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- PDB-8qt9: Cryo-EM structure of stably reduced Streptococcus pneumoniae NADP... -

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Basic information

Entry
Database: PDB / ID: 8qt9
TitleCryo-EM structure of stably reduced Streptococcus pneumoniae NADPH oxidase in complex with NADH
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 / 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / FAD-binding FR-type domain-containing protein
Similarity search - Component
Biological speciesStreptococcus pneumoniae (bacteria)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 2.36 Å
AuthorsDubach, V.R.A. / San Segundo-Acosta, P. / Murphy, B.J.
Funding support Germany, 1items
OrganizationGrant numberCountry
Max Planck Society Germany
CitationJournal: To Be Published / Year: 2024
Title: Structural and mechanistic insight into Streptococcus pneumoniae NADPH oxidase
Authors: Dubach, V.R.A. / San Segundo-Acosta, P. / Murphy, B.J.
History
DepositionOct 12, 2023Deposition site: PDBE / Processing site: PDBE
Revision 1.0Jul 24, 2024Provider: repository / Type: Initial release

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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)48,7455
Polymers46,0611
Non-polymers2,6844
Water1,15364
1


  • Idetical with deposited unit
  • defined by author&software
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_555x,y,z1

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Components

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


Mass: 46060.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Streptococcus pneumoniae (bacteria) / Gene: spr0531 / Production host: Escherichia coli BL21(DE3) (bacteria) / Variant (production host): C41 / References: UniProt: Q8CZ28
#2: 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
#3: 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
#4: Chemical ChemComp-NAI / 1,4-DIHYDRONICOTINAMIDE ADENINE DINUCLEOTIDE / NADH


Mass: 665.441 Da / Num. of mol.: 1 / Source method: obtained synthetically / Formula: C21H29N7O14P2 / Feature type: SUBJECT OF INVESTIGATION
#5: Water ChemComp-HOH / water


Mass: 18.015 Da / Num. of mol.: 64 / 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: Streptococcus pneumoniae NADPH oxidase with FAD and heme cofactors and NADH bound
Type: COMPLEX / Entity ID: #1 / Source: RECOMBINANT
Molecular weightUnits: MEGADALTONS / Experimental value: NO
Source (natural)Organism: Streptococcus pneumoniae (bacteria)
Source (recombinant)Organism: Escherichia coli BL21(DE3) (bacteria) / Strain: C41
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 phosphateNADH1
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: 277 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: 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 opticsEnergyfilter name: TFS Selectris X / Energyfilter slit width: 10 eV
Image scansWidth: 4096 / Height: 4096

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Processing

EM software
IDNameCategory
7UCSF ChimeraXmodel fitting
9Cootmodel refinement
10PHENIXmodel refinement
Image processingDetails: Movies were collected in EER format and not gain corrected
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 14339155
Details: These are the combined particles from two different Topaz models and a crYOLO model and contains duplicate particles.
3D reconstructionResolution: 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 buildingB 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
Refine-IDTypeDev idealNumber
ELECTRON MICROSCOPYf_bond_d0.0033566
ELECTRON MICROSCOPYf_angle_d0.6274874
ELECTRON MICROSCOPYf_dihedral_angle_d10.815467
ELECTRON MICROSCOPYf_chiral_restr0.048509
ELECTRON MICROSCOPYf_plane_restr0.003579

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