Electrical Characteristics of CMOS-Compatible SiOx-Based Resistive-Switching Devices.

The electrical characteristics and resistive switching properties of memristive devices have been studied in a wide temperature range. The insulator and electrode materials of these devices (silicon oxide and titanium nitride, respectively) are fully compatible with conventional complementary metal-oxide-semiconductor (CMOS) fabrication processes. Silicon oxide is also obtained through the low-temperature chemical vapor deposition method. It is revealed that the as-fabricated devices do not require electroforming but their resistance state cannot be stored before thermal treatment. After the thermal treatment, the devices exhibit bipolar-type resistive switching with synaptic behavior. The conduction mechanisms in the device stack are associated with the effect of traps in the insulator, which form filaments in the places where the electric field is concentrated. The filaments shortcut the capacitance of the stack to different degrees in the high-resistance state (HRS) and in the low-resistance state (LRS). As a result, the electron transport possesses an activation nature with relatively low values of activation energy in an HRS. On the contrary, Ohm's law and tunneling are observed in an LRS. CMOS-compatible materials and low-temperature fabrication techniques enable the easy integration of the studied resistive-switching devices with traditional analog-digital circuits to implement new-generation hardware neuromorphic systems.

Structure and dynamics of an archetypal DNA nanoarchitecture revealed via cryo-EM and molecular dynamics simulations.

DNA can be folded into rationally designed, unique, and functional materials. To fully realise the potential of these DNA materials, a fundamental understanding of their structure and dynamics is necessary, both in simple solvents as well as more complex and diverse anisotropic environments. Here we analyse an archetypal six-duplex DNA nanoarchitecture with single-particle cryo-electron microscopy and molecular dynamics simulations in solvents of tunable ionic strength and within the anisotropic environment of biological membranes. Outside lipid bilayers, the six-duplex bundle lacks the designed symmetrical barrel-type architecture. Rather, duplexes are arranged in non-hexagonal fashion and are disorted to form a wider, less elongated structure. Insertion into lipid membranes, however, restores the anticipated barrel shape due to lateral duplex compression by the bilayer. The salt concentration has a drastic impact on the stability of the inserted barrel-shaped DNA nanopore given the tunable electrostatic repulsion between the negatively charged duplexes. By synergistically combining experiments and simulations, we increase fundamental understanding into the environment-dependent structural dynamics of a widely used nanoarchitecture. This insight will pave the way for future engineering and biosensing applications.

CryoEM structure and assembly mechanism of a bacterial virus genome gatekeeper.

Numerous viruses package their dsDNA genome into preformed capsids through a portal gatekeeper that is subsequently closed. We report the structure of the DNA gatekeeper complex of bacteriophage SPP1 (gp612gp1512gp166) in the post-DNA packaging state at 2.7 Å resolution obtained by single particle cryo-electron microscopy. Comparison of the native SPP1 complex with assembly-naïve structures of individual components uncovered the complex program of conformational changes leading to its assembly. After DNA packaging, gp15 binds via its C-terminus to the gp6 oligomer positioning gp15 subunits for oligomerization. Gp15 refolds its inner loops creating an intersubunit ß-barrel that establishes different types of contacts with six gp16 subunits. Gp16 binding and oligomerization is accompanied by folding of helices that close the portal channel to keep the viral genome inside the capsid. This mechanism of assembly has broad functional and evolutionary implications for viruses of the prokaryotic tailed viruses-herpesviruses lineage.

Suppression of amyloid-ß fibril growth by drug-engineered polymorph transformation.

Fibrillization of the protein amyloid ß is assumed to trigger Alzheimer's pathology. Approaches that target amyloid plaques, however, have garnered limited clinical success, and their failures may relate to the scarce understanding of the impact of potential drugs on the intertwined stages of fibrillization. Here, we demonstrate that bexarotene, a T-cell lymphoma medication with known antiamyloid activity both in vitro and in vivo, suppresses amyloid fibrillization by promoting an alternative fibril structure. We employ time-resolved in situ atomic force microscopy to quantify the kinetics of growth of individual fibrils and supplement it with structure characterization by cryo-EM. We show that fibrils with structure engineered by the drug nucleate and grow substantially slower than "normal" fibrils; remarkably, growth remains stunted even in drug-free solutions. We find that the suppression of fibril growth by bexarotene is not because of the drug binding to the fibril tips or to the peptides in the solution. Kinetic analyses attribute the slow growth of drug-enforced fibril polymorph to the distinctive dynamics of peptide chain association to their tips. As an additional benefit, the bexarotene fibrils kill primary rat hippocampal neurons less efficiently than normal fibrils. In conclusion, the suggested drug-driven polymorph transformation presents a mode of action to irreversibly suppress toxic aggregates not only in Alzheimer's but also potentially in myriad diverse pathologies that originate with protein condensation.

