MISO: microfluidic protein isolation enables single-particle cryo-EM structure determination from a single cell colony.

Cryo-EM structure and functional analyses show that human iron exporter ferroportin (Fpn) can bind and transport calcium ions and the transport activity is reduced when the concentration of ferrous ions is high.

The cryo-EM structure of ETB-Gi complex, obtained using the Fusion-G system, provides insights into the distinct activation mechanism and G-protein promiscuity of ETB, aiding in the design of ETB agonists for therapeutic applications.

A host of new technologies are under development to improve the quality and reproducibility of cryoEM grid preparation. Here we have systematically investigated the preparation of three macromolecular complexes using three different vitrification devices (Vitrobot™, chameleon and a time-resolved cryoEM device) on various timescales, including grids made within 6 ms, (the fastest reported to date), to interrogate particle behaviour at the air-water interface for different timepoints. Results demonstrate that different macromolecular complexes can respond to the thin film environment formed during cryoEM sample preparation in highly variable ways, shedding light on why cryoEM sample preparation can be difficult to optimise. We demonstrate that reducing time between sample application and vitrification is just one tool to improve cryoEM grid quality, but that it is unlikely to be a generic ‘silver bullet’ for improving the quality of every cryoEM sample preparation.

A simple and robust procedure for preparing graphene-oxide cryo-EM grids.

Bacteroidetes are abundant members of the human microbiota, utilising a myriad of diet- and host-derived glycans in the distal gut. Glycan uptake across the bacterial outer membrane (OM) of these bacteria is mediated by SusCD protein complexes, comprising a membrane-embedded barrel and a lipoprotein lid, that is thought to open and close to facilitate substrate binding and transport. However, surface-exposed glycan binding proteins and glycoside hydrolases also play critical roles in the capture, processing and transport of large glycan chains. The interactions between these components in the OM are poorly understood, despite being crucial for nutrient acquisition by our colonic microbiota. Here we show that for both the levan and dextran utilisation systems of Bacteroides thetaiotaomicron, the additional OM components assemble on the core SusCD transporter, forming stable glycan utilising machines which we term ‘utilisomes’. Single particle cryogenic electron microscopy (cryoEM) structures in the absence and presence of substrate reveal concerted conformational changes that demonstrate the mechanism of substrate capture, and rationalise the role of each component in the utilisome.

Lateral diffusion of proteins in the plane of a biological membrane is important for many vital processes, including energy conversion, signaling, chemotaxis, cell division, protein insertion, and secretion. In bacteria, all these functions are located in a single membrane. Therefore, quantitative measurements of protein diffusion in bacterial membranes can provide insight into many important processes. Diffusion of membrane proteins in eukaryotes has been studied in detail using various experimental techniques, including fluorescence correlation spectroscopy (FCS), fluorescence recovery after photobleaching (FRAP), and particle tracking using single-molecule fluorescence (SMF) microscopy. In case of bacteria, such experiments are intrinsically difficult due to the small size of the cells. Here, we review these experimental approaches to quantify diffusion in general and their strengths and weaknesses when applied to bacteria. In addition, we propose a method to extract multiple diffusion coefficients from trajectories obtained from SMF data, using cumulative probability distributions (CPDs). We demonstrate the power of this approach by quantifying the heterogeneous diffusion of the bacterial membrane protein TatA, which forms a pore for the translocation of folded proteins. Using computer simulations, we study the effect of cell dimensions and membrane curvature on measured CPDs. We find that at least two mobile populations with distinct diffusion coefficients (of 7 and 169 nm(2) ms(-1) , respectively) are necessary to explain the experimental data. The approach described here should be widely applicable for the quantification of membrane-protein diffusion in living bacteria.

Due to their unique properties, expression and study of membrane proteins in heterologous systems remains difficult. Among the bacterial systems available, the Gram-positive lactic bacterium, Lactococcus lactis, traditionally used in food fermentations, is nowadays widely used for large-scale production and functional characterization of bacterial and eukaryotic membrane proteins. The aim of this chapter is to describe the different possibilities for the functional characterization of peripheral or intrinsic membrane proteins expressed in Lactococcus lactis.

BackgroundDespite their functional and biotechnological importance, the study of membrane proteins remains difficult due to their hydrophobicity and their low natural abundance in cells. Furthermore, into established heterologous systems, these proteins are frequently only produced at very low levels, toxic and mis- or unfolded. Lactococcus lactis, a Gram-positive lactic bacterium, has been traditionally used in food fermentations. This expression system is also widely used in biotechnology for large-scale production of heterologous proteins. Various expression vectors, based either on constitutive or inducible promoters, are available for this system. While previously used to produce bacterial and eukaryotic membrane proteins, the ability of this system to produce plant membrane proteins was until now not tested.Methodology/Principal FindingsThe aim of this work was to test the expression, in Lactococcus lactis, of either peripheral or intrinsic Arabidopsis membrane proteins that could not be produced, or in too low amount, using more classical heterologous expression systems. In an effort to easily transfer genes from Gateway-based Arabidopsis cDNA libraries to the L. lactis expression vector pNZ8148, we first established a cloning strategy compatible with Gateway entry vectors. Interestingly, the six tested Arabidopsis membrane proteins could be produced, in Lactococcus lactis, at levels compatible with further biochemical analyses. We then successfully developed solubilization and purification processes for three of these proteins. Finally, we questioned the functionality of a peripheral and an intrinsic membrane protein, and demonstrated that both proteins were active when produced in this system.Conclusions/SignificanceAltogether, these data suggest that Lactococcus lactis might be an attractive system for the efficient and functional production of difficult plant membrane proteins.

