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Showing papers on "Structural biology published in 2018"


Journal ArticleDOI
TL;DR: A combination of techniques from cell biology, biophysics, physical chemistry, structural biology, and bioinformatics are starting to help establish the molecular principles of an emerging field, thus paving the way for exciting discoveries, including novel therapeutic approaches for the treatment of age-related disorders.

1,317 citations


Journal ArticleDOI
04 Jul 2018-Nature
TL;DR: High-resolution structural studies of GPCRs have led to insights into the role of allostery in GPCR-mediated signal transduction, as well as highlighting how the wide diversity of druggable allosteric sites across these receptors present opportunities for developing new classes of therapeutics.
Abstract: G-protein-coupled receptors (GPCRs) are key cell-surface proteins that transduce external environmental cues into biochemical signals across the membrane. GPCRs are intrinsically allosteric proteins; they interact via spatially distinct yet conformationally linked domains with both endogenous and exogenous proteins, nutrients, metabolites, hormones, small molecules and biological agents. Here we explore recent high-resolution structural studies, which are beginning to unravel the atomic details of allosteric transitions that govern GPCR biology, as well as highlighting how the wide diversity of druggable allosteric sites across these receptors present opportunities for developing new classes of therapeutics.

233 citations


Journal ArticleDOI
04 May 2018-eLife
TL;DR: The structure by electron cryo-microscopy of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35 is determined.
Abstract: The adenosine A2A receptor (A2AR) is a prototypical G protein-coupled receptor (GPCR) that couples to the heterotrimeric G protein GS. Here, we determine the structure by electron cryo-microscopy (cryo-EM) of A2AR at pH 7.5 bound to the small molecule agonist NECA and coupled to an engineered heterotrimeric G protein, which contains mini-GS, the βγ subunits and nanobody Nb35. Most regions of the complex have a resolution of ~3.8 A or better. Comparison with the 3.4 A resolution crystal structure shows that the receptor and mini-GS are virtually identical and that the density of the side chains and ligand are of comparable quality. However, the cryo-EM density map also indicates regions that are flexible in comparison to the crystal structures, which unexpectedly includes regions in the ligand binding pocket. In addition, an interaction between intracellular loop 1 of the receptor and the β subunit of the G protein was observed.

192 citations


Journal ArticleDOI
24 May 2018-eLife
TL;DR: An in vitro selection platform based on synthetic single domain antibodies named sybodies, designed to target the limited hydrophilic surfaces of membrane proteins, and designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface.
Abstract: Mechanistic and structural studies of membrane proteins require their stabilization in specific conformations. Single domain antibodies are potent reagents for this purpose, but their generation relies on immunizations, which impedes selections in the presence of ligands typically needed to populate defined conformational states. To overcome this key limitation, we developed an in vitro selection platform based on synthetic single domain antibodies named sybodies. To target the limited hydrophilic surfaces of membrane proteins, we designed three sybody libraries that exhibit different shapes and moderate hydrophobicity of the randomized surface. A robust binder selection cascade combining ribosome and phage display enabled the generation of conformation-selective, high affinity sybodies against an ABC transporter and two previously intractable human SLC transporters, GlyT1 and ENT1. The platform does not require access to animal facilities and builds exclusively on commercially available reagents, thus enabling every lab to rapidly generate binders against challenging membrane proteins.

160 citations


Journal ArticleDOI
TL;DR: It is shown that i‐motifs formed from naturally occurring C‐rich sequences in the human genome are stable and persist in the nuclei of living human cells, the first to interlink the stability of DNA i‐Motifs in vitro with their stability in’vivo.
Abstract: C-rich DNA has the capacity to form a tetra-stranded structure known as an i-motif. The i-motifs within genomic DNA have been proposed to contribute to the regulation of DNA transcription. However, direct experimental evidence for the existence of these structures invivo has been missing. Whether i-motif structures form in complex environment of living cells is not currently known. Herein, using state-of-the-art in-cell NMR spectroscopy, we evaluate the stabilities of i-motif structures in the complex cellular environment. We show that i-motifs formed from naturally occurring C-rich sequences in the human genome are stable and persist in the nuclei of living human cells. Our data show that i-motif stabilities invivo are generally distinct from those invitro. Our results are the first to interlink the stability of DNA i-motifs invitro with their stability invivo and provide essential information for the design and development of i-motif-based DNA biosensors for intracellular applications.

