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- EMDB-18647: Cryo-EM structure of Streptococcus pneumoniae NADPH oxidase F397A... -

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Basic information

Entry
Database: EMDB / ID: EMD-18647
TitleCryo-EM structure of Streptococcus pneumoniae NADPH oxidase F397A mutant in complex with NADPH
Map dataMain map used for atomic model building and figures
Sample
  • Complex: Protein-substrate complex of NADPH oxidase with NADPH
    • Protein or peptide: FAD-binding FR-type domain-containing protein
  • Ligand: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: water
KeywordsNADPH oxidase / ROS producing / flavoprotein / heme protein / MEMBRANE 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
FAD-binding FR-type domain-containing protein
Similarity search - Component
Biological speciesStreptococcus pneumoniae (bacteria)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.64 Å
AuthorsDubach VRA / San Segundo-Acosta P / Murphy BJ
Funding support Germany, 1 items
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, 2023-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateJul 31, 2024-
Current statusJul 31, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18647.png

Downloads & links

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Map

FileDownload / File: emd_18647.map.gz / Format: CCP4 / Size: 38.4 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationMain map used for atomic model building and figures
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.11 Å/pix.
x 216 pix.
= 240.667 Å		1.11 Å/pix.
x 216 pix.
= 240.667 Å		1.11 Å/pix.
x 216 pix.
= 240.667 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.1142 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.596
Minimum - Maximum-3.6757724 - 5.956163
Average (Standard dev.)0.00041987997 (±0.09912834)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions216216216
Spacing216216216
CellA=B=C: 240.6672 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18647_msk_1.map
Projections & Slices
AxesZYX

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Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw unsharp map obtained after merging the half maps

Fileemd_18647_additional_1.map
AnnotationRaw unsharp map obtained after merging the half maps
Projections & Slices
AxesZYX

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Slices (1/2)
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Histogram

Histogram (log scale)

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Half map: Half map A used during refinement and FSC...

Fileemd_18647_half_map_1.map
AnnotationHalf map A used during refinement and FSC gold standard resolution estimation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B used during refinement and FSC...

Fileemd_18647_half_map_2.map
AnnotationHalf map B used during refinement and FSC gold standard resolution estimation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Protein-substrate complex of NADPH oxidase with NADPH

EntireName: Protein-substrate complex of NADPH oxidase with NADPH
Components
  • Complex: Protein-substrate complex of NADPH oxidase with NADPH
    • Protein or peptide: FAD-binding FR-type domain-containing protein
  • Ligand: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
  • Ligand: FLAVIN-ADENINE DINUCLEOTIDE
  • Ligand: PROTOPORPHYRIN IX CONTAINING FE
  • Ligand: water

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Supramolecule #1: Protein-substrate complex of NADPH oxidase with NADPH

SupramoleculeName: Protein-substrate complex of NADPH oxidase with NADPH / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Molecular weightTheoretical: 46 KDa

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

MacromoleculeName: FAD-binding FR-type domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Molecular weightTheoretical: 45.984457 KDa
Recombinant expressionOrganism: Escherichia coli BL21(DE3) (bacteria)
SequenceString: EFSMKSVKGL LFIIASFILT LLTWMNTSPQ FMIPGLALTS LSLTFILATR LPLLESWFHS LEKVYTVHKF TAFLSIILLI FHNFSMGGL WGSRLAAQFG NLAIYIFASI ILVAYLGKYI QYEAWRWIHR LVYLAYILGL FHIYMIMGNR LLTFNLLSFL V GSYALLGL ...String:
EFSMKSVKGL LFIIASFILT LLTWMNTSPQ FMIPGLALTS LSLTFILATR LPLLESWFHS LEKVYTVHKF TAFLSIILLI FHNFSMGGL WGSRLAAQFG NLAIYIFASI ILVAYLGKYI QYEAWRWIHR LVYLAYILGL FHIYMIMGNR LLTFNLLSFL V GSYALLGL LAGFYIIFLY QKISFPYLGK ITHLKRLNHD TREIQIHLSR PFNYQSGQFA FLKIFQEGFE SAPHPFSISG GH GQTLYFT VKTSGDHTKN IYDNLQAGSK VTLDRAYGHM IIEEGRENQV WIAGGIGITP FISYIREHPI LDKQVHFYYS FRG DENAVY LDLLRNYAQK NPNFELHLID STKDGYLNFE QKEVPEHATV YMCGPISMMK ALAKQIKKQN PKTELIYEGA KFK

UniProtKB: FAD-binding FR-type domain-containing protein

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Macromolecule #2: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

MacromoleculeName: NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE
type: ligand / ID: 2 / Number of copies: 1 / Formula: NDP
Molecular weightTheoretical: 745.421 Da
Chemical component information

ChemComp-NDP:
NADPH DIHYDRO-NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE

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Macromolecule #3: FLAVIN-ADENINE DINUCLEOTIDE

MacromoleculeName: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 3 / Number of copies: 1 / Formula: FAD
Molecular weightTheoretical: 785.55 Da
Chemical component information

ChemComp-FAD:
FLAVIN-ADENINE DINUCLEOTIDE / FAD*YM

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Macromolecule #4: PROTOPORPHYRIN IX CONTAINING FE

MacromoleculeName: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 4 / Number of copies: 2 / Formula: HEM
Molecular weightTheoretical: 616.487 Da
Chemical component information

ChemComp-HEM:
PROTOPORPHYRIN IX CONTAINING FE

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Macromolecule #5: water

MacromoleculeName: water / type: ligand / ID: 5 / Number of copies: 27 / Formula: HOH
Molecular weightTheoretical: 18.015 Da
Chemical component information

ChemComp-HOH:
WATER

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation stateparticle

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Sample preparation

Concentration6 mg/mL
BufferpH: 7.5
Component:
ConcentrationFormulaName
250.0 mMNaClsodium chloride
50.0 mMTris-HClTris-HCl
0.002 %LMNGLauryl Maltose Neopentyl Glycol
1.0 mMFADFlavin-adenine-dinucleotide
5.0 mMNADPHNicotinamide adenine dinucleotide phosphate
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY ARRAY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 4 K / Instrument: FEI VITROBOT MARK III

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Electron microscopy

MicroscopeTFS KRIOS
Specialist opticsEnergy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV
Image recordingFilm or detector model: FEI FALCON IV (4k x 4k) / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Number grids imaged: 2 / Number real images: 45181 / Average exposure time: 4.0 sec. / Average electron dose: 70.0 e/Å2
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.
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 2.1 µm / Nominal defocus min: 0.7000000000000001 µm / Nominal magnification: 215000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

DetailsMovies were collected in EER format and not gain corrected
Particle selectionNumber selected: 13817533
Details: These are the combined picked particles from a topaz model and a crYOLO model and contain duplicate particles
Startup modelType of model: NONE
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.64 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Final refinement was local refinement in cryoSPARC 4.1 with a mask excluding the detergent micelle
Number images used: 546234
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

Initial modelChain - Source name: Other / Chain - Initial model type: experimental model
Details: Initial model was the previousy resolved WT protein with NADPH bound
DetailsInitial model was fit using ChimeraX and refined with Coot and PHENIX
RefinementSpace: REAL / Protocol: RIGID BODY FIT / Target criteria: Cross-correlation coefficient
Output model

PDB-8qta:
Cryo-EM structure of Streptococcus pneumoniae NADPH oxidase F397A mutant in complex with NADPH

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