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- EMDB-22444: Chlamydomonas reinhardtii radial spoke minimal head complex -

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Basic information

Entry
Database: EMDB / ID: EMD-22444
TitleChlamydomonas reinhardtii radial spoke minimal head complex
Map data
Sample
  • Complex: radial spoke minimal head complex
    • Protein or peptide: Radial spoke protein 9
    • Protein or peptide: Flagellar radial spoke protein 4
    • Protein or peptide: Flagellar radial spoke protein 6
    • Protein or peptide: Flagellar radial spoke protein 6
    • Protein or peptide: Flagellar radial spoke protein 10
    • Protein or peptide: Radial spoke protein 10
Function / homology
Function and homology information


radial spoke head / radial spoke / kinocilium / cilium movement involved in cell motility / motile cilium assembly / axoneme assembly / motile cilium / axoneme / cilium assembly / membrane
Similarity search - Function
Radial spokehead-like protein / Radial spokehead-like protein / Possible plasma membrane-binding motif in junctophilins, PIP-5-kinases and protein kinases. / MORN motif / MORN repeat
Similarity search - Domain/homology
Flagellar radial spoke protein 4 / Flagellar radial spoke protein 6 / Radial spoke protein 10 / Radial spoke head protein 9 homolog
Similarity search - Component
Biological speciesChlamydomonas reinhardtii (plant)
Methodsingle particle reconstruction / cryo EM / Resolution: 2.95 Å
AuthorsGrossman-Haham I / Coudray N / Yu Z / Wang F / Bhabha G / Vale RD
Funding support United States, 3 items
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)GM103310 United States
National Institutes of Health/National Institute of General Medical Sciences (NIH/NIGMS)R35GM118106 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 11, 2020-
Header (metadata) releaseDec 16, 2020-
Map releaseDec 16, 2020-
UpdateJan 27, 2021-
Current statusJan 27, 2021Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-7jr9
  • Surface level: 0.015
  • Imaged by UCSF Chimera
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_22444.png

Downloads & links

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Map

FileDownload / File: emd_22444.map.gz / Format: CCP4 / Size: 125 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.855 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.04072325 - 0.08759396
Average (Standard dev.)-5.58329e-05 (±0.0021981997)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions320320320
Spacing320320320
CellA=B=C: 273.6 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8550.8550.855
M x/y/z320320320
origin x/y/z0.0000.0000.000
length x/y/z273.600273.600273.600
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS320320320
D min/max/mean-0.0410.088-0.000

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Supplemental data

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Sample components

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Entire : radial spoke minimal head complex

EntireName: radial spoke minimal head complex
Components
  • Complex: radial spoke minimal head complex
    • Protein or peptide: Radial spoke protein 9
    • Protein or peptide: Flagellar radial spoke protein 4
    • Protein or peptide: Flagellar radial spoke protein 6
    • Protein or peptide: Flagellar radial spoke protein 6
    • Protein or peptide: Flagellar radial spoke protein 10
    • Protein or peptide: Radial spoke protein 10

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Supramolecule #1: radial spoke minimal head complex

SupramoleculeName: radial spoke minimal head complex / type: complex / ID: 1 / Parent: 0 / Macromolecule list: all
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)

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Macromolecule #1: Radial spoke protein 9

MacromoleculeName: Radial spoke protein 9 / type: protein_or_peptide / ID: 1 / Number of copies: 2 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 29.572348 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MVQLEPNITL VLKHLASCGA VVSAEQQAAL DHSIPIKRIE AGLRSLTLWG RLTTLNGKDY LVAEGYNVAS SKEGAAVYET KYFYSQDGA RWSDLQPVDS ETATRCARIK GMLSGDPAKN YELEEKDPNA PEPSPEAEEE VKPLVFQIPE LAVLRCRVDA I ATATSVIP ...String:
MVQLEPNITL VLKHLASCGA VVSAEQQAAL DHSIPIKRIE AGLRSLTLWG RLTTLNGKDY LVAEGYNVAS SKEGAAVYET KYFYSQDGA RWSDLQPVDS ETATRCARIK GMLSGDPAKN YELEEKDPNA PEPSPEAEEE VKPLVFQIPE LAVLRCRVDA I ATATSVIP TDSTILNAAS QVVPNRLFAG AAYPEKLESY QHRFSLPGSG VTLSQDLRGT WAVQYDAFKG VAQVRSLLFP GY FFYYAAN ELTWGSLYVG DGLRNNDLIF ML

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Macromolecule #2: Flagellar radial spoke protein 4

