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- PDB-8qta: Cryo-EM structure of Streptococcus pneumoniae NADPH oxidase F397A... -

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Basic information

Entry
Database: PDB / ID: 8qta
TitleCryo-EM structure of Streptococcus pneumoniae NADPH oxidase F397A mutant in complex with NADPH
ComponentsFAD-binding FR-type domain-containing protein
KeywordsMEMBRANE PROTEIN / NADPH oxidase / ROS producing / flavoprotein / heme protein
Function / homology
Function and homology information


2 iron, 2 sulfur cluster binding / flavin adenine dinucleotide binding / oxidoreductase activity / membrane
Similarity search - Function
: / FAD-binding 8 / FAD-binding domain / Oxidoreductase FAD/NAD(P)-binding / Oxidoreductase NAD-binding domain / FAD-binding domain, ferredoxin reductase-type / Ferredoxin-NADP reductase (FNR), nucleotide-binding domain / Ferredoxin reductase-type FAD binding domain profile. / Riboflavin synthase-like beta-barrel
Similarity search - Domain/homology
FLAVIN-ADENINE DINUCLEOTIDE / PROTOPORPHYRIN IX CONTAINING FE / Chem-NDP / FAD-binding FR-type domain-containing protein
Similarity search - Component
Biological speciesStreptococcus pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsDubach, V.R.A. / San Segundo-Acosta, P. / Murphy, B.J.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: Nat Struct Mol Biol / Year: 2024
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
DepositionOct 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release
Revision 1.1Jul 31, 2024Group: Data collection / Database references / Category: citation / citation_author / em_admin
Item: _citation.country / _citation.journal_abbrev ..._citation.country / _citation.journal_abbrev / _citation.journal_id_CSD / _citation.journal_id_ISSN / _citation.pdbx_database_id_PubMed / _citation.title / _citation_author.identifier_ORCID / _citation_author.name / _em_admin.last_update

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

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Assembly

Deposited unit
A: FAD-binding FR-type domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)48,7485
Polymers45,9841
Non-polymers2,7644
Water48627
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

#1: Protein FAD-binding FR-type domain-containing protein


Mass: 45984.457 Da / Num. of mol.: 1 / Mutation: F397A
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae (bacteria) / Gene: spr0531 / Production host: Escherichia coli BL21(DE3) (bacteria) / References: UniProt: Q8CZ28
#2: Chemical ChemComp-NDP / NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE


Mass: 745.421 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H30N7O17P3 / Feature type: SUBJECT OF INVESTIGATION
#3: Chemical ChemComp-FAD / FLAVIN-ADENINE DINUCLEOTIDE


Mass: 785.550 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C27H33N9O15P2 / Feature type: SUBJECT OF INVESTIGATION / Comment: FAD*YM
#4: Chemical ChemComp-HEM / PROTOPORPHYRIN IX CONTAINING FE / HEME


Mass: 616.487 Da / Num. of mol.: 2 / Source method: obtained synthetically / Formula: C34H32FeN4O4 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 27 / Source method: isolated from a natural source / Formula: H2O
Has ligand of interestY

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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: Protein-substrate complex of NADPH oxidase with NADPH / Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightValue: 0.046 MDa / Experimental value: NO
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria)
Buffer solutionpH: 7.5
Buffer component
IDConc.NameFormulaBuffer-ID
1250 mMsodium chlorideNaCl1
250 mMTris-HClTris-HCl1
30.002 %Lauryl Maltose Neopentyl GlycolLMNG1
41 mMFlavin-adenine-dinucleotideFAD1
55 mMNicotinamide adenine dinucleotide phosphateNADPH1
SpecimenConc.: 6 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 400 divisions/in. / Grid type: UltrAuFoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK III / Cryogen name: ETHANE / Humidity: 100 % / Chamber temperature: 4 K

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: TFS KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: 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 holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingAverage exposure time: 4 sec. / Electron dose: 70 e/Å2 / Film or detector model: FEI FALCON IV (4k x 4k) / Num. of grids imaged: 2 / Num. of real images: 45181
Details: Movies were collected in EER format. Hole selection was done with help from plasmon imaging (WJH Hagen, 2022). 2 grids were imaged over the course of 4 independent microscope sessions.
EM imaging opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameVersionCategory
1Topazparticle selection
2crYOLOparticle selection
3EPUimage acquisition
5CTFFIND4.1CTF correction
8UCSF ChimeraXmodel fitting
10PHENIXmodel refinement
11Cootmodel refinement
12cryoSPARCinitial Euler assignment
13cryoSPARCfinal Euler assignment
14cryoSPARCclassification
15cryoSPARC3D reconstruction
Image processingDetails: Movies were collected in EER format and not gain corrected
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 13817533
Details: These are the combined picked particles from a topaz model and a crYOLO model and contain duplicate particles
3D reconstructionResolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 546234 / Algorithm: FOURIER SPACE
Details: Final refinement was local refinement in cryoSPARC 4.1 with a mask excluding the detergent micelle
Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL / Target criteria: Cross-correlation coefficient
Details: Initial model was fit using ChimeraX and refined with Coot and PHENIX
Atomic model buildingDetails: Initial model was the previousy resolved WT protein with NADPH bound
Source name: Other / Type: experimental model
Refine LS restraints
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0023563
ELECTRON MICROSCOPYf_angle_d0.4834872
ELECTRON MICROSCOPYf_dihedral_angle_d10.701466
ELECTRON MICROSCOPYf_chiral_restr0.037509
ELECTRON MICROSCOPYf_plane_restr0.003580

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