Immunogold labeling in transmission electron microscopy (TEM) utilizes the high electron density of gold nanoparticles conjugated to proteins to identify specific antigens in biological samples. In this work we applied the concept of immunogold labeling for the labeling of negatively charged phospholipids, namely phosphatidylserine, by a simple protocol, performed entirely in the liquid-phase, from which cryo-TEM specimens can be directly prepared. Labeling included a two-step process using biotinylated annexin-V and gold-conjugated streptavidin. We initially applied it on liposomal systems demonstrating its specificity and selectivity, differentiating between 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and 1,2-dioleoyl-sn-glycero-3-phospho-l-serine (DOPS) membranes. We also observed specific labeling on extracellular vesicle samples isolated from THP1 cells and from MDA-468 cells that underwent stimulations. Finally, we compared the levels of annexin-V labeling on the cells vs. on their isolated EVs by flow cytometry and found a good correlation with cryo-TEM results. This simple, yet effective labeling technique makes it possible to differentiate between negatively charged to non-negatively charged membranes, thus shedding light on their possible EV shedding mechanism.

NADPH-dependent assimilatory sulfite reductase (SiR) from Escherichia coli performs a six-electron reduction of sulfite to the bioavailable sulfide. SiR is composed of a flavoprotein (SiRFP) reductase subunit and a hemoprotein (SiRHP) oxidase subunit. There is no known high-resolution structure of SiR or SiRFP, thus we do not yet fully understand how the subunits interact to perform their chemistry. Here, we used small angle neutron scattering to understand the impact of conformationally restricting the highly mobile SiRFP octamer into an electron accepting (closed) or electron donating (open) conformation, showing that SiR remains active, flexible, and asymmetric even with these conformational restrictions. From these scattering data, we model the first solution structure of SiRFP. Further, computational modeling of the N-terminal 52 amino acids that are responsible for SiRFP oligomerization suggests an eight-helical bundle tethers together the SiRFP subunits to form the SiR core. Finally, mass spectrometry analysis of the closed SiRFP variant show that SiRFP is capable of inter-molecular domain swapping, in which the electron donating domain from one polypeptide is able to interact directly with the domain accepting domain of another polypeptide. This structural characterization suggests that SiR performs its high-volume electron transfer through both inter- and intramolecular pathways between SiRFP domains and, thus, cis or trans transfer from reductase to oxidase subunits. Such highly redundant potential for electron transfer makes this system a potential target for designing synthetic enzymes.

Single particle analysis (SPA) in cryo-electron microscopy (cryo-EM) is highly used to obtain the near-atomic structure of biological macromolecules. The current methods allow users to produce high-resolution maps from many samples. However, there are still challenging cases that require extra processing to obtain high resolution. This is the case when the macromolecule of the sample is composed of different components and we want to focus just on one of them. For example, if the macromolecule is composed of several flexible subunits and we are interested in a specific one, if it is embedded in a viral capsid environment, or if it has additional components to stabilize it, such as nanodiscs. The signal from these components, which in principle we are not interested in, can be removed from the particles using a projection subtraction method. Currently, there are two projection subtraction methods used in practice and both have some limitations. In fact, after evaluating their results, we consider that the problem is still open to new solutions, as they do not fully remove the signal of the components that are not of interest. Our aim is to develop a new and more precise projection subtraction method, improving the performance of state-of-the-art methods. We tested our algorithm with data from public databases and an in-house data set. In this work, we show that the performance of our algorithm improves the results obtained by others, including the localization of small ligands, such as drugs, whose binding location is unknown a priori.

