Electron cryotomography sample preparation using the Vitrobot

Iancu, Cristina V.; Tivol, William F.; Schooler, Jordan B; Dias, D. Prabha; Henderson, Gregory P.; Murphy, Gavin E.; Wright, Elizabeth; Li, Zhuo; Yu, Zhiheng; Briegel, Ariane; Gan, Lu; He, Yongning; Jensen, Grant J.

doi:10.1038/nprot.2006.432

Cited by 189 publications

(145 citation statements)

References 18 publications

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“…(36) and T. maritima cells were treated with 1 mg/mL polymyxin B for 10 h on ice. Cultures were plunge-frozen across EM grids as described (37). Standard EM tilt series were collected on 300 kV electron cryo-microscopes and 3D reconstructions were calculated as described (38,39).…”

Section: Resultsmentioning

confidence: 99%

Universal architecture of bacterial chemoreceptor arrays

Briegel

Ortega

Tocheva³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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301

405

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Chemoreceptors are key components of the high-performance signal transduction system that controls bacterial chemotaxis. Chemoreceptors are typically localized in a cluster at the cell pole, where interactions among the receptors in the cluster are thought to contribute to the high sensitivity, wide dynamic range, and precise adaptation of the signaling system. Previous structural and genomic studies have produced conflicting models, however, for the arrangement of the chemoreceptors in the clusters. Using whole-cell electron cryo-tomography, here we show that chemoreceptors of different classes and in many different species representing several major bacterial phyla are all arranged into a highly conserved, 12-nm hexagonal array consistent with the proposed ''trimer of dimers'' organization. The various observed lengths of the receptors confirm current models for the methylation, flexible bundle, signaling, and linker sub-domains in vivo. Our results suggest that the basic mechanism and function of receptor clustering is universal among bacterial species and was thus conserved during evolution.bacterial ultrastructure ͉ chemotaxis ͉ electron cryo-tomography

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Section: Resultsmentioning

confidence: 99%

Universal architecture of bacterial chemoreceptor arrays

Briegel

Ortega

Tocheva³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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301

405

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“…Other plungers that can be used and the protocols to use them are the Vitrobot (http://www.fei.com/) (see ref. 23), the Gatan cryoplunge (http://www.gatan.com/) and a time-resolved plunge-freezing apparatus. The time-resolved unit, which is made to order, is useful when attempting to resolve reactions that occur in the 10-ms timescale24.…”

Section: The Freeze-plungermentioning

confidence: 99%

Preparation of macromolecular complexes for cryo-electron microscopy

2007

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This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a threedimensional reconstruction of the macromolecular complex.

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“…The cryo-samples were prepared as reported previously (34). Briefly, 3 l of isolated axonemes (labeled or unlabeled for control) in HMEEK buffer and 1 l of 5-fold concentrated and BSA-treated 10 nm colloidal gold (Sigma) (35) were applied to Quantifoil holey carbon grids (copper R2/2, Quantifoil Micro Tools GmbH, Jena, Germany) that were previously coated with 10 nm colloidal gold. Excess fluid was blotted from the grid for ϳ2 s with Whatman filter paper and then plunge frozen in liquid ethane using a homemade plunge freezer to achieve sample vitrification.…”

Section: Methodsmentioning

confidence: 99%

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

Song

Awata

Tritschler

et al. 2015

Journal of Biological Chemistry

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Background: Techniques to localize proteins in situ at high resolution are important but limited. Results: Combining SNAP tag technology with cryo-electron tomography, we precisely localized proteins within the N-DRC that are important for ciliary motility. Conclusion: The developed method was applied to localize proteins with ϳ3 nm resolution without interfering with the complex function. Significance: The method is a powerful tool for studies of proteins in situ.

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Electron cryotomography sample preparation using the Vitrobot

Cited by 189 publications

References 18 publications

Universal architecture of bacterial chemoreceptor arrays

Universal architecture of bacterial chemoreceptor arrays

Preparation of macromolecular complexes for cryo-electron microscopy

In Situ Localization of N and C Termini of Subunits of the Flagellar Nexin-Dynein Regulatory Complex (N-DRC) Using SNAP Tag and Cryo-electron Tomography

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