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- EMDB-20602: Cryo-EM structure of the 48-nm repeat unit of the doublet microtu... -

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Basic information

Entry
Database: EMDB / ID: EMD-20602
TitleCryo-EM structure of the 48-nm repeat unit of the doublet microtubule from Tetrahymena thermophila
Map data48-nm repeating unit of Tetrahymena doublet
Sample
  • Organelle or cellular component: 48nm-repeat of microtubule doublet
    • Complex: Tubulin lattice
      • Protein or peptide: Tubulin alpha chain
      • Protein or peptide: Tubulin beta chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordscilia / doublet / axoneme / microtubule inner protein / STRUCTURAL PROTEIN
Function / homology
Function and homology information


microtubule-based process / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / structural constituent of cytoskeleton / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
RIB43A / RIB43A / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site ...RIB43A / RIB43A / Tubulin-beta mRNA autoregulation signal. / Alpha tubulin / Beta tubulin, autoregulation binding site / Beta tubulin / Tubulin / Tubulin, C-terminal / Tubulin C-terminal domain / Tubulin, conserved site / Tubulin subunits alpha, beta, and gamma signature. / Tubulin/FtsZ family, C-terminal domain / Tubulin/FtsZ-like, C-terminal domain / Tubulin/FtsZ, C-terminal / Tubulin/FtsZ, 2-layer sandwich domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ family, GTPase domain / Tubulin/FtsZ, GTPase domain / Tubulin/FtsZ, GTPase domain superfamily
Similarity search - Domain/homology
RIB43A protein / Tubulin alpha chain / Tubulin beta chain / Protofilament ribbon protein
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.3 Å
AuthorsIchikawa M / Khalifa AAZ / Vargas J / Basu K / Bui KH
Funding support Canada, 3 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)DG69462 Canada
Canadian Institutes of Health Research (CIHR)PG388933 Canada
Natural Sciences and Engineering Research Council (NSERC, Canada)RGPIN-2018-04813 Canada
CitationJournal: Proc Natl Acad Sci U S A / Year: 2019
Title: Tubulin lattice in cilia is in a stressed form regulated by microtubule inner proteins.
Authors: Muneyoshi Ichikawa / Ahmad Abdelzaher Zaki Khalifa / Shintaroh Kubo / Daniel Dai / Kaustuv Basu / Mohammad Amin Faghfor Maghrebi / Javier Vargas / Khanh Huy Bui /
Abstract: Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic ...Cilia, the hair-like protrusions that beat at high frequencies to propel a cell or move fluid around are composed of radially bundled doublet microtubules. In this study, we present a near-atomic resolution map of the doublet microtubule by cryoelectron microscopy. The map demonstrates that the network of microtubule inner proteins weaves into the tubulin lattice and forms an inner sheath. From mass spectrometry data and de novo modeling, we identified Rib43a proteins as the filamentous microtubule inner proteins in the protofilament ribbon region. The Rib43a-tubulin interaction leads to an elongated tubulin dimer distance every 2 dimers. In addition, the tubulin lattice structure with missing microtubule inner proteins (MIPs) by sarkosyl treatment shows significant longitudinal compaction and lateral angle change between protofilaments. These results are evidence that the MIPs directly affect and stabilize the tubulin lattice. It suggests that the doublet microtubule is an intrinsically stressed filament and that this stress could be manipulated in the regulation of ciliary waveforms.
History
DepositionAug 14, 2019-
Header (metadata) releaseSep 11, 2019-
Map releaseSep 25, 2019-
UpdateMar 20, 2024-
Current statusMar 20, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
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  • Surface view colored by cylindrical radius
  • Surface level: 0.045
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  • Surface view with fitted model
  • Atomic models: PDB-6u0h
  • Surface level: 0.045
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  • Surface view with fitted model
  • Atomic models: PDB-6u0t
  • Surface level: 0.045
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  • Surface view with fitted model
  • Atomic models: PDB-6u0u
  • Surface level: 0.045
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u0h
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u0t
  • Imaged by Jmol
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-6u0u
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20602.png