Recent advances in cryo electron microscopy (cryo-EM) and image processing provide new opportunities to analyse drug targets at high resolution. However, structural heterogeneity limits resolution in many practical cases, hence restricting the level at which structural details can be analysed and drug design be performed. As structural disorder is not spread throughout the entire structure of a given macromolecular complex but instead is found in certain regions that move with respect to others and covering molecular scales from domain conformational changes up to the level of side chain conformations in ligand binding pockets, it is possible to focus the attention on those regions and the associated relative movements. Here we show how the usage of focused classifications and refinements provide insights into global conformational arrangements, exemplified on the human ribosome and on the cannabinoid G protein coupled receptor (GPCR), and how they can improve the local map resolution from an essentially disordered region to the 3-4 Å and finally to the 2 Å resolution range. A systematic analysis with variable spherical masks during focused refinement is presented showing that the choice of an optimal mask size helps refining to high resolution. This study covers several practical approaches on 4 examples illustrating how important mask size & shape and including neighbouring structural elements are for a focused analysis of a macromolecular complex. Such methods will be crucial for cryo-EM structure-based drug design of various medical targets and are applicable to single particle cryo-EM and electron tomography data.

This article was initially published in the Journal of Structural Biology, instead of the Journal of Structural Biology: X, due to a publisher error. We regret the inconvenience. The link to the article published in Journal of Structural Biology: X is presented below: https://www.sciencedirect.com/science/article/pii/S2590152423000090. The full Elsevier Policy on Article Withdrawal can be found at https://www.elsevier.com/about/policies/article-withdrawal.

Cryo-electron microscopy (cryo-EM) single-particle analysis is a revolutionary imaging technique to resolve and visualize biomacromolecules. Image alignment in cryo-EM is an important and basic step to improve the precision of the image distance calculation. However, it is a very challenging task due to high noise and low signal-to-noise ratio. Therefore, we propose a new deep unsupervised difference learning (UDL) strategy with novel pseudo-label guided learning network architecture and apply it to pair-wise image alignment in cryo-EM. The training framework is fully unsupervised. Furthermore, a variant of UDL called joint UDL (JUDL), is also proposed, which is capable of utilizing the similarity information of the whole dataset and thus further increase the alignment precision. Assessments on both real-world and synthetic cryo-EM single-particle image datasets suggest the new unsupervised joint alignment method can achieve more accurate alignment results. Our method is highly efficient by taking advantages of GPU devices. The source code of our methods is publicly available at "http://www.csbio.sjtu.edu.cn/bioinf/JointUDL/" for academic use.

Ramachandran plots, which describe protein structures by plotting the dihedral angle pairs of the backbone on a two-dimensional plane, have played an important role in structural biology over the past few decades. However, despite continued discovery of new protein structures to date, the Ramachandran plot is still constructed by only a small number of data points, and further it cannot reflect the steric information of proteins. Here, we investigated the secondary structure of proteins in terms of static and dynamic characteristics. As for static feature, the Ramachandran plot was revisited for the dataset consisting of 9,148 non-redundant high-resolution protein structures released in the protein data bank until April 1, 2022. By calculating amino acid propensities, it was found that the proportion of secondary structures with respect to residue depth is directly related to their hydrophobicity. As for dynamic feature, normal mode analysis (NMA) based on an elastic network model (ENM) was carried out for the dataset using our KOSMOS web server (http://bioengineering.skku.ac.kr/kosmos/). All ENM-based NMA results were stored in the KOSMOS database, allowing researchers to use them in various ways. In this process, it was commonly found that high B-factors appeared at the edge of the alpha helix region, which was elucidated by introducing residue depth. In addition, by investigating the change in dihedral angle, it was possible to quantitatively survey the contribution of structural change of protein on the Ramachandran plot. In conclusion, our statistical analysis of protein characteristics will provide insight into a range of protein structural studies.

O-Glycosylation of hydroxylysine (Hyl) in collagen occurs at an early stage of biosynthesis before the triple-helix has formed. This simple post-translational modification (PTM) of lysine by either a galactosyl or glucosylgalactosyl moiety is highly conserved in collagens and depends on the species, type of tissue and the collagen amino acid sequence. The structural/functional reason why only specific lysines are modified is poorly understood, and has led to increased efforts to map the sites of PTMs on collagen sequences from different species and to ascertain their potential role in vivo. To investigate this, we purified collagen type I (Col1) from the skins of four animals, then used mass spectrometry and proteomic techniques to identify lysines that were oxidised, galactosylated, glucosylgalactosylated, or glycated in its mature sequence. We found 18 out of the 38 lysines in collagen type Iα1, (Col1A1) and 7 of the 30 lysines in collagen type Iα2 (Col1A2) were glycosylated. Six of these modifications had not been reported before, and included a lysine involved in crosslinking collagen molecules. A Fourier transform analysis of the positions of the glycosylated hydroxylysines showed they display a regular axial distribution with the same d-period observed in collagen fibrils. The significance of this finding in terms of the assembly of collagen molecules into fibrils and of potential restrictions on the growth of the collagen fibrils is discussed.

