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- PDB-7jrj: Chlamydomonas reinhardtii radial spoke head and neck (recombinant) -

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Basic information

Entry
Database: PDB / ID: 7jrj
TitleChlamydomonas reinhardtii radial spoke head and neck (recombinant)
Components
  • (Flagellar radial spoke protein ...) x 10
  • (Radial spoke protein ...) x 2
  • Nucleoside diphosphate kinase 6Nucleoside-diphosphate kinase
  • unknown protein
KeywordsSTRUCTURAL PROTEIN / Cilia / Radial spoke / Mechano-regulation
Function / homology
Function and homology information


radial spoke head / radial spoke / positive regulation of cilium-dependent cell motility / kinocilium / cilium movement involved in cell motility / motile cilium assembly / axoneme assembly / Set1C/COMPASS complex / UTP biosynthetic process / CTP biosynthetic process ...radial spoke head / radial spoke / positive regulation of cilium-dependent cell motility / kinocilium / cilium movement involved in cell motility / motile cilium assembly / axoneme assembly / Set1C/COMPASS complex / UTP biosynthetic process / CTP biosynthetic process / Oxidoreductases / motile cilium / GTP biosynthetic process / nucleoside diphosphate kinase activity / axoneme / cilium assembly / oxidoreductase activity / cytoskeleton / calmodulin binding / phosphorylation / ATP binding / membrane / metal ion binding / cytoplasm
Similarity search - Function
Nucleoside diphosphate kinase 6 / Radial spokehead-like protein / Radial spoke 3 / Radial spokehead-like protein / Radial spoke protein 3 / Sdc1/DPY30 / Dpy-30 motif / Dpy-30 motif / Possible plasma membrane-binding motif in junctophilins, PIP-5-kinases and protein kinases. / MORN motif ...Nucleoside diphosphate kinase 6 / Radial spokehead-like protein / Radial spoke 3 / Radial spokehead-like protein / Radial spoke protein 3 / Sdc1/DPY30 / Dpy-30 motif / Dpy-30 motif / Possible plasma membrane-binding motif in junctophilins, PIP-5-kinases and protein kinases. / MORN motif / MORN repeat / Nucleoside diphosphate kinase, active site / Nucleoside diphosphate kinase (NDPK) active site signature. / Nucleoside diphosphate kinase / Nucleoside diphosphate kinase-like domain / Nucleoside diphosphate kinase / NDK / Nucleoside diphosphate kinase-like domain superfamily / IQ calmodulin-binding motif / Short calmodulin-binding motif containing conserved Ile and Gln residues. / IQ motif profile. / NADP-dependent oxidoreductase domain / Aldo/keto reductase family / NADP-dependent oxidoreductase domain superfamily / IQ motif, EF-hand binding site
Similarity search - Domain/homology
Flagellar radial spoke protein 3 / Flagellar radial spoke protein 4 / Flagellar radial spoke protein 6 / Flagellar radial spoke protein 1 / Radial spoke protein 10 / Radial spoke head protein 9 homolog / Flagellar radial spoke protein 5 / Nucleoside diphosphate kinase 6 / Flagellar radial spoke protein 2
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
MethodELECTRON MICROSCOPY / single particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsGrossman-Haham, I. / Coudray, N. / Yu, Z. / Wang, F. / Zhang, N. / Bhabha, G. / Vale, R.D.
Funding support United States, 3items
OrganizationGrant numberCountry
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R00GM112982 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118106 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)GM103310 United States
CitationJournal: Nat Struct Mol Biol / Year: 2021
Title: Structure of the radial spoke head and insights into its role in mechanoregulation of ciliary beating.
Authors: Iris Grossman-Haham / Nicolas Coudray / Zanlin Yu / Feng Wang / Nan Zhang / Gira Bhabha / Ronald D Vale /
Abstract: Motile cilia power cell locomotion and drive extracellular fluid flow by propagating bending waves from their base to tip. The coordinated bending of cilia requires mechanoregulation by the radial ...Motile cilia power cell locomotion and drive extracellular fluid flow by propagating bending waves from their base to tip. The coordinated bending of cilia requires mechanoregulation by the radial spoke (RS) protein complexes and the microtubule central pair (CP). Despite their importance for ciliary motility across eukaryotes, the molecular function of the RSs is unknown. Here, we reconstituted the Chlamydomonas reinhardtii RS head that abuts the CP and determined its structure using single-particle cryo-EM to 3.1-Å resolution, revealing a flat, negatively charged surface supported by a rigid core of tightly intertwined proteins. Mutations in this core, corresponding to those involved in human ciliopathies, compromised the stability of the recombinant complex, providing a molecular basis for disease. Partially reversing the negative charge on the RS surface impaired motility in C. reinhardtii. We propose that the RS-head architecture is well-suited for mechanoregulation of ciliary beating through physical collisions with the CP.
History
DepositionAug 12, 2020Deposition site: RCSB / Processing site: RCSB
Revision 1.0Dec 16, 2020Provider: repository / Type: Initial release
Revision 1.1Dec 30, 2020Group: Database references / Category: citation / Item: _citation.pdbx_database_id_PubMed / _citation.title
Revision 1.2Jan 27, 2021Group: Database references / Category: citation
Item: _citation.journal_volume / _citation.page_first ..._citation.journal_volume / _citation.page_first / _citation.page_last / _citation.year

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Structure visualization

Movie
  • Deposited structure unit
  • Imaged by Jmol
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  • Superimposition on EM map
  • EMDB-22446
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Structure viewerMolecule:
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Assembly