Cryo-EM structure of a type IV secretion system.

Bacterial conjugation is the fundamental process of unidirectional transfer of DNAs, often plasmid DNAs, from a donor cell to a recipient cell1. It is the primary means by which antibiotic resistance genes spread among bacterial populations2,3. In Gram-negative bacteria, conjugation is mediated by a large transport apparatus-the conjugative type IV secretion system (T4SS)-produced by the donor cell and embedded in both its outer and inner membranes. The T4SS also elaborates a long extracellular filament-the conjugative pilus-that is essential for DNA transfer4,5. Here we present a high-resolution cryo-electron microscopy (cryo-EM) structure of a 2.8 megadalton T4SS complex composed of 92 polypeptides representing 8 of the 10 essential T4SS components involved in pilus biogenesis. We added the two remaining components to the structural model using co-evolution analysis of protein interfaces, to enable the reconstitution of the entire system including the pilus. This structure describes the exceptionally large protein-protein interaction network required to assemble the many components that constitute a T4SS and provides insights on the unique mechanism by which they elaborate pili.

Features of the Territorial Distribution, Composition and Structure of Phytocenoses with the Participation of Fraxinus excelsior, Their Resource Qualities, Ecological and Economic Importance (Southeastern Part of the East European Plain).

At present, the distribution area of Fraxinus excelsior L. in the forest ecosystems of the Volga Region is rather low and ranges from 0.01% to 2.5%. In the Middle Volga Region, using the example of the Penza region, five types of deciduous forests were identified in the composition with Fraxinus excelsior L.: oak forest aegopodium, oak forest nettle, oak forest hazel-linden, oak forest aegopodium-motley grass, oak forest carex-motley grass. In the forest phytocenoses of the Moksha River basin, the quality of Fraxinus excelsior L. is 1.5-1.7. In the forest phytocenoses of the Khoper River basin, the average quality value reaches 2.4-2.8, and in the forest tracts of the Sura river basin it is 2.8-3.2. In the western part of the study area, individuals of age class II-III (21-40, 41-60 years) predominate, in the central part-age class I (1-20 years), in the eastern part-age class V (81-100 years). This circumstance allows us to conclude that its populations in the western regions are represented by stands of different ages; the presence of young stands and middle-aged stands indicates the presence of conditions for reproduction and distribution. At the border of its range, Fraxinus excelsior L. grows in a stable population; in the western part of the Middle Volga Region, the number of species in forest stands with a predominance of Fraxinus excelsior L. is 26-30% higher than this indicator in more eastern regions. In the direction from east to west, the number of species in the composition of forest stands increases (up to 8.4), with a predominance of Fraxinus excelsior L. The number of plant associations increases in the direction from east to west. If in the east of the Penza region Fraxinus excelsior L. occurs in 6-7 plant associations, then in the west of the region-in 18-25 associations. The maximum timber stock for 100 years of Fraxinus excelsior L. stands reaches 380 m3/ha. Such a natural bioresource potential is of importance for the conduct of the national economy. Forest management in phytocenoses with the participation of this tree species is a strategic branch direction. It is expedient to restore populations of Fraxinus excelsior L. everywhere and to cultivate them in the territory of the East European Plain and especially in its south-eastern part. This is fully consistent with the principles of sustainable ecological and economic development against the background of local natural, climatic and geographical conditions. This type is necessary when solving environmental, resource-saving and economic problems in the territory under consideration.

Cryo-EM Structures of Two Bacteriophage Portal Proteins Provide Insights for Antimicrobial Phage Engineering.