155 citations


Journal ArticleDOI
TL;DR: This Minireview will focus on the cross-linking/MS strategy, which is currently one of the most promising MS-based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins.
Abstract: Structural mass spectrometry (MS) is gaining increasing importance for deriving valuable three-dimensional structural information on proteins and protein complexes, and it complements existing techniques, such as NMR spectroscopy and X-ray crystallography. Structural MS unites different MS-based techniques, such as hydrogen/deuterium exchange, native MS, ion-mobility MS, protein footprinting, and chemical cross-linking/MS, and it allows fundamental questions in structural biology to be addressed. In this Minireview, I will focus on the cross-linking/MS strategy. This method not only delivers tertiary structural information on proteins, but is also increasingly being used to decipher protein interaction networks, both in vitro and in vivo. Cross-linking/MS is currently one of the most promising MS-based approaches to derive structural information on very large and transient protein assemblies and intrinsically disordered proteins.

143 citations


Journal ArticleDOI
TL;DR: In the past few years, significant technological breakthroughs in single particle cryo-electron microscopy enabled a 'resolution revolution' of this technique, which changed structural biology in an unprecedented way.

115 citations


Journal ArticleDOI
TL;DR: In this article, a multistage tandem MS approach was used to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode.
Abstract: Protein complexes exhibit great diversity in protein membership, post-translational modifications and noncovalent cofactors, enabling them to function as the actuators of many important biological processes. The exposition of these molecular features using current methods lacks either throughput or molecular specificity, ultimately limiting the use of protein complexes as direct analytical targets in a wide range of applications. Here, we apply native proteomics, enabled by a multistage tandem MS approach, to characterize 125 intact endogenous complexes and 217 distinct proteoforms derived from mouse heart and human cancer cell lines in discovery mode. The native conditions preserved soluble protein-protein interactions, high-stoichiometry noncovalent cofactors, covalent modifications to cysteines, and, remarkably, superoxide ligands bound to the metal cofactor of superoxide dismutase 2. These data enable precise compositional analysis of protein complexes as they exist in the cell and demonstrate a new approach that uses MS as a bridge to structural biology.

114 citations


Journal ArticleDOI
07 Jun 2018-eLife
TL;DR: The ensemble nature of macromolecular structure can elucidate allosteric mechanisms and open new doors for long-range control of protein function by combining multiple-temperature X-ray crystallography experiments and structure determination from hundreds of individual small-molecule fragment soaks.
Abstract: Proteins perform many important jobs in each of the cells in our bodies, such as transporting other molecules and helping chemical reactions to occur. The part of the protein directly involved in these tasks is called the active site. Other areas of the protein can communicate with the active site to switch the protein on or off. This method of control is known as allostery. Switching proteins on and off could help us to develop treatments for certain diseases. For example, a protein called PTP1B reduces how well cells can respond to insulin. Switching this protein off could therefore help to treat diabetes. However, much like it’s hard to guess how a light switch is wired to a light bulb without seeing behind the walls, it is hard to predict which remote areas of a protein are ‘wired’ to the active site. Keedy, Hill et al. have now used two complementary methods to examine the structure of PTP1B and find new allosteric sites. The first method captured a series of X-ray images from crystallized molecules of the protein held at different temperatures. This revealed areas of PTP1B that can move like windshield wipers to communicate with each other. The second method soaked PTP1B crystals in trays with hundreds of drug-sized molecules and assessed which sites on the protein the molecules bound to. The molecules generally bound to just a few sites of the protein. Further tests on one of these sites showed that it can communicate with the active site to turn the protein on or off. Further work will be needed to develop drugs that could treat diabetes by binding to the newly identified allosteric sites in PTP1B. More generally, the methods developed by Keedy, Hill et al. could be used to study allostery in other important proteins.

