
Cryo-electron tomography analysis of purified and membrane-associated ciliary rootlets reveals a complex architecture consisting of flexible longitudinal filaments, two types of cross-striations, and membrane-connecting protrusions, providing insights into the structural basis for rootlet functions.


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Ciliary rootlets are striated bundles of filaments that connect the base of cilia to internal cellular structures. Rootlets are critical for the sensory and motile functions of cilia. However, the mechanisms underlying these functions remain unknown, in part due to a lack of structural information of rootlet organization. In this study, we obtain 3D reconstructions of membrane-associated and purified rootlets using cryo-electron tomography (cryo-ET). We show that flexible protrusions on the rootlet surface, which emanate from the cross-striations, connect to intracellular membranes. In purified rootlets, the striations were classified into amorphous (A)-bands, associated with accumulations on the rootlet surface, and discrete (D)-bands corresponding to punctate lines of density that run through the rootlet. These striations connect a flexible network of longitudinal filaments. Subtomogram averaging suggests the filaments consist of two intertwined coiled coils. The rootlet’s filamentous architecture, with frequent membrane-connecting cross-striations, lends itself well for anchoring large membranes in the cell.

### Competing Interest Statement

The authors have declared no competing interest.

A cryo-ET study of ciliary rootlet organization

cryo-electron-tomography-reveals-the-intricate-ultrastructure-and-membrane-interactions-of-ciliary-rootlets


Cryo-Electron Tomography Reveals the Intricate Ultrastructure and Membrane Interactions of Ciliary Rootlets


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C型肝炎ウイルスのエンベロープタンパク質E1とE2は二量体の複合体を形成し、中和抗体の標的となっている。本研究では、この複合体の高次構造を初めて明らかにした。


Structural studies of a homodimeric arrangement of&nbsp;hepatitis C virus envelope E1/E2 heterodimers reveals the molecular basis&nbsp;of&nbsp;intracomplex interactions and &nbsp;provides mechanistic insights into neutralizing antibody evasion and membrane fusion with host cells.

The hepatitis C virus envelope protein complex is a dimer of heterodimers - Nature

慢性c型肝炎ウイルス感染の主要な原因となるエンベロープタンパク質複合体の二量体構造の解明


慢性C型肝炎ウイルス感染の主要な原因となるエンベロープタンパク質複合体の二量体構造の解明



Covalent inhibitors GW9662 and T0070907 do not prevent binding of synthetic ligands to PPARγ; instead, they allosterically stabilize a repressive conformation that reduces the binding affinity of synthetic ligands, which can still adopt orthosteric or alternate binding modes.


Peroxisome proliferator-activated receptor gamma (PPARγ) is a nuclear receptor transcription factor that regulates gene expression programs in response to ligand binding. Endogenous lipids and synthetic ligands, including covalent antagonist inhibitors such as GW9662 and T0070907, are thought to compete for the orthosteric pocket in the ligand-binding domain (LBD). However, we previously showed that synthetic PPARγ ligands can cooperatively cobind with and reposition a bound endogenous orthosteric ligand to an alternate site, synergistically regulating PPARγ structure and function (Shang et al., 2018). Here, we reveal the structural mechanism of cobinding between a synthetic covalent antagonist inhibitor with other synthetic ligands. Biochemical and NMR data show that covalent antagonist inhibitors weaken—but do not prevent—the binding of other synthetic ligands via an allosteric mechanism rather than direct ligand clashing. The covalent ligands shift the LBD ensemble toward a transcriptionally repressive conformation, which structurally clashes with and reduces the orthosteric binding affinity of non-covalent synthetic ligands. Crystal structures reveal different non-covalent synthetic ligand-specific cobinding mechanisms ranging from alternate site binding to unexpectedly adopting an orthosteric binding mode by altering the covalent ligand binding pose. Our findings not only highlight the significant flexibility of the PPARγ orthosteric pocket and its ability to accommodate multiple ligands simultaneously, but also demonstrate that GW9662 and T0070907 should not be used as reliable chemical tools to inhibit the binding of other ligands to PPARγ.

### Competing Interest Statement

The authors have declared no competing interest.

Crystal structures generated during the current study are available in the Protein Data Bank (PDB) under accession codes 8ZFN, 8ZFO, 8ZFP, 8ZFQ, 8ZFR, 8ZFS, 8ZFT as detailed in Supplementary Table S1 . All other datasets generated and/ or analyzed during the current study are available from the corresponding author on reasonable request.

Unanticipated mechanisms of covalent inhibitor and synthetic ligand cobinding to PPARγ

structural-mechanisms-reveal-unexpected-cobinding-of-covalent-inhibitors-and-synthetic-ligands-to-the-nuclear-receptor-pparγ


Structural Mechanisms Reveal Unexpected Cobinding of Covalent Inhibitors and Synthetic Ligands to the Nuclear Receptor PPARγ



The aggregation of α-synuclein protein into amyloid fibrils is closely associated with neurodegenerative disorders. The structural polymorphism of these fibrils is highly dependent on the pH and buffer conditions, with distinct polymorphs favored at different pH levels. Even in the presence of seeds, the polymorph selection during aggregation is largely determined by the environmental factors rather than the seed structure.