Widespread antibiotic resistance has returned attention to bacteriophages as a means of managing bacterial pathogenesis. Synthetic biology approaches to engineer phages have demonstrated genomic editing to broaden natural host ranges, or to optimise microbicidal action. Gram positive pathogens cause serious pastoral animal and human infections that are especially lethal in newborns. Such pathogens are targeted by the obligate lytic phages of the Salasmaviridae and Guelinviridae families. These phages have relatively small ~20 kb linear protein-capped genomes and their compact organisation, relatively few structural elements, and broad host range, are appealing from a phage-engineering standpoint. In this study, we focus on portal proteins, which are core elements for the assembly of such tailed phages. The structures of dodecameric portal complexes from Salasmaviridae phage GA1, which targets Bacillus pumilus, and Guelinviridae phage phiCPV4 that infects Clostridium perfringens, were determined at resolutions of 3.3 Å and 2.9 Å, respectively. Both are found to closely resemble the related phi29 portal protein fold. However, the portal protein of phiCPV4 exhibits interesting differences in the clip domain. These structures provide new insights on structural diversity in Caudovirales portal proteins and will be essential for considerations in phage structural engineering.

Unwinding of a DNA replication fork by a hexameric viral helicase.

Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged ß-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.

Image-Based Wheat Fungi Diseases Identification by Deep Learning.

Diseases of cereals caused by pathogenic fungi can significantly reduce crop yields. Many cultures are exposed to them. The disease is difficult to control on a large scale; thus, one of the relevant approaches is the crop field monitoring, which helps to identify the disease at an early stage and take measures to prevent its spread. One of the effective control methods is disease identification based on the analysis of digital images, with the possibility of obtaining them in field conditions, using mobile devices. In this work, we propose a method for the recognition of five fungal diseases of wheat shoots (leaf rust, stem rust, yellow rust, powdery mildew, and septoria), both separately and in case of multiple diseases, with the possibility of identifying the stage of plant development. A set of 2414 images of wheat fungi diseases (WFD2020) was generated, for which expert labeling was performed by the type of disease. More than 80% of the images in the dataset correspond to single disease labels (including seedlings), more than 12% are represented by healthy plants, and 6% of the images labeled are represented by multiple diseases. In the process of creating this set, a method was applied to reduce the degeneracy of the training data based on the image hashing algorithm. The disease-recognition algorithm is based on the convolutional neural network with the EfficientNet architecture. The best accuracy (0.942) was shown by a network with a training strategy based on augmentation and transfer of image styles. The recognition method was implemented as a bot on the Telegram platform, which allows users to assess plants by lesions in the field conditions.

Sulfate-Containing Composite Based on Ni-Rich Layered Oxide LiNi0.8Mn0.1Co0.1O2 as High-Performance Cathode Material for Li-ion Batteries.

Composite positive electrode materials (1-x) LiNi0.8Mn0.1Co0.1O2∙xLi2SO4 (x = 0.002-0.005) for Li-ion batteries have been synthesized via conventional hydroxide or carbonate coprecipitation routes with subsequent high-temperature lithiation in either air or oxygen atmosphere. A comparative study of the materials prepared from transition metal sulfates (i.e., containing sulfur) and acetates (i.e., sulfur-free) with powder X-ray diffraction, electron diffraction, high angle annular dark field transmission electron microscopy, energy-dispersive X-ray spectroscopy, and electron energy loss spectroscopy revealed that the sulfur-containing species occur as amorphous Li2SO4 at the grain boundaries and intergranular contacts of the primary NMC811 crystallites. This results in a noticeable enhancement of rate capability and capacity retention over prolonged charge/discharge cycling compared to their sulfur-free analogs. The improvement is attributed to suppressing the high voltage phase transition and the associated accumulation of anti-site disorder upon cycling and improving the secondary agglomerates' mechanical integrity by increasing interfacial fracture toughness through linking primary NMC811 particles with soft Li2SO4 binder, as demonstrated with nanoindentation experiments. As the synthesis of the (1-x) LiNi0.8Mn0.1Co0.1O2∙xLi2SO4 composites do not require additional operational steps to introduce sulfur, these electrode materials might demonstrate high potential for commercialization.

The structural basis for Z α1-antitrypsin polymerization in the liver.

