Biophysics

eLife assessment

This paper presents a single-molecule polarization microscopy study aimed at monitoring the arginine/agmatine antiporter AdiC as it transiently exchanges between conformational states. This approach measures how a bis-TMR fluorophore anchored onto helix 6a changes its orientation in the microscope, and the authors identify four states that they propose correspond to the key steps in the transport cycle (inward-open, inward occluded, outward occluded and outward open). This is a cutting-edge and challenging approach that sets the stage for direct measurements of conformational equilibria and will thus be of interest to anyone studying transport mechanisms. However, additional investigation is required to validate the robustness of the post-processing of the single-molecule data to yield the four-state model compared to alternate models, to test the robustness of the data with transport mutants/conditions that would slow or eliminate states, and to consolidate transitions that are observed that conflict with previous observations of obligatory coupling in AdiC.

eLife assessment

The manuscript reports a new structure of the small conductance mechanosensitive channel MscS from E. coli in the open state, together with coarse-grained and atomistic molecular dynamics simulations of MscS and the related channel MSL1 of plant mitochondria in closed and open states. The important finding is that the surrounding lipid bilayer is severely distorted in the closed state only, with the protein inducing high curvature in the inner leaflet due to the membrane protruding into the cytoplasm. The authors argue convincingly that the role of membrane tension is to increase the energy of the protein-membrane system in this closed state compared to the relatively flat-membrane open state, in contrast to the previous proposal that tension-induced gating is driven by expansion of the in-plane area of the protein. The finding may be relevant for the understanding of ion channel mechano-sensation more generally, including of the PIEZO1 channel.

eLife assessment

This important paper provides a convincing mechanism for relative binding specificity of Type II inhibitors to kinases. The combination of a sequence-derived Potts-model with experimental dissociation constants and calculated free energies of binding to the DFG-out state is highly compelling and goes beyond the current state of the art. Given the importance of kinases in pathophysiological processes, the results will be of interest to a broad audience and, in addition, the combination of computational methods can be applicable to a wide variety of other biophysical processes that involve conformational rearrangements.

eLife assessment

This manuscript provides compelling evidence that in response to calcium, the C-lobe of calmodulin changes its interaction with the C-terminal domain of an SK2 small-conductance calcium-activated potassium channel. These findings will be of interest to those in the field of ion channels and calcium signaling as they are valuable to understanding the molecular mechanics by which calcium activates SK2 channels, which are important for a wide variety of physiological signaling processes.

eLife assessment

This valuable work, of interest to ion channel physiologists, identifies regions involved in the desensitization of the proton-activated chloride channel (PAC), a widely expressed ion channel involved in organelle pH homeostasis and acid-induced cell death. At the present stage the data only incompletely support the interpretations, and further experiments will be required to consolidate some of the authors' claims.

eLife assessment

Single-particle tomography (SPT) is a useful method to determine the structure of proteins imaged in situ. This important work presents an easy-to-use tool for SPT that approximates the use of 2D tomographic projections using a "pseudo-subtomogram" data structure, chosen to facilitate implementation within the existing Relion codebase. The examples shown provide solid support for the claims about the efficacy of the approach.

eLife assessment

The goal of this study is to identify allosteric modulators of a SLC-1 amino acid transporter, ASCT2, which has been implicated in cancer progression. By combining computational and docking methods with functional measurements, this study provides solid evidence for specific aspects of allosteric SLC-1 inhibition mechanisms. The findings are important to transporter mechanism and pharmacology.

eLife assessment

The authors measure the work output of shrinking mammalian microtubules, reporting results of fundamental importance that advance our mechanistic understanding of how shrinking microtubules exert forces on chromosomes during cell division. Carefully performed, technically advanced experiments and model-based quantitative data analysis provide compelling evidence for the authors' conclusions. This work will be of interest for cell biologists interested in cell division, biophysicists interested in force production by biopolymers, and structural biologists interested in microtubule dynamics.

eLife assessment

This paper, which provides useful information on the assembly of volume-regulated anions channels formed by LRRC8 proteins, will be of interest scientists in the field of ion channels. The authors report the structure of a LRRC8C-LRRC8A chimera with native functional properties as a heptameric complex with a lipid-filled pore. This is very interesting and well-presented work, but the evidence supporting the physiological relevance of the heptameric assembly and the hypothesized role of lipids is still incomplete.

eLife Assessment:

Predicting the effect of mutations on protein stability is important both for protein engineering and for helping to decipher the effects of genetic and clinical mutations. The machine learning methodology introduce here is timely in view of the millions of AlphaFold model structures that are now becoming available, which could hypothetically be examined through approaches such as this one. The methodology presented is valuable, but the manuscript would benefit from a substantial amount of comparative data to provide more compelling evidence for the validity of the methods.