Downloads & links

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Map

FileDownload / File: emd_20602.map.gz / Format: CCP4 / Size: 247.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Annotation48-nm repeating unit of Tetrahymena doublet
Voxel sizeX=Y=Z: 1.751 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.10797777 - 0.20274499
Average (Standard dev.)0.0025466024 (±0.012814775)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions402402402
Spacing402402402
CellA=B=C: 703.90204 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.7511.7511.751
M x/y/z402402402
origin x/y/z0.0000.0000.000
length x/y/z703.902703.902703.902
α/β/γ90.00090.00090.000
start NX/NY/NZ1548552
NX/NY/NZ198170217
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS402402402
D min/max/mean-0.1080.2030.003

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Supplemental data

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Sample components

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Entire : 48nm-repeat of microtubule doublet

EntireName: 48nm-repeat of microtubule doublet
Components
  • Organelle or cellular component: 48nm-repeat of microtubule doublet
    • Complex: Tubulin lattice
      • Protein or peptide: Tubulin alpha chain
      • Protein or peptide: Tubulin beta chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATEGuanosine triphosphate
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: 48nm-repeat of microtubule doublet

SupramoleculeName: 48nm-repeat of microtubule doublet / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB255 / Organelle: cilia / Location in cell: cilia

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Supramolecule #2: Tubulin lattice

SupramoleculeName: Tubulin lattice / type: complex / ID: 2 / Parent: 1 / Macromolecule list: #1-#2
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB255 / Organelle: cilia / Location in cell: cilia

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Macromolecule #1: Tubulin alpha chain

MacromoleculeName: Tubulin alpha chain / type: protein_or_peptide / ID: 1 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 49.639035 KDa
Recombinant expressionOrganism: Tetrahymena thermophila (eukaryote)
SequenceString: MREVISIHVG QGGIQVGNAC WELFCLEHGI QPDGQMPSDK TIGGGDDAFN TFFSETGAGK HVPRAVFLDL EPTVIDEVRT GTYRQLFHP EQLISGKEDA ANNFARGHYT IGKEIVDLCL DRIRKLADNC TGLQGFLVFN SVGGGTGSGL GSLLLERLSV D YGKKSKLG ...String:
MREVISIHVG QGGIQVGNAC WELFCLEHGI QPDGQMPSDK TIGGGDDAFN TFFSETGAGK HVPRAVFLDL EPTVIDEVRT GTYRQLFHP EQLISGKEDA ANNFARGHYT IGKEIVDLCL DRIRKLADNC TGLQGFLVFN SVGGGTGSGL GSLLLERLSV D YGKKSKLG FTIYPSPQVS TAVVEPYNSI LSTHSLLEHT DVAVMLDNEA IYDICRRNLD IERPTYTNLN RLIAQVISSL TA SLRFDGA LNVDITEFQT NLVPYPRIHF MLSSYAPIIS AEKAYHEQLS VAEITNSAFE PANMMAKCDP RHGKYMACSM MYR GDVVPK DVNASIATIK TKRTIQFVDW CPTGFKVGIN YQPPTVVPGG DLAKVMRAVC MISNSTAIAE VFSRLDHKFD LMYA KRAFV HWYVGEGMEE GEFSEAREDL AALEKDYEEV GIETAEGEGE EEGY

UniProtKB: Tubulin alpha chain

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Macromolecule #2: Tubulin beta chain