The serpinopathies are among a diverse set of conformational diseases that involve the aberrant self-association of proteins into ordered aggregates. α1-Antitrypsin deficiency is the archetypal serpinopathy and results from the formation and deposition of mutant forms of α1-antitrypsin as "polymer" chains in liver tissue. No detailed structural analysis has been performed of this material. Moreover, there is little information on the relevance of well-studied artificially induced polymers to these disease-associated molecules. We have isolated polymers from the liver tissue of Z α1-antitrypsin homozygotes (E342K) who have undergone transplantation, labeled them using a Fab fragment, and performed single-particle analysis of negative-stain electron micrographs. The data show structural equivalence between heat-induced and ex vivo polymers and that the intersubunit linkage is best explained by a carboxyl-terminal domain swap between molecules of α1-antitrypsin.

Detection of Genomic Regions Associated with Resistance to Stem Rust in Russian Spring Wheat Varieties and Breeding Germplasm.

Stem rust caused by Puccinia graminis f. sp. tritici Eriks. is a dangerous disease of common wheat worldwide. Development and cultivation of the varieties with genetic resistance is one of the most effective and environmentally important ways for protection of wheat against fungal pathogens. Field phytopathological screening and genome-wide association study (GWAS) were used for assessment of the genetic diversity of a collection of spring wheat genotypes on stem rust resistance loci. The collection consisting of Russian varieties of spring wheat and introgression lines with alien genetic materials was evaluated over three seasons (2016, 2017 and 2018) for resistance to the native population of stem rust specific to the West Siberian region of Russia. The results indicate that most varieties displayed from moderate to high levels of susceptibility to P. graminis; 16% of genotypes had resistance or immune response. In total, 13,006 single-nucleotide polymorphism (SNP) markers obtained from the Infinium 15K array were used to perform genome-wide association analysis. GWAS detected 35 significant marker-trait associations (MTAs) with SNPs located on chromosomes 1A, 2A, 2B, 3B, 5A, 5B, 6A, 7A and 7B. The most significant associations were found on chromosomes 7A and 6A where known resistance genes Sr25 and Sr6Ai = 2 originated from Thinopyrum ssp. are located. Common wheat lines containing introgressed fragments from Triticum timopheevii and Triticum kiharae were found to carry Sr36 gene on 2B chromosome. It has been suggested that the quantitative trait loci (QTL) mapped to the chromosome 5BL may be new loci inherited from the T. timopheevii. It can be inferred that a number of Russian wheat varieties may contain the Sr17 gene, which does not currently provide effective protection against pathogen. This is the first report describing the results of analysis of the genetic factors conferring resistance of Russian spring wheat varieties to stem rust.

Lysis Performance of Bacteriophages with Different Plaque Sizes and Comparison of Lysis Kinetics After Simultaneous and Sequential Phage Addition.

Background: Although bacteriophages see a revival for specifically removing undesired bacteria, there is still much uncertainty about how to achieve the most rapid and long-lasting clearance. Materials and Methods: This study investigated the lysis kinetics of three distinct environmental coliphages, reproducibly forming different plaque sizes (big, medium, and small). Lysis performance by individual phages was compared with the one obtained after simultaneous or sequential addition of all three phages. Kinetics was monitored by density absorbance or by flow cytometry, with the latter having the advantage of providing higher sensitivity. Results: Plaque size happened to correlate with lysis kinetics in liquid suspensions, with phages producing big (phage B), medium (phage M), and small (phage S) plaques showing maximal bacterial clearance under the chosen conditions within ∼6, 12, and 18 h, respectively. Use of a phage cocktail (all three phages added simultaneously) resulted in slower initial lysis compared with the fastest lysing phage with the greatest plaque size alone, but it showed longer efficacy in suppression. When adding phages sequentially, overall lysis kinetics could be influenced by administering phages at different time points. The lowest bacterial concentration after 36 h was obtained when administering phages in the sequence S, M, and B although this combination initially took the longest to achieve bacterial clearance. Conclusions: Results support that timing and order of phage addition can modulate strength and duration of bacterial suppression and, thus, influence the overall success of phage treatment.

Structural transitions during the scaffolding-driven assembly of a viral capsid.

