Optimizing Contrast in Automated 4D STEM Cryotomography.

4D STEM is an emerging approach to electron microscopy. While it was developed principally for high-resolution studies in materials science, the possibility to collect the entire transmitted flux makes it attractive for cryomicroscopy in application to life science and radiation-sensitive materials where dose efficiency is of utmost importance. We present a workflow to acquire tomographic tilt series of 4D STEM data sets using a segmented diode and an ultrafast pixelated detector, demonstrating the methods using a specimen of a T4 bacteriophage. Full integration with the SerialEM platform conveniently provides all the tools for grid navigation and automation of the data collection. Scripts are provided to convert the raw data to mrc format files and further to generate a variety of modes representing both scattering and phase contrasts, including incoherent and annular bright field, integrated center of mass, and parallax decomposition of a simulated integrated differential phase contrast. Principal component analysis of virtual annular detectors proves particularly useful, and axial contrast is improved by 3D deconvolution with an optimized point spread function. Contrast optimization enables visualization of irregular features such as DNA strands and thin filaments of the phage tails, which would be lost upon averaging or imposition of an inappropriate symmetry.

Unraveling the multifaceted resilience of arsenic resistant bacterium Deinococcus indicus.

Arsenic (As) is a toxic heavy metal widely found in the environment that severely undermines the integrity of water resources. Bioremediation of toxic compounds is an appellative sustainable technology with a balanced cost-effective setup. To pave the way for the potential use of Deinococcus indicus, an arsenic resistant bacterium, as a platform for arsenic bioremediation, an extensive characterization of its resistance to cellular insults is paramount. A comparative analysis of D. indicus cells grown in two rich nutrient media conditions (M53 and TGY) revealed distinct resistance patterns when cells are subjected to stress via UV-C and methyl viologen (MV). Cells grown in M53 demonstrated higher resistance to both UV-C and MV. Moreover, cells grow to higher density upon exposure to 25 mM As(V) in M53 in comparison with TGY. This analysis is pivotal for the culture of microbial species in batch culture bioreactors for bioremediation purposes. We also demonstrate for the first time the presence of polyphosphate granules in D. indicus which are also found in a few Deinococcus species. To extend our analysis, we also characterized DiArsC2 (arsenate reductase) involved in arsenic detoxification and structurally determined different states, revealing the structural evidence for a catalytic cysteine triple redox system. These results contribute for our understanding into the D. indicus resistance mechanism against stress conditions.

Visualization of Organelles In Situ by Cryo-STEM Tomography.

Cryogenic electron microscopy (cryo-EM) relies on the imaging of biological or organic specimens embedded in their native aqueous medium; water is solidified into a glass (i.e., vitrified) without crystallization. The cryo-EM method is widely used to determine the structure of biological macromolecules recently at a near-atomic resolution. The approach has been extended to the study of organelles and cells using tomography, but the conventional mode of wide-field transmission EM imaging suffers a severe limitation in the specimen thickness. This has led to a practice of milling thin lamellae using a focused ion beam; the high resolution is obtained by subtomogram averaging from the reconstructions, but three-dimensional relations outside the remaining layer are lost. The thickness limitation can be circumvented by scanned probe imaging, similar to the scanning EM or the confocal laser scanning microscope. While scanning transmission electron microscopy (STEM) in materials science provides atomic resolution in single images, the sensitivity of cryogenic biological specimens to electron irradiation requires special considerations. This protocol presents a setup for cryo-tomography using STEM. The basic topical configuration of the microscope is described for both two- and three-condenser systems, while automation is provided by the non-commercial SerialEM software. Enhancements for batch acquisition and correlative alignment to previously-acquired fluorescence maps are also described. As an example, we show the reconstruction of a mitochondrion, pointing out the inner and outer membrane and calcium phosphate granules, as well as surrounding microtubules, actin filaments, and ribosomes. Cryo-STEM tomography excels in revealing the theater of organelles in the cytoplasm and, in some cases, even the nuclear periphery of adherent cells in culture.

Correlating cryo-super resolution radial fluctuations and dual-axis cryo-scanning transmission electron tomography to bridge the light-electron resolution gap.

Visualization of organelles and their interactions with other features in the native cell remains a challenge in modern biology. We have introduced cryo-scanning transmission electron tomography (CSTET), which can access 3D volumes on the scale of 1 micron with a resolution of nanometers, making it ideal for this task. Here we introduce two relevant advances: (a) we demonstrate the utility of multi-color super-resolution radial fluctuation light microscopy under cryogenic conditions (cryo-SRRF), and (b) we extend the use of deconvolution processing for dual-axis CSTET data. We show that cryo-SRRF nanoscopy is able to reach resolutions in the range of 100 nm, using commonly available fluorophores and a conventional widefield microscope for cryo-correlative light-electron microscopy. Such resolution aids in precisely identifying regions of interest before tomographic acquisition and enhances precision in localizing features of interest within the 3D reconstruction. Dual-axis CSTET tilt series data and application of entropy regularized deconvolution during post-processing results in close-to-isotropic resolution in the reconstruction without averaging. The integration of cryo-SRRF with deconvolved dual-axis CSTET provides a versatile workflow for studying unique objects in a cell.

Structural and functional characterization of SiiA, an auxiliary protein from the SPI4-encoded type 1 secretion system from Salmonella enterica.

Salmonella invasion is mediated by a concerted action of the Salmonella pathogenicity island 4 (SPI4)-encoded type one secretion system (T1SS) and the SPI1-encoded type three secretion system (T3SS-1). The SPI4-encoded T1SS consists of five proteins (SiiABCDF) and secretes the giant adhesin SiiE. Here, we investigated structure-function relationships in SiiA, a non-canonical T1SS subunit. We show that SiiA consists of a membrane domain, an intrinsically disordered periplasmic linker region and a folded globular periplasmic domain (SiiA-PD). The crystal structure of SiiA-PD displays homology to that of MotB and other peptidoglycan (PG)-binding domains. SiiA-PD binds PG in vitro, albeit at an acidic pH, only. Mutation of Arg162 impedes PG binding of SiiA and reduces Salmonella invasion efficacy. SiiA forms a complex with SiiB at the inner membrane (IM), and the observed SiiA-MotB homology is paralleled by a predicted SiiB-MotA homology. We show that, similar to MotAB, SiiAB translocates protons across the IM. Mutating Asp13 in SiiA impairs proton translocation. Overall, SiiA shares numerous properties with MotB. However, MotAB uses the proton motif force (PMF) to energize the bacterial flagellum, it remains to be shown how usage of the PMF by SiiAB assists T1SS function and Salmonella invasion.

Design of an allosterically modulated doxycycline and doxorubicin drug-binding protein.

The allosteric interplay between distant functional sites present in a single protein provides for one of the most important regulatory mechanisms in biological systems. While the design of ligand-binding sites into proteins remains challenging, this holds even truer for the coupling of a newly engineered binding site to an allosteric mechanism that regulates the ligand affinity. Here it is shown how computational design algorithms enabled the introduction of doxycycline- and doxorubicin-binding sites into the serine proteinase inhibitor (serpin) family member α1-antichymotrypsin. Further engineering allowed exploitation of the proteinase-triggered serpin-typical S-to-R transition to modulate the ligand affinities. These design variants follow strategies observed in naturally occurring plasma globulins that allow for the targeted delivery of hormones in the blood. By analogy, we propose that the variants described in the present study could be further developed to allow for the delivery of the antibiotic doxycycline and the anticancer compound doxorubicin to tissues/locations that express specific proteinases, such as bacterial infection sites or tumor cells secreting matrix metalloproteinases.