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- EMDB-22446: Chlamydomonas reinhardtii radial spoke head and neck (recombinant) -

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Basic information

Entry
Database: EMDB / ID: EMD-22446
TitleChlamydomonas reinhardtii radial spoke head and neck (recombinant)
Map data
Sample
  • Complex: Chlamydomonas reinhardtii radial spoke head and neck
    • Protein or peptide: x 14 types
Function / homology
Function and homology information


radial spoke head / radial spoke / positive regulation of cilium-dependent cell motility / cilium movement involved in cell motility / motile cilium assembly / axoneme assembly / Set1C/COMPASS complex / UTP biosynthetic process / CTP biosynthetic process / Oxidoreductases ...radial spoke head / radial spoke / positive regulation of cilium-dependent cell motility / 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 / membrane / 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 kinases active site. / 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)
Methodsingle particle reconstruction / cryo EM / Resolution: 3.03 Å
AuthorsGrossman-Haham I / Coudray N / Yu Z / Wang F / Zhang N / 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 12, 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
  • Download
  • Surface view colored by radius
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
  • Surface view with fitted model
  • Atomic models: PDB-7jrj
  • Surface level: 0.015
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

Downloads & links

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Map

FileDownload / File: emd_22446.map.gz / Format: CCP4 / Size: 600.7 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Voxel sizeX=Y=Z: 0.835 Å
Density
Contour LevelBy AUTHOR: 0.015 / Movie #1: 0.015
Minimum - Maximum-0.035352327 - 0.0930655
Average (Standard dev.)0.0001230415 (±0.0018453321)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions540540540
Spacing540540540
CellA=B=C: 450.9 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z0.8350.8350.835
M x/y/z540540540
origin x/y/z0.0000.0000.000
length x/y/z450.900450.900450.900
α/β/γ90.00090.00090.000
start NX/NY/NZ1081340
NX/NY/NZ13584349
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS540540540
D min/max/mean-0.0350.0930.000

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Supplemental data

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Additional map: Focused map for the RSP1 protrusion

Fileemd_22446_additional_1.map
AnnotationFocused map for the RSP1 protrusion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Focused map for the RSP3 protrusion

Fileemd_22446_additional_2.map
AnnotationFocused map for the RSP3 protrusion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Focused map for the RSP2 protrusion

Fileemd_22446_additional_3.map
AnnotationFocused map for the RSP2 protrusion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Focused map for the RSP10 protrusion

Fileemd_22446_additional_4.map
AnnotationFocused map for the RSP10 protrusion
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: Focused map for the radial spoke arm region

Fileemd_22446_additional_5.map
AnnotationFocused map for the radial spoke arm region
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Additional map: composite map

Fileemd_22446_additional_6.map
Annotationcomposite map
Projections & Slices
AxesZYX

Projections

Slices (1/2)
Density Histograms

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Sample components

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Entire : Chlamydomonas reinhardtii radial spoke head and neck

EntireName: Chlamydomonas reinhardtii radial spoke head and neck
Components
  • Complex: Chlamydomonas reinhardtii radial spoke head and neck
    • Protein or peptide: Radial spoke protein 10
    • Protein or peptide: Flagellar radial spoke protein 1
    • Protein or peptide: Nucleoside diphosphate kinase 6Nucleoside-diphosphate kinase
    • Protein or peptide: Flagellar radial spoke protein 2
    • Protein or peptide: unknown protein
    • Protein or peptide: Flagellar radial spoke protein 3
    • Protein or peptide: Flagellar radial spoke protein 5
    • 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: Flagellar radial spoke protein 1
    • Protein or peptide: Flagellar radial spoke protein 2

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Supramolecule #1: Chlamydomonas reinhardtii radial spoke head and neck

SupramoleculeName: Chlamydomonas reinhardtii radial spoke head and neck / 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 10

MacromoleculeName: Radial spoke protein 10 / type: protein_or_peptide / ID: 1 / 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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Macromolecule #2: Flagellar radial spoke protein 1