MacromoleculeName: Tubulin beta chain / type: protein_or_peptide / ID: 2 / Number of copies: 1 / Enantiomer: LEVO
Source (natural)Organism: Tetrahymena thermophila (eukaryote)
Molecular weightTheoretical: 49.617676 KDa
Recombinant expressionOrganism: Tetrahymena thermophila (eukaryote)
SequenceString: MREIVHIQGG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLERINVY YNEATGGRYV PRAILMDLEP GTMDSVRAGP FGQLFRPDN FVFGQTGAGN NWAKGHYTEG AELIDSVLDV VRKEAEGCDC LQGFQITHSL GGGTGSGMGT LLISKVREEY P DRIMETFS ...String:
MREIVHIQGG QCGNQIGAKF WEVISDEHGI DPTGTYHGDS DLQLERINVY YNEATGGRYV PRAILMDLEP GTMDSVRAGP FGQLFRPDN FVFGQTGAGN NWAKGHYTEG AELIDSVLDV VRKEAEGCDC LQGFQITHSL GGGTGSGMGT LLISKVREEY P DRIMETFS VVPSPKVSDT VVEPYNATLS VHQLVENADE CMVIDNEALY DICFRTLKLT TPTYGDLNHL VSAAMSGVTC CL RFPGQLN SDLRKLAVNL IPFPRLHFFM IGFAPLTSRG SQQYRALTVP ELTQQMFDAK NMMCAADPRH GRYLTASALF RGR MSTKEV DEQMLNVQNK NSSYFVEWIP NNIKSSICDI PPKGLKMAVT FVGNSTAIQE MFKRVAEQFT AMFRRKAFLH WYTG EGMDE MEFTEAESNM NDLVSEYQQY QDATAEEEGE FEEEEGEN

UniProtKB: Tubulin beta chain

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Macromolecule #3: GUANOSINE-5'-TRIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-TRIPHOSPHATE / type: ligand / ID: 3 / Number of copies: 1 / Formula: GTP
Molecular weightTheoretical: 523.18 Da
Chemical component information

ChemComp-GTP:
GUANOSINE-5'-TRIPHOSPHATE / GTP, energy-carrying molecule*YM / Guanosine triphosphate

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Macromolecule #4: MAGNESIUM ION

MacromoleculeName: MAGNESIUM ION / type: ligand / ID: 4 / Number of copies: 1 / Formula: MG
Molecular weightTheoretical: 24.305 Da

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Macromolecule #5: GUANOSINE-5'-DIPHOSPHATE

MacromoleculeName: GUANOSINE-5'-DIPHOSPHATE / type: ligand / ID: 5 / Number of copies: 1 / Formula: GDP
Molecular weightTheoretical: 443.201 Da
Chemical component information

ChemComp-GDP:
GUANOSINE-5'-DIPHOSPHATE / GDP, energy-carrying molecule*YM / Guanosine diphosphate

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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

Concentration2 mg/mL
BufferpH: 7.4
GridModel: Quantifoil R2/2 / Material: COPPER / Mesh: 200 / Support film - Material: CARBON / Support film - topology: HOLEY / Pretreatment - Type: GLOW DISCHARGE / Pretreatment - Time: 30 sec.
VitrificationCryogen name: ETHANE / Chamber humidity: 100 % / Chamber temperature: 298 K / Instrument: FEI VITROBOT MARK IV / Details: Blot force 3 for 5 seconds.

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Electron microscopy

MicroscopeFEI TITAN KRIOS
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELDBright-field microscopy / Cs: 2.7 mm / Nominal defocus max: 3.8000000000000003 µm / Nominal defocus min: 1.2 µm / Nominal magnification: 59000
Sample stageSpecimen holder model: FEI TITAN KRIOS AUTOGRID HOLDER / Cooling holder cryogen: NITROGEN
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Frames/image: 1-7 / Number real images: 5983 / Average exposure time: 1.4 sec. / Average electron dose: 45.0 e/Å2
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 60386
Startup modelType of model: EMDB MAP
EMDB ID:

8537

Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER (ver. 19.02) / Details: Hel
Final 3D classificationNumber classes: 3 / Software - Name: FREALIGN (ver. 9.03)
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: RELION (ver. 3.0), FREALIGN (ver. 9.03))
Final reconstructionNumber classes used: 1 / Applied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.3 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Details: Relion Gold Standard Refinement / Number images used: 60386
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 190
Output model

PDB-6u0h:
Tubulin lattice of the ciliary doublet microtubule from Tetrahymena thermophila

PDB-6u0t:
Protofilament Ribbon Flagellar Proteins Rib43a-S

PDB-6u0u:
Protofilament Ribbon Flagellar Proteins Rib43a-L

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