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- EMDB-28804: Subtomographic average of flagellar axoneme doublet from Trypanos... -

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Basic information

Entry
Database: EMDB / ID: EMD-28804
TitleSubtomographic average of flagellar axoneme doublet from Trypanosoma brucei MC8 Knockdown mutant
Map dataSubtomographic average of flagellar axoneme doublet from Trypanosoma brucei MC8 Knockdown mutant
Sample
  • Organelle or cellular component: Detergent-extracted flagellum of Trypanosoma brucei
Keywordsflagella / axoneme / MIPs / MC8 Knockdown / STRUCTURAL PROTEIN
Biological speciesTrypanosoma brucei (eukaryote)
Methodsubtomogram averaging / cryo EM / Resolution: 16.5 Å
AuthorsShimogawa MM / Wang H
Funding support United States, 1 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute Of Allergy and Infectious Diseases (NIH/NIAID)AI052348 United States
CitationJournal: Nat Commun / Year: 2023
Title: FAP106 is an interaction hub for assembling microtubule inner proteins at the cilium inner junction.
Authors: Michelle M Shimogawa / Angeline S Wijono / Hui Wang / Jiayan Zhang / Jihui Sha / Natasha Szombathy / Sabeeca Vadakkan / Paula Pelayo / Keya Jonnalagadda / James Wohlschlegel / Z Hong Zhou / Kent L Hill /
Abstract: Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) ...Motility of pathogenic protozoa depends on flagella (synonymous with cilia) with axonemes containing nine doublet microtubules (DMTs) and two singlet microtubules. Microtubule inner proteins (MIPs) within DMTs influence axoneme stability and motility and provide lineage-specific adaptations, but individual MIP functions and assembly mechanisms are mostly unknown. Here, we show in the sleeping sickness parasite Trypanosoma brucei, that FAP106, a conserved MIP at the DMT inner junction, is required for trypanosome motility and functions as a critical interaction hub, directing assembly of several conserved and lineage-specific MIPs. We use comparative cryogenic electron tomography (cryoET) and quantitative proteomics to identify MIP candidates. Using RNAi knockdown together with fitting of AlphaFold models into cryoET maps, we demonstrate that one of these candidates, MC8, is a trypanosome-specific MIP required for parasite motility. Our work advances understanding of MIP assembly mechanisms and identifies lineage-specific motility proteins that are attractive targets to consider for therapeutic intervention.
History
DepositionNov 6, 2022-
Header (metadata) releaseSep 6, 2023-
Map releaseSep 6, 2023-
UpdateSep 6, 2023-
Current statusSep 6, 2023Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_28804.png

Downloads & links

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Map

FileDownload / File: emd_28804.map.gz / Format: CCP4 / Size: 3.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationSubtomographic average of flagellar axoneme doublet from Trypanosoma brucei MC8 Knockdown mutant
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
6.76 Å/pix.
x 100 pix.
= 675.9 Å		6.76 Å/pix.
x 80 pix.
= 540.72 Å		6.76 Å/pix.
x 120 pix.
= 811.08 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

generated in cubic-lattice coordinate

Voxel sizeX=Y=Z: 6.759 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.46
Minimum - Maximum-3.433434 - 3.9919567
Average (Standard dev.)-0.000000033531368 (±0.6610114)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin-40-60-50
Dimensions80120100
Spacing12080100
CellA: 811.07996 Å / B: 540.72 Å / C: 675.89996 Å
α=β=γ: 90.0 °

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Supplemental data

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Half map: Subtomographic average of flagellar axoneme doublet from Trypanosoma...

Fileemd_28804_half_map_1.map
AnnotationSubtomographic average of flagellar axoneme doublet from Trypanosoma brucei MC8 Knockdown mutant(half-map 1)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Subtomographic average of flagellar axoneme doublet from Trypanosoma...

Fileemd_28804_half_map_2.map
AnnotationSubtomographic average of flagellar axoneme doublet from Trypanosoma brucei MC8 Knockdown mutant(half-map 2)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Detergent-extracted flagellum of Trypanosoma brucei

EntireName: Detergent-extracted flagellum of Trypanosoma brucei
Components
  • Organelle or cellular component: Detergent-extracted flagellum of Trypanosoma brucei

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Supramolecule #1: Detergent-extracted flagellum of Trypanosoma brucei

SupramoleculeName: Detergent-extracted flagellum of Trypanosoma brucei / type: organelle_or_cellular_component / ID: 1 / Parent: 0
Source (natural)Organism: Trypanosoma brucei (eukaryote) / Organelle: flagella

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Experimental details

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Structure determination

Methodcryo EM
Processingsubtomogram averaging
Aggregation statecell

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Sample preparation

BufferpH: 7.4
VitrificationCryogen name: ETHANE

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Average electron dose: 2.68 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Nominal defocus max: 5.0 µm / Nominal defocus min: 3.0 µm
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 16.5 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: PEET / Details: The FSC was performed by calcUnbiasedFSC from PEET / Number subtomograms used: 1936
ExtractionNumber tomograms: 52 / Number images used: 3092
Final angle assignmentType: ANGULAR RECONSTITUTION / Software - Name: PEET

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