Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity

Nogales, Eva; Scheres, Sjors H.W.

doi:10.1016/j.molcel.2015.02.019

Cited by 330 publications

(233 citation statements)

References 106 publications

(115 reference statements)

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“…3E,F) of large molecular assemblies from small amounts of sample and without the need for crystallization (Bai et al 2015a;Nogales and Scheres 2015). Here, I review these recent structural studies of the biogenesis of the 60S ribosomal subunit in the context of existing functional data and discuss how they have contributed to a more complete mechanistic understanding of 60S ribosomal subunit biogenesis.…”

Section: Introductionmentioning

confidence: 99%

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

Greber

2016

RNA

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Eukaryotic ribosomes, the protein-producing factories of the cell, are composed of four ribosomal RNA molecules and roughly 80 proteins. Their biogenesis is a complex process that involves more than 200 biogenesis factors that facilitate the production, modification, and assembly of ribosomal components and the structural transitions along the maturation pathways of the preribosomal particles. Here, I review recent structural and mechanistic insights into the biogenesis of the large ribosomal subunit that were furthered by cryo-electron microscopy of natively purified pre-60S particles and in vitro reconstituted ribosome assembly factor complexes. Combined with biochemical, genetic, and previous structural data, these structures have provided detailed insights into the assembly and maturation of the central protuberance of the 60S subunit, the network of biogenesis factors near the ribosomal tunnel exit, and the functional activation of the large ribosomal subunit during cytoplasmic maturation.

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Section: Introductionmentioning

confidence: 99%

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

Greber

2016

RNA

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“…However, there are many technical limitations that make examining protein complexes challenging by standard structural approaches. Massive advances have been made in the use of cryo electron microscopy to study large dynamic protein complexes,1 however even this approach is limited by disorder, flexibility and sample heterogeneity. Dynamic regions within proteins with either no or limited secondary structure are a major contributor to flexibility and sample heterogeneity, and they are a major impediment to generating constructs that are amenable to higher‐resolution methods.…”

Section: Introductionmentioning

confidence: 99%

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

et al. 2016

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The ability of proteins to bind and interact with protein partners plays fundamental roles in many cellular contexts. X‐ray crystallography has been a powerful approach to understand protein‐protein interactions; however, a challenge in the crystallization of proteins and their complexes is the presence of intrinsically disordered regions. In this article, we describe an application of hydrogen deuterium exchange mass spectrometry (HDX‐MS) to identify dynamic regions within type III phosphatidylinositol 4 kinase beta (PI4KIIIβ) in complex with the GTPase Rab11. This information was then used to design deletions that allowed for the production of diffraction quality crystals. Importantly, we also used HDX‐MS to verify that the new construct was properly folded, consistent with it being catalytically and functionally active. Structures of PI4KIIIβ in an Apo state and bound to the potent inhibitor BQR695 in complex with both GTPγS and GDP loaded Rab11 were determined. This hybrid HDX‐MS/crystallographic strategy revealed novel aspects of the PI4KIIIβ‐Rab11 complex, as well as the molecular mechanism of potency of a PI4K specific inhibitor (BQR695). This approach is widely applicable to protein‐protein complexes, and is an excellent strategy to optimize constructs for high‐resolution structural approaches.

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“…In addition, the structure of the full-length P2X receptor with its intracellular domains has not been developed. With the help of newly improved technology, such as cryo-EM [137,138] , discoveries of more P2X structures are expected.…”

Section: Questions To Be Answeredmentioning

confidence: 99%

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

Wang

2016

Acta Pharmacol Sin

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P2X receptors, as ATP-gated non-selective trimeric ion channels, are permeable to Na + , K + and Ca 2+ . Comparing with other ligand-gated ion channel families, P2X receptors are distinct in their unique gating properties and pathophysiological roles, and have attracted attention as promising drug targets for a variety of diseases, such as neuropathic pain, multiple sclerosis, rheumatoid arthritis and thrombus. Several small molecule inhibitors for distinct P2X subtypes have entered into clinical trials. However, many questions regarding the gating mechanism of P2X remain unsolved. The structural determinations of P2X receptors at the resting and ATPbound open states revealed that P2X receptor gating is a cooperative allosteric process involving multiple domains, which marks the beginning of the post-structure era of P2X research at atomic level. Here, we review the current knowledge on the structure-function relationship of P2X receptors, depict the whole picture of allosteric changes during the channel gating, and summarize the active sites that may contribute to new strategies for developing novel allosteric drugs targeting P2X receptors.

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Cryo-EM: A Unique Tool for the Visualization of Macromolecular Complexity

Cited by 330 publications

References 106 publications

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

Mechanistic insight into eukaryotic 60S ribosomal subunit biogenesis by cryo-electron microscopy

Using hydrogen deuterium exchange mass spectrometry to engineer optimized constructs for crystallization of protein complexes: Case study of PI4KIIIβ with Rab11

Insights into the channel gating of P2X receptors from structures, dynamics and small molecules

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