105 citations


Journal ArticleDOI
15 Aug 2018-eLife
TL;DR: The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.
Abstract: Membrane proteins are difficult to work with due to their insolubility in aqueous solution and quite often their poor stability in detergent micelles. Here, we present the peptidisc for their facile capture into water-soluble particles. Unlike the nanodisc, which requires scaffold proteins of different lengths and precise amounts of matching lipids, reconstitution of detergent solubilized proteins in peptidisc only requires a short amphipathic bi-helical peptide (NSPr) and no extra lipids. Multiple copies of the peptide wrap around to shield the membrane-exposed part of the target protein. We demonstrate the effectiveness of this 'one size fits all' method using five different membrane protein assemblies (MalFGK2, FhuA, SecYEG, OmpF, BRC) during 'on-column', 'in-gel', and 'on-bead' reconstitution embedded within the membrane protein purification protocol. The peptidisc method is rapid and cost-effective, and it may emerge as a universal tool for high-throughput stabilization of membrane proteins to advance modern biological studies.

101 citations


Journal ArticleDOI
TL;DR: How structural biology has illuminated the agonist-induced cascade of conformational changes that culminate in a cellular response to GPCR activation is reviewed.
Abstract: G protein–coupled receptors (GPCRs), which mediate processes as diverse as olfaction and maintenance of metabolic homeostasis, have become the single most effective class of therapeutic drug targets. As a result, understanding the molecular basis for their activity is of paramount importance. Recent technological advances have made GPCR structural biology increasingly tractable, offering views of these receptors in unprecedented atomic detail. Structural and biophysical data have shown that GPCRs function as complex allosteric machines, communicating ligand-binding events through conformational change. Changes in receptor conformation lead to activation of effector proteins, such as G proteins and arrestins, which are themselves conformational switches. Here, we review how structural biology has illuminated the agonist-induced cascade of conformational changes that culminate in a cellular response to GPCR activation. Expected final online publication date for the Annual Review of Biophysics Volume 47 is M...

Journal ArticleDOI
TL;DR: The SNARE proteins, their structures and functions in eukaryotic cells, are introduced, and recent breakthroughs in elucidating the regulation of SNARE assembly and disassembly through the use of high-resolution structural biology and biophysical techniques are discussed.

Journal ArticleDOI
02 May 2018-eLife
TL;DR: In this paper, the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 A was reported.
Abstract: Canonical transient receptor channels (TRPC) are non-selective cation channels. They are involved in receptor-operated Ca2+ signaling and have been proposed to act as store-operated channels (SOC). Their malfunction is related to cardiomyopathies and their modulation by small molecules has been shown to be effective against renal cancer cells. The molecular mechanism underlying the complex activation and regulation is poorly understood. Here, we report the electron cryo-microscopy structure of zebrafish TRPC4 in its unliganded (apo), closed state at an overall resolution of 3.6 A. The structure reveals the molecular architecture of the cation conducting pore, including the selectivity filter and lower gate. The cytoplasmic domain contains two key hubs that have been shown to interact with modulating proteins. Structural comparisons with other TRP channels give novel insights into the general architecture and domain organization of this superfamily of channels and help to understand their function and pharmacology.

Journal ArticleDOI
TL;DR: The structure of the Escherichia coli multidrug efflux transporter AcrB in a SMALP scaffold to sub-nm resolution is reported, with the resulting map being consistent with high resolution crystal structures and other EM derived maps.

Journal ArticleDOI
TL;DR: The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin’s negative cooperativity of binding likely arises and provides insight into the high affinity of certain super-mitogenic insulins.
Abstract: Understanding the structural biology of the insulin receptor and how it signals is of key importance in the development of insulin analogs to treat diabetes. We report here a cryo-electron microscopy structure of a single insulin bound to a physiologically relevant, high-affinity version of the receptor ectodomain, the latter generated through attachment of C-terminal leucine zipper elements to overcome the conformational flexibility associated with ectodomain truncation. The resolution of the cryo-electron microscopy maps is 3.2 A in the insulin-binding region and 4.2 A in the membrane-proximal region. The structure reveals how the membrane proximal domains of the receptor come together to effect signalling and how insulin's negative cooperativity of binding likely arises. Our structure further provides insight into the high affinity of certain super-mitogenic insulins. Together, these findings provide a new platform for insulin analog investigation and design.

