Single-particle Electron Microscopy Research Articles

Biochemical and Structural Characterization of Human Core Elongator and Its Subassemblies.

Conserved from yeast to humans and composed of six core subunits (Elp1–Elp6), Elongator is a multiprotein complex that catalyzes the modification of the anticodon loop of transfer RNAs (tRNAs) and in turn regulates messenger RNA decoding efficiency. Previous studies showed that yeast Elongator consists of two subassemblies (yElp1/2/3 and yElp4/5/6) and adopts an asymmetric overall architecture. Yet, much less is known about the structural properties of the orthologous human Elongator. Furthermore, the order in which the different Elongator subunits come together to form the full assembly as well as how they coordinate with one another to catalyze tRNA modification is not fully understood. Here, we purified recombinant human Elongator subunits and subassemblies and examined them by single-particle electron microscopy. We found that the human Elongator complex is assembled from two subcomplexes that share similar overall morphologies as their yeast counterparts. Complementary co-purification and pulldown assays revealed that the scaffolding subunit human ELP1 (hELP1) has stabilizing effects on the human ELP3 catalytic subunit. Furthermore, the peripheral hELP2 subunit appears to enhance the integrity and substrate-binding ability of the dimeric hELP1/2/3. Lastly, we found that hELP4/5/6 is recruited to hELP1/2/3 via hELP3. Collectively, our work generated insights into the assembly process of core human Elongator and the coordination of different subunits within this complex.

Correlating structure, morphology and properties of metal nanostructures by combining single-particle optical spectroscopy and electron microscopy.

The nanoscale morphology of metal nanostructures directly defines their optical, catalytic and electronic properties and even small morphological changes can cause significant property variations. On the one hand, this dependence allows for precisely tuning and exploring properties by shape engineering; next to advanced synthesis protocols, post-synthesis modification through tailored laser modification has become an emerging tool to do so. On the other hand, with this interconnection also comes the quest for detailed structure-property correlation and understanding of laser-induced reshaping processes on the individual nanostructure level beyond ensemble averages. With the development of single-particle (ultrafast) optical spectroscopy techniques and advanced electron microscopy such understanding can in principle be gained at the femtosecond temporal and atomic spatial scale, respectively. However, accessing both on the same individual nanostructure is far from straightforward as it requires the combination of optical spectroscopy and electron microscopy. In this Minireview, we highlight key studies from recent years that performed such correlative measurements on the same individual metal nanostructure either in a consecutive ex situ manner or in situ inside the electron microscope. We demonstrate that such a detailed correlation is critical for revealing the full picture of the structure-property relationship and the physics behind light-induced nanostructure modifications. We put emphasis on the advantages and disadvantages of each methodology as well as on the unique information that one can gain only by correlative studies performed on the same individual nanostructure and end with an outlook on possible further development of this field in the near future.

Customizable nano-sized colloidal tetrahedra by polymerization-induced particle self-assembly (PIPA)

Nanoscale colloidal tetrahedra are synthesized by polymerization-induced particle self-assembly, with high purity, which allows the 3D reconstruction by single particle electron microscopy analysis.

Putting on molecular weight: Enabling cryo-EM structure determination of sub-100-kDa proteins.

Significant advances in the past decade have enabled high-resolution structure determination of a vast variety of proteins by cryogenic electron microscopy single particle analysis. Despite improved sample preparation, next-generation imaging hardware, and advanced single particle analysis algorithms, small proteins remain elusive for reconstruction due to low signal-to-noise and lack of distinctive structural features. Multiple efforts have therefore been directed at the development of size-increase techniques for small proteins. Here we review the latest methods for increasing effective molecular weight of proteins <100 ​kDa through target protein binding or target protein fusion - specifically by using nanobody-based assemblies, fusion tags, and symmetric scaffolds. Finally, we summarize these state-of-the-art techniques into a decision-tree to facilitate the design of tailored future approaches, and thus for further exploration of ever-smaller proteins that make up the largest part of the human genome.

Structural Analysis of Botulinum Neurotoxins Type B and E by Cryo-EM.

