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Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA - Nature (2010)

Bottom Line: The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template.The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins.The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, and Keck Advanced Microscopy Center, University of California, San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

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γTuSC filament structurea) Cryo-electron micrograph of γTuSC filaments. Scalebar, 50 nm. b) The resolution of the structure is estimated at 8 Å by the Fourier shell correlation. c) A segment of the reconstructed filament filtered to 8 Å. d) A cutaway view of the filament, illustrating the lack of connection between helical layers. e) One turn of the helix, coloured by γTuSC. The filament has six and a half γTuSCs per turn, with a half γTuSC overlap. f) A single γTuSC/Spc110p1-220 subunit from the filament, with the approximate boundaries between the individual proteins indicated by colour.
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Figure 2: γTuSC filament structurea) Cryo-electron micrograph of γTuSC filaments. Scalebar, 50 nm. b) The resolution of the structure is estimated at 8 Å by the Fourier shell correlation. c) A segment of the reconstructed filament filtered to 8 Å. d) A cutaway view of the filament, illustrating the lack of connection between helical layers. e) One turn of the helix, coloured by γTuSC. The filament has six and a half γTuSCs per turn, with a half γTuSC overlap. f) A single γTuSC/Spc110p1-220 subunit from the filament, with the approximate boundaries between the individual proteins indicated by colour.

Mentions: The N-terminal 220 residues of Spc110p (Spc110p1-220), which attaches γTuSC to the nuclear face of the yeast spindle pole body, dramatically increased the stability and length of γTuSC assemblies (Fig. 1d). Copurification with Spc110p1-220 yielded a continuum of γTuSC oligomers ranging from dimers to long, well-ordered helical filaments, even under conditions where γTuSC alone fails to assemble (Supplementary Fig. 2d–f). We determined the three-dimensional structure of γTuSC filaments from cryo-electron micrographs (Fig. 2a), using iterative helical real space reconstruction, a single particle approach to helical structure determination17. The resolution of the reconstruction, which included about 25,000 γTuSC subunits, was estimated at 8 Å by the Fourier shell correlation 0.5 cutoff (Fig. 2b).


Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.

Kollman JM, Polka JK, Zelter A, Davis TN, Agard DA - Nature (2010)

γTuSC filament structurea) Cryo-electron micrograph of γTuSC filaments. Scalebar, 50 nm. b) The resolution of the structure is estimated at 8 Å by the Fourier shell correlation. c) A segment of the reconstructed filament filtered to 8 Å. d) A cutaway view of the filament, illustrating the lack of connection between helical layers. e) One turn of the helix, coloured by γTuSC. The filament has six and a half γTuSCs per turn, with a half γTuSC overlap. f) A single γTuSC/Spc110p1-220 subunit from the filament, with the approximate boundaries between the individual proteins indicated by colour.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2921000&req=5

Figure 2: γTuSC filament structurea) Cryo-electron micrograph of γTuSC filaments. Scalebar, 50 nm. b) The resolution of the structure is estimated at 8 Å by the Fourier shell correlation. c) A segment of the reconstructed filament filtered to 8 Å. d) A cutaway view of the filament, illustrating the lack of connection between helical layers. e) One turn of the helix, coloured by γTuSC. The filament has six and a half γTuSCs per turn, with a half γTuSC overlap. f) A single γTuSC/Spc110p1-220 subunit from the filament, with the approximate boundaries between the individual proteins indicated by colour.
Mentions: The N-terminal 220 residues of Spc110p (Spc110p1-220), which attaches γTuSC to the nuclear face of the yeast spindle pole body, dramatically increased the stability and length of γTuSC assemblies (Fig. 1d). Copurification with Spc110p1-220 yielded a continuum of γTuSC oligomers ranging from dimers to long, well-ordered helical filaments, even under conditions where γTuSC alone fails to assemble (Supplementary Fig. 2d–f). We determined the three-dimensional structure of γTuSC filaments from cryo-electron micrographs (Fig. 2a), using iterative helical real space reconstruction, a single particle approach to helical structure determination17. The resolution of the reconstruction, which included about 25,000 γTuSC subunits, was estimated at 8 Å by the Fourier shell correlation 0.5 cutoff (Fig. 2b).

Bottom Line: The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template.The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins.The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, Howard Hughes Medical Institute, and Keck Advanced Microscopy Center, University of California, San Francisco, San Francisco, California 94158, USA.

ABSTRACT
Microtubules are nucleated in vivo by gamma-tubulin complexes. The 300-kDa gamma-tubulin small complex (gamma-TuSC), consisting of two molecules of gamma-tubulin and one copy each of the accessory proteins Spc97 and Spc98, is the conserved, essential core of the microtubule nucleating machinery. In metazoa multiple gamma-TuSCs assemble with other proteins into gamma-tubulin ring complexes (gamma-TuRCs). The structure of gamma-TuRC indicated that it functions as a microtubule template. Because each gamma-TuSC contains two molecules of gamma-tubulin, it was assumed that the gamma-TuRC-specific proteins are required to organize gamma-TuSCs to match 13-fold microtubule symmetry. Here we show that Saccharomyces cerevisiae gamma-TuSC forms rings even in the absence of other gamma-TuRC components. The yeast adaptor protein Spc110 stabilizes the rings into extended filaments and is required for oligomer formation under physiological buffer conditions. The 8-A cryo-electron microscopic reconstruction of the filament reveals 13 gamma-tubulins per turn, matching microtubule symmetry, with plus ends exposed for interaction with microtubules, implying that one turn of the filament constitutes a microtubule template. The domain structures of Spc97 and Spc98 suggest functions for conserved sequence motifs, with implications for the gamma-TuRC-specific proteins. The gamma-TuSC filaments nucleate microtubules at a low level, and the structure provides a strong hypothesis for how nucleation is regulated, converting this less active form to a potent nucleator.

Show MeSH
Related in: MedlinePlus