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- EMDB-20603: Cryo-EM map of the A-tubule prepared by sonication -

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Basic information

Entry
Database: EMDB / ID: EMD-20603
TitleCryo-EM map of the A-tubule prepared by sonication
Map dataCryo-EM map of the A-tubule prepared by sonication
Sample
  • Organelle or cellular component: 48nm-repeat of A-tubule from the doublet microtubule
    • Complex: Tubulin lattice
    • Complex: Microtubule Inner Proteins
Biological speciesTetrahymena thermophila (eukaryote)
Methodsingle particle reconstruction / cryo EM / Resolution: 4.4 Å
AuthorsIchikawa M / Khalifa AAZ / Vargas J / Basu K / Bui KH
Funding support Canada, 3 items
OrganizationGrant numberCountry
Natural Sciences and Engineering Research Council (Canada)DG69462 Canada
Natural Sciences and Engineering Research Council (Canada)RGPIN-2018-04813 Canada
Canadian Institutes of Health ResearchPG388933 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 25, 2019-
Map releaseSep 25, 2019-
UpdateOct 23, 2019-
Current statusOct 23, 2019Processing site: RCSB / Status: Released

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Structure visualization

Movie
  • Surface view with section colored by density value
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
  • Surface view colored by cylindrical radius
  • Surface level: 0.045
  • Imaged by UCSF Chimera
  • Download
Movie viewer
Structure viewerEM map:
SurfViewMolmilJmol/JSmol
Supplemental images

emd_20603.png

Downloads & links

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Map

FileDownload / File: emd_20603.map.gz / Format: CCP4 / Size: 247.8 MB / Type: IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
AnnotationCryo-EM map of the A-tubule prepared by sonication
Voxel sizeX=Y=Z: 1.751 Å
Density
Contour LevelBy AUTHOR: 0.045 / Movie #1: 0.045
Minimum - Maximum-0.08865335 - 0.1864288
Average (Standard dev.)0.0018866525 (±0.01074398)
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
MAP C/R/S123
start NC/NR/NS000
NC/NR/NS402402402
D min/max/mean-0.0890.1860.002

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Supplemental data

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Sample components

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Entire : 48nm-repeat of A-tubule from the doublet microtubule

EntireName: 48nm-repeat of A-tubule from the doublet microtubule
Components
  • Organelle or cellular component: 48nm-repeat of A-tubule from the doublet microtubule
    • Complex: Tubulin lattice
    • Complex: Microtubule Inner Proteins

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Supramolecule #1: 48nm-repeat of A-tubule from the doublet microtubule

SupramoleculeName: 48nm-repeat of A-tubule from the doublet microtubule / 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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Supramolecule #3: Microtubule Inner Proteins

SupramoleculeName: Microtubule Inner Proteins / type: complex / ID: 3 / Parent: 1
Source (natural)Organism: Tetrahymena thermophila (eukaryote) / Strain: SB255 / Organelle: cilia / Location in cell: cilia

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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
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 - Frames/image: 1-7 / Number real images: 7838 / 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: 100.0 µm / Illumination mode: FLOOD BEAM / Imaging mode: BRIGHT FIELD / Cs: 2.7 mm / Nominal defocus max: 3.8 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: 36375
CTF correctionSoftware - Name: RELION (ver. 3.0)
Startup modelType of model: EMDB MAP
EMDB ID:

8537

Final reconstructionApplied symmetry - Point group: C1 (asymmetric) / Algorithm: BACK PROJECTION / Resolution.type: BY AUTHOR / Resolution: 4.4 Å / Resolution method: FSC 0.143 CUT-OFF / Software - Name: RELION (ver. 3.0) / Number images used: 36375
Initial angle assignmentType: PROJECTION MATCHING / Software - Name: SPIDER (ver. 19.02) / Details: Hel
Final angle assignmentType: PROJECTION MATCHING / Software: (Name: RELION (ver. 3.0), FREALIGN (ver. 9.03))
Final 3D classificationNumber classes: 3 / Software - Name: FREALIGN (ver. 9.03)
FSC plot (resolution estimation)

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Atomic model buiding 1

RefinementSpace: REAL / Protocol: AB INITIO MODEL / Overall B value: 179

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