Botulinum neurotoxins (BoNTs) are the causative agents of a potentially lethal paralytic disease targeting cholinergic nerve terminals. Multiple BoNT serotypes exist, with types A, B and E being the main cause of human botulism. Their extreme toxicity has been exploited for cosmetic and therapeutic uses to treat a wide range of neuromuscular disorders. Although naturally occurring BoNT types share a common end effect, their activity varies significantly based on the neuronal cell-surface receptors and intracellular SNARE substrates they target. These properties are the result of structural variations that have traditionally been studied using biophysical methods such as X-ray crystallography. Here, we determined the first structures of botulinum neurotoxins using single-particle cryogenic electron microscopy. The maps obtained at 3.6 and 3.7 Å for BoNT/B and /E, respectively, highlight the subtle structural dynamism between domains, and of the binding domain in particular. This study demonstrates how the recent advances made in the field of single-particle electron microscopy can be applied to bacterial toxins of clinical relevance and the botulinum neurotoxin family in particular.

Conserved and divergent features of neuronal CaMKII holoenzyme structure, function, and high-order assembly.

SUMMARYNeuronal CaMKII holoenzymes (α and β isoforms) enable molecular signal computation underlying learning and memory but also mediate excitotoxic neuronal death. Here, we provide a comparative analysis of these signaling devices, using single-particle electron microscopy (EM) in combination with biochemical and live-cell imaging studies. In the basal state, both isoforms assemble mainly as 12-mers (but also 14-mers and even 16-mers for the β isoform). CaMKIIα and β isoforms adopt an ensemble of extended activatable states (with average radius of 12.6 versus 16.8 nm, respectively), characterized by multiple transient intra- and inter-holoenzyme interactions associated with distinct functional properties. The extended state of CaMKIIβ allows direct resolution of intra-holoenzyme kinase domain dimers. These dimers could enable cooperative activation by calmodulin, which is observed for both isoforms. High-order CaMKII clustering mediated by inter-holoenzyme kinase domain dimerization is reduced for the β isoform for both basal and excitotoxicity-induced clusters, both in vitro and in neurons.

Single Extracellular Vesicle Analysis Performed by Imaging Flow Cytometry and Nanoparticle Tracking Analysis Evaluate the Accuracy of Urinary Extracellular Vesicle Preparation Techniques Differently.

Small extracellular vesicles isolated from urine (uEVs) are increasingly recognized as potential biomarkers. Meanwhile, different uEV preparation strategies exist. Conventionally, the performance of EV preparation methods is evaluated by single particle quantification, Western blot, and electron microscopy. Recently, we introduced imaging flow cytometry (IFCM) as a next-generation single EV analysis technology. Here, we analyzed uEV samples obtained with different preparation procedures using nanoparticle tracking analysis (NTA), semiquantitative Western blot, and IFCM. IFCM analyses demonstrated that urine contains a predominant CD9+ sEV population, which exceeds CD63+ and CD81+ sEV populations. Furthermore, we demonstrated that the storage temperature of urine samples negatively affects the recovery of CD9+ sEVs. Although overall reduced, the highest CD9+ sEV recovery was obtained from urine samples stored at −80 °C and the lowest from those stored at −20 °C. Upon comparing the yield of the different uEV preparations, incongruencies between NTA and IFCM data became apparent. Results obtained by both NTA and IFCM were consistent with Western blot analyses for EV marker proteins; however, NTA results correlated with the amount of the impurity marker uromodulin. Despite demonstrating that the combination of ultrafiltration and size exclusion chromatography appears as a reliable uEV preparation technique, our data challenge the soundness of traditional NTA for the evaluation of different EV preparation methods.

The structure of natively iodinated bovine thyroglobulin.

