Advancements in the field of cryo-electron tomography have greatly contributed to our current understanding of prokaryotic cell organization and revealed intracellular structures with remarkable architecture. In this review, we present some of the prominent advancements in cryo-electron tomography, illustrated by a subset of structural examples to demonstrate the power of the technique. More specifically, we focus on technical advances in automation of data collection and processing, sample thinning approaches, correlative cryo-light and electron microscopy, and sub-tomogram averaging methods. In turn, each of these advances enabled new insights into bacterial cell architecture, cell cycle progression, and the structure and function of molecular machines. Taken together, these significant advances within the cryo-electron tomography workflow have led to a greater understanding of prokaryotic biology. The advances made the technique available to a wider audience and more biological questions and provide the basis for continued advances in the near future.
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http://dx.doi.org/10.1016/j.yjsbx.2022.100065 | DOI Listing |
Commun Biol
January 2025
Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA.
Rapid structural analysis of purified proteins and their complexes has become increasingly common thanks to key methodological advances in cryo-electron microscopy (cryo-EM) and associated data processing software packages. In contrast, analogous structural analysis in cells via cryo-electron tomography (cryo-ET) remains challenging due to critical technical bottlenecks, including low-throughput sample preparation and imaging, and laborious data processing methods. Here, we describe a rapid in situ cryo-ET sample preparation and data analysis workflow that results in the routine determination of sub-nm resolution ribosomal structures.
View Article and Find Full Text PDFNat Struct Mol Biol
January 2025
Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Cilia are motile or sensory organelles present on many eukaryotic cells. Their formation and function rely on axonemal microtubules, which exhibit very slow dynamics, but the underlying mechanisms are largely unexplored. Here we reconstituted in vitro the individual and collective activities of the ciliary tip module proteins CEP104, CSPP1, TOGARAM1, ARMC9 and CCDC66, which interact with each other and with microtubules and, when mutated in humans, cause ciliopathies such as Joubert syndrome.
View Article and Find Full Text PDFNat Commun
January 2025
Department of Chemistry, Korea Advanced Institute of Science and Technology, Daejeon, Republic of Korea.
Phase separation of specific proteins into liquid-like condensates is a key mechanism for forming membrane-less organelles, which organize diverse cellular processes in space and time. These protein condensates hold immense potential as biomaterials capable of containing specific sets of biomolecules with high densities and dynamic liquid properties. Despite their appeal, methods to manipulate protein condensate materials remain largely unexplored.
View Article and Find Full Text PDFSTAR Protoc
January 2025
Key Laboratory for Protein Sciences of Ministry of Education, School of Life Sciences, Tsinghua University, Beijing 100084, China; State Key Laboratory of Membrane Biology, School of Life Sciences, Tsinghua University, Beijing 100084, China; Tsinghua-Peking Joint Center for Life Sciences, Beijing 100084, China; Beijing Frontier Research Center for Biological Structure, Beijing 100084, China; School of Life Sciences, Tsinghua University, Beijing 100084, China. Electronic address:
Contrast transfer function (CTF) estimation is essential to the data processing workflow of cryo-electron tomography (cryoET). Here, we present a protocol for CTF estimation of the cryoET tilt series with CTFMeasure. CTFMeasure can estimate the CTF parameters together with the absolute tilt angle offset of the sample.
View Article and Find Full Text PDFJ Cell Biol
March 2025
Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Centrioles are microtubule-based organelles required for the formation of centrosomes and cilia. Centriolar microtubules, unlike their cytosolic counterparts, are stable and grow very slowly, but the underlying mechanisms are poorly understood. Here, we reconstituted in vitro the interplay between the proteins that cap distal centriole ends and control their elongation: CP110, CEP97, and CPAP/SAS-4.
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