Protocols in Current Issue
    Purification of Mitochondrial Ribosomal Complexes from Trypanosoma cruzi and Leishmania tarentolae for Cryo-EM Analysis
    Authors:  Stéphanie Durrieu-Gaillard, Marie Sissler and Yaser Hashem, date: 05/20/2022, view: 32, Q&A: 0

    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,

    Bsoft: Image Processing for Structural Biology
    Author:  J. Bernard Heymann, date: 04/20/2022, view: 1061, Q&A: 0

    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

    Purification of Mitochondrial Ribosomes with the Translocase Oxa1L from HEK Cells
    Authors:  Hanting Yang and Nirupa Desai, date: 08/05/2021, view: 2090, Q&A: 0

    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

    Purification and Cryo-electron Microscopy Analysis of Plant Mitochondrial Ribosomes
    Authors:  Florent Waltz, Philippe Giegé and Yaser Hashem, date: 08/05/2021, view: 1597, Q&A: 0

    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

    Preparation of Doublet Microtubule Fraction for Single Particle Cryo-electron Microscopy
    Authors:  Corbin Black, Daniel Chen Dai, Katya Peri, Muneyoshi Ichikawa and Khanh Huy Bui, date: 06/05/2021, view: 2537, Q&A: 0

    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

    Assembly and Imaging Set up of PIE-Scope
    [Abstract] 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 ...
    Expression and Purification of Functionally Active Serotonin 5-HT2A Receptor in Insect Cells Using Low-titer Viral Stock
    [Abstract] 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 ...
    Protocols for Processing and Interpreting cryoEM Data Using Bsoft: A Case Study of the Retinal Adhesion Protein, Retinoschisin
    Author:  J. Bernard Heymann, date: 01/20/2020, view: 3143, Q&A: 0
    [Abstract] 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 ...
    Super-resolution Microscopy at Cryogenic Temperatures Using Solid Immersion Lenses
    [Abstract] 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 ...

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