Thyroglobulin is a homodimeric glycoprotein that is essential for the generation of thyroid hormones in vertebrates. Upon secretion into the lumen of follicles in the thyroid gland, tyrosine residues within the protein become iodinated to produce monoiodotyrosine (MIT) and diiodotyrosine (DIT). A subset of evolutionarily conserved pairs of DIT (and MIT) residues can then engage in oxidative coupling reactions that yield either thyroxine (T4; produced from coupling of a DIT `acceptor' with a DIT `donor') or triiodothyronine (T3; produced from coupling of a DIT acceptor with an MIT donor). Although multiple iodotyrosine residues have been identified as potential donors and acceptors, the specificity and structural context of the pairings (i.e. which donor is paired with which acceptor) have remained unclear. Here, single-particle cryogenic electron microscopy (cryoEM) was used to generate a high-resolution reconstruction of bovine thyroglobulin (2.3 Å resolution in the core region and 2.6 Å overall), allowing the structural characterization of two post-reaction acceptor-donor pairs as well as tyrosine residues modified as MIT and DIT. A substantial spatial separation between donor Tyr149 and acceptor Tyr24 was observed, suggesting that for thyroxine synthesis significant peptide motion is required for coupling at the evolutionarily conserved thyroglobulin amino-terminus.

Human antibody recognition of H7N9 influenza virus HA following natural infection.

Avian H7N9 influenza viruses cause sporadic outbreaks of human infections and threaten to cause a major pandemic. The breadth of B cell responses to natural infection and the dominant antigenic sites recognized during first exposure to H7 HA following infection are incompletely understood. Here, we studied the B cell response to H7 HA of 2 individuals who had recovered from natural H7N9 virus infection. We used competition binding, hydrogen-deuterium mass spectrometry, and single-particle negative stain electron microscopy to identify the patterns of molecular recognition of the antibody responses to H7 HA. We found that circulating H7-reactive B cells recognized a diverse antigenic landscape on the HA molecule, including HA head domain epitopes in antigenic sites A and B and in the trimer interface-II region and epitopes in the stem region. Most H7 antibodies exhibited little heterosubtypic breadth, but many recognized a wide diversity of unrelated H7 strains. We tested the antibodies for functional activity and identified clones with diverse patterns of inhibition, including neutralizing, hemagglutination- or egress-inhibiting, or HA trimer–disrupting activities. Thus, the human B cell response to primary H7 natural infection is diverse, highly functional, and broad for recognition of diverse H7 strains.

Thermodynamic and structural basis of temperature-dependent gating in TRP channels.

Living organisms require detecting the environmental thermal clues for survival, allowing them to avoid noxious stimuli or find prey moving in the dark. In mammals, the Transient Receptor Potential ion channels superfamily is constituted by 27 polymodal receptors whose activity is controlled by small ligands, peptide toxins, protons and voltage. The thermoTRP channels subgroup exhibits unparalleled temperature dependence -behaving as heat and cold sensors. Functional studies have dissected their biophysical features in detail, and the advances of single-particle Cryogenic Electron microscopy provided the structural framework required to propose detailed channel gating mechanisms. However, merging structural and functional evidence for temperature-driven gating of thermoTRP channels has been a hard nut to crack, remaining an open question nowadays. Here we revisit the highlights on the study of heat and cold sensing in thermoTRP channels in the light of the structural data that has emerged during recent years.

Extraction and quantification of metal-containing nanoparticles in marine shellfish based on single particle inductively coupled plasma-mass spectrometry technique

Quantitative characterization of nanoparticles (NPs) in marine shellfish is critical to understanding the risks of bio-accumulation. Based on single particle (sp)ICP-MS and electron microscopy, a standardized protocol was developed to extract Ag, Au, and indigenous Ti-containing NPs from mussels. The optimal parameters are: dry sample extraction with tetramethylammonium hydroxide (TMAH), 5% (v/v) final concentration of TMAH, extraction at 25 ℃ for 12 h, and separation by centrifugation (3000 rpm for 5 min). The particle number recoveries of spiked Ag and Au NPs were 88 ± 0.9% and 95 ± 1.1%, respectively, while Ti-containing NPs had a particle number concentration of 8.2 × 106 particles/mg and an average size of 70 nm in tested mussels. Furthermore, titanium oxide NPs, including rutile, anatase, and Magnéli phases (TixO2x-1) were found ubiquitously in 10 shellfish based on the optimal method. The particle number concentrations and average sizes of the Ti-containing NPs were 2.1 × 106–8.4 × 106 particles/mg and 70–80 nm, respectively. These Ti-containing NPs, such as TiO2, accounted for about half of the Ti mass in shellfish, indicating that marine shellfish may be a significant sink for Ti-containing NPs.

