Cryo-electron microscopy snapshots of the spliceosome: structural insights into a dynamic ribonucleoprotein machine

An unresolved issue in RAF kinase signaling is how binding of autoinhibited RAF monomers to activated RAS initiates the conformational changes required to form active RAF dimers. Here, we present cryo-electron microscopy structures of full-length BRAF complexes derived from mammalian cells: autoinhibited monomeric BRAF:14-3-32:MEK and BRAF:14-3-32 complexes and an inhibitor-bound, dimeric BRAF2:14-3-32 complex, at 3.7, 4.1, and 3.9 Å resolution, respectively. The RAS binding domain (RBD) of BRAF is resolved in the autoinhibited structures, and we find that neither MEK nor ATP binding is required to stabilize the autoinhibited complexes. Notably, the RBD was found to interact extensively with the 14-3-3 protomer bound to the BRAF C-terminal site. Moreover, through structure-guided mutational studies, our findings indicate that RAS-RAF binding is a dynamic process and that RBD residues at the 14-3-3 interface have a dual function, first stabilizing RBD orientation in the autoinhibited state and then contributing to full RAS contact.

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Structural insights into stressosome assembly

IUCrJ ◽

10.1107/s205225251900945x ◽

2019 ◽

Vol 6 (5) ◽

pp. 938-947 ◽

Cited By ~ 2

Author(s):

Eunju Kwon ◽

Deepak Pathak ◽

Han-ul Kim ◽

Pawan Dahal ◽

Sung Chul Ha ◽

...

Keyword(s):

Crystal Structure ◽

Electron Microscopy ◽

Environmental Stress ◽

Assembly Model ◽

Bacterial Viability ◽

Binding Interface ◽

Cryo Electron Microscopy ◽

Stress Signals ◽

Structural Insights

The stressosome transduces environmental stress signals to SigB to upregulate SigB-dependent transcription, which is required for bacterial viability. The stressosome core is composed of RsbS and at least one of the RsbR paralogs. A previous cryo-electron microscopy (cryo-EM) structure of the RsbRA–RsbS complex determined under a D2 symmetry restraint showed that the stressosome core forms a pseudo-icosahedron consisting of 60 STAS domains of RsbRA and RsbS. However, it is still unclear how RsbS and one of the RsbR paralogs assemble into the stressosome. Here, an assembly model of the stressosome is presented based on the crystal structure of the RsbS icosahedron and cryo-EM structures of the RsbRA–RsbS complex determined under diverse symmetry restraints (nonsymmetric C1, dihedral D2 and icosahedral I envelopes). 60 monomers of the crystal structure of RsbS fitted well into the I-restrained cryo-EM structure determined at 4.1 Å resolution, even though the STAS domains in the I envelope were averaged. This indicates that RsbS and RsbRA share a highly conserved STAS fold. 22 protrusions observed in the C1 envelope, corresponding to dimers of the RsbRA N-domain, allowed the STAS domains of RsbRA and RsbS to be distinguished in the stressosome core. Based on these, the model of the stressosome core was reconstructed. The mutation of RsbRA residues at the binding interface in the model (R189A/Q191A) significantly reduced the interaction between RsbRA and RsbS. These results suggest that nonconserved residues in the conserved STAS folds between RsbS and RsbR paralogs determine stressosome assembly.

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Structural Insights into TOR Signaling

Genes ◽

10.3390/genes11080885 ◽

2020 ◽

Vol 11 (8) ◽

pp. 885

Author(s):

Lucas Tafur ◽

Jennifer Kefauver ◽

Robbie Loewith

Keyword(s):

Electron Microscopy ◽

Protein Kinase ◽

Molecular Biology ◽

Cellular Responses ◽

Therapeutic Strategies ◽

Cellular Growth ◽

Tor Signaling ◽

Cellular Homeostasis ◽

Cryo Electron Microscopy ◽

Structural Insights

The Target of Rapamycin (TOR) is a highly conserved serine/threonine protein kinase that performs essential roles in the control of cellular growth and metabolism. TOR acts in two distinct multiprotein complexes, TORC1 and TORC2 (mTORC1 and mTORC2 in humans), which maintain different aspects of cellular homeostasis and orchestrate the cellular responses to diverse environmental challenges. Interest in understanding TOR signaling is further motivated by observations that link aberrant TOR signaling to a variety of diseases, ranging from epilepsy to cancer. In the last few years, driven in large part by recent advances in cryo-electron microscopy, there has been an explosion of available structures of (m)TORC1 and its regulators, as well as several (m)TORC2 structures, derived from both yeast and mammals. In this review, we highlight and summarize the main findings from these reports and discuss both the fascinating and unexpected molecular biology revealed and how this knowledge will potentially contribute to new therapeutic strategies to manipulate signaling through these clinically relevant pathways.

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Structural Insights Into the Initiation and Elongation of Ubiquitination by Ubr1

10.1101/2021.04.12.439291 ◽

2021 ◽

Author(s):

Man Pan ◽

Qingyun Zheng ◽

Tian Wang ◽

Lujun Liang ◽

Junxiong Mao ◽

...