As structural determination of protein complexes approaches atomic resolution, there is an increasing focus on conformational dynamics. Here we conceptualize the combination of two techniques which have become established in recent years: microcrystal electron diffraction and ultrafast electron microscopy. We show that the extremely low dose of pulsed photoemission still enables microED due to the strength of the electron bunching from diffraction of the protein crystals. Indeed, ultrafast electron diffraction experiments on protein crystals have already been demonstrated to be effective in measuring intermolecular forces in protein microcrystals. We discuss difficulties that may arise in the acquisition and processing of data and the overall feasibility of the experiment, paying specific attention to dose and signal-to-noise ratio. In doing so, we outline a detailed workflow that may be effective in minimizing the dose on the specimen. A series of model systems that would be good candidates for initial experiments is provided.

Small GTPase RhoA switches from GTP-bound state to GDP-bound state by hydrolyzing GTP, which is accelerated by GTPases activating proteins (GAPs). However, less study of RhoA structural dynamic changes was conducted during this process, which is essential for understanding the molecular mechanism of GAP dissociation. Here, we solved a RhoA structure in GDP-bound state with switch II flipped outward. Because lacking the intermolecular interactions with guanine nucleotide, we proposed this conformation of RhoA could be an intermediate after GAP dissociation. Further molecular dynamics simulations found the conformational changes of switch regions are indeed existing in RhoA and involved in the regulation of GAP dissociation and GEF recognition. Besides, the guanine nucleotide binding pocket extended to switch II region, indicating a potential "druggable" cavity for RhoA. Taken together, our study provides a deeper understanding of the dynamic properties of RhoA switch regions and highlights the direction for future drug development.

Tandem Repeat Proteins (TRPs) are a class of proteins with repetitive amino acid sequences that have been studied extensively for over two decades. Different features at the level of sequence, structure, function and evolution have been attributed to them by various authors. And yet many of its salient features appear only when looking at specific subclasses of protein tandem repeats. Here, we attempt to rationalize the existing knowledge on Tandem Repeat Proteins (TRPs) by pointing out several dichotomies. The emerging picture is more nuanced than generally assumed and allows us to draw some boundaries of what is not a "proper" TRP. We conclude with an operational definition of a specific subset, which we have denominated STRPs (Structural Tandem Repeat Proteins), which separates a subclass of tandem repeats with distinctive features from several other less well-defined types of repeats. We believe that this definition will help researchers in the field to better characterize the biological meaning of this large yet largely understudied group of proteins.

Recent technological advances in cryo electron microscopy (cryo-EM) have led to new opportunities in the structural biology field. Here we benchmark the performance of two 300 kV latest-generation cryo electron microscopes, Titan Krios G4 from Thermofisher Scientific and CRYO ARM 300 from Jeol, with regards to achieving high resolution single particle reconstructions on a real case sample. We compare potentially limiting factors such as drift rates, astigmatism & coma aberrations and performance during image processing and show that both microscopes, while comprising rather different technical setups & parameter settings and equipped with different types of energy filters & cameras, achieve a resolution of around 2 Å on the human ribosome, a non-symmetric object which constitutes a key drug target. Astigmatism correction, CTF refinement and correction of higher order aberrations through refinement in separate optics groups helped to account for astigmatism/coma caused by beam tilting during multi-spot and multi-hole acquisition in neighbouring holes without stage movement. The obtained maps resolve Mg2+ ions, water molecules, inhibitors and side-chains including chemical modifications. The fact that both instruments can resolve such detailed features will greatly facilitate understanding molecular mechanisms of various targets and helps in cryo-EM structure based drug design. The methods and analysis tools used here will be useful also to characterize existing instruments and optimize data acquisition settings and are applicable broadly to other drug targets in structural biology.