TXNIP interaction with GLUT1 depends on PI(4,5)P2

GLUT1 is a major glucose facilitator expressed ubiquitously among tissues. Upregulation of its expression plays an important role in the development of many types of cancer and metabolic diseases. Thioredoxin-interacting protein (TXNIP) is an α-arrestin that acts as an adaptor for GLUT1 in clathrin-mediated endocytosis. It regulates cellular glucose uptake in response to both intracellular and extracellular signals via its control on GLUT1‐4. In order to understand the interaction between GLUT1 and TXNIP, we generated GLUT1 lipid nanodiscs and carried out isothermal titration calorimetry and single-particle electron microscopy experiments. We found that GLUT1 lipid nanodiscs and TXNIP interact in a 1:1 ratio and that this interaction requires phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2 or PIP2).

Structure of Usutu virus SAAR-1776 displays fusion loop asymmetry

Usutu virus (USUV) is an emerging arbovirus in Europe that has been increasingly identified in asymptomatic humans and donated blood samples and is a cause of increased incidents of neuroinvasive human disease. Treatment or prevention options for USUV disease are currently nonexistent, the result of a lack of understanding of the fundamental elements of USUV pathogenesis. Here, we report two structures of the mature USUV virus, determined at a resolution of 2.4 Å, using single-particle cryogenic electron microscopy. Mature USUV is an icosahedral shell of 180 copies of envelope (E) and membrane (M) proteins arranged in the classic herringbone pattern. However, unlike previous reports of flavivirus structures, we observe virus subpopulations and differences in the fusion loop disulfide bond. Presence of a second, unique E glycosylation site could elucidate host interactions, contributing to the broad USUV tissue tropism. The structures provide a basis for exploring USUV interactions with glycosaminoglycans and lectins, the role of the RGD motif as a receptor, and the inability of West Nile virus therapeutic antibody E16 to neutralize the mature USUV strain SAAR-1776. Finally, we identify three lipid binding sites and predict key residues that likely participate in virus stability and flexibility during membrane fusion. Our findings provide a framework for the development of USUV therapeutics and expand the current knowledge base of flavivirus biology.

Titanium nanoparticles fate in small-sized watersheds under different land-uses

Surface waters from three catchments having contrasting land-uses (forested, agricultural, and urban) were sampled monthly and analysed for nanoparticulate titanium dioxide (NPs-TiO2) by single particle ICPMS and electron microscopy. We report one-year of data for NPs-TiO2 having average number and mass concentrations of 9.1 × 108 NPs-TiO2 particles L-1 and 11 µg NPs-TiO2 L-1 respectively. An increase in concentration during warmer months is observed in the forested and agricultural catchments. Both concentrations of NPs-TiO2 are within the range of recently reported values using similar analytical approaches. The positive correlations for NPs-TiO2 mass concentration or particle number with the concentration of some trace elements and DOC in the forested and agricultural catchments suggest the detected NPs-TiO2 in these two systems are mostly from geogenic origin. Additionally, microscopy imaging confirmed the presence of NPs in the three catchments. Furthermore, the land-area normalized annual flux of NPs-TiO2 (1.65 kg TiO2 year-1 km-2) was highest for the agricultural catchment, suggesting that agricultural practices have a different impact on the NPs-TiO2 dynamics and exports than other land-uses (urban or forestry). A similar trend is also found by the reanalysis of recent literature data.

TMEM120A is a coenzyme A-binding membrane protein with structural similarities to ELOVL fatty acid elongase.