The aggregation of the protein α-synuclein is closely associated with several neurodegenerative disorders and as such the structures of the amyloid fibril aggregates have high scientific and medical significance. However, there are dozens of unique atomic-resolution structures of these aggregates, and such a highly polymorphic nature of the α-synuclein fibrils hampers efforts in disease-relevant in vitro studies on α-synuclein amyloid aggregation. In order to better understand the factors that affect polymorph selection, we studied the structures of α-synuclein fibrils in vitro as a function of pH and buffer using cryo-EM helical reconstruction. We find that in the physiological range of pH 5.8-7.4 a pH-dependent selection between Types 1, 2 and 3 polymorphs occurs. Our results indicate that even in the presence of seeds, the polymorph selection during aggregation is highly dependent on the buffer conditions, attributed to the non-polymorph-specific nature of secondary nucleation. We also uncovered two new polymorphs that occur at pH 7.0 in phosphate-buffered saline. The first is a monofilament Type 1 fibril that highly resembles the structure of the juvenile-onset synucleinopathy polymorph found in patient-derived material. The second is a new Type 5 polymorph that resembles a polymorph that has been recently reported in a study that used diseased tissues to seed aggregation. Taken together, our results highlight the shallow amyloid energy hypersurface that can be altered by subtle changes in the environment, including the pH which is shown to play a major role in polymorph selection and in many cases appears to be the determining factor in seeded aggregation. The results also suggest the possibility of producing disease-relevant structure in vitro .

### Competing Interest Statement

The authors have declared no competing interest.

On the pH-dependence of α-synuclein amyloid polymorphism and the role of secondary nucleation in seed-based amyloid propagation

structural-polymorphism-of-α-synuclein-amyloid-fibrils-is-strongly-influenced-by-ph-and-buffer-conditions


Structural Polymorphism of α-Synuclein Amyloid Fibrils is Strongly Influenced by pH and Buffer Conditions



The cryo-EM structures of the mammalian voltage-gated potassium channel Kv1.2 reveal distinct ion-occupancy patterns in the selectivity filter under different functional states, including open, C-type inactivated, toxin-blocked, and sodium-bound conditions.


We present near-atomic-resolution cryo-EM structures of the mammalian voltage-gated potassium channel Kv1.2 in open, C-type inactivated, toxin-blocked and sodium-bound states at 3.2 Å, 2.5 Å, 3.2 Å, and 2.9Å. These structures, all obtained at nominally zero membrane potential in detergent micelles, reveal distinct ion-occupancy patterns in the selectivity filter. The first two structures are very similar to those reported in the related Shaker channel and the much-studied Kv1.2-2.1 chimeric channel. On the other hand, two new structures show unexpected patterns of ion occupancy. First, the toxin α- Dendrotoxin, like Charybdotoxin, is seen to attach to the negatively-charged channel outer mouth, and a lysine residue penetrates into the selectivity filter, with the terminal amine coordinated by carbonyls, partially disrupting the outermost ion-binding site. In the remainder of the filter two densities of bound ions are observed, rather than three as observed with other toxin-blocked Kv channels. Second, a structure of Kv1.2 in Na+ solution does not show collapse or destabilization of the selectivity filter, but instead shows an intact selectivity filter with ion density in each binding site. We also attempted to image the C-type inactivated Kv1.2 W366F channel in Na+ solution, but the protein conformation was seen to be highly variable and only a low-resolution structure could be obtained. These findings present new insights into the stability of the selectivity filter and the mechanism of toxin block of this intensively studied, voltage-gated potassium channel.

### Competing Interest Statement

The authors have declared no competing interest.

Cryo-EM structures of Kv1.2 potassium channels, conducting and non-conducting

cryo-em-structures-of-kv1-2-potassium-channels-in-conducting-and-non-conducting-states


Cryo-EM Structures of Kv1.2 Potassium Channels in Conducting and Non-Conducting States



The article presents a model for the sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5, revealing distinct receptor conformations upon agonist, allosteric modulator, and G protein binding.


We propose a model for a sequential, multistep activation mechanism of metabotropic glutamate receptor subtype 5, including a series of structures in lipid nanodiscs, from inactive to fully active, with agonist-bound intermediate states.

Stepwise activation of a metabotropic glutamate receptor - Nature

structural-insights-into-the-stepwise-activation-mechanism-of-metabotropic-glutamate-receptor-subtype-5


Structural Insights into the Stepwise Activation Mechanism of Metabotropic Glutamate Receptor Subtype 5