MacromoleculeName: Flagellar radial spoke protein 1 / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 87.873445 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MALGPFVLPF RGDQYSFGIN FKSSPEEKLN FDLSCVAFDV KGQLHDTLHA RKPTALDGAL VKGFEKQALP EETVQVEGDD VIYMFPKKF ERQVEVLLFV ASAPSIPGKK HDLDSSSKLE FAVSYSDVGG QAFNQSFDLK PLAAQGGVSS IIVAVMYLQA E GGWTLRSV ...String:
MALGPFVLPF RGDQYSFGIN FKSSPEEKLN FDLSCVAFDV KGQLHDTLHA RKPTALDGAL VKGFEKQALP EETVQVEGDD VIYMFPKKF ERQVEVLLFV ASAPSIPGKK HDLDSSSKLE FAVSYSDVGG QAFNQSFDLK PLAAQGGVSS IIVAVMYLQA E GGWTLRSV GDCHPFDSPG LIVPELKQTI LNLRDHHGVQ LDAADAIQAI DPAERVPVTR QFQDQSLDEA SAGRAAEPAP VK KLRIDLS WTFWPPPPPT EEGEEPPEEP ALEYNLVMYN KDGEEVQSIS TGNREATGAR AGRPEPEEDE EEEKEEEKEE PEE GEEGEE GEGGEPKEPP PPPPAPKVDP YEFKERDVIY LDVPDLPAEV RSMVLLVTNY DEENGFTRVR TVRCRLVDVS NGEA PLPGS KAAVAAAAAA AEQGLAAPPN PERVLADYGV LSKYEDDKAT TQVALMKLYK EYADSAFNVF RGAGVDNVAA FIGQE PDTI INQLKAYLEA TKKQKAAEAA AAAAAEESGE EITADPKPHV WRFRALGLNF GGDSLEAIEH DLKNLFAFDG DLAPGA ARD SDTSRSSFPN GDTYFGSYAD DVKHGPGLYA FATGAGYAGE YAGGKRHGRG VMVFPDGGTY VGEFVADKFE GQGQYRY PD GSVYTGSWAA GQKHGPGVYW DTARGCLRGE WKKGLLVGKG TYEQPALRFE GEFVRGMPAG TATYTLTGHR TLDMPCFA A QHIQAEEGPT LALPCAYGIP PGSGDEPQLD EEGQPIEDTD KPPLPAHPKY EGLTFTAEQL PGAAPDTVFP PEEGKPVPI TAVPAFSVST GLVA

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Macromolecule #3: Nucleoside diphosphate kinase 6

MacromoleculeName: Nucleoside diphosphate kinase 6 / type: protein_or_peptide / ID: 3 / Number of copies: 1 / Enantiomer: LEVO / EC number: nucleoside-diphosphate kinase
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 61.422211 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAELEKTFAL IKPDAVRAGK AQEIMQLIEL NGFTIIAKQK LQLTRARAEE FYGEHKGKEF FPKLVNFMTS GPIWALVLAK PGAILAWRA LMGPTNVFKA RAEQPKCLRA LYGTDGTQNA THGSDSPISA AREIKFFFPT LSGDPTIYAE PTAAAEYITK R IQPALAKA ...String:
MAELEKTFAL IKPDAVRAGK AQEIMQLIEL NGFTIIAKQK LQLTRARAEE FYGEHKGKEF FPKLVNFMTS GPIWALVLAK PGAILAWRA LMGPTNVFKA RAEQPKCLRA LYGTDGTQNA THGSDSPISA AREIKFFFPT LSGDPTIYAE PTAAAEYITK R IQPALAKA LAALAREKPS ADKFEAITFV AGYLLQNNPN KPKVLMPDEW DPALMGDDED DEADFINARL AAPTGNDGAT KA EFDAMVE AAKADTGGAA PAQEFVYPDS KPTTPVVPAP PAPGSKPPSA SGARPQSARP TSARPPSASA APPAPLAPVP PPA SSSRPA SGSGRPPSAT ARPPSATPPP PPPAVELEEA DDPAQLDEAA TKVQAAFRGY QARKEVAVMR SEAQPGEAAA EPEQ EAELQ PEAEPEPQPE GEQEPQPQAS ASSSFLPDGV TEEMAAEAAT RVQAHMRGHL ARKQVAAIKA QQAAPAVAES SEALA EPEP QPEAEAEPQP QASVSSSFLP DGVTEEMAAE AATLVQAHMR GHLARKQVAA IKAQQAAPAV AESSEAQAEA EQTEAE AEP QPDAEAEAGA AEGEAEPEPE AAE

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Macromolecule #4: Flagellar radial spoke protein 2

