Cryo-electron tomography: gaining insight into cellular processes by structural approaches

Yahav, Tal; Maimon, Tal; Grossman, Einat; Dahan, Idit; Medalia, Ohad

doi:10.1016/j.sbi.2011.07.004

Cited by 44 publications

(27 citation statements)

References 66 publications

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“…Cryo-ET offers the most general means to acquire 3D information on large pleomorphic structures by using vitrification without any addition of fixatives to the sample (Ben-Harush et al, 2010;Lucic et al, 2005;Mader et al, 2010;Yahav et al, 2011). Hence, we applied cryo-ET to ice-embedded Xenopus spread NEs in order to dissect the supramolecular organization of Ce-lamin.…”

Section: Ce-lamin Assembles Into 5-6 Nm Protofilamentsmentioning

confidence: 99%

Filaments assembly of ectopically expressed Caenorhabditis elegans lamin within Xenopus oocytes

Grossman

Dahan

Stick

et al. 2012

Journal of Structural Biology

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Section: Ce-lamin Assembles Into 5-6 Nm Protofilamentsmentioning

confidence: 99%

Filaments assembly of ectopically expressed Caenorhabditis elegans lamin within Xenopus oocytes

Grossman

Dahan

Stick

et al. 2012

Journal of Structural Biology

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“…2D electron crystallography requires a planar or tubular crystal; the crystal's electron diffraction is used for 3D reconstruction potentially leading to atomic resolution 32 . In electron tomography of vitrified or traditionally fixed specimens, a macromolecule or sub-cellular component is rotated inside the TEM for subsequent tomographic reconstruction 33 , with the advantage that singular objects can be reconstructed; however at present this technique has a resolution limit ranging from 40 to 20 Å 34,35 . Among all these molecular TEM techniques, SPA of NS and cryoEM data has been the most widely used.…”

Section: Discussionmentioning

confidence: 99%

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Cabra

Samsó

2015

JoVE

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Citation: Cabra, V., Samsó, M. Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction. J. Vis. Exp. (95), e52311, doi:10.3791/52311 (2015). AbstractCryo-electron microscopy (cryoEM) entails flash-freezing a thin layer of sample on a support, and then visualizing the sample in its frozen hydrated state by transmission electron microscopy (TEM). This can be achieved with very low quantity of protein and in the buffer of choice, without the use of any stain, which is very useful to determine structure-function correlations of macromolecules. When combined with singleparticle image processing, the technique has found widespread usefulness for 3D structural determination of purified macromolecules.The protocol presented here explains how to perform cryoEM and examines the causes of most commonly encountered problems for rational troubleshooting; following all these steps should lead to acquisition of high quality cryoEM images. The technique requires access to the electron microscope instrument and to a vitrification device. Knowledge of the 3D reconstruction concepts and software is also needed for computerized image processing. Importantly, high quality results depend on finding the right purification conditions leading to a uniform population of structurally intact macromolecules.The ability of cryoEM to visualize macromolecules combined with the versatility of single particle image processing has proven very successful for structural determination of large proteins and macromolecular machines in their near-native state, identification of their multiple components by 3D difference mapping, and creation of pseudo-atomic structures by docking of x-ray structures. The relentless development of cryoEM instrumentation and image processing techniques for the last 30 years has resulted in the possibility to generate de novo 3D reconstructions at atomic resolution level.

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“…En optimisant les conditions d'imagerie, les deux feuillets de phospholipides peuvent alors être mis en évidence : les membranes se présentent comme deux lignes parallèles, denses aux électrons, correspondant aux têtes polaires des phospholipides, et séparées par une ligne claire correspondant à leurs chaînes aliphatiques (Figure 1). Au-delà de cette structure en bicouche déjà bien connue, on peut ainsi analyser les dissymétries d'épais-seur et de composition des deux feuillets, et découvrir la présence de membranes finement accolées au sein de plusieurs par cryo-tomographie [10,11] (qui permet de s'abstraire des problèmes de superposition, mais au prix d'une limitation de la résolution à quelques 4-7 nm [ Figure 2D] …”

Section: Visualiser Les Bicouches De Phospholipidesunclassified

Observer la cellule et ses structures membranaires à l’échelle du nanomètre

2012

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Cryo-electron tomography: gaining insight into cellular processes by structural approaches

Cited by 44 publications

References 66 publications

Filaments assembly of ectopically expressed Caenorhabditis elegans lamin within Xenopus oocytes

Filaments assembly of ectopically expressed Caenorhabditis elegans lamin within Xenopus oocytes

Do's and Don'ts of Cryo-electron Microscopy: A Primer on Sample Preparation and High Quality Data Collection for Macromolecular 3D Reconstruction

Observer la cellule et ses structures membranaires à l’échelle du nanomètre

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