scholarly journals The in situ structures of mono-, di-, and trinucleosomes in human heterochromatin

2018 ◽  
Vol 29 (20) ◽  
pp. 2450-2457 ◽  
Author(s):  
Shujun Cai ◽  
Désirée Böck ◽  
Martin Pilhofer ◽  
Lu Gan
Keyword(s):  
Basic Principle ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Higher Order ◽  
Conformational Variability ◽  
Repressive Chromatin ◽  
Cryo Electron Tomography ◽  
Chromatin Folding

The in situ three-dimensional organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.

scholarly journals The in situ structures of mono-, di-, and tri-nucleosomes in human heterochromatin

2018 ◽  
Author(s):  
Shujun Cai ◽  
Désirée Böck ◽  
Martin Pilhofer ◽  
Lu Gan
Keyword(s):  
Basic Principle ◽  
Electron Tomography ◽  
Higher Order ◽  
Conformational Variability ◽  
Repressive Chromatin ◽  
Cryo Electron Tomography ◽  
Chromatin Folding

AbstractThe in situ 3-D organization of chromatin at the nucleosome and oligonucleosome levels is unknown. Here we use cryo-electron tomography (cryo-ET) to determine the in situ structures of HeLa nucleosomes, which have canonical core structures and asymmetric, flexible linker DNA. Subtomogram remapping suggests that sequential nucleosomes in heterochromatin follow irregular paths at the oligonucleosome level. This basic principle of higher-order repressive chromatin folding is compatible with the conformational variability of the two linker DNAs at the single-nucleosome level.

scholarly journals A guided approach for subtomogram averaging of challenging macromolecular assemblies

2020 ◽  
Author(s):  
Danielle Grotjahn ◽  
Saikat Chowdhury ◽  
Gabriel C. Lander
Keyword(s):  
Electron Tomography ◽  
A Priori ◽  
Three Dimensional ◽  
Structural Heterogeneity ◽  
Dimensional Structure ◽  
Diverse Range ◽  
Macromolecular Assemblies ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

scholarly journals Molecular architecture of inner dynein arms in situ in Chlamydomonas reinhardtii flagella

2008 ◽  
Vol 183 (5) ◽  
pp. 923-932 ◽  
Author(s):  
Khanh Huy Bui ◽  
Hitoshi Sakakibara ◽  
Tandis Movassagh ◽  
Kazuhiro Oiwa ◽  
Takashi Ishikawa
Keyword(s):  
Chlamydomonas Reinhardtii ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Molecular Architecture ◽  
Heavy Chains ◽  
Distal Side ◽  
Cryo Electron Tomography ◽  
Dynein Arms

The inner dynein arm regulates axonemal bending motion in eukaryotes. We used cryo-electron tomography to reconstruct the three-dimensional structure of inner dynein arms from Chlamydomonas reinhardtii. All the eight different heavy chains were identified in one 96-nm periodic repeat, as expected from previous biochemical studies. Based on mutants, we identified the positions of the AAA rings and the N-terminal tails of all the eight heavy chains. The dynein f dimer is located close to the surface of the A-microtubule, whereas the other six heavy chain rings are roughly colinear at a larger distance to form three dyads. Each dyad consists of two heavy chains and has a corresponding radial spoke or a similar feature. In each of the six heavy chains (dynein a, b, c, d, e, and g), the N-terminal tail extends from the distal side of the ring. To interact with the B-microtubule through stalks, the inner-arm dyneins must have either different handedness or, more probably, the opposite orientation of the AAA rings compared with the outer-arm dyneins.

scholarly journals In situ structure of virus capsids within cell nuclei by correlative light and cryo-electron tomography

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Swetha Vijayakrishnan ◽  
Marion McElwee ◽  
Colin Loney ◽  
Frazer Rixon ◽  
David Bhella
Keyword(s):  
Electron Microscopy ◽  
Structure Determination ◽  
Electron Tomography ◽  
3D Structure ◽  
Three Dimensional ◽  
Cell Nuclei ◽  
Nuclear Egress ◽  
Virus Capsids ◽  
Cryo Electron Tomography