Assembly of tailed bacteriophages and herpesviruses starts with formation of procapsids (virion precursors without DNA). Scaffolding proteins (SP) drive assembly by chaperoning the major capsid protein (MCP) to build an icosahedral lattice. Here we report near-atomic resolution cryo-EM structures of the bacteriophage SPP1 procapsid, the intermediate expanded procapsid with partially released SPs, and the mature capsid with DNA. In the intermediate state, SPs are bound only to MCP pentons and to adjacent subunits from hexons. SP departure results in the expanded state associated with unfolding of the MCP N-terminus and straightening of E-loops. The newly formed extensive inter-capsomere bonding appears to compensate for release of SPs that clasp MCP capsomeres together. Subsequent DNA packaging instigates bending of MCP A domain loops outwards, closing the hexons central opening and creating the capsid auxiliary protein binding interface. These findings provide a molecular basis for the sequential structural rearrangements during viral capsid maturation.

Unravelling Ribosome Function Through Structural Studies.

Ribosomes are biological nanomachine that synthesise all proteins within a cell. It took decades to reveal the architecture of this essential cellular component. To understand the structure -function relationship of this nanomachine needed the utilisisation of different biochemical, biophysical and structural techniques. Structural studies combined with mutagenesis of the different ribosomal complexes comprising various RNAs and proteins enabled us to understand how this machine works inside a cell. Nowadays quite a number of ribosomal structures were published that confirmed biochemical studies on particular steps of protein synthesis by the ribosome . Four major steps were identified: initiation , elongation, termination and recycling. These steps lead us to the important question how the ribosome function can be regulated. Advances in technology for cryo electron microscopy: sample preparations, image recording, developments in algorithms for image analysis and processing significantly helped in revelation of structural details of the ribosome . We now have a library of ribosome structures from prokaryotes to eukaryotes that enable us to understand the complex mechanics of this nanomachine. As this structural library continues to grow, we gradually improve our understanding of this process and how it can be regulated and how the specific ribosomes can be stalled or activated, or completely disabled. This article provides a comprehensive overview of ribosomal structures that represent structural snapshots of the ribosome at its different functional states. Better understanding rises more particular questions that have to be addressed by determination structures of more complexes.Synopsis: Structural biology of the ribosome.

MAP3K7 is recurrently deleted in pediatric T-lymphoblastic leukemia and affects cell proliferation independently of NF-κB.

BACKGROUND: Deletions of 6q15-16.1 are recurrently found in pediatric T-cell acute lymphoblastic leukemia (T-ALL). This chromosomal region includes the mitogen-activated protein kinase kinase kinase 7 (MAP3K7) gene which has a crucial role in innate immune signaling and was observed to be functionally and prognostically relevant in different cancer entities. Therefore, we correlated the presence of MAP3K7 deletions with clinical parameters in a cohort of 327 pediatric T-ALL patients and investigated the function of MAP3K7 in the T-ALL cell lines CCRF-CEM, Jurkat and MOLT-4. METHODS: MAP3K7 deletions were detected by multiplex ligation-dependent probe amplification (MLPA). T-ALL cell lines were transduced with adeno-associated virus (AAV) vectors expressing anti-MAP3K7 shRNA or a non-silencing shRNA together with a GFP reporter. Transduction efficiency was measured by flow cytometry and depletion efficiency by RT-PCR and Western blots. Induction of apoptosis was measured by flow cytometry after staining with PE-conjugated Annexin V. In order to assess the contribution of NF-κB signaling to the effects of MAP3K7 depletion, cells were treated with TNF-α and cell lysates analyzed for components of the NF-κB pathway by Western blotting and for expression of the NF-κB target genes BCL2, CMYC, FAS, PTEN and TNF-α by RT-PCR. RESULTS: MAP3K7 is deleted in approximately 10% and point-mutated in approximately 1% of children with T-ALL. In 32 of 33 leukemias the deletion of MAP3K7 also included the adjacent CASP8AP2 gene. MAP3K7 deletions were associated with the occurrence of SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and outcome. Depletion of MAP3K7 expression in T-ALL cell lines by shRNAs slowed down proliferation and induced apoptosis, but neither changed protein levels of components of NF-κB signaling nor NF-κB target gene expression after stimulation with TNF-α. CONCLUSIONS: This study revealed that the recurrent deletion of MAP3K7/CASP8AP2 is associated with SIL-TAL1 fusions and a mature immunophenotype, but not with response to treatment and risk of relapse. Homozygous deletions of MAP3K7 were not observed, and efficient depletion of MAP3K7 interfered with viability of T-ALL cells, indicating that a residual expression of MAP3K7 is indispensable for T-lymphoblasts.

