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- EMDB-8539: Cryo-EM map of protofilament of microtubule doublet -

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Basic information

Entry
Database: EMDB / ID: EMD-8539
TitleCryo-EM map of protofilament of microtubule doublet
Map data
Sample
  • Organelle or cellular component: Protofilament from ciliary microtubule doublet
    • Protein or peptide: Tubulin alpha chain
    • Protein or peptide: Tubulin beta chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE
Keywordscilia / doublet / protofilament / tubulin / STRUCTURAL PROTEIN
Function / homology
Function and homology information


microtubule-based process / structural constituent of cytoskeleton / Hydrolases; Acting on acid anhydrides; Acting on GTP to facilitate cellular and subcellular movement / microtubule / hydrolase activity / GTPase activity / GTP binding / metal ion binding / cytoplasm
Similarity search - Function
Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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 ...Alpha tubulin / Tubulin-beta mRNA autoregulation signal. / 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
Tubulin alpha chain / Tubulin beta chain
Similarity search - Component
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.6 Å
AuthorsIchikawa M / Liu D
Funding support Canada, 1 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (NSERC, Canada)69462 Canada
CitationJournal: Nat Commun / Year: 2017
Title: Subnanometre-resolution structure of the doublet microtubule reveals new classes of microtubule-associated proteins.
Authors: Muneyoshi Ichikawa / Dinan Liu / Panagiotis L Kastritis / Kaustuv Basu / Tzu Chin Hsu / Shunkai Yang / Khanh Huy Bui /
Abstract: Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the ...Cilia are ubiquitous, hair-like appendages found in eukaryotic cells that carry out functions of cell motility and sensory reception. Cilia contain an intriguing cytoskeletal structure, termed the axoneme that consists of nine doublet microtubules radially interlinked and longitudinally organized in multiple specific repeat units. Little is known, however, about how the axoneme allows cilia to be both actively bendable and sturdy or how it is assembled. To answer these questions, we used cryo-electron microscopy to structurally analyse several of the repeating units of the doublet at sub-nanometre resolution. This structural detail enables us to unambiguously assign α- and β-tubulins in the doublet microtubule lattice. Our study demonstrates the existence of an inner sheath composed of different kinds of microtubule inner proteins inside the doublet that likely stabilizes the structure and facilitates the specific building of the B-tubule.
History
DepositionDec 22, 2016-
Header (metadata) releaseFeb 15, 2017-
Map releaseMay 10, 2017-
UpdateDec 25, 2024-
Current statusDec 25, 2024Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view colored by height
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Surface view with fitted model
  • Atomic models: PDB-5ucy
  • Surface level: 0.06
  • Imaged by UCSF Chimera
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  • Simplified surface model + fitted atomic model
  • Atomic modelsPDB-5ucy
  • Imaged by Jmol
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Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_8539.png

Downloads & links

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Map

FileDownload / File: emd_8539.map.gz / Format: CCP4 / Size: 27.9 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
Projections & slices

Image control

Size
Brightness
Contrast
Others
AxesZ (Sec.)Y (Row.)X (Col.)
1.38 Å/pix.
x 194 pix.
= 266.75 Å		1.38 Å/pix.
x 194 pix.
= 266.75 Å		1.38 Å/pix.
x 194 pix.
= 266.75 Å

Surface

Projections

Slices (1/3)

Slices (1/2)

Slices (2/3)

Images are generated by Spider.

Voxel sizeX=Y=Z: 1.375 Å
Density

Histogram

Histogram (log scale)
Contour LevelBy AUTHOR: 0.06 / Movie #1: 0.06
Minimum - Maximum-0.03319637 - 0.14551778
Average (Standard dev.)0.0012695125 (±0.008444522)
SymmetrySpace group: 1
Details

EMDB XML:

Map geometry
Axis orderXYZ
Origin000
Dimensions194194194
Spacing194194194
CellA=B=C: 266.75 Å
α=β=γ: 90.0 °

CCP4 map header:

modeImage stored as Reals
Å/pix. X/Y/Z1.3751.3751.375
M x/y/z194194194
origin x/y/z0.0000.0000.000
length x/y/z266.750266.750266.750
α/β/γ90.00090.00090.000
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS194194194
D min/max/mean-0.0330.1460.001

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Supplemental data

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Sample components

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Entire : Protofilament from ciliary microtubule doublet

EntireName: Protofilament from ciliary microtubule doublet
Components
  • Organelle or cellular component: Protofilament from ciliary microtubule doublet
    • Protein or peptide: Tubulin alpha chain
    • Protein or peptide: Tubulin beta chain
  • Ligand: GUANOSINE-5'-TRIPHOSPHATE
  • Ligand: MAGNESIUM ION
  • Ligand: GUANOSINE-5'-DIPHOSPHATE

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Supramolecule #1: Protofilament from ciliary microtubule doublet

SupramoleculeName: Protofilament from ciliary microtubule doublet / type: organelle_or_cellular_component / ID: 1 / Parent: 0 / Macromolecule list: #1-#2
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB255

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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: 48.788254 KDa
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 GIETAE

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: 48.009199 KDa
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 QDAT

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

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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

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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

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
Image recordingFilm or detector model: FEI FALCON II (4k x 4k) / Detector mode: INTEGRATING / Digitization - Dimensions - Width: 4096 pixel / Digitization - Dimensions - Height: 4096 pixel / Digitization - Frames/image: 1-7 / Number grids imaged: 4 / Number real images: 5983 / Average exposure time: 1.4 sec. / Average electron dose: 45.0 e/Å2
Electron beamAcceleration voltage: 300 kV / Electron source: FIELD EMISSION GUN
Electron opticsC2 aperture diameter: 50.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / 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
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company

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Image processing

Particle selectionNumber selected: 127428
Startup modelType of model: EMDB MAP
EMDB ID:

8528

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Resolution.type: BY AUTHOR / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: AV3
Details: 23 boxes of the protofilament are boxed out from the doublet map (EMD-8528), aligned and averaged using subtomogram averaging software.
Number images used: 23
Initial angle assignmentType: OTHER / Software - Name: MATLAB / Details: Geometry calculated from the microtubule doublet
Final angle assignmentType: OTHER / Software - Name: AV3 / Details: Alignment using Tom AV3 software

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: BACKBONE TRACE / Target criteria: minimization global
Output model

PDB-5ucy:
Cryo-EM map of protofilament of microtubule doublet

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