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- PDB-9b4h: Chlamydomonas reinhardtii mastigoneme filament -

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Basic information

Entry
Database: PDB / ID: 9b4h
TitleChlamydomonas reinhardtii mastigoneme filament
Components
  • C-type lectin domain-containing protein
  • Tyrosine-protein kinase ephrin type A/B receptor-like domain-containing protein
KeywordsSTRUCTURAL PROTEIN / Mastigoneme / Glycosylated hydroxyproline
Function / homology
Function and homology information


Tyrosine-protein kinase ephrin type A/B receptor-like / Tyrosine-protein kinase ephrin type A/B receptor-like / Putative ephrin-receptor like / Lectin C-type domain / C-type lectin domain profile. / C-type lectin-like / C-type lectin (CTL) or carbohydrate-recognition domain (CRD) / C-type lectin-like/link domain superfamily / C-type lectin fold / Growth factor receptor cysteine-rich domain superfamily / Immunoglobulin-like fold
Similarity search - Domain/homology
: / alpha-D-galactopyranose / C-type lectin domain-containing protein / Tyrosine-protein kinase ephrin type A/B receptor-like domain-containing protein
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.1 Å
AuthorsDai, J. / Ma, M. / Zhang, R. / Brown, A.
Funding support United States, 7items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R01GM141109 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)R01GM138854 United States
National Institutes of Health/National Heart, Lung, and Blood Institute (NIH/NHLBI)R01HL128370 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM131909 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)R35GM152057 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
DepositionMar 20, 2024Deposition site: RCSB / Processing site: RCSB
Revision 1.0Apr 10, 2024Provider: repository / Type: Initial release

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Structure visualization

Structure viewerMolecule:
MolmilJmol/JSmol

Downloads & links

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Assembly

Deposited unit
A: Tyrosine-protein kinase ephrin type A/B receptor-like domain-containing protein
X: C-type lectin domain-containing protein
B: Tyrosine-protein kinase ephrin type A/B receptor-like domain-containing protein
hetero molecules


Theoretical massNumber of molelcules
Total (without water)1,340,328248
Polymers1,280,6543
Non-polymers59,674245
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: electron microscopy, not applicable
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Protein , 2 types, 3 molecules ABX

#1: Protein Tyrosine-protein kinase ephrin type A/B receptor-like domain-containing protein / MST1


Mass: 206316.203 Da / Num. of mol.: 2 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / Strain: CC-4402 / References: UniProt: A8J9H7
#2: Protein C-type lectin domain-containing protein / MST3


Mass: 868021.500 Da / Num. of mol.: 1 / Source method: isolated from a natural source / Source: (natural) Chlamydomonas reinhardtii (plant) / Strain: CC-4402 / References: UniProt: A0A2K3DRP3

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Sugars , 8 types, 245 molecules

#3: Polysaccharide
2-acetamido-2-deoxy-beta-D-glucopyranose-(1-4)-2-acetamido-2-deoxy-beta-D-glucopyranose


Type: oligosaccharide / Mass: 424.401 Da / Num. of mol.: 6
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
DGlcpNAcb1-4DGlcpNAcb1-Glycam Condensed SequenceGMML 1.0
[]{[(4+1)][b-D-GlcpNAc]{[(4+1)][b-D-GlcpNAc]{}}}LINUCSPDB-CARE
#4: Polysaccharide...
beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose


Type: oligosaccharide / Mass: 282.245 Da / Num. of mol.: 86
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafb1-2LArafb1-ROHGlycam Condensed SequenceGMML 1.0
[][L-1-deoxy-Araf]{[(2+1)][b-L-Araf]{}}LINUCSPDB-CARE
#5: Polysaccharide
beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose


Type: oligosaccharide / Mass: 546.474 Da / Num. of mol.: 10
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafb1-2LArafb5-5LArafb1-2LArafb1-ROHGlycam Condensed SequenceGMML 1.0
#6: Polysaccharide beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L- ...beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose


Type: oligosaccharide / Mass: 1074.934 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafb1-2LArafb5-5LArafb1-2LArafb5-5LArafb1-2LArafb5-5LArafb1-2LArafb1-ROHGlycam Condensed SequenceGMML 1.0
#7: Polysaccharide beta-L-arabinofuranose-(1-4)-beta-L-ribulofuranose


Type: oligosaccharide / Mass: 282.245 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafb1-4LRulfb2-Glycam Condensed SequenceGMML 1.0
[][L-1-deoxy-Araf]{[(2+1)][b-L-Araf]{}}LINUCSPDB-CARE
#8: Polysaccharide beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L- ...beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose-(5-5)-beta-L-arabinofuranose-(1-2)-beta-L-arabinofuranose


Type: oligosaccharide / Mass: 810.704 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
DescriptorTypeProgram
LArafb1-2LArafb5-5LArafb1-2LArafb5-5LArafb1-2LArafb1-ROHGlycam Condensed SequenceGMML 1.0
#9: Sugar...
ChemComp-A1AIO / beta-L-glucofuranose


Type: L-saccharide / Mass: 180.156 Da / Num. of mol.: 114 / Source method: obtained synthetically / Formula: C6H12O6 / Feature type: SUBJECT OF INVESTIGATION
#10: Sugar...
ChemComp-GLA / alpha-D-galactopyranose / alpha-D-galactose / D-galactose / galactose / ALPHA D-GALACTOSE / Galactose


Type: D-saccharide, alpha linking / Mass: 180.156 Da / Num. of mol.: 26 / Source method: obtained synthetically / Formula: C6H12O6 / Feature type: SUBJECT OF INVESTIGATION
IdentifierTypeProgram
DGalpaCONDENSED IUPAC CARBOHYDRATE SYMBOLGMML 1.0
a-D-galactopyranoseCOMMON NAMEGMML 1.0
a-D-GalpIUPAC CARBOHYDRATE SYMBOLPDB-CARE 1.0
GalSNFG CARBOHYDRATE SYMBOLGMML 1.0

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Details

Has ligand of interestY

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Experimental details

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Experiment

ExperimentMethod: ELECTRON MICROSCOPY
EM experimentAggregation state: FILAMENT / 3D reconstruction method: single particle reconstruction

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Sample preparation

ComponentName: Mastigoneme filament / Type: COMPLEX
Details: Mastigoneme filaments copurified with doublet microtubules isolated from C. reinhardtii cilia
Entity ID: #1-#2 / Source: NATURAL
Molecular weightExperimental value: NO
Source (natural)Organism: Chlamydomonas reinhardtii (plant) / Strain: CC-4402
Buffer solutionpH: 7.4 / Details: Buffer contained protease inhibitors
Buffer component
IDConc.NameFormulaBuffer-ID
130 mMHEPES1
25 mMmagnesium sulfateMgSO41
31 mMDTT1
40.5 mMEGTA1
525 mMpotassium chlorideKCl1
SpecimenEmbedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Details: Unknown concentration. Copurified with doublet microtubules.
Specimen supportGrid material: COPPER / Grid type: Quantifoil
VitrificationCryogen name: ETHANE / Humidity: 100 %

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Electron microscopy imaging

Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
MicroscopyModel: FEI TITAN KRIOS
Electron gunElectron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Illumination mode: FLOOD BEAM
Electron lensMode: BRIGHT FIELDBright-field microscopy / Nominal defocus max: 3000 nm / Nominal defocus min: 1000 nm
Specimen holderCryogen: NITROGEN / Specimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER
Image recordingElectron dose: 39 e/Å2 / Film or detector model: GATAN K3 BIOQUANTUM (6k x 4k)

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Processing

EM softwareName: PHENIX / Version: 1.21_5207: / Category: model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 735338
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.1 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 687452 / Algorithm: FOURIER SPACE / Symmetry type: POINT
Atomic model buildingSpace: REAL
Atomic model buildingSource name: AlphaFold / Type: in silico model

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