Keyword(s):

Electron Microscopy ◽

E3 Ligase ◽

Degradation Pathways ◽

Single Chain ◽

Cryo Electron Microscopy ◽

N Terminus ◽

Specific Manner ◽

Ubiquitin Conjugating Enzyme ◽

Structural Insights ◽

Conjugating Enzyme

The N-end rule pathway was one of the first ubiquitin (Ub)-dependent degradation pathways to be identified. Ubr1, a single-chain E3 ligase, targets proteins bearing a destabilizing residue at the N-terminus (N-degron) for rapid K48-linked ubiquitination and proteasome-dependent degradation. How Ubr1 catalyses the initiation of ubiquitination on the substrate and elongation of the Ub chain in a linkage-specific manner through a single E2 ubiquitin-conjugating enzyme (Ubc2) remains unknown. Here, we report the cryo-electron microscopy structures of two complexes representing the initiation and elongation intermediates of Ubr1 captured using chemical approaches. In these two structures, Ubr1 adopts different conformations to facilitate the transfer of Ub from Ubc2 to either an N-degron peptide or a monoubiquitinated degron. These structures not only reveal the architecture of the Ubr1 complex but also provide mechanistic insights into the initiation and elongation steps of ubiquitination catalyzed by Ubr1.

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Structural insights into the mechanism of human NPC1L1-mediated cholesterol uptake

Science Advances ◽

10.1126/sciadv.abg3188 ◽

2021 ◽

Vol 7 (29) ◽

pp. eabg3188

Author(s):

Miaoqing Hu ◽

Fan Yang ◽

Yawen Huang ◽

Xin You ◽

Desheng Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Cholesterol Level ◽

Cholesterol Absorption ◽

Bound State ◽

Intestinal Cholesterol Absorption ◽

Cholesterol Uptake ◽

Cryo Electron Microscopy ◽

Niemann Pick ◽

Structural Insights ◽

Apo State

Niemann-Pick C1-like 1 (NPC1L1) protein plays a central role in the intestinal cholesterol absorption and is the target of a drug, ezetimibe, which inhibits NPC1L1 to reduce cholesterol absorption. Here, we present cryo–electron microscopy structures of human NPC1L1 in apo state, cholesterol-enriched state, and ezetimibe-bound state to reveal molecular details of NPC1L1-mediated cholesterol uptake and ezetimibe inhibition. Comparison of these structures reveals that the sterol-sensing domain (SSD) could respond to the cholesterol level alteration by binding different number of cholesterol molecules. Upon increasing cholesterol level, SSD binds more cholesterol molecules, which, in turn, triggers the formation of a stable structural cluster in SSD, while binding of ezetimibe causes the deformation of the SSD and destroys the structural cluster, leading to the inhibition of NPC1L1 function. These results provide insights into mechanisms of NPC1L1 function and ezetimibe action and are of great significance for the development of new cholesterol absorption inhibitors.

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New challenges to image processing posed by cryo-Electron microscopy of single particles

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100086416 ◽

1991 ◽

Vol 49 ◽

pp. 422-423

Author(s):

Joachim Frank

Keyword(s):

Electron Microscopy ◽

Phase Contrast ◽

Particle Orientation ◽

Single Particles ◽

Contrast Transfer Function ◽

Virtual Absence ◽

Cryo Electron Microscopy ◽

Spatial Frequencies ◽

New Challenges ◽

Particle Images

Compared with images of negatively stained single particle specimens, those obtained by cryo-electron microscopy have the following new features: (a) higher “signal” variability due to a higher variability of particle orientation; (b) reduced signal/noise ratio (S/N); (c) virtual absence of low-spatial-frequency information related to elastic scattering, due to the properties of the phase contrast transfer function (PCTF); and (d) reduced resolution due to the efforts of the microscopist to boost the PCTF at low spatial frequencies, in his attempt to obtain recognizable particle images.

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Enhanced information from biological materials through technological improvements in cryo-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100124343 ◽

1992 ◽

Vol 50 (1) ◽

pp. 788-789

Author(s):

Marc J.C. de Jong ◽

Wim M. Busing ◽

Max T. Otten

Keyword(s):

Electron Microscopy ◽

Electron Beam ◽

Biological Materials ◽

Electron Crystallography ◽

Cryo Electron Microscopy ◽

Transmission Electron ◽

The Many ◽

Technological Improvements ◽

Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

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Study of eukaryotic flagellar components by cryo-electron microscopy

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100142141 ◽

1986 ◽

Vol 44 ◽

pp. 98-101

Author(s):

John M. Murray ◽

Rob Ward

Keyword(s):

Electron Microscopy ◽

Primary Motion ◽

Cryo Electron Microscopy ◽

Cross Links ◽

Cilia And Flagella

The eukaryotic flagellum is constructed from 11 parallel tubular elements arranged as 9 peripheral fibers (doublet microtubules) and 2 central fibers (singlet microtubules). The primary motion generating component has been found to be arranged as axially periodic “arms” bridging the adjacent doublets. The dynein, comprising the arms, has been isolated and characterized from several different cilia and flagella. Various radial and azimuthal cross-links stabilize the axially aligned microtubules, and probably play some role in controlling the form of the flagella beat cycle.

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