Journal ArticleDOI
26 Jul 2018
TL;DR: The application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein is illustrated, showing high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.
Abstract: Advances in structural biology have yielded exponential growth in G protein-coupled receptor (GPCR) structure solution. Nonetheless, the instability of fully active GPCR complexes with cognate heterotrimeric G proteins has made them elusive. Existing structures have been limited to nanobody-stabilized GPCR:Gs complexes. Here we present methods for enhanced GPCR:G protein complex stabilization via engineering G proteins with reduced nucleotide affinity, limiting Gα:Gβγ dissociation. We illustrate the application of dominant negative G proteins of Gαs and Gαi2 to the purification of stable complexes where this was not possible with wild-type G protein. Active state complexes of adenosine:A1 receptor:Gαi2βγ and calcitonin gene-related peptide (CGRP):CLR:RAMP1:Gαsβγ:Nb35 were purified to homogeneity and were stable in negative stain electron microscopy. These were suitable for structure determination by cryo-electron microscopy at 3.6 and 3.3 A resolution, respectively. The dominant negative Gα-proteins are thus high value tools for structure determination of agonist:GPCR:G protein complexes that are critical for informed translational drug discovery.

Journal ArticleDOI
TL;DR: The contribution of structural biology is focused on to understand the roles of STAT3, to design new inhibitors and to suggest new strategies of pharmacological intervention.
Abstract: Transcription factors are proteins able to bind DNA and induce the transcription of specific genes. Consequently, they play a pivotal role in multiple cellular pathways and are frequently over-expressed or dysregulated in cancer. Here, we will focus on a specific “signal transducer and activator of transcription” (STAT3) factor that is involved in several pathologies, including cancer. For long time, the mechanism by which STAT3 exerts its cellular functions has been summarized by a three steps process: (1) Protein phosphorylation by specific kinases, (2) dimerization promoted by phosphorylation, (3) activation of gene expression by the phosphorylated dimer. Consequently, most of the inhibitors reported in literature aimed at blocking phosphorylation and dimerization. However, recent observations reopened the debate and the entire functional mechanism has been revisited stimulating the scientific community to pursue new inhibition strategies. In particular, the dimerization of the unphosphorylated species has been experimentally demonstrated and specific roles proposed also for these dimers. Despite difficulties in the expression and purification of the full length STAT3, structural biology investigations allowed the determination of atomistic structures of STAT3 dimers and several protein domains. Starting from this information, computational methods have been used both to improve the understanding of the STAT3 functional mechanism and to design new inhibitors to be used as anticancer drugs. In this review, we will focus on the contribution of structural biology to understand the roles of STAT3, to design new inhibitors and to suggest new strategies of pharmacological intervention.

Journal ArticleDOI
31 Jul 2018-eLife
TL;DR: A model of TMC1 is generated based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway.
Abstract: The hair cell mechanotransduction (MET) channel complex is essential for hearing, yet it's molecular identity and structure remain elusive. The transmembrane channel-like 1 (TMC1) protein localizes to the site of the MET channel, interacts with the tip-link responsible for mechanical gating, and genetic alterations in TMC1 alter MET channel properties and cause deafness, supporting the hypothesis that TMC1 forms the MET channel. We generated a model of TMC1 based on X-ray and cryo-EM structures of TMEM16 proteins, revealing the presence of a large cavity near the protein-lipid interface that also harbors the Beethoven mutation, suggesting that it could function as a permeation pathway. We also find that hair cells are permeable to 3 kDa dextrans, and that dextran permeation requires TMC1/2 proteins and functional MET channels, supporting the presence of a large permeation pathway and the hypothesis that TMC1 is a pore forming subunit of the MET channel complex.

Journal ArticleDOI
TL;DR: In this opinion article, recent successes in the field of CLMS with protein structure modeling and challenges it still holds are highlighted.

Journal ArticleDOI
TL;DR: The development of data standards and a prototype system for archiving integrative/hybrid structural models of macromolecular assemblies, which provide the definitions required for representing I/H models that span multiple spatiotemporal scales and conformational states are presented.

Journal ArticleDOI
01 Sep 2018-Methods
TL;DR: Application and refinement of hyphenated mass spectrometry-based methods have yielded unprecedented insights in many areas, including membrane protein conformation, dynamics, lipid/ligand binding, and conformational perturbations due to ligandbinding, which can be challenging to study using other methods.

