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The authors reported the first pseudoatomic model of a telomerase complex, calculated from mass spectrometry, negative-staining EM, cryoEM, X-ray crystallography and NMR data. This complex contains the integral telomerase RNA (TER), the telomerase reverse transcriptase (TERT) and several additional proteins including a telomeric G-strand binding protein. The NMR structure of the TER pseudoknot, as well as the X-ray structures of two of the additional proteins, were determined. Altogether, this study revealed the path of TER on TERT and suggested an exit path for the telomeric repeat DNA.
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This study represents one of the few examples in which NMR is used to identify the disordered fragments in a free monomeric ligand and cryoEM is used to position these fragments in the 3D structure of the ligand bound to a complex. Here chimera of the ligand were produced, in which a globular domain is inserted into each disordered fragment, and observation of these chimera by cryoEM identified the engineered fragments in the complex.
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31 Lu, A., Magupalli, V.G., Ruan, J., Yin, Q., Atianand, M.K., Vos, M.R., Schroder, G.F., Fitzgerald, K.A., Wu, H., Egelman, E.H., Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Cell 156 (2014), 1193–1206.
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The authors used a panel of peptide labeling schemes and solid-state NMR experiments, together with single-particle and helical cryoEM approaches, to determine the overall structure of three polymorphs formed by a 11-residue peptide of transthyretin. Description of the subsets of fibril populations was further supported by X-ray diffraction, scanning transmission electron microscopy and atomic force microscopy measurements.
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This article illustrates how cryoEM and liquid-state NMR can be combined to describe a population distribution of conformers in a large molecular machine. Here key features of the cryoEM model are validated using biochemical as well as methyl-transverse relaxation optimized spectroscopy NMR experiments.
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