The absence or presence of a lytic coliphage affects the response of Escherichia coli to heat, chlorine, or UV exposure.

Disinfection aims at maximal inactivation of target organisms and the sustainable suppression of their regrowth. Whereas many disinfection efforts achieve efficient inactivation when the effect is measured directly after treatment, there are questions about the sustainability of this effect. One aspect is that the treated bacteria might recover and regain the ability to grow. In an environmental context, another question is how amenable surviving bacteria are to predation by omnipresent bacteriophages. Provisional data suggested that bacteria when subjected to sublethal heat stress might develop a phage-resistant phenotype. The result made us wonder about the susceptibility to phage-mediated lysis for bacteria exposed to a gradient of chlorine and UV-LED disinfection strengths. Whereas bacteria exposed to low sublethal chlorine doses still underwent phage-mediated lysis, the critical chlorine Ct of 0.5 mg min/L eliminated this susceptibility and induced phage resistance in the cells that survived treatment. In the case of UV, even the smallest tested dose of 2.8 mJ/cm2 abolished phage lysis leading to direct regrowth. Results suggest that bacteria surviving disinfection might have higher environmental survival chances directly after treatment compared to non-treated cells. A reason could possibly lie in their compromised metabolism that is essential for phage replication.

Structure of a VirD4 coupling protein bound to a VirB type IV secretion machinery.

Type IV secretion (T4S) systems are versatile bacterial secretion systems mediating transport of protein and/or DNA T4S systems are generally composed of 11 VirB proteins and 1 VirD protein (VirD4). The VirB1-11 proteins assemble to form a secretion machinery and a pilus while the VirD4 protein is responsible for substrate recruitment. The structure of VirD4 in isolation is known; however, its structure bound to the VirB1-11 apparatus has not been determined. Here, we purify a T4S system with VirD4 bound, define the biochemical requirements for complex formation and describe the protein-protein interaction network in which VirD4 is involved. We also solve the structure of this complex by negative stain electron microscopy, demonstrating that two copies of VirD4 dimers locate on both sides of the apparatus, in between the VirB4 ATPases. Given the central role of VirD4 in type IV secretion, our study provides mechanistic insights on a process that mediates the dangerous spread of antibiotic resistance genes among bacterial populations.

Alternative splicing of telomerase catalytic subunit hTERT generated by apoptotic endonuclease EndoG induces human CD4+ T cell death.

Telomerase activity is regulated by alternative splicing of its catalytic subunit human Telomerase Reverse Transcriptase (hTERT) mRNA. Induction of a non-active spliced hTERT leads to inhibition of telomerase activity. However, very little is known about the mechanism of hTERT mRNA alternative splicing. The aim of this study was to determine the role of the apoptotic endonuclease EndoG in alternative splicing of hTERT and telomerase activity. A strong correlation was identified between EndoG expression levels and hTERT splice variants in human CD4+ and CD8+ T lymphocytes. Overexpression of EndoG in CD4+ T cells down-regulated the expression of the active full-length hTERT variant and up-regulated expression of the non-active spliced variant. A reduction in full-length hTERT transcripts down-regulated telomerase activity. Long-term in vitro cultivation of EndoG-overexpressing CD4+ T cells led to dramatically shortened telomeres, conversion of cells into a replicative senescence state, and activation of the BCL2/BAX-associated apoptotic pathway finally leading to cell death. These data indicated the participation of EndoG in alternative mRNA splicing of the telomerase catalytic subunit hTERT, regulation of telomerase activity and determination of cell fate.

Structural Analysis of Protein Complexes by Cryo Electron Microscopy.

Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3-5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147-1151, 2051; Bai et al., Nature 525(7568):212-217, 2015; Vinothkumar et al., Nature 515(7525):80-84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265-273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System.