Journal ArticleDOI
20 Feb 2018-eLife
TL;DR: This work directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies and establishes that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures.
Abstract: Molecular recognition is integral to biological function and frequently involves preferred binding of a molecule to one of several exchanging ligand conformations in solution. In such a process the bound structure can be selected from the ensemble of interconverting ligands a priori (conformational selection, CS) or may form once the ligand is bound (induced fit, IF). Here we focus on the ubiquitous and conserved Hsp70 chaperone which oversees the integrity of the cellular proteome through its ATP-dependent interaction with client proteins. We directly quantify the flux along CS and IF pathways using solution NMR spectroscopy that exploits a methyl TROSY effect and selective isotope-labeling methodologies. Our measurements establish that both bacterial and human Hsp70 chaperones interact with clients by selecting the unfolded state from a pre-existing array of interconverting structures, suggesting a conserved mode of client recognition among Hsp70s and highlighting the importance of molecular dynamics in this recognition event.

Journal ArticleDOI
TL;DR: Protein expression in HEK293T cells was applied to recapitulate the maturation steps of intracellular superoxide dismutase 1 (SOD1) and to study the effect of mutations linked to familial amyotrophic lateral sclerosis (fALS) by in-cell NMR.
Abstract: ConspectusCellular structural biology methods are needed to characterize biological processes at atomic resolution in the physiological environment of the cell. Toward this goal, solution in-cell NMR is a powerful approach because it provides structural and dynamic data on macromolecules inside living cells. Several approaches have been developed for in-cell NMR in cultured human cells, which are needed to study processes related to human diseases that rely on the delivery of exogenous macromolecules to the cells. Such strategies, however, may not be applicable to proteins that are sensitive to the external environment or prone to aggregate and can introduce artifacts during protein purification or delivery.As a complementary approach, direct protein expression for in-cell NMR in human cells was developed. This strategy is especially useful when studying processes like protein folding, maturation, and post-translational modification, starting right after protein synthesis. Compared with the protein expres...

Journal ArticleDOI
TL;DR: This analysis provides novel insights into ligand binding, allosteric modulation, and signaling of the AR family by mapping onto the relevant crystal structures all site-directed mutagenesis data, curated and deposited at the GPCR database.

Journal ArticleDOI
TL;DR: This work designs protein–protein interfaces between dimers and trimers to generate dodecameric protein assemblies with dihedral point group symmetry and shows that information about stoichiometry, intersubunit connectivity, and complex topology is rapidly gained by this SID-IM-MS methodology.
Abstract: Computational protein design provides the tools to expand the diversity of protein complexes beyond those found in nature. Understanding the rules that drive proteins to interact with each other enables the design of protein–protein interactions to generate specific protein assemblies. In this work, we designed protein–protein interfaces between dimers and trimers to generate dodecameric protein assemblies with dihedral point group symmetry. We subsequently analyzed the designed protein complexes by native MS. We show that the use of ion mobility MS in combination with surface-induced dissociation (SID) allows for the rapid determination of the stoichiometry and topology of designed complexes. The information collected along with the speed of data acquisition and processing make SID ion mobility MS well-suited to determine key structural features of designed protein complexes, thereby circumventing the requirement for more time- and sample-consuming structural biology approaches.

Journal ArticleDOI
21 Feb 2018-eLife
TL;DR: A battery of spectroscopic methods are used to show that the ~100 fold activation of the mitotic kinase Aurora A by phosphorylation occurs without a population shift from the DFG-Out to the D FG-In state, and that the activation loop of the activated kinase remains highly dynamic.
Abstract: The transfer of phosphate groups onto proteins (protein phosphorylation) is one of the most important methods used to send signals inside cells. The enzymes that catalyze this process, called protein kinases, are themselves controlled by the phosphorylation of a flexible region called the activation loop. For many years it had been thought that the purpose of activation loop phosphorylation was to clamp the otherwise flexible activation loop in an active state that allows molecules that need to be phosphorylated to bind to the kinase. This assumption was based on static pictures of protein kinases obtained by X-ray crystallography, in which individual states are trapped and visualized in a crystal lattice. However, new methods and approaches now mean it is possible to visualize how the position of the activation loop changes as it moves in solution. By applying these techniques, Ruff et al. show that the static model is incorrect in a protein kinase called Aurora A. In this enzyme, the phosphorylated activation loop continues to switch back and forth between active and inactive states. Phosphorylation instead enhances the catalytic activity of the active state. Aurora A regulates several important steps in cell division, and plays important roles in several kinds of cancer. The discovery that activated forms of Aurora A can have different dynamic properties raises the possibility that inhibitor molecules could be designed to exploit these differences and block specific activities of Aurora A in cancer cells. To realize this goal we need to better understand how a kinase switching between active and inactive states affects the ability of inhibitors to interact with it.