TMEM120A, also named as TACAN, is a novel membrane protein highly conserved in vertebrates and was recently proposed to be a mechanosensitive channel involved in sensing mechanical pain. Here we present the single-particle cryogenic electron microscopy (cryo-EM) structure of human TMEM120A, which forms a tightly packed dimer with extensive interactions mediated by the N-terminal coiled coil domain (CCD), the C-terminal transmembrane domain (TMD), and the re-entrant loop between the two domains. The TMD of each TMEM120A subunit contains six transmembrane helices (TMs) and has no clear structural feature of a channel protein. Instead, the six TMs form an α-barrel with a deep pocket where a coenzyme A (CoA) molecule is bound. Intriguingly, some structural features of TMEM120A resemble those of elongase for very long-chain fatty acids (ELOVL) despite the low sequence homology between them, pointing to the possibility that TMEM120A may function as an enzyme for fatty acid metabolism, rather than a mechanosensitive channel.

Structure and function at the lipid–protein interface of a pentameric ligand-gated ion channel

Although it has long been proposed that membrane proteins may contain tightly bound lipids, their identity, the structure of their binding sites, and their functional and structural relevance have remained elusive. To some extent, this is because tightly bound lipids are often located at the periphery of proteins, where the quality of density maps is usually poorer, and because they may be outcompeted by detergent molecules used during standard purification procedures. As a step toward characterizing natively bound lipids in the superfamily of pentameric ligand-gated ion channels (pLGICs), we applied single-particle cryogenic electron microscopy to fragments of native membrane obtained in the complete absence of detergent-solubilization steps. Because of the heterogeneous lipid composition of membranes in the secretory pathway of eukaryotic cells, we chose to study a bacterial pLGIC (ELIC) expressed in Escherichia coli's inner membrane. We obtained a three-dimensional reconstruction of unliganded ELIC (2.5-Å resolution) that shows clear evidence for two types of tightly bound lipid at the protein-bulk-membrane interface. One of them was consistent with a "regular" diacylated phospholipid, in the cytoplasmic leaflet, whereas the other one was consistent with the tetra-acylated structure of cardiolipin, in the periplasmic leaflet. Upon reconstitution in E. coli polar-lipid bilayers, ELIC retained the functional properties characteristic of members of this superfamily, and thus, the fitted atomic model is expected to represent the (long-debated) unliganded-closed, "resting" conformation of this ion channel. Notably, the addition of cardiolipin to phosphatidylcholine membranes restored the ion-channel activity that is largely lost in phosphatidylcholine-only bilayers.

Abstract P-28: Structural Dynamics of DNA-Associated Chaperon Facilitates Chromatine Transcription

Background: Histone chaperon FACT (“FAcilitates Chromatin Transcription”) is a multifunctional and conserved eukaryotic protein involved in DNA transcription, replication and repair; which can reversibly unfold nucleosomes in presence of ATP. FACT is necessary for the viability and growth of breast tumor cells meanwhile in normal cells it can be knocked out without loss of vitality. Human FACT (hFACT) is a target for promising anticancer drug curaxins, which causes FACT trapping in chromatin of cancer cells and destabilizes the nucleosome. The nucleosome-unfolding activity is an important function of hFACT in vivo; however, the mechanism of FACT-dependent nucleosome unfolding remains unknown. Methods: Here, we studied negative stained hFACT structure using single particle electron microscopy using JEOL 2100 TEM. Micrographs were captured with 25k magnification, and 4.1 Å pixel size. EM images pre-processing and single particles collection were performed in EMAN2.3, followed by 2D-particles analysis in RELION2.0. Final 2D-classes included ~70 000 single particles images. Results: Based on 2D-classess data analysis we evaluated several states of hFACT reflecting its conformational flexibility: the “closed” complex is characterized by four domains localized close to each other and forming a compact structure; “intermediate” state represented by classes with identified three domains having compact structure and more disordered fourth domain, and the “open” complex, represented by three domains forming almost linear structure. The “closed” and “open” states are present in comparable amounts and significantly outnumber the “intermediate” state. It has been shown that hFACT domains are connected through flexible linkers and SPT16 and SSRP1 dimerization domains (DDs) form the “joint”-like connection between the two subunits. In the “сlosed” conformation, the DNA-binding surface of FACT is covered by its two C-terminal and middle domains (MDs). The N-terminal domain (NTD) of SPT16 was not resolved previously, but it is the best candidate for the forth domain that is clearly visible only in the “closed” conformation of hFACT, based on its dimensions and the longest linker length. Conclusion: We propose that during conversion to the “open” complexes SPT16 NTD is moving away from the other subunits leading to formation of the first intermediate state with the NTD domain poorly resolved or not resolved, while less mobile DDs and MDs maintain more compact structure and the DNA-binding site is still protected by the CTDs. In the “open” state SPT16/SSRP1 visible MDs and DDs form almost linear structure, unmasking the DNA-binding sites and making them accessible for the interaction with a nucleosome.