MacromoleculeName: Flagellar radial spoke protein 4 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 52.159195 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MGSSHHHHHH GGSAENLYFQ GMAAVDSVAQ ALAYLQVHSP QDGTSMYDHL VKLVSKVLED QPKNAVDLLE TSLLVKKSTF DPKESSPLV PIPVAPDATQ TQAAVSIFGD PELPINPATG EPVPADPPNE FEAENMLGAA AVLDCLGVGL GRELGVNIAL A AKRIGEDP ...String:
MGSSHHHHHH GGSAENLYFQ GMAAVDSVAQ ALAYLQVHSP QDGTSMYDHL VKLVSKVLED QPKNAVDLLE TSLLVKKSTF DPKESSPLV PIPVAPDATQ TQAAVSIFGD PELPINPATG EPVPADPPNE FEAENMLGAA AVLDCLGVGL GRELGVNIAL A AKRIGEDP KLAVRSVRFF GKFLGLYSDY FVFEVAFKKE AAKEAAPAAP APERVEGEAA SSSAPEVPVE EPGKGANKFT YL VCSSLGG PLTRLPDVTP AQVKASRRIK KLLTGRLTSH VSTYPAFPGN EANYLRALIA RISAATVVAP SDLFSLNDET GEL ERAEDW EPPAGREMAA PTAWVHVRPH LKSQGRCEVH KRELPEDADE DEFYNEDELE EGPDLLAALE EDAQLPGEQA AWTP IYSSA SEAVKTQAGG LRSLVWPGAV CGGRGSEWTC VYVGWGVKNA PFVPLPPPPV AQEFAWGEVE TQELELKPAP PPPEE EAEA DE

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Macromolecule #3: Flagellar radial spoke protein 6

MacromoleculeName: Flagellar radial spoke protein 6 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 48.884551 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAADVGQALA FLQQVKTTQG ASIYEGLKAA LAKVLEDRPV NAVEALETSV LSTPPAANLS VPLVPAASAA AAAAAVAKAS LFGDPEPVL DPESGEPIDP DAPNEFECED VEGDGDLLDG LGVGLGRQEM YAAMLAVKRL GEDAKRGVST VRFFGKFFGT Q ADYYVFET ...String:
MAADVGQALA FLQQVKTTQG ASIYEGLKAA LAKVLEDRPV NAVEALETSV LSTPPAANLS VPLVPAASAA AAAAAVAKAS LFGDPEPVL DPESGEPIDP DAPNEFECED VEGDGDLLDG LGVGLGRQEM YAAMLAVKRL GEDAKRGVST VRFFGKFFGT Q ADYYVFET TLQSNPDMPE APEGTIPLEP YGEGVNAYIY FVSNTLGGPL QQLPYVTPEQ IKASRLLRRY LTGRLDAPVS AF PAFPGNE ANYLRALIAR ISAATVCCPR GFFTADDDSA ELSANDEWVP LKGREMALPV NWSHRYAHLK GQGRTVTHKR DPP DEEEEP EKNFWTAEEM EAGPPPLATL DTDAPLPAAT GDKVPPPAWS PVFASASVTT RNQVAGVRSN RWPGAVCACA GRHF TSMYV GWGIKAGGEW SPCPPPPPVP QWGAPAAGVE GGQQLLLECN DLPPKPAPPE EEDE

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Macromolecule #4: Flagellar radial spoke protein 6

MacromoleculeName: Flagellar radial spoke protein 6 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 698.854 Da
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #5: Flagellar radial spoke protein 10

MacromoleculeName: Flagellar radial spoke protein 10 / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 528.644 Da
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #6: Radial spoke protein 10

MacromoleculeName: Radial spoke protein 10 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 23.553943 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MADDELPPQP VWEGPLDEDG KPHGLGKMEY PPPPMGEDDE EEKPGDKFEG TMEHGVRTGK GTYTWGVSGA VYTGDYVNGK KHGKGKMVY PDKGVYEGDW VEDVMQGQGT YTYPNGDIYQ GAFWAGKRHG KGMYHYKGPC CQLVGDWADG GFTYGRWVYA D GSMFMGKF ...String:
MADDELPPQP VWEGPLDEDG KPHGLGKMEY PPPPMGEDDE EEKPGDKFEG TMEHGVRTGK GTYTWGVSGA VYTGDYVNGK KHGKGKMVY PDKGVYEGDW VEDVMQGQGT YTYPNGDIYQ GAFWAGKRHG KGMYHYKGPC CQLVGDWADG GFTYGRWVYA D GSMFMGKF GGAAADSKPT AGSYFYSSSS LVQEGHFAKD GSWVGHRDPA VGKEFSVA

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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

Concentration0.1 mg/mL
BufferpH: 7.4 / Details: 30 mM HEPES, 150 mM NaCl, 1 mM TCEP
GridModel: Quantifoil R1.2/1.3 / Material: GOLD / Mesh: 300 / Support film - Material: GRAPHENE OXIDE
VitrificationCryogen name: ETHANE / Chamber humidity: 95 % / Chamber temperature: 293 K / Instrument: FEI VITROBOT MARK IV

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsIllumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 0.001 mm / Nominal defocus max: 2.5 µm / Nominal defocus min: 1.0 µm / Nominal magnification: 130000
Specialist opticsEnergy filter - Slit width: 30 eV
Image recordingFilm or detector model: GATAN K2 SUMMIT (4k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 2886 / Average electron dose: 73.3 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 1775590
CTF correctionSoftware - Name: Gctf
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9)
Final 3D classificationNumber classes: 3 / Avg.num./class: 150000 / Software - Name: cryoSPARC (ver. 2.9)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionResolution.type: BY AUTHOR / Resolution: 2.95 Å / Resolution method: FSC 0.143 CUT-OFF / Number images used: 251088

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Atomic model buiding 1

RefinementProtocol: AB INITIO MODEL
Output model

PDB-7jr9:
Chlamydomonas reinhardtii radial spoke minimal head complex

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