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- EMDB-43891: Chlamydomonas reinhardtii mastigoneme (constituent map 3) -

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Basic information

Entry
Database: EMDB / ID: EMD-43891
TitleChlamydomonas reinhardtii mastigoneme (constituent map 3)
Map dataChlamydomonas reinhardtii mastigoneme (constituent map 3, sharpened)
Sample
  • Complex: Native mastigonemes
KeywordsMastigoneme / cilia / STRUCTURAL PROTEIN
Biological speciesChlamydomonas reinhardtii (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsDai J / Ma M / Zhang R / Brown A
Funding support United States, 6 items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM141109 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM143183 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)1R01GM138854 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM139856 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131909 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL128370 United States
CitationJournal: Cell / Year: 2024
Title: Mastigoneme structure reveals insights into the O-linked glycosylation code of native hydroxyproline-rich helices.
Authors: Jin Dai / Meisheng Ma / Qingwei Niu / Robyn J Eisert / Xiangli Wang / Poulomi Das / Karl F Lechtreck / Susan K Dutcher / Rui Zhang / Alan Brown /
Abstract: Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or ...Hydroxyproline-rich glycoproteins (HRGPs) are a ubiquitous class of protein in the extracellular matrices and cell walls of plants and algae, yet little is known of their native structures or interactions. Here, we used electron cryomicroscopy (cryo-EM) to determine the structure of the hydroxyproline-rich mastigoneme, an extracellular filament isolated from the cilia of the alga Chlamydomonas reinhardtii. The structure demonstrates that mastigonemes are formed from two HRGPs (a filament of MST1 wrapped around a single copy of MST3) that both have hyperglycosylated poly(hydroxyproline) helices. Within the helices, O-linked glycosylation of the hydroxyproline residues and O-galactosylation of interspersed serine residues create a carbohydrate casing. Analysis of the associated glycans reveals how the pattern of hydroxyproline repetition determines the type and extent of glycosylation. MST3 possesses a PKD2-like transmembrane domain that forms a heteromeric polycystin-like cation channel with PKD2 and SIP, explaining how mastigonemes are tethered to ciliary membranes.
History
DepositionFeb 29, 2024-
Header (metadata) releaseApr 10, 2024-
Map releaseApr 10, 2024-
UpdateApr 24, 2024-
Current statusApr 24, 2024Processing site: RCSB / Status: Released

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Structure visualization

Supplemental images

emd_43891.png

Downloads & links

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Map

FileDownload / File: emd_43891.map.gz / Format: CCP4 / Size: 512 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationChlamydomonas reinhardtii mastigoneme (constituent map 3, sharpened)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.39 Å/pix.
x 512 pix.
= 711.68 Å		1.39 Å/pix.
x 512 pix.
= 711.68 Å		1.39 Å/pix.
x 512 pix.
= 711.68 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.39 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.5
Minimum - Maximum-1.4273642 - 3.1546953
Average (Standard dev.)-0.00044822236 (±0.054235723)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions512512512
Spacing512512512
CellA=B=C: 711.68 Å
α=β=γ: 90.0 °

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Supplemental data

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Mask #1

Fileemd_43891_msk_1.map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Additional map: Chlamydomonas reinhardtii mastigoneme (constituent map 3, unsharpened)...

Fileemd_43891_additional_1.map
AnnotationChlamydomonas reinhardtii mastigoneme (constituent map 3, unsharpened)
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map A

Fileemd_43891_half_map_1.map
AnnotationHalf map A
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Half map: Half map B

Fileemd_43891_half_map_2.map
AnnotationHalf map B
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

Histogram

Histogram (log scale)

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Sample components

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Entire : Native mastigonemes

EntireName: Native mastigonemes
Components
  • Complex: Native mastigonemes

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Supramolecule #1: Native mastigonemes

SupramoleculeName: Native mastigonemes / type: complex / ID: 1 / Parent: 0 / Macromolecule list: #1
Source (natural)Organism: Chlamydomonas reinhardtii (plant)

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Experimental details

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Structure determination

Methodcryo EM
Processingsingle particle reconstruction
Aggregation statefilament

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Sample preparation

Concentration0.01 mg/mL
BufferpH: 7.4
Details: HMDEKP buffer (30 mM HEPES, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 25 mM KCl, pH 7.4) containing 1x ProteaseArrest protease inhibitors (G-Biosciences)
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 277.15 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: COUNTING / Digitization - Dimensions - Width: 3838 pixel / Digitization - Dimensions - Height: 3710 pixel / Number real images: 19601 / Average exposure time: 9.0 sec. / Average electron dose: 38.9 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 0.01 mm / Nominal defocus max: 3.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 81000
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

Startup modelType of model: OTHER / Details: ab initio by cryoSPARC
Final reconstructionResolution.type: BY AUTHOR / Resolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: cryoSPARC / Number images used: 687452
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC
Final angle assignmentType: PROJECTION MATCHING / Software - Name: cryoSPARC (ver. 3.4)
Final 3D classificationNumber classes: 2 / Software - Name: cryoSPARC
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL

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