[English] 日本語
![](img/lk-miru.gif)
- EMDB-18646: Cryo-EM structure of stably reduced Streptococcus pneumoniae NADP... -
+
Open data
-
Basic information
Entry | ![]() | |||||||||
---|---|---|---|---|---|---|---|---|---|---|
Title | Cryo-EM structure of stably reduced Streptococcus pneumoniae NADPH oxidase in complex with NADH | |||||||||
![]() | B-factor sharpened map used for atomic model building and figures. | |||||||||
![]() |
| |||||||||
![]() | NADPH oxidase / ROS producing / flavoprotein / heme protein / MEMBRANE PROTEIN | |||||||||
Function / homology | ![]() 2 iron, 2 sulfur cluster binding / flavin adenine dinucleotide binding / oxidoreductase activity / membrane Similarity search - Function | |||||||||
Biological species | ![]() ![]() | |||||||||
Method | single particle reconstruction / cryo EM / Resolution: 2.36 Å | |||||||||
![]() | Dubach VRA / San Segundo-Acosta P / Murphy BJ | |||||||||
Funding support | ![]()
| |||||||||
![]() | ![]() 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 |
|
-
Structure visualization
Supplemental images |
---|
-
Downloads & links
-EMDB archive
Map data | ![]() | 97.1 MB | ![]() | |
---|---|---|---|---|
Header (meta data) | ![]() ![]() | 21.6 KB 21.6 KB | Display Display | ![]() |
FSC (resolution estimation) | ![]() | 10.4 KB | Display | ![]() |
Images | ![]() | 117.2 KB | ||
Masks | ![]() | 103 MB | ![]() | |
Filedesc metadata | ![]() | 6.8 KB | ||
Others | ![]() ![]() ![]() | 52.1 MB 95.7 MB 95.7 MB | ||
Archive directory | ![]() ![]() | HTTPS FTP |
-Related structure data
Related structure data | ![]() 8qt9MC ![]() 8qt6C ![]() 8qt7C ![]() 8qtaC M: atomic model generated by this map C: citing same article ( |
---|---|
Similar structure data | Similarity search - Function & homology ![]() |
-
Links
EMDB pages | ![]() ![]() |
---|---|
Related items in Molecule of the Month |
-
Map
File | ![]() | ||||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | B-factor sharpened map used for atomic model building and figures. | ||||||||||||||||||||
Voxel size | X=Y=Z: 0.955 Å | ||||||||||||||||||||
Density |
| ||||||||||||||||||||
Symmetry | Space group: 1 | ||||||||||||||||||||
Details | EMDB XML:
|
-Supplemental data
-Mask #1
File | ![]() | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Projections & Slices |
| ||||||||||||
Density Histograms |
-Additional map: Raw unsharpened map obtained after merging half maps.
File | emd_18646_additional_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Raw unsharpened map obtained after merging half maps. | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half map A obtained during final refinement in cryoSPARC.
File | emd_18646_half_map_1.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half map A obtained during final refinement in cryoSPARC. | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-Half map: Half map B obtained during final refinement in cryoSPARC.
File | emd_18646_half_map_2.map | ||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Annotation | Half map B obtained during final refinement in cryoSPARC. | ||||||||||||
Projections & Slices |
| ||||||||||||
Density Histograms |
-
Sample components
-Entire : Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactor...
Entire | Name: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors and NADH bound |
---|---|
Components |
|
-Supramolecule #1: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactor...
Supramolecule | Name: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors and NADH bound type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1 |
---|---|
Source (natural) | Organism: ![]() ![]() |
-Macromolecule #1: FAD-binding FR-type domain-containing protein
Macromolecule | Name: FAD-binding FR-type domain-containing protein / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO |
---|---|
Source (natural) | Organism: ![]() ![]() |
Molecular weight | Theoretical: 46.060551 KDa |
Recombinant expression | Organism: ![]() ![]() |
Sequence | String: 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 |
-Macromolecule #2: FLAVIN-ADENINE DINUCLEOTIDE
Macromolecule | Name: FLAVIN-ADENINE DINUCLEOTIDE / type: ligand / ID: 2 / Number of copies: 1 / Formula: FAD |
---|---|
Molecular weight | Theoretical: 785.55 Da |
Chemical component information | ![]() ChemComp-FAD: |
-Macromolecule #3: PROTOPORPHYRIN IX CONTAINING FE
Macromolecule | Name: PROTOPORPHYRIN IX CONTAINING FE / type: ligand / ID: 3 / Number of copies: 2 / Formula: HEM |
---|---|
Molecular weight | Theoretical: 616.487 Da |
Chemical component information | ![]() ChemComp-HEM: |
-Macromolecule #4: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE
Macromolecule | Name: 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / type: ligand / ID: 4 / Number of copies: 1 / Formula: NAI |
---|---|
Molecular weight | Theoretical: 665.441 Da |
Chemical component information | ![]() ChemComp-NAI: |
-Macromolecule #5: water
Macromolecule | Name: water / type: ligand / ID: 5 / Number of copies: 64 / Formula: HOH |
---|---|
Molecular weight | Theoretical: 18.015 Da |
Chemical component information | ![]() ChemComp-HOH: |
-Experimental details
-Structure determination
Method | cryo EM |
---|---|
![]() | single particle reconstruction |
Aggregation state | particle |
-
Sample preparation
Concentration | 6 mg/mL | ||||||||||||||||||
---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
Buffer | pH: 7.5 Component:
| ||||||||||||||||||
Grid | Model: 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. | ||||||||||||||||||
Vitrification | Cryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 277 K / Instrument: FEI VITROBOT MARK III |
-
Electron microscopy
Microscope | TFS KRIOS |
---|---|
Specialist optics | Energy filter - Name: TFS Selectris X / Energy filter - Slit width: 10 eV |
Image recording | Film 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 beam | Acceleration voltage: 300 kV / Electron source: ![]() |
Electron optics | C2 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 stage | Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN |
Experimental equipment | ![]() Model: Titan Krios / Image courtesy: FEI Company |
+
Image processing
-Atomic model buiding 1
Details | Model was fit using ChimeraX. Coot and PHENIX were used for model refinement. |
---|---|
Refinement | Space: REAL / Protocol: RIGID BODY FIT / Overall B value: 26.54 / Target criteria: Cross-correlation coefficient |
Output model | ![]() PDB-8qt9: |