Journal: 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.
Name: GUANOSINE-5'-TRIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
Mass
Theoretical: 0.52318 kDa
+
Component #5: ligand, MAGNESIUM ION
Ligand
Name: MAGNESIUM ION / Number of Copies: 1 / Recombinant expression: No
Mass
Theoretical: 2.430505 MDa
+
Component #6: ligand, GUANOSINE-5'-DIPHOSPHATE
Ligand
Name: GUANOSINE-5'-DIPHOSPHATE / Number of Copies: 1 / Recombinant expression: No
Mass
Theoretical: 0.443201 kDa
-
Experimental details
-
Sample preparation
Specimen state
filament
Sample solution
pH: 7.4
Vitrification
Instrument: FEI VITROBOT MARK IV / Cryogen name: ETHANE / Temperature: 298 K / Humidity: 100 % / Details: Blot force 3 for 5 seconds
-
Electron microscopy imaging
Experimental equipment
Model: Titan Krios / Image courtesy: FEI Company
Imaging
Microscope: FEI TITAN KRIOS
Electron gun
Electron source: FIELD EMISSION GUN / Accelerating voltage: 300 kV / Electron dose: 45 e/Å2 / Illumination mode: FLOOD BEAM
Lens
Magnification: 59000 X (nominal) / Cs: 2.7 mm / Imaging mode: BRIGHT FIELD / Defocus: 1200 - 3800 nm
Specimen Holder
Model: FEI TITAN KRIOS AUTOGRID HOLDER
Camera
Detector: FEI FALCON II (4k x 4k)
-
Image acquisition
Image acquisition
Number of digital images: 5983
-
Image processing
Processing
Method: single particle reconstruction / Applied symmetry: C1 (asymmetric) / Number of projections: 23
3D reconstruction
Software: Tom AV3 / Resolution: 4.6 Å / Resolution method: FSC 0.143 CUT-OFF Details: 23 boxes of the protofilament are boxed out from the doublet map (EMD-8528), aligned and averaged using subtomogram averaging software. Euler angles: Alignment using Tom AV3 software
wwPDB released updated PDB data conforming to the new PDBx/mmCIF dictionary. This is a major update changing the version number from 4 to 5, and with Remediation, in which all the entries are updated. See below links for details.
In this update, many items about electron microscopy experimental information are reorganized (e.g. em_software). Now, EM Navigator and Yorodumi are based on the updated data.
Yorodumi is a browser for structure data from EMDB, PDB, SASBDB, etc.
All the functionalities will be ported from the levgacy version.
This page is also the successor to EM Navigator detail page, and also detail information page/front-end page for Omokage search.
Related info.: Yorodumi (legacy version) / EMDB / PDB / SASBDB / Comparison of 3 databanks / Yorodumi Search / Yorodumi Papers / Jmol/JSmol / Function and homology information / Changes in new EM Navigator and Yorodumi