI TITAN KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 48000 X / Nominal defocus max: -3000 nm / Nominal defocus min: -1500 nm / Cs: 2.7 mm |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Electron 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 scans | Movie frames/image: 40 / Used frames/image: 1-40 |
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Processing
EM software |
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CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||||||
Particle selection | Num. of particles selected: 684000 | ||||||||||||||||||||||||||||
Symmetry | Point symmetry: C1 (asymmetric) | ||||||||||||||||||||||||||||
3D reconstruction | Resolution: 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 building | Protocol: FLEXIBLE FIT / Space: REAL | ||||||||||||||||||||||||||||
Atomic model building | PDB-ID: 6QQ6 Pdb chain-ID: A / Accession code: 6QQ6 / Source name: PDB / Type: experimental model |