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- EMDB-25787: C2-symmetric single-particle cryo-EM map of T. vaginalis FDPF3 -

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Basic information

Entry
Database: EMDB / ID: EMD-25787
TitleC2-symmetric single-particle cryo-EM map of T. vaginalis FDPF3
Map dataC2-symmetric, sharpened, masked map of TvFDPF3
Sample
  • Complex: Dimeric assembly of TvFDPF3 N-terminal FDP-like domains
Keywordsoxygen scavenging / fmn / nadh / flavodiiron protein / OXIDOREDUCTASE
Biological speciesTrichomonas vaginalis (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 6.8 Å
AuthorsBell TA / Chao LH
Funding support2 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM142495
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM121856
CitationJournal: J Biol Chem / Year: 2022
Title: A natural fusion of flavodiiron, rubredoxin, and rubredoxin oxidoreductase domains is a self-sufficient water-forming oxidase of Trichomonas vaginalis.
Authors: Evana N Abdulaziz / Tristan A Bell / Bazlur Rashid / Mina L Heacock / Tarik Begic / Owen S Skinner / Mohammad A Yaseen / Luke H Chao / Vamsi K Mootha / Antonio J Pierik / Valentin Cracan /
Abstract: Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. ...Microaerophilic pathogens such as Giardia lamblia, Entamoeba histolytica, and Trichomonas vaginalis have robust oxygen consumption systems to detoxify oxygen and maintain intracellular redox balance. This oxygen consumption results from HO-forming NADH oxidase (NOX) activity of two distinct flavin-containing systems: HO-forming NOXes and multicomponent flavodiiron proteins (FDPs). Neither system is membrane bound, and both recycle NADH into oxidized NAD while simultaneously removing O from the local environment. However, little is known about the specific contributions of these systems in T. vaginalis. In this study, we use bioinformatics and biochemical analyses to show that T. vaginalis lacks a NOX-like enzyme and instead harbors three paralogous genes (FDPF1-3), each encoding a natural fusion product between the N-terminal FDP, central rubredoxin (Rb), and C-terminal NADH:Rb oxidoreductase domains. Unlike a "stand-alone" FDP that lacks Rb and oxidoreductase domains, this natural fusion protein with fully populated flavin redox centers directly accepts reducing equivalents of NADH to catalyze the four-electron reduction of oxygen to water within a single polypeptide with an extremely high turnover. Furthermore, using single-particle cryo-EM, we present structural insights into the spatial organization of the FDP core within this multidomain fusion protein. Together, these results contribute to our understanding of systems that allow protozoan parasites to maintain optimal redox balance and survive transient exposure to oxic conditions.
History
DepositionDec 21, 2021-
Header (metadata) releaseJul 6, 2022-
Map releaseJul 6, 2022-
UpdateJan 17, 2024-
Current statusJan 17, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_25787.png

Downloads & links

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Map

FileDownload / File: emd_25787.map.gz / Format: CCP4 / Size: 64 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationC2-symmetric, sharpened, masked map of TvFDPF3
Voxel sizeX=Y=Z: 0.825 Å
Density
Contour LevelBy AUTHOR: 0.014
Minimum - Maximum-0.06294447 - 0.09495563
Average (Standard dev.)0.00012491332 (±0.0026372948)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions256256256
Spacing256256256
CellA=B=C: 211.2 Å
α=β=γ: 90.0 °

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Supplemental data

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Additional map: C2-symmetric, sharpened, unmasked map of TvFDPF3

Fileemd_25787_additional_1.map
AnnotationC2-symmetric, sharpened, unmasked map of TvFDPF3
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 1 from masked refinement of C2-symmetric map...

Fileemd_25787_half_map_1.map
AnnotationHalf-map 1 from masked refinement of C2-symmetric map prior to sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half-map 2 from masked refinement of C2-symmetric map...

Fileemd_25787_half_map_2.map
AnnotationHalf-map 2 from masked refinement of C2-symmetric map prior to sharpening
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Dimeric assembly of TvFDPF3 N-terminal FDP-like domains

EntireName: Dimeric assembly of TvFDPF3 N-terminal FDP-like domains
Components
  • Complex: Dimeric assembly of TvFDPF3 N-terminal FDP-like domains

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Supramolecule #1: Dimeric assembly of TvFDPF3 N-terminal FDP-like domains

SupramoleculeName: Dimeric assembly of TvFDPF3 N-terminal FDP-like domains
type: complex / ID: 1 / Parent: 0 / Details: Resolved portion of full-lengh TvFDPF3
Source (natural)Organism: Trichomonas vaginalis (eukaryote) / Strain: C-1:NIH

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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

Concentration0.8 mg/mL
BufferpH: 7.5
Component:
ConcentrationNameFormula
50.0 mMHEPES
150.0 mMSodium ChlorideNaClSodium chloride
GridModel: Quantifoil R1.2/1.3 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Support film - Film thickness: 12 / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec. / Pretreatment - Atmosphere: AIR / Pretreatment - Pressure: 3.9 kPa
Details: Grid was washed twice with chloroform and air dried prior to glow discharge and sample application.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 295 K / Instrument: FEI VITROBOT MARK IV / Details: Blotting condition: 5 s at +15 blotting force..
DetailsMonodisperse sample

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: OTHER / Imaging mode: OTHER / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.3 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Number grids imaged: 1 / Number real images: 7398 / Average electron dose: 54.5 e/Å2 / Details: Images were collected as 51-frame movies.
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1511632
Details: All micrographs were imported into CryoSPARC and preprocessed using the CryoSPARC internal Patch Motion Correction and Patch CTF Estimation jobs. An initial particle set was generated by ...Details: All micrographs were imported into CryoSPARC and preprocessed using the CryoSPARC internal Patch Motion Correction and Patch CTF Estimation jobs. An initial particle set was generated by blob picking particles between 80 and 180 angstroms in diameter from all micrographs. These particles were twice sequentially subjected to 2D classification, and the best classes selected. The micrographs were then manually curated to remove images with minimum CTF fits of 9 angstrom or greater. After curation, 6,938 of the original 7,398 movies remained under consideration. The 2D classes selected from blob picking were used to train a Topaz model, and the model was used to pick particles from the curated micrographs. Duplicate particles were removed by excluding particles with a center within 125 angstrom of another particle.
Startup modelType of model: NONE / Details: Model generated ab initio in RELION
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final 3D classificationNumber classes: 6 / Software - Name: RELION (ver. 3.1.3)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.1.3)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C2 (2 fold cyclic) / Resolution.type: BY AUTHOR / Resolution: 6.8 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.1.3) / Number images used: 53709
FSC plot (resolution estimation)

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