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- PDB-8qt9: Cryo-EM structure of stably reduced Streptococcus pneumoniae NADP... -
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Open data
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Basic information
Entry | Database: PDB / ID: 8qt9 | ||||||
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Title | Cryo-EM structure of stably reduced Streptococcus pneumoniae NADPH oxidase in complex with NADH | ||||||
![]() | FAD-binding FR-type domain-containing protein | ||||||
![]() | MEMBRANE PROTEIN / NADPH oxidase / ROS producing / flavoprotein / heme protein | ||||||
Function / homology | ![]() 2 iron, 2 sulfur cluster binding / flavin adenine dinucleotide binding / oxidoreductase activity / membrane Similarity search - Function | ||||||
Biological species | ![]() ![]() | ||||||
Method | ELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.36 Å | ||||||
![]() | Dubach, V.R.A. / San Segundo-Acosta, P. / Murphy, B.J. | ||||||
Funding support | ![]()
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![]() | ![]() Title: Structural and mechanistic insights into Streptococcus pneumoniae NADPH oxidase. Authors: Victor R A Dubach / Pablo San Segundo-Acosta / Bonnie J Murphy / ![]() ![]() Abstract: Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant ...Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) have a major role in the physiology of eukaryotic cells by mediating reactive oxygen species production. Evolutionarily distant proteins with the NOX catalytic core have been found in bacteria, including Streptococcus pneumoniae NOX (SpNOX), which is proposed as a model for studying NOXs because of its high activity and stability in detergent micelles. We present here cryo-electron microscopy structures of substrate-free and nicotinamide adenine dinucleotide (NADH)-bound SpNOX and of NADPH-bound wild-type and F397A SpNOX under turnover conditions. These high-resolution structures provide insights into the electron-transfer pathway and reveal a hydride-transfer mechanism regulated by the displacement of F397. We conducted structure-guided mutagenesis and biochemical analyses that explain the absence of substrate specificity toward NADPH and suggest the mechanism behind constitutive activity. Our study presents the structural basis underlying SpNOX enzymatic activity and sheds light on its potential in vivo function. | ||||||
History |
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Structure visualization
Structure viewer | Molecule: ![]() ![]() |
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Downloads & links
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Download
PDBx/mmCIF format | ![]() | 101.6 KB | Display | ![]() |
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PDB format | ![]() | 73.8 KB | Display | ![]() |
PDBx/mmJSON format | ![]() | Tree view | ![]() | |
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-Validation report
Arichive directory | ![]() ![]() | HTTPS FTP |
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-Related structure data
Related structure data | ![]() 18646MC ![]() 8qt6C ![]() 8qt7C ![]() 8qtaC M: map data used to model this data C: citing same article ( |
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Similar structure data | Similarity search - Function & homology ![]() |
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Links
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Assembly
Deposited unit | ![]()
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Components
#1: Protein | Mass: 46060.551 Da / Num. of mol.: 1 Source method: isolated from a genetically manipulated source Source: (gene. exp.) ![]() ![]() ![]() ![]() | ||||||
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#2: Chemical | ChemComp-FAD / | ||||||
#3: Chemical | #4: Chemical | ChemComp-NAI / | #5: Water | ChemComp-HOH / | Has ligand of interest | Y | |
-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: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors and NADH bound Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT | ||||||||||||||||||||||||||||||
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Molecular weight | Units: MEGADALTONS / Experimental value: NO | ||||||||||||||||||||||||||||||
Source (natural) | Organism: ![]() ![]() | ||||||||||||||||||||||||||||||
Source (recombinant) | Organism: ![]() ![]() | ||||||||||||||||||||||||||||||
Buffer solution | pH: 7.5 | ||||||||||||||||||||||||||||||
Buffer component |
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Specimen | Conc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES | ||||||||||||||||||||||||||||||
Specimen support | Grid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R1.2/1.3 | ||||||||||||||||||||||||||||||
Vitrification | Instrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / 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: TFS KRIOS |
Electron gun | Electron source: ![]() |
Electron lens | Mode: BRIGHT FIELD / Nominal magnification: 215000 X / Nominal defocus max: 2100 nm / Nominal defocus min: 700 nm / Cs: 2.7 mm / C2 aperture diameter: 50 µm / Alignment procedure: COMA FREE |
Specimen holder | Cryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER |
Image recording | Average exposure time: 3.96 sec. / Electron dose: 70 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 1 / Num. of real images: 22259 / Details: Movies were collected in EER format. |
EM imaging optics | Energyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV |
Image scans | Width: 4096 / Height: 4096 |
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Processing
EM software |
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Image processing | Details: Movies were collected in EER format and not gain corrected | ||||||||||||||||||||||||
CTF correction | Type: PHASE FLIPPING AND AMPLITUDE CORRECTION | ||||||||||||||||||||||||
Particle selection | Num. of particles selected: 14339155 Details: These are the combined particles from two different Topaz models and a crYOLO model and contains duplicate particles. | ||||||||||||||||||||||||
3D reconstruction | Resolution: 2.36 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 697211 / Algorithm: FOURIER SPACE Details: Final refinement is local refinement in cryoSPARC 4.0 using a tight mask excluding the detergent micelle. Symmetry type: POINT | ||||||||||||||||||||||||
Atomic model building | B value: 26.54 / Protocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient Details: Model was fit using ChimeraX. Coot and PHENIX were used for model refinement. | ||||||||||||||||||||||||
Refine LS restraints |
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