Cryo-electron tomography (cryo-ET) and subtomogram averaging (STA) can resolve protein complexes at near atomic resolution, and when combined with focused ion beam (FIB) milling, macromolecules can be observed within their native context. Unlike single particle acquisition (SPA), cryo-ET can be slow, which may reduce overall project throughput. We here propose a fast, multi-position tomographic acquisition scheme based on beam-tilt corrected beam-shift imaging along the tilt axis, which yields sub-nanometer in situ STA averages.

Structural and functional characterization of proteins as well as the design of targeted drugs heavily rely on recombinant protein expression and purification. The polyhistidine-tag (His-tag) is among the most prominent examples of affinity tags used for the isolation of recombinant proteins from their expression hosts. Short peptide tags are commonly considered not to interfere with the structure of the tagged protein and tag removal is frequently neglected. This study demonstrates the formation of higher-order oligomers based on the example of two His-tagged membrane proteins, the dimeric arginine-agmatine antiporter AdiC and the pentameric light-driven proton pump proteorhodopsin. Size exclusion chromatography revealed the formation of tetrameric AdiC and decameric as well as pentadecameric proteorhodopsin through specific interactions between their His-tags. In addition, single particle cryo-electron microscopy (cryo-EM) allowed structural insights into the three-dimensional arrangement of the higher-order oligomers and the underlying His-tag-mediated interactions. These results reinforce the importance of considering the length and removal of affinity purification tags and illustrate how neglect can lead to potential interference with downstream biophysical or biochemical characterization of the target protein.

The foraminiferal order Rotaliida represents one third of the extant genera of foraminifers. The shells of these organisms are extensively used to decipher characteristics of marine ecosystems and global climate events. It was shown that shell calcite of benthic Rotaliida is twinned. We extend our previous work on microstructure and texture characterization of benthic Rotaliida and investigate shell calcite organization for planktonic rotaliid species. Based on results gained from electron backscattered diffraction (EBSD) and field emission electron microscopy (FESEM) imaging of chemically etched/fixed shell surfaces we show for the planktonic species Globigerinoides sacculifer, Pulleniatina obliquiloculata, Orbulina universa (belonging to the two main planktonic, the globigerinid and globorotaliid, clades): very extensive 60°-{001}-twinning of the calcite and describe a new and specific microstructure for the twinned crystals. We address twin and crystal morphology development from nucleation within a biopolymer template (POS) to outermost shell surfaces. We demonstrate that the calcite of the investigated planktonic Rotaliida forms through competitive growth. We complement the structural knowledge gained on the clade 1 and clade 2 species with EBSD results of Globigerinita glutinata and Candeina nitida shells (clade 3 planktonic species). The latter are significantly less twinned and have a different shell calcite microstructure. We demonstrate that the calcite of all rotaliid species is twinned, however, to different degrees. We discuss for the species of the three planktonic clades characteristics of the twinned calcite and of other systematic misorientations. We address the strong functionalization of foraminiferal calcite and indicate how the twinning affects biocalcite material properties.

Staphylococcal protein A (SpA) domain B (the basis of affibody) has been widely used in affinity chromatography and found therapeutic applications against inflammatory diseases through targeting the Fc part of immunoglobulin G (IgG). We have performed extensive molecular dynamics simulation of 41 SpA mutants and compared their dynamics and conformations to wild type. The simulations revealed the molecular details of structural and dynamics changes that occurred due to introducing point mutations and helped to explain the SPR results. It was observed in some variants a point mutation caused extensive structural changes far from the mutation site, while an effect of some other mutations was limited to the site of the mutated residue. Also, the pattern of hydrogen bond networks and hydrophobic core arrangements were investigated. We figured out mutations that occurred at positions 128, 136, 150 and 153, affected two hydrophobic cores at the interface as well as mutations introduced at positions 129 and 154 interrupted two hydrogen bond networks of the interface, SPR data showed all of these mutations reduced binding affinity significantly. Overall, by scanning the SpA-Fc interface through the large numbers of introduced mutations, the new insights have been gained which would help to design high- affinity ligands of IgG.