Journal ArticleDOI
TL;DR: These mesophases are used to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology.
Abstract: In meso crystallization of membrane proteins from lipidic mesophases is central to protein structural biology but limited to membrane proteins with small extracellular domains (ECDs), comparable to the water channels (3-5 nm) of the mesophase. Here we present a strategy expanding the scope of in meso crystallization to membrane proteins with very large ECDs. We combine monoacylglycerols and phospholipids to design thermodynamically stable ultra-swollen bicontinuous cubic phases of double-gyroid (Ia3d), double-diamond (Pn3m), and double-primitive (Im3m) space groups, with water channels five times larger than traditional lipidic mesophases, and showing re-entrant behavior upon increasing hydration, of sequences Ia3d→Pn3m→Ia3d and Pn3m→Im3m→Pn3m, unknown in lipid self-assembly. We use these mesophases to crystallize membrane proteins with ECDs inaccessible to conventional in meso crystallization, demonstrating the methodology on the Gloeobacter ligand-gated ion channel (GLIC) protein, and show substantial modulation of packing, molecular contacts and activation state of the ensued proteins crystals, illuminating a general strategy in protein structural biology.

Journal ArticleDOI
TL;DR: A brief introduction to the method is attempted, the latest advances in cryo-EM structure determination of TRP channels are reviewed, and molecular insights into the channel function are discussed based on the wealth of TRPs structures.
Abstract: Cryo-electron microscopy (cryo-EM) permits the determination of atomic protein structures by averaging large numbers of individual projection images recorded at cryogenic temperatures—a method termed single-particle analysis. The cryo-preservation traps proteins within a thin glass-like ice layer, making literally a freeze image of proteins in solution. Projections of randomly adopted orientations are merged to reconstruct a 3D density map. While atomic resolution for highly symmetric viruses was achieved already in 2009, the development of new sensitive and fast electron detectors has enabled cryo-EM for smaller and asymmetrical proteins including fragile membrane proteins. As one of the most important structural biology methods at present, cryo-EM was awarded in October 2017 with the Nobel Prize in Chemistry. The molecular understanding of Transient-Receptor-Potential (TRP) channels has been boosted tremendously by cryo-EM single-particle analysis. Several near-atomic and atomic structures gave important mechanistic insights, e.g., into ion permeation and selectivity, gating, as well as into the activation of this enigmatic and medically important membrane protein family by various chemical and physical stimuli. Lastly, these structures have set the starting point for the rational design of TRP channel-targeted therapeutics to counteract life-threatening channelopathies. Here, we attempt a brief introduction to the method, review the latest advances in cryo-EM structure determination of TRP channels, and discuss molecular insights into the channel function based on the wealth of TRP channel cryo-EM structures.

Journal ArticleDOI
TL;DR: This work uses a hybrid method to study membrane proteins in their native membranes, combining high-resolution solid-state nuclear magnetic resonance spectroscopy and electron cryotomography using the same sample, and demonstrates that the bacterial membrane protein YidC adopts a different conformation in native membranes.

Journal ArticleDOI
TL;DR: This work has developed the first such tool, which incorporates mass spectrometry derived protection factors from HRF labeling as a new centroid score term for the Rosetta scoring function to improve the prediction of protein tertiary structures.
Abstract: In recent years mass spectrometry-based covalent labeling techniques such as hydroxyl radical footprinting (HRF) have emerged as valuable structural biology techniques, yielding information on protein tertiary structure. These data, however, are not sufficient to predict protein structure unambiguously, as they provide information only on the relative solvent exposure of certain residues. Despite some recent advances, no software currently exists that can utilize covalent labeling mass spectrometry data to predict protein tertiary structure. We have developed the first such tool, which incorporates mass spectrometry derived protection factors from HRF labeling as a new centroid score term for the Rosetta scoring function to improve the prediction of protein tertiary structures. We tested our method on a set of four soluble benchmark proteins with known crystal structures and either published HRF experimental results or internally acquired data. Using the HRF labeling data, we rescored large decoy sets of ...