Fully automated, sequential focused ion beam milling for cryo-electron tomography
- ETH Zürich, Switzerland
- Carl Zeiss Microscopy GmbH, Germany
Abstract
Cryo-electron tomography (cryoET) has become a powerful technique at the interface of structural biology and cell biology, due to its unique ability for imaging cells in their native state and determining structures of macromolecular complexes in their cellular context. A limitation of cryoET is its restriction to relatively thin samples. Sample thinning by cryo-focused ion beam (cryoFIB) milling has significantly expanded the range of samples that can be analyzed by cryoET. Unfortunately, cryoFIB milling is low-throughput, time-consuming and manual. Here we report a method for fully automated sequential cryoFIB preparation of high-quality lamellae, including rough milling and polishing. We reproducibly applied this method to eukaryotic and bacterial model organisms, and show that the resulting lamellae are suitable for cryoET imaging and subtomogram averaging. Since our method reduces the time required for lamella preparation and minimizes the need for user input, we envision the technique will render previously inaccessible projects feasible.
Data availability
The tomography data are deposited at EMDB/EMPIAR.EMPIAR-10376, EMD-10707, EMD-10708, EMD-10710
Article and author information
Author details
Funding
Swiss National Science Foundation (#31003A_179255)
- Martin Pilhofer
European Research Council (#679209)
- Martin Pilhofer
Nomis Foundation (nd)
- Martin Pilhofer
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Andrew P Carter, MRC Laboratory of Molecular Biology, United Kingdom
Version history
- Received: September 28, 2019
- Accepted: March 7, 2020
- Accepted Manuscript published: March 9, 2020 (version 1)
- Version of Record published: March 19, 2020 (version 2)
Copyright
© 2020, Zachs et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
Metrics
-
- 6,200
- Page views
-
- 766
- Downloads
-
- 60
- Citations
Article citation count generated by polling the highest count across the following sources: Scopus, Crossref, PubMed Central.
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Fully automated, sequential focused ion beam milling for cryo-electron tomography
eLife 9:e52286.
https://doi.org/10.7554/eLife.52286
Further reading
-
- Microbiology and Infectious Disease
- Structural Biology and Molecular Biophysics
The transcriptional regulator SsrB acts as a switch between virulent and biofilm lifestyles of non-typhoidal Salmonella enterica serovar Typhimurium. During infection, phosphorylated SsrB activates genes on Salmonella Pathogenicity Island-2 (SPI-2) essential for survival and replication within the macrophage. Low pH inside the vacuole is a key inducer of expression and SsrB activation. Previous studies demonstrated an increase in SsrB protein levels and DNA-binding affinity at low pH; the molecular basis was unknown (Liew et al., 2019). This study elucidates its underlying mechanism and in vivo significance. Employing single-molecule and transcriptional assays, we report that the SsrB DNA binding domain alone (SsrBc) is insufficient to induce acid pH-sensitivity. Instead, His12, a conserved residue in the receiver domain, confers pH sensitivity to SsrB allosterically. Acid-dependent DNA binding was highly cooperative, suggesting a new configuration of SsrB oligomers at SPI-2-dependent promoters. His12 also plays a role in SsrB phosphorylation; substituting His12 reduced phosphorylation at neutral pH and abolished pH-dependent differences. Failure to flip the switch in SsrB renders Salmonella avirulent and represents a potential means of controlling virulence.
-
- Neuroscience
- Structural Biology and Molecular Biophysics
Ca2+/calmodulin-dependent protein kinase II (CaMKII) is essential for long-term potentiation (LTP) of excitatory synapses that is linked to learning and memory. In this study, we focused on understanding how interactions between CaMKIIα and the actin-crosslinking protein α-actinin-2 underlie long-lasting changes in dendritic spine architecture. We found that association of the two proteins was unexpectedly elevated within 2 minutes of NMDA receptor stimulation that triggers structural LTP in primary hippocampal neurons. Furthermore, disruption of interactions between the two proteins prevented the accumulation of enlarged mushroom-type dendritic spines following NMDA receptor activation. α-Actinin-2 binds to the regulatory segment of CaMKII. Calorimetry experiments, and a crystal structure of α-actinin-2 EF hands 3 and 4 in complex with the CaMKII regulatory segment, indicate that the regulatory segment of autoinhibited CaMKII is not fully accessible to α-actinin-2. Pull-down experiments show that occupation of the CaMKII substrate-binding groove by GluN2B markedly increases α-actinin-2 access to the CaMKII regulatory segment. Furthermore, in situ labelling experiments are consistent with the notion that recruitment of CaMKII to NMDA receptors contributes to elevated interactions between the kinase and α-actinin-2 during structural LTP. Overall, our study provides new mechanistic insight into the molecular basis of structural LTP and reveals an added layer of sophistication to the function of CaMKII.