Abstract Cryo electron microscopy (cryo-EM), a key method for structure determination involves imaging purified material embedded in vitreous ice. Images are then computationally processed to obtain three-dimensional structures approaching atomic resolution. There is increasing interest in extending structural studies by cryo-EM into the cell, where biological structures and processes may be imaged in context. The limited penetrating power of electrons prevents imaging of thick specimens (> 500 nm) however. Cryo-sectioning methods employed to overcome this are technically challenging, subject to artefacts or involve specialised and costly equipment. Here we describe the first structure of herpesvirus capsids determined by sub-tomogram averaging from nuclei of eukaryotic cells, achieved by cryo-electron tomography (cryo-ET) of re-vitrified cell sections prepared using the Tokuyasu method. Our reconstructions confirm that the capsid associated tegument complex is present on capsids prior to nuclear egress. We demonstrate that this method is suited to both 3D structure determination and correlative light/electron microscopy, thus expanding the scope of cryogenic cellular imaging.

scholarly journals Correlative cryogenic montage electron tomography for comprehensive in-situ whole-cell structural studies

2022 ◽  
Author(s):  
Jie E Yang ◽  
Matthew R Larson ◽  
Bryan S Sibert ◽  
Joseph Y Kim ◽  
Daniel Parrell ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Low Dose ◽  
Structural Information ◽  
Electron Tomography ◽  
Ion Beam ◽  
Three Dimensional ◽  
Structural Studies ◽  
Cryo Electron Tomography ◽  
Efficient Data

Imaging large fields of view while preserving high-resolution structural information remains a challenge in low-dose cryo-electron tomography. Here, we present robust tools for montage electron tomography tailored for vitrified specimens. The integration of correlative cryo-fluorescence microscopy, focused-ion beam milling, and micropatterning produces contextual three-dimensional architecture of cells. Montage tilt series may be processed in their entirety or as individual tiles suitable for sub-tomogram averaging, enabling efficient data processing and analysis.

Cryo electron tomography of vitrified fibroblasts: Microtubule plus ends in situ

2008 ◽  
Vol 161 (3) ◽  
pp. 459-468 ◽  
Author(s):  
Roman I. Koning ◽  
Sandra Zovko ◽  
Montserrat Bárcena ◽  
Gert T. Oostergetel ◽  
Henk K. Koerten ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Cryo Electron Tomography

Three-Dimensional Imaging of In Situ Specimens with Low-Dose Electron Tomography to Analyze Protein Conformation

2004 ◽  
Vol 2 (5) ◽  
pp. 561-567 ◽  
Author(s):  
Martina Banyay ◽  
Fredrik Gilstring ◽  
Elenor Hauzenberger ◽  
Lars-Göran Öfverstedt ◽  
Anders B. Eriksson ◽  
...  
Keyword(s):  
Protein Conformation ◽  
Low Dose ◽  
Electron Tomography ◽  
Three Dimensional ◽  
Three Dimensional Imaging

scholarly journals Structural insights into flagellar stator–rotor interactions

eLife ◽  
2019 ◽  
Vol 8 ◽  
Author(s):  
Yunjie Chang ◽  
Ki Hwan Moon ◽  
Xiaowei Zhao ◽  
Steven J Norris ◽  
MD A Motaleb ◽  
...  
Keyword(s):  
Conformational Change ◽  
High Speed ◽  
Electron Tomography ◽  
Deletion Mutants ◽  
Flagellar Motor ◽  
Wild Type ◽  
Cryo Electron Tomography ◽  
Flagellar Filament ◽  
Structural Insights

The bacterial flagellar motor is a molecular machine that can rotate the flagellar filament at high speed. The rotation is generated by the stator–rotor interaction, coupled with an ion flux through the torque-generating stator. Here we employed cryo-electron tomography to visualize the intact flagellar motor in the Lyme disease spirochete, Borrelia burgdorferi. By analyzing the motor structures of wild-type and stator-deletion mutants, we not only localized the stator complex in situ, but also revealed the stator–rotor interaction at an unprecedented detail. Importantly, the stator–rotor interaction induces a conformational change in the flagella C-ring. Given our observation that a non-motile mutant, in which proton flux is blocked, cannot generate the similar conformational change, we propose that the proton-driven torque is responsible for the conformational change required for flagellar rotation.

Sign in / Sign up

Export Citation Format

Share Document