Deposited unit
E: Radial spoke protein 10
F: Flagellar radial spoke protein 1
K: Nucleoside diphosphate kinase 6
I: Flagellar radial spoke protein 2
J: unknown protein
G: Flagellar radial spoke protein 3
H: Flagellar radial spoke protein 5
A: Radial spoke protein 9
B: Radial spoke protein 9
C: Flagellar radial spoke protein 4
D: Flagellar radial spoke protein 6
L: Flagellar radial spoke protein 6
M: Flagellar radial spoke protein 10
N: Flagellar radial spoke protein 1
O: Flagellar radial spoke protein 2


Theoretical massNumber of molelcules
Total (without water)518,32815
Polymers518,32815
Non-polymers00
Water0
1


  • Idetical with deposited unit
  • defined by author
  • Evidence: gel filtration
TypeNameSymmetry operationNumber
identity operation1_5551

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Components

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Radial spoke protein ... , 2 types, 3 molecules EAB

#1: Protein Radial spoke protein 10 /


Mass: 23553.943 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP10, CHLRE_01g005450v5, CHLREDRAFT_185792 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q27YU4
#8: Protein Radial spoke protein 9 /


Mass: 29572.348 Da / Num. of mol.: 2
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP9, CHLRE_07g330200v5, CHLREDRAFT_182960 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q27YU5

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Flagellar radial spoke protein ... , 10 types, 10 molecules FIGHCDLMNO

#2: Protein Flagellar radial spoke protein 1


Mass: 87873.445 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP1, CHLREDRAFT_196412 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q27YU0
#4: Protein Flagellar radial spoke protein 2


Mass: 77445.297 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP2, CHLREDRAFT_186281 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q6UBQ3
#6: Protein Flagellar radial spoke protein 3


Mass: 39361.293 Da / Num. of mol.: 1 / Fragment: UNP residues 159-516
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP3 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: P12759
#7: Protein Flagellar radial spoke protein 5


Mass: 55928.180 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP5, CHLREDRAFT_190792 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q27YU7, Oxidoreductases
#9: Protein Flagellar radial spoke protein 4


Mass: 49900.820 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP4 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q01656
#10: Protein Flagellar radial spoke protein 6


Mass: 48884.551 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP6 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q01657
#11: Protein/peptide Flagellar radial spoke protein 6


Mass: 528.644 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Production host: Spodoptera frugiperda (fall armyworm)
#12: Protein/peptide Flagellar radial spoke protein 10


Mass: 443.539 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Production host: Spodoptera frugiperda (fall armyworm)
#13: Protein Flagellar radial spoke protein 1


Mass: 7592.350 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Production host: Spodoptera frugiperda (fall armyworm)
#14: Protein Flagellar radial spoke protein 2


Mass: 5890.252 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Production host: Spodoptera frugiperda (fall armyworm)

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Protein / Protein/peptide , 2 types, 2 molecules KJ

#3: Protein Nucleoside diphosphate kinase 6 / Nucleoside-diphosphate kinase / radial spoke protein 23


Mass: 61422.211 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Gene: RSP23, CHLRE_16g654300v5, CHLREDRAFT_139197 / Production host: Spodoptera frugiperda (fall armyworm) / References: UniProt: Q69B19, nucleoside-diphosphate kinase
#5: Protein/peptide unknown protein


Mass: 358.434 Da / Num. of mol.: 1
Source method: isolated from a genetically manipulated source
Source: (gene. exp.) Chlamydomonas reinhardtii (plant) / Production host: Spodoptera frugiperda (fall armyworm)

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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: Chlamydomonas reinhardtii radial spoke head and neck / Type: COMPLEX / Entity ID: all / Source: RECOMBINANT
Molecular weightExperimental value: NO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Source (recombinant)Organism: Spodoptera frugiperda (fall armyworm)
Buffer solutionpH: 7.4 / Details: 30 mM HEPES, 300 mM NaCl, 1 mM TCEP
SpecimenConc.: 0.1 mg/ml / Embedding applied: NO / Shadowing applied: NO / Staining applied: NO / Vitrification applied: YES
Specimen supportGrid material: GOLD / Grid mesh size: 300 divisions/in. / Grid type: Quantifoil R1.2/1.3
VitrificationInstrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Humidity: 95 % / Chamber temperature: 295 K

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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 magnification: 105000 X / Nominal defocus max: 1500 nm / Nominal defocus min: 800 nm
Image recordingElectron dose: 70 e/Å2 / Detector mode: SUPER-RESOLUTION / Film or detector model: GATAN K3 (6k x 4k) / Num. of grids imaged: 2 / Num. of real images: 7572
Image scansMovie frames/image: 35

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Processing

EM software
IDNameVersionCategory
1cryoSPARC2.9particle selection
2SerialEMimage acquisition
4GctfCTF correction
7Coot0.8.9model fitting
9cryoSPARC2.9initial Euler assignment
10RELION3final Euler assignment
11RELION3classification
12RELION33D reconstruction
19PHENIX1.17.1-3660model refinement
CTF correctionType: PHASE FLIPPING AND AMPLITUDE CORRECTION
Particle selectionNum. of particles selected: 942591
SymmetryPoint symmetry: C1 (asymmetric)
3D reconstructionResolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Num. of particles: 136659 / Num. of class averages: 1 / Symmetry type: POINT
Atomic model buildingProtocol: RIGID BODY FIT / Space: REAL
Atomic model buildingPDB-ID: 7JR9

7jr9

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