Cytoskeletal Architecture and Macromolecular Structure in Intact Eukaryotic Cells Revealed by Cryo-Electron Tomography

2003 ◽  
Vol 9 (S03) ◽  
pp. 380-381
Author(s):  
O. Medalia ◽  
G. Gerisch ◽  
W. Baumeister
Keyword(s):  
Electron Tomography ◽  
Eukaryotic Cells ◽  
Macromolecular Structure ◽  
Cryo Electron Tomography

scholarly journals A flexible framework for multi-particle refinement in cryo-electron tomography

PLoS Biology ◽  
2021 ◽  
Vol 19 (8) ◽  
pp. e3001319
Author(s):  
Alister Burt ◽  
Lorenzo Gaifas ◽  
Tom Dendooven ◽  
Irina Gutsche
Keyword(s):  
Software Package ◽  
Electron Tomography ◽  
Macromolecular Structure ◽  
Computational Tools ◽  
Cryo Electron Tomography ◽  
Flexible Framework ◽  
Subtomogram Averaging ◽  
Particle Refinement ◽  
Hiv 1

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) are increasingly used for macromolecular structure determination in situ. Here, we introduce a set of computational tools and resources designed to enable flexible approaches to STA through increased automation and simplified metadata handling. We create a bidirectional interface between the Dynamo software package and the Warp-Relion-M pipeline, providing a framework for ab initio and geometrical approaches to multiparticle refinement in M. We illustrate the power of working within this framework by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles (VLPs), and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we provide a comprehensive, step-by-step guide to obtaining a 3.4-Å reconstruction from EMPIAR-10164. The guide is hosted on https://teamtomo.org/, a collaborative online platform we establish for sharing knowledge about cryo-ET.

scholarly journals Tools enabling flexible approaches to high-resolution subtomogram averaging

2021 ◽  
Author(s):  
Alister Burt ◽  
Lorenzo Gaifas ◽  
Tom Dendooven ◽  
Irina Gutsche
Keyword(s):  
Software Package ◽  
State Of The Art ◽  
Electron Tomography ◽  
Macromolecular Structure ◽  
Computational Tools ◽  
Cryo Electron Tomography ◽  
Subtomogram Averaging ◽  
Tomography Data ◽  
Hiv 1

AbstractCryo-electron tomography and subtomogram averaging are increasingly used for macromolecular structure determination in situ. Here we introduce a set of computational tools and resources designed to enable flexible approaches to subtomogram averaging. In particular, our tools simplify metadata handling, increase automation, and interface the Dynamo software package with the Warp-Relion-M pipeline. We provide a framework for ab initio and geometrical approaches to subtomogram averaging combining tools from these packages. We illustrate the power of working within the framework enabled by our developments by applying it to EMPIAR-10164, a publicly available dataset containing immature HIV-1 virus-like particles, and a challenging in situ dataset containing chemosensory arrays in bacterial minicells. Additionally, we establish an open and collaborative online platform for sharing knowledge and tools related to cryo-electron tomography data processing. To this platform, we contribute a comprehensive guide to obtaining state-of-the-art results from EMPIAR-10164.

scholarly journals Cryo-Electron Tomography of Frozen-Hydrated Sections of Eukaryotic Cells

2003 ◽  
Vol 9 (S02) ◽  
pp. 1166-1167 ◽  
Author(s):  
C. Schwartz ◽  
D. Nicastro ◽  
M.S. Ladinsky ◽  
D. Mastronarde ◽  
E. O'Toole ◽  
...  
Keyword(s):  
Electron Tomography ◽  
Eukaryotic Cells ◽  
Cryo Electron Tomography

scholarly journals Opening Windows into the Cell: Focused Ion Beam Micromachining of Eukaryotic Cells for Cryo-Electron Tomography

2013 ◽  
Vol 104 (2) ◽  
pp. 353a-354a ◽  
Author(s):  
Elizabeth Villa ◽  
Miroslava Schaffer ◽  
Alexander Rigort ◽  
Felix Bauerlein ◽  
Juergen Plitzko ◽  
...  
Keyword(s):  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Eukaryotic Cells ◽  
Cryo Electron Tomography

scholarly journals Reproducibility in Focused Ion Beam sample preparation - a Key Requirement for Cryo-Electron Tomography of Eukaryotic Cells

2013 ◽  
Vol 19 (S2) ◽  
pp. 876-877
Author(s):  
M. Schaffer ◽  
E. Villa ◽  
B. Engel ◽  
Y. Fukuda ◽  
T. Laugks ◽  
...  
Keyword(s):  
Sample Preparation ◽  
Focused Ion Beam ◽  
Electron Tomography ◽  
Ion Beam ◽  
Eukaryotic Cells ◽  
Beam Sample ◽  
Cryo Electron Tomography

Extended abstract of a paper presented at Microscopy and Microanalysis 2013 in Indianapolis, Indiana, USA, August 4 – August 8, 2013.

scholarly journals Whole-cell cryo-electron tomography of cultured and primary eukaryotic cells on micropatterned TEM grids

2021 ◽  
Author(s):  
Bryan S Sibert ◽  
Joseph Y Kim ◽  
Jie E Yang ◽  
Elizabeth R Wright
Keyword(s):  
Electron Tomography ◽  
Ion Beam ◽  
Physiological State ◽  
Light And Electron Microscopy ◽  
Eukaryotic Cells ◽  
Host Pathogen Interactions ◽  
Whole Cell ◽  
Wide Range ◽  
Cryo Electron Tomography ◽  
Host Pathogen

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technique that can provide nanometer-level resolution of biological structures within the cellular context and in a near-native frozen-hydrated state. It remains a challenge to culture or adhere cells on TEM grids in a manner that is suitable for tomography while preserving the physiological state of the cells. Here, we demonstrate the versatility of micropatterning to direct and promote growth of both cultured and primary eukaryotic cells on TEM grids. We show that micropatterning is compatible with and can be used to enhance studies of host-pathogen interactions using respiratory syncytial virus infected BEAS-2B cells as an example. We demonstrate the ability to use whole-cell tomography of primary Drosophila neuronal cells to identify organelles and cytoskeletal stuctures in cellular axons and the potential for micropatterning to dramatically increase throughput for these studies. During micropatterning, cell growth is targeted by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design make micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (FIB-SEM).

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