Zinc transporter 8 (ZnT8) is mainly expressed in pancreatic islet β cells and is responsible for H+-coupled uptake (antiport) of Zn2+ into the lumen of insulin secretory granules. Structures of human ZnT8 and its prokaryotic homolog YiiP have provided structural basis for constructing a plausible transport cycle for Zn2+. However, the mechanistic role that protons play in the transport process remains unclear. Here we present a lumen-facing cryo-EM structure of ZnT8 from Xenopus tropicalis (xtZnT8) in the presence of Zn2+ at a luminal pH (5.5). Compared to a Zn2+-bound xtZnT8 structure at a cytosolic pH (7.5), the low-pH structure displays an empty transmembrane Zn2+-binding site with a disrupted coordination geometry. Combined with a Zn2+-binding assay our data suggest that protons may disrupt Zn2+ coordination at the transmembrane Zn2+-binding site in the lumen-facing state, thus facilitating Zn2+ release from ZnT8 into the lumen.

Eukaryotic initiation factor 2 (eIF2) plays a key role in protein synthesis and in its regulation. The assembly of this heterotrimeric factor is facilitated by Cdc123, a member of the ATP grasp family that binds the γ subunit of eIF2. Notably, some mutations related to MEHMO syndrome, an X-linked intellectual disability, affect Cdc123-mediated eIF2 assembly. The mechanism of action of Cdc123 is unclear and structural information for the human protein is awaited. Here, the crystallographic structure of human Cdc123 (Hs-Cdc123) bound to domain 3 of human eIF2γ (Hs-eIF2γD3) was determined. The structure shows that the domain 3 of eIF2γ is bound to domain 1 of Cdc123. In addition, the long C-terminal region of Hs-Cdc123 provides a link between the ATP and Hs-eIF2γD3 binding sites. A thermal shift assay shows that ATP is tightly bound to Cdc123 whereas the affinity of ADP is much smaller. Yeast cell viability experiments, western blot analysis and two-hybrid assays show that ATP is important for the function of Hs-Cdc123 in eIF2 assembly. These data and recent findings allow us to propose a refined model to explain the mechanism of action of Cdc123 in eIF2 assembly.

Cryo-electron tomography and subtomogram averaging are rising and fast-evolving imaging techniques to study biological events, providing structural information at an unprecedented resolution while preserving spatial correlation in their native contexts. The latest technology and methodology development ranging from sample preparation to data collection and data processing, has enabled significant advancement in its applications to various biological systems. This review provides an overview of the current technology development enabling high-resolution structural study in situ, highlighting the use of a priori information of biological samples to assess the quality of subtomogram averaging pipeline. We exemplify the applications of this technique to understanding viruses and principles of macromolecule assembly using different biological systems, ranging from in vitro to in situ samples, which provide structural information at different resolutions and contexts.

Leucine Rich Repeat (LRR) domains, are present in hundreds of thousands of proteins across all kingdoms of life and are typically involved in protein-protein interactions and ligand recognition. LRR domains are classified into eight classes and when examined in three dimensions seven, of them form curved solenoid-like super-helices, also described as toruses, with a beta sheet on the concave (inside) and stacked alpha-helices on the convex (outside) of the torus. Here we present an overview of the least characterized 8th class of LRR proteins, the TpLRR-like LRRs, named after the Treponema pallidum protein Tp0225. Proteins from the TpLRR class differ from the proteins in all other known LRR classes by having a flipped curvature, with the beta sheet on the convex side of the torus and irregular secondary structure instead of helices on the opposite, now concave site. TpLRR proteins also present highly divergent sequence pattern of individual repeats and can associate with specific types of additional domains. Several of the characterized proteins from this class, specifically the BspA-like proteins, were found in human bacterial and protozoan pathogens, playing an important role in the interactions between the pathogens and the host immune system. In this paper we surveyed all existing experimental structures and selected AlphaFold models of the best-known proteins containing this class of LRR repeats, analyzing the relation between the pattern of conserved residues, specific structural features and functions of these proteins.