Using cryoelectron microscopy of vitreous sections, we investigated in situ the ultrastructure of biological membranes, selected from several cell types for their diverse biological functions. Here we describe how to visualize the two membrane leaflets and tightly apposed membranes, lying as close as 1.1 nm apart, by tuning the imaging conditions. We show how defects in membrane stacks may be clues to resolving their structure. Details of membrane proteins are also resolved, as well as protein lattices with correlations between stacked membranes. Imaging the cell in its native hydrated state can now be done in the nanometer resolution range, which should open unique routes for investigating structure-function relationships.
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http://dx.doi.org/10.1073/pnas.1200881109 | DOI Listing |
Elife
January 2025
Laboratory of Biophysical Chemistry of Macromolecules, Institute of Chemical Sciences and Engineering (ISIC), School of Basic Sciences, École Polytechnique Fédérale de Lausanne (EPFL), Lausanne, Switzerland.
Specialized magnetic beads that bind target proteins to a cryogenic electron microscopy grid make it possible to study the structure of protein complexes from dilute samples.
View Article and Find Full Text PDFNucleic Acids Res
January 2025
Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5, Kashiwanoha, Kashiwa, Chiba 277-8562, Japan.
U6 snRNA (small nuclear ribonucleic acid) is a ribozyme that catalyzes pre-messenger RNA (pre-mRNA) splicing and undergoes epitranscriptomic modifications. After transcription, the 3'-end of U6 snRNA is oligo-uridylylated by the multi-domain terminal uridylyltransferase (TUTase), TUT1. The 3'- oligo-uridylylated tail of U6 snRNA is crucial for U4/U6 di-snRNP (small nuclear ribonucleoprotein) formation and pre-mRNA splicing.
View Article and Find Full Text PDFBiophys Rev
December 2024
Randall Centre for Cell & Molecular Biophysics, New Hunt's House, Guy's Campus, King's College London, London, UK.
Calcium binding to troponin triggers the contraction of skeletal and heart muscle through structural changes in the thin filaments that allow myosin motors from the thick filaments to bind to actin and drive filament sliding. Here, we review studies in which those changes were determined in demembranated fibres of skeletal and heart muscle using fluorescence for in situ structure (FISS), which determines domain orientations using polarised fluorescence from bifunctional rhodamine attached to cysteine pairs in the target domain. We describe the changes in the orientations of the N-terminal lobe of troponin C (TnC) and the troponin IT arm in skeletal and cardiac muscle cells associated with contraction and compare the orientations with those determined in isolated cardiac thin filaments by cryo-electron microscopy.
View Article and Find Full Text PDFUnlabelled: Myosin-IC (myo1c) is a class-I myosin that supports transport and remodeling of the plasma membrane and membrane-bound vesicles. Like other members of the myosin family, its biochemical kinetics are altered in response to changes in mechanical loads that resist the power stroke. However, myo1c is unique in that the primary force-sensitive kinetic transition is the isomerization that follows ATP binding, not ADP release as in other slow myosins.
View Article and Find Full Text PDFThe bile acid-sensitive ion channel (BASIC) is the least understood member of the mammalian epithelial Na channel/degenerin (ENaC/DEG) superfamily of ion channels, which are involved in a variety of physiological processes. While some members of this superfamily, including BASIC, are inhibited by extracellular Ca (Ca ), the molecular mechanism underlying Ca modulation remains unclear. Here, by determining the structure of human BASIC in the presence and absence of Ca using single particle cryo-electron microscopy (cryo-EM), we reveal Ca -dependent conformational changes in the transmembrane domain and β-linkers.
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