Generation of ordered protein assemblies using rigid three-body fusion

Protein nanomaterial design is an emerging discipline with applications in medicine and beyond. A long-standing design approach uses genetic fusion to join protein homo-oligomer subunits via α-helical linkers to form more complex symmetric assemblies, but this method is hampered by linker flexibility and a dearth of geometric solutions. Here, we describe a general computational method for rigidly fusing homo-oligomer and spacer building blocks to generate user-defined architectures that generates far more geometric solutions than previous approaches. The fusion junctions are then optimized using Rosetta to minimize flexibility. We apply this method to design and test 92 dihedral symmetric protein assemblies using a set of designed homodimers and repeat protein building blocks. Experimental validation by native mass spectrometry, small-angle X-ray scattering, and negative-stain single-particle electron microscopy confirms the assembly states for 11 designs. Most of these assemblies are constructed from designed ankyrin repeat proteins (DARPins), held in place on one end by α-helical fusion and on the other by a designed homodimer interface, and we explored their use for cryogenic electron microscopy (cryo-EM) structure determination by incorporating DARPin variants selected to bind targets of interest. Although the target resolution was limited by preferred orientation effects and small scaffold size, we found that the dual anchoring strategy reduced the flexibility of the target-DARPIN complex with respect to the overall assembly, suggesting that multipoint anchoring of binding domains could contribute to cryo-EM structure determination of small proteins.

Structural and Functional Analyses of the Tridomain-Nonribosomal Peptide Synthetase FmoA3 for 4-Methyloxazoline Ring Formation.

Nonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α-methyl-l-serine to synthesize a 4-methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head-to-tail homodimeric structures of FmoA3 by X-ray crystallography (apo-form, with adenylyl-imidodiphosphate and α-methyl-l-seryl-AMP) and cryogenic electron microscopy single particle analysis, and performed site-directed mutagenesis experiments. The data revealed that α-methyl-l-serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3's Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.

Structural and Functional Analyses of the Tridomain‐Nonribosomal Peptide Synthetase FmoA3 for 4‐Methyloxazoline Ring Formation

AbstractNonribosomal peptide synthetases (NRPSs) are attractive targets for bioengineering to generate useful peptides. FmoA3 is a single modular NRPS composed of heterocyclization (Cy), adenylation (A), and peptidyl carrier protein (PCP) domains. It uses α‐methyl‐l‐serine to synthesize a 4‐methyloxazoline ring, probably with another Cy domain in the preceding module FmoA2. Here, we determined the head‐to‐tail homodimeric structures of FmoA3 by X‐ray crystallography (apo‐form, with adenylyl‐imidodiphosphate and α‐methyl‐l‐seryl‐AMP) and cryogenic electron microscopy single particle analysis, and performed site‐directed mutagenesis experiments. The data revealed that α‐methyl‐l‐serine can be accommodated in the active site because of the extra space around Ala688. The Cy domains of FmoA2 and FmoA3 catalyze peptide bond formation and heterocyclization, respectively. FmoA3’s Cy domain seems to lose its donor PCP binding activity. The collective data support a proposed catalytic cycle of FmoA3.