MacromoleculeName: Flagellar radial spoke protein 2 / type: protein_or_peptide / ID: 4 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 77.445297 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MAPTQAGHDT AYLKETVGEA LARGCAAAIS AQPNDPVEYL GLWLLKYVKN AEVEGNFYRE RQQDLQKKKD RLVKEAQSEQ AAKSVALTR KEAADALALV TAEPRELLEA AVKLVKQHTA AGAAYAAVVA EPEEPDWVAP EDDEAAAVET EDEAAGGAAL A EGEEPPPE ...String:
MAPTQAGHDT AYLKETVGEA LARGCAAAIS AQPNDPVEYL GLWLLKYVKN AEVEGNFYRE RQQDLQKKKD RLVKEAQSEQ AAKSVALTR KEAADALALV TAEPRELLEA AVKLVKQHTA AGAAYAAVVA EPEEPDWVAP EDDEAAAVET EDEAAGGAAL A EGEEPPPE PEPEPEAAPE DGEGDAPAPK IPRPVDYSKK YFAYVAASAG QEHVLEADLY RPAPPPEDAD EDFKPEPLPY SF RVLDEKL PMLYVPNVAA EERVKFFRKF PKIGSYQACG VALPASGEFK ALLAADTLFP EGSGQPLSAD DRDFVWEVSQ SLS RALEAV QARAAEALAA TSAAEAVEEL KAKVAELREQ AAAEAAAAAP PPPAEGEEGE GEAPPAEEEP PAEEEAEEEE EEAE EGAEE GAEEGEEGEE APPKPKKKKK VFNPIPGLQA AIEKLTAAAE AATEADARAQ AAVALEKQAL DEVVALASSH SDATL SSLR NMLSVPQGTY HVVKALLHLL GRPAASFSTW KRAHSHFSPR LFEDMAAYDA TAERDMAVWG RVRSCYKAAP AAKKLD AEM PNTLFGSVAL MYIKQVRRVA RKAVLQRELA AKLAKAQQDL ADKQAALVEA ERVKAEREAE EARLAAEAEA AAAAEAE AA ARAAAEAEAA AAAEAAAEAA AEAAAAAAEA AAEAGEGEAV AEREAAPAEA EAAPAEGEAA PPAEGEGEAQ PAQEGSNS S SSSSDSSSSE ESKAAAE

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Macromolecule #5: unknown protein

MacromoleculeName: unknown protein / type: protein_or_peptide / ID: 5 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 358.434 Da
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
(UNK)(UNK)(UNK)(UNK)

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Macromolecule #6: Flagellar radial spoke protein 3

MacromoleculeName: Flagellar radial spoke protein 3 / type: protein_or_peptide / ID: 6 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 39.361293 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GAAAEPILEV LVGKVLEQGL MEVLEEEELA AMRAHQEHFE QIRNAELVAT QRMEAAERRK LEEKERRMQQ ERERVERERV VRQKVAASA FARGYLSGIV NTVFDRLVSS GYIYDPVMRE VETAFMPWLK EQAIGYLARG VVARRVVDKL VEDAAAALAA N RSTLADKA ...String:
GAAAEPILEV LVGKVLEQGL MEVLEEEELA AMRAHQEHFE QIRNAELVAT QRMEAAERRK LEEKERRMQQ ERERVERERV VRQKVAASA FARGYLSGIV NTVFDRLVSS GYIYDPVMRE VETAFMPWLK EQAIGYLARG VVARRVVDKL VEDAAAALAA N RSTLADKA ASTAATVDAW AERQAKMEAE LQGKELEAVR RRPTFVLREL KPAVASADAV EAAAAELTAQ AEEAANAKWE AD KAEAAEK ARAEAEAAAE EQKALLEELA ATAAAEAEER GEEPPAEPPS LPDGVEPVDV EAEVAKAVEA VPKPPVKEVT DID ILSYMM DKGAITKDAI IQALAVHALG DKAYTNHPAF AEAEGA

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Macromolecule #7: Flagellar radial spoke protein 5

MacromoleculeName: Flagellar radial spoke protein 5 / type: protein_or_peptide / ID: 7 / Number of copies: 1 / Enantiomer: LEVO / EC number: Oxidoreductases
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 55.92818 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: MSEPGEEPVA APAGPAPDPV LNELYGSERP AVELLPGVPL SPIVNSCWLP ADAKAMLAES WIPVPPEDAG EEAGPPPPAF EAAAPEYNE LVRRLAKTAP FRKWNELTIQ AKQLEQEVAG LKGPDAEAKQ AELENVKVQI ADAEAAVAEV KQSFSDDPLS L TGWMQALT ...String:
MSEPGEEPVA APAGPAPDPV LNELYGSERP AVELLPGVPL SPIVNSCWLP ADAKAMLAES WIPVPPEDAG EEAGPPPPAF EAAAPEYNE LVRRLAKTAP FRKWNELTIQ AKQLEQEVAG LKGPDAEAKQ AELENVKVQI ADAEAAVAEV KQSFSDDPLS L TGWMQALT DLADGGMTTF EVSGQGWPYC SLRQLFGEMP SAAPPAGFFD GVERVLGTFK RRYEKERGPG SVQLMLKLAP NV FSDAWST GGAPAAVAAV EAYVERARAN VFGPDGGVTP EGVPEPLDLV QLVWWDFAAA DPLPVLKALQ RMATDQLQVD EDS GEVSVS EPKKIRGIGL VDFPADRLKA AIQAGVPITC VQVEHSVLVR SAQPVLDLCA KYGIKVLARG GTLGGLLSAK YLGA PPPDP VRGDADLDSV PGCLDAVNNV GGWARLQAAL AVIKGIADKH GVKPETVALR WQIDAGCFPL VTTRWSSRVW RQFGY EGWS SFEVSGGRPG VDGPLFQVES FLDVEDVRAL AGLAAVHLGP KAG

