0 Q&A 1261 Views Oct 20, 2022

The ribosome is a complex cellular machinery whose solved structure allowed for an incredible leap in structural biology research. Different ions bind to the ribosome, stabilizing inter-subunit interfaces and structurally linking rRNAs, proteins, and ligands. Besides cations such as K+ and Mg2+, polyamines are known to stabilize the folding of RNA and overall structure. The bacterial ribosome is composed of a small (30S) subunit containing the decoding center and a large (50S) subunit devoted to peptide bond formation. We have previously shown that the small ribosomal subunit of Staphylococcus aureus is sensitive to changes in ionic conditions and polyamines concentration. In particular, its decoding center, where mRNA codons and tRNA anticodons interact, is prone to structural deformations in the absence of spermidine. Here, we report a detailed protocol for the purification of the intact and functional 30S, achieved through specific ionic conditions and the addition of spermidine. Using this protocol, we obtained the cryo-electron microscopy (cryo-EM) structure of the 30S–mRNA complex from S. aureus at 3.6 Å resolution. The 30S–mRNA complex formation was verified by a toeprinting assay. In this article, we also include a description of toeprinting and cryo-EM protocols. The described protocols can be further used to study the process of translation regulation.

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1 Q&A 1890 Views May 20, 2022

Kinetoplastids are unicellular eukaryotic parasites responsible for human pathologies such as Chagas disease, sleeping sickness or Leishmaniasis, caused by Trypanosoma cruzi, Trypanosoma brucei, and various Leishmania spp., respectively. They harbor a single large mitochondrion that is essential for the survival of the parasite. Interestingly, most of the mitochondrial gene expression machineries and processes present significant differences from their nuclear and cytosolic counterparts. A striking example concerns their mitochondrial ribosomes, in charge of translating the few essential mRNAs encoded by mitochondrial genomes. Here, we present a detailed protocol including the specific procedures to isolate mitochondria from two species of kinetoplastids, T. cruzi and L. tarentolae, by differential centrifugations. Then, we detail the protocol to purify mitochondrial ribosomal complexes from these two species of parasites (including ribosomal maturating complexes) by a sucrose gradient approach. Finally, we describe how to prepare cryo-electron microscopy (cryo-EM) grids from these two sorts of samples. This protocol will be useful for further studies aiming at analyzing mitochondrial translation regulation.

0 Q&A 2168 Views Apr 20, 2022

Bsoft is a software package primarily developed for processing electron micrographs, with the goal of determining the structures of biologically relevant molecules, molecular assemblies, and parts of cells. However, it incorporates many ways to deal with images, from the mundane to very sophisticated algorithms. This article is an introduction into its use, illustrating that it is an extensive toolbox, for manipulating and understanding images. Bsoft has over 150 programs, allowing the user an infinite number of ways to process images. These programs can be executed on the command line, or through the interactive program called brun. The main visualization program is bshow, providing numerous ways to manipulate and interpret images. The primary aim is to provide the user with powerful capabilities, including processing large numbers of images. An important additional aim is to make it as accessible as possible, making it easier to deal with image formats and features, and enhance productivity.

0 Q&A 3460 Views Aug 5, 2021

Mitochondrial ribosomes (mitoribosomes) perform protein synthesis inside mitochondria, the organelles responsible for energy conversion and adenosine triphosphate (ATP) production in eukaryotic cells. To investigate their functions and structures, large-scale purification of intact mitoribosomes from mitochondria-rich animal tissues or HEK cells have been developed. However, the fast purification of mitoribosomes anchored to the mitochondrial inner membrane in complex with the Oxa1L translocase remains particularly challenging. Herein, we present a protocol recently developed and modified in our lab that provides details for the efficient isolation of intact mitoribosomes with its translocase Oxa1L. We combined the cell culture of PDE12-/- or wild-type HEK293 cell lines with the isolation of mitochondria and the purification steps used for the biochemical and structural studies of mitoribosomes and Oxa1L.

Graphic abstract:

Schematic procedure for the purification of mitoribosomes from HEK cells. The protocol described herein includes two main sections: 1) isolation of mitochondria from HEK cells; and 2) purification of mitoribosome-Oxa1L from mitochondria. RB: Resuspension Buffer (see Recipes) (Created with BioRender.com).

