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- EMDB-18645: Cryo-EM structure of Streptococcus pneumoniae NADPH oxidase in co... -

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Basic information

Entry
Database: EMDB / ID: EMD-18645
TitleCryo-EM structure of Streptococcus pneumoniae NADPH oxidase in complex with NADPH
Map dataMain map used for atomic model building and figures.
Sample
  • Complex: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors
    • 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: 2.2 Å
AuthorsDubach VRA / San Segundo-Acosta P / Murphy BJ
Funding support Germany, 1 items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: To Be Published / Year: 2024
Title: Structural and mechanistic insight into Streptococcus pneumoniae NADPH oxidase
Authors: Dubach VRA / San Segundo-Acosta P / Murphy BJ
History
DepositionOct 12, 2023-
Header (metadata) releaseJul 24, 2024-
Map releaseJul 24, 2024-
UpdateJul 24, 2024-
Current statusJul 24, 2024Processing site: PDBe / Status: Released

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Structure visualization

Supplemental images

emd_18645.png

Downloads & links

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Map

FileDownload / File: emd_18645.map.gz / Format: CCP4 / Size: 83.7 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.)
0.92 Å/pix.
x 280 pix.
= 256.704 Å		0.92 Å/pix.
x 280 pix.
= 256.704 Å		0.92 Å/pix.
x 280 pix.
= 256.704 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 0.9168 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.62
Minimum - Maximum-2.984938 - 5.4654517
Average (Standard dev.)-0.00019740645 (±0.08166799)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions280280280
Spacing280280280
CellA=B=C: 256.704 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_18645_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Raw unsharpened map obtained in cryoSPARC after merging...

Fileemd_18645_additional_1.map
AnnotationRaw unsharpened map obtained in cryoSPARC after merging of the half maps.
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 gold...

Fileemd_18645_half_map_1.map
AnnotationHalf map B used during refinement and gold standard FSC resolution estimation.
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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

Fileemd_18645_half_map_2.map
AnnotationHalf map A used during refinement and gold standard FSC 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 : Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors

EntireName: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors
Components
  • Complex: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors
    • 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: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors

SupramoleculeName: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors
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: 46.060551 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 PKTELIYEGF 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: 65 / 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
1.0 mMNADPHnicotinamide adenine dinucleotide phosphate
GridModel: UltrAuFoil R1.2/1.3 / Material: GOLD / Mesh: 400 / Support film - Material: GOLD / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 90 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 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: 1 / Number real images: 22259 / Average exposure time: 3.96 sec. / Average electron dose: 70.0 e/Å2 / Details: Movies were collected in EER format.
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: 14458708
Details: These are the combined particles from two different Topaz models and a crYOLO model and contains duplicate particles.
Startup modelType of model: NONE
Final reconstructionAlgorithm: FOURIER SPACE / Resolution.type: BY AUTHOR / Resolution: 2.2 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC
Details: Final refinement was local refinement in cryoSPARC 4.0 with a mask excluding the detergent micelle.
Number images used: 591137
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC
Final 3D classificationSoftware - 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 previously made SpNOX model without substrate 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-8qt7:
Cryo-EM structure of Streptococcus pneumoniae NADPH oxidase in complex with NADPH

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