CryoEM single particle reconstruction with a complex-valued particle stack.

Single particle reconstruction (SPR) in cryoEM is an image processing task with an elaborate hierarchy that starts with many very noisy multi-frame images. Efficient representation of the intermediary image structures is critical for keeping the calculations manageable. One such intermediary structure is called a particle stack and contains cut-out images of particles in square boxes of predefined size. The micrograph that is the source of the boxed images is usually corrected for motion between frames prior to particle stack creation. However, the contrast transfer function (CTF) or its Fourier Transform point spread function (PSF) are not considered at this step. Historically, the particle stack was intended for large particles and for a tighter PSF, which is characteristic of lower resolution data. The field now performs analyses of smaller particles and to higher resolution, and these conditions result in a broader PSF that requires larger padding and slower calculations to integrate information for each particle. Consequently, the approach to handling structures such as the particle stack should be reexamined to optimize data processing. Here we propose to use as a source image for the particle stack a complex-valued image, in which CTF correction is implicitly applied as a real component of the image. We can achieve it by applying an initial CTF correction to the entire micrograph first and perform box cutouts as a subsequent step. The final CTF correction that we refine and apply later has a very narrow PSF, and so cutting out particles from micrographs that were approximately corrected for CTF does not require extended buffering, i.e. the boxes during the analysis only have to be large enough to encompass the particle. The Fourier Transform of an exit-wave reconstruction creates an image that has complex values. This is a complex value image considered in real space, opposed to standard SPR data processing where complex numbers appear only in Fourier space. This extension of the micrograph concept provides multiple advantages because the particle box size can be small and calculations crucial for high resolution reconstruction such as Ewald sphere correction, aberration refinement, and particle-specific defocus refinement can be performed on the small box data.

Monomer and dimer structures of cytochrome bo3 ubiquinol oxidase from Escherichia coli.

The Escherichia coli cytochrome bo3 ubiquinol oxidase is a four-subunit heme-copper oxidase that serves as a proton pump in the E. coli aerobic respiratory chain. Despite many mechanistic studies, it is unclear whether this ubiquinol oxidase functions as a monomer, or as a dimer in a manner similar to its eukaryotic counterparts-the mitochondrial electron transport complexes. In this study, we determined the monomeric and dimeric structures of the E. coli cytochrome bo3 ubiquinol oxidase reconstituted in amphipol by cryogenic electron microscopy single particle reconstruction (cryo-EM SPR) to a resolution of 3.15 and 3.46 Å, respectively. We have discovered that the protein can form a dimer with C2 symmetry, with the dimerization interface maintained by interactions between the subunit II of one monomer and the subunit IV of the other monomer. Moreover, the dimerization does not induce significant structural changes in the monomers, except the movement of a loop in subunit IV (residues 67-74).

The His-tag as a decoy modulating preferred orientation in cryoEM.

The His-tag is a widely used affinity tag that facilitates purification by means of affinity chromatography of recombinant proteins for functional and structural studies. We show here that His-tag presence affects how coproheme decarboxylase interacts with the air-water interface during grid preparation for cryoEM. Depending on His-tag presence or absence, we observe significant changes in patterns of preferred orientation. Our analysis of particle orientations suggests that His-tag presence can mask the hydrophobic and hydrophilic patches on a protein's surface that mediate the interactions with the air-water interface, while the hydrophobic linker between a His-tag and the coding sequence of the protein may enhance other interactions with the air-water interface. Our observations suggest that tagging, including rational design of the linkers between an affinity tag and a protein of interest, offer a promising approach to modulating interactions with the air-water interface.

Structural and functional analysis of EntV reveals a 12 amino acid fragment protective against fungal infections.

Fungal pathogens are a continuing challenge due to few effective antifungals and a rise in resistance. In previous work, we described the inhibition of Candida albicans virulence following exposure to the 68 amino acid bacteriocin, EntV, secreted by Enterococcus faecalis. Here, to optimize EntV as a potential therapeutic and better understand its antifungal features, an X-ray structure is obtained. The structure consists of six alpha helices enclosing a seventh 16 amino acid helix (α7). The individual helices are tested for antifungal activity using in vitro and nematode infection assays. Interestingly, α7 retains antifungal, but not antibacterial activity and is also effective against Candida auris and Cryptococcus neoformans. Further reduction of α7 to 12 amino acids retains full antifungal activity, and excellent efficacy is observed in rodent models of C. albicans oropharyngeal, systemic, and venous catheter infections. Together, these results showcase EntV-derived peptides as promising candidates for antifungal therapeutic development.

Molecular analysis and essentiality of Aro1 shikimate biosynthesis multi-enzyme in Candida albicans.

In the human fungal pathogen Candida albicans, ARO1 encodes an essential multi-enzyme that catalyses consecutive steps in the shikimate pathway for biosynthesis of chorismate, a precursor to folate and the aromatic amino acids. We obtained the first molecular image of C. albicans Aro1 that reveals the architecture of all five enzymatic domains and their arrangement in the context of the full-length protein. Aro1 forms a flexible dimer allowing relative autonomy of enzymatic function of the individual domains. Our activity and in cellulo data suggest that only four of Aro1's enzymatic domains are functional and essential for viability of C. albicans, whereas the 3-dehydroquinate dehydratase (DHQase