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Macromolecule #8: Radial spoke protein 9

MacromoleculeName: Radial spoke protein 9 / type: protein_or_peptide / ID: 8 / 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 #9: Flagellar radial spoke protein 4

MacromoleculeName: Flagellar radial spoke protein 4 / type: protein_or_peptide / ID: 9 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 49.90082 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: GMAAVDSVAQ ALAYLQVHSP QDGTSMYDHL VKLVSKVLED QPKNAVDLLE TSLLVKKSTF DPKESSPLVP IPVAPDATQT QAAVSIFGD PELPINPATG EPVPADPPNE FEAENMLGAA AVLDCLGVGL GRELGVNIAL AAKRIGEDPK LAVRSVRFFG K FLGLYSDY ...String:
GMAAVDSVAQ ALAYLQVHSP QDGTSMYDHL VKLVSKVLED QPKNAVDLLE TSLLVKKSTF DPKESSPLVP IPVAPDATQT QAAVSIFGD PELPINPATG EPVPADPPNE FEAENMLGAA AVLDCLGVGL GRELGVNIAL AAKRIGEDPK LAVRSVRFFG K FLGLYSDY FVFEVAFKKE AAKEAAPAAP APERVEGEAA SSSAPEVPVE EPGKGANKFT YLVCSSLGGP LTRLPDVTPA QV KASRRIK KLLTGRLTSH VSTYPAFPGN EANYLRALIA RISAATVVAP SDLFSLNDET GELERAEDWE PPAGREMAAP TAW VHVRPH LKSQGRCEVH KRELPEDADE DEFYNEDELE EGPDLLAALE EDAQLPGEQA AWTPIYSSAS EAVKTQAGGL RSLV WPGAV CGGRGSEWTC VYVGWGVKNA PFVPLPPPPV AQEFAWGEVE TQELELKPAP PPPEEEAEAD E

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Macromolecule #10: Flagellar radial spoke protein 6

MacromoleculeName: Flagellar radial spoke protein 6 / type: protein_or_peptide / ID: 10 / 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 #11: Flagellar radial spoke protein 6

MacromoleculeName: Flagellar radial spoke protein 6 / type: protein_or_peptide / ID: 11 / 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 #12: Flagellar radial spoke protein 10

MacromoleculeName: Flagellar radial spoke protein 10 / type: protein_or_peptide / ID: 12 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 443.539 Da
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString:
(UNK)(UNK)(UNK)(UNK)(UNK)

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Macromolecule #13: Flagellar radial spoke protein 1

MacromoleculeName: Flagellar radial spoke protein 1 / type: protein_or_peptide / ID: 13 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 7.59235 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)

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Macromolecule #14: Flagellar radial spoke protein 2

MacromoleculeName: Flagellar radial spoke protein 2 / type: protein_or_peptide / ID: 14 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Chlamydomonas reinhardtii (plant)
Molecular weightTheoretical: 5.890252 KDa
Recombinant expressionOrganism: Spodoptera frugiperda (fall armyworm)
SequenceString: (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK) ...String:
(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK)(UNK)(UNK)(UNK)(UNK)(UNK) (UNK)(UNK) (UNK)(UNK)(UNK)

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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, 300 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: 295 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 / Nominal defocus max: 1.5 µm / Nominal defocus min: 0.8 µm / Nominal magnification: 105000
Image recordingFilm or detector model: GATAN K3 (6k x 4k) / Detector mode: SUPER-RESOLUTION / Number grids imaged: 2 / Number real images: 7572 / Average electron dose: 70.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 942591
CTF correctionSoftware - Name: Gctf
Startup modelType of model: INSILICO MODEL
In silico model: A model for radial spoke protein 23 (chain K) was generated in Phyre2. The rest was built de-novo.
Initial angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: cryoSPARC (ver. 2.9)
Final 3D classificationNumber classes: 8 / Avg.num./class: 100000 / Software - Name: RELION (ver. 3.0)
Final angle assignmentType: MAXIMUM LIKELIHOOD / Software - Name: RELION (ver. 3.0)
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 3.03 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 136659

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Atomic model buiding 1

Initial modelPDB ID:
RefinementSpace: REAL / Protocol: RIGID BODY FIT
Output model

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

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