This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Axonemal dyneins are large, microtubule-based motor enzymes that convert the chemical energy derived from ATP binding and hydrolysis into the mechanical forces that bend cilia and flagella. While great progress has been made in understanding some aspects of flagellar mechanisms, much remains to be learned about the function of individual dynein subunits, their structural organization within the axoneme s 96 nm axial repeat, and the relationship between the motors and the regulatory machinery that coordinates their pattern of beat. We are therefore studying the 3-D structure of wild-type and mutant flagella using cryo-electron tomography in combination with correlation averaging (see Subproject T0066) to improve the signal-to-noise ratio in the tomographic reconstructions, and thus to enhance their resolution. We have recorded several tilt series of demembranated and rapidly frozen axonemes isolated from wild-type Chlamydomonas and from different inner dynein arm mutant strains (pf9, 6F5, pf10). The 3-D reconstructions and volume averages of the 96nm repeat of Chlamydomonas wild type axonemes confirm the main structural features of the inner and outer dynein arm complexes. However, in comparison to the 2-D pictures provided by previous structural studies, the tomographic volumes allow a 3D analysis and reveal new structural details, such as two novel linker structures between the inner and outer dynein arm complexes. We are using 3-D difference maps of averaged wild type and mutant axonemes to determine in situ the structural alterations that result from specific polypeptide defects.
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| Zhao, Xiaowei; Schwartz, Cindi L; Pierson, Jason et al. (2017) Three-Dimensional Structure of the Ultraoligotrophic Marine Bacterium ""Candidatus Pelagibacter ubique"". Appl Environ Microbiol 83: |
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| Park, J Genevieve; Palmer, Amy E (2015) Properties and use of genetically encoded FRET sensors for cytosolic and organellar Ca2+ measurements. Cold Spring Harb Protoc 2015:pdb.top066043 |
| McCoy, Kelsey M; Tubman, Emily S; Claas, Allison et al. (2015) Physical limits on kinesin-5-mediated chromosome congression in the smallest mitotic spindles. Mol Biol Cell 26:3999-4014 |
| Höög, Johanna L; Lötvall, Jan (2015) Diversity of extracellular vesicles in human ejaculates revealed by cryo-electron microscopy. J Extracell Vesicles 4:28680 |
| Marc, Robert E; Anderson, James R; Jones, Bryan W et al. (2014) The AII amacrine cell connectome: a dense network hub. Front Neural Circuits 8:104 |
| Weber, Britta; Tranfield, Erin M; Höög, Johanna L et al. (2014) Automated stitching of microtubule centerlines across serial electron tomograms. PLoS One 9:e113222 |
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