RETURN TO ISSUEPREVNewsNEXTMISTIC BOOSTS PROTEIN YIELDSDiscovery allows eukaryotic membrane proteins to be made in high yieldsAMANDA YARNELLCite this: Chem. Eng. News 2005, 83, 9, 13Publication Date (Print):February 28, 2005Publication History Published online14 November 2010Published inissue 28 February 2005https://pubs.acs.org/doi/10.1021/cen-v083n009.p013https://doi.org/10.1021/cen-v083n009.p013newsACS PublicationsCopyright © 2005 American Chemical SocietyArticle Views8Altmetric-Citations1LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InRedditEmail Other access options SUBJECTS:Peptides and proteins Get e-Alerts

Although membrane proteins are abundant and fulfill many important functions, only a few high-resolution conformations of this class of proteins have been published (reviewed in [1; 2]). Structural investigations are hampered by the problems that are encountered during their large-scale expression and purification as well as during application of diffraction and NMR techniques. Since publication of the high-resolution diffraction map of the photoreaction centre [3], the conformations of other bacterial membrane proteins have been solved at a relatively slow pace. Most of these proteins already occur at high concentrations in nature and some of them even in ordered arrays [1; 2]. More recently high resolution structures of eukaryotic membrane proteins have been added to the structural data base, including rhodopsin, a G protein-coupled receptor [4; 5]. Improvements in crystallisation as well as in X-ray and electron diffraction techniques hold promise that these developments will accelerate, and structures of an increasing number of membrane proteins will become available in the future. Some of this work illuminates structural changes that occur during functional activities in a stroboscopic manner [4; 6].

Persistent hurdles impede the successful determination of high-resolution crystal structures of eukaryotic integral membrane proteins (IMP). We designed a high-throughput structural genomics oriented pipeline that seeks to minimize effort in uncovering high-quality, responsive non-redundant targets for crystallization. This "discovery-oriented" pipeline sidesteps two significant bottlenecks in the IMP structure determination pipeline: expression and membrane extraction with detergent. In addition, proteins that enter the pipeline are then rapidly vetted by their presence in the included volume on a size-exclusion column--a hallmark of well-behaved IMP targets. A screen of 384 rationally selected eukaryotic IMPs in baker's yeast Saccharomyces cerevisiae is outlined to demonstrate the results expected when applying this discovery-oriented pipeline to whole-organism membrane proteomes.

Ryanodine receptors (RyRs) are large intracellular calcium release channels that play a crucial role in coupling excitation to contraction in both cardiac and skeletal muscle cells. In addition, they are expressed in other cell types where their function is less well understood. Hundreds of mutations in the different isoforms of RyR have been associated with inherited myopathies and cardiac arrhythmia disorders. The structure of these important drug targets remained elusive for a long time, despite decades of intensive research. In the recent years, a technical revolution in the field of single particle cryogenic electron microscopy (SP cryo-EM) allowed solving high-resolution structures of the skeletal and cardiac RyR isoforms. Together with the structures of individual domains solved by X-ray crystallography, this resulted in an unprecedented understanding of the structure, gating and regulation of these largest known ion channels. In this chapter we describe the recently solved high-resolution structures of RyRs, discuss molecular details of the channel gating, regulation and the disease mutations. Additionally, we highlight important questions that require further progress in structural studies of RyRs.

Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution1-3. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships4, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.

Knowing the subcellular locations of proteins is an important step in understanding the protein functions. The experimental methods for determining the protein subcellular localization are usually costly and time consuming. Thus, in the last decade, many computational methods were introduced to determine the protein subcellular location in sillico. Although the physicochemical properties of membrane proteins and globular proteins are different, most of these methods treated the globular proteins and membrane proteins equally. Thus, developing method for determining the subcellular locations for them separately would be necessary. We proposed an algorithm called Weighted Gene Ontology Scores (WGOS) to predict the protein subcellular location of membrane proteins. By comparing the prediction performance of WGOS to MemLoci, which is the only existing method for predicting membrane protein localizations, WGOS performed significantly better than the MemLoci, indicating WGOS might be able to provide better results in practical applications.

VDAC constitutes the primary pathway for the exchange of metabolites across the outer mitochondrial membrane. It acts as a scaffold for modulator proteins and is implicated in mitochondria‐dependent cell death. Because of its critical role in mitochondrial biology, we sought to obtain the crystal structure of VDAC as a first step to understanding its gating mechanism, in vivo regulation and interaction with other proteins. mVDAC1 was expressed in E. coli, purified from inclusion body and refolded. The purified protein was biochemically characterized to ensure that its identity, proper fold and electrophysiological properties are the same as the native protein.We present the structure of mVDAC1 at 2.3 Åresolution crystallized in lipidic bicelles to preserve the native conformation. mVDAC1 adopts a β‐barrel architecture composed of 19 β‐strands, thus displaying a new fold for β‐barrel membrane proteins resulting from an odd number of strands. The N‐terminus is located inside the barrel and forms an α‐helix halfway down the pore resulting in a partial narrowing at the center. This orientation of the helix is ideally suited to gate metabolite flux through the channel. The mVDAC1 structure presented here represents the first high resolution crystal structure of a eukaryotic β‐barrel membrane protein providing critical information for our understanding of its role in mitochondrial physiology. Funding: NIH and AHA.