0 Q&A 2395 Views Aug 5, 2021

Plants make up by far the largest part of biomass on Earth. They are the primary source of food and the basis of most drugs used for medicinal purposes. Similarly to all eukaryotes, plant cells also use mitochondria for energy production. Among mitochondrial gene expression processes, translation is the least understood; although, recent advances have revealed the specificities of its main component, the mitochondrial ribosome (mitoribosome). Here, we present a detailed protocol to extract highly pure cauliflower mitochondria by differential centrifugation for the purification of mitochondrial ribosomes using a sucrose gradient and the preparation of cryo-electron microscopy (cryo-EM) grids. Finally, the specific bioinformatics pipeline used for image acquisition, the processing steps, and the data analysis used for cryo-EM of the plant mitoribosome are described. This protocol will be used for further analysis of the critical steps of mitochondrial translation, such as its initiation and regulation.

0 Q&A 3183 Views Jun 5, 2021

Over the years, studying the ultrastructure of the eukaryotic cilia/flagella using electron microscopy (EM) has contributed significantly toward our understanding of ciliary function. Major complexes in the cilia, such as inner and outer dynein arms, radial spokes, and dynein regulatory complexes, were originally discovered by EM. Classical resin-embedding EM or cryo-electron tomography can be performed directly on the isolated cilia or in some cases, cilia directly attached to the cell body. Recently, single particle cryo-EM has emerged as a powerful structural technique to elucidate high-resolution structures of macromolecular complexes; however, single particle cryo-EM requires non-overlapping complexes, i.e., the doublet microtubule of the cilia. Here, we present a protocol to separate the doublet microtubule from the isolated cilia bundle of two species, Tetrahymena thermophila and Chlamydomonas reinhardtii, using ATP reactivation and sonication. Our approach produces good distribution and random orientation of the doublet microtubule fragments, which is suitable for single particle cryo-EM analysis.

0 Q&A 3809 Views Oct 5, 2020
Cryo-Electron Tomography (cryo-ET) is a method that enables resolving the structure of macromolecular complexes directly in the cellular environment. However, sample preparation for in situ Cryo-ET is labour-intensive and can require both cryo-lamella preparation through cryo-Focused Ion Beam (FIB) milling and correlative light microscopy to ensure that the event of interest is present in the lamella. Here, we present an integrated cryo-FIB and light microscope setup called the Photon Ion Electron microscope (PIE-scope) that enables direct and rapid isolation of cellular regions containing protein complexes of interest. The PIE-scope can be retrofitted on existing microscopes, although the drawings we provide are meant to work on ThermoFisher DualBeams with small mechanical modifications those can be adapted on other brands.
0 Q&A 3398 Views Aug 5, 2020
The serotonin 5-HT2A receptor (5-HT2AR) is a member of the GPCR family that is important for various neurological functions and whose dysregulation causes many mental health disorders. Structural investigations of 5-HT2AR require the production of functionally active receptors expressed from eukaryotic cell cultures. In this protocol, we describe a step-by-step method to express and purify serotonin 5-HT2AR using a baculoviral expression vector system in Sf9 cell cultures, derived from our work with the rat (matching Uniprot ID P14842) and human (matching Uniprot ID P28223) 5-HT2ARs. A unique feature of this method is the utilization of cell culture additives to infect cells at low multiplicity of infection, thereby using several fold less quantity of viral titer compared to prior methods without the additive. This protocol can be tweaked to selectively over-express glycosylated or non-glycosylated forms of the receptor by varying the post-infection harvest times.
0 Q&A 3666 Views Jan 20, 2020
The goal of cryoEM is to determine the structures of biomolecules from electron micrographs. In many cases the processing is straightforward and can be handled with routine protocols. In other cases, the properties and behavior of the specimen require adaptions to properly interpret the data. Here I describe the protocols for examining the higher order assemblies of the retinal adhesion protein, retinoschisin (RS1), using the Bsoft package. The protocols for micrograph preprocessing, 2D classification and 3D alignment and reconstruction follow the usual patterns for the majority of cryoEM specimens. The interpretation of the results is specific to the branched network of RS1 filaments. The 2D class averages are used to determine the relative positions of the RS1 molecules, thus defining the interacting interfaces in the network. The major interface of the linear filament is then further examined by reconstructing the “unit cell” and fitting the molecular models.
0 Q&A 4933 Views Nov 20, 2019
Our mechanistic understanding of cell function depends on imaging biological processes in cells with molecular resolution. Super-resolution fluorescence microscopy plays a crucial role by reporting cellular ultrastructure with 20-30 nm resolution. However, this resolution is insufficient to image macro-molecular machinery at work. A path to improve resolution is to image under cryogenic conditions, which substantially increases the brightness of most fluorophores and preserves native ultrastructure much better than chemical fixatives. Cryogenic conditions are, however, underutilized because of the lack of compatible high numerical aperture (NA) objectives. Here we describe a protocol for the use of super-hemispherical solid immersion lenses (superSILs) to achieve super-resolution imaging at cryogenic temperatures with an effective NA of 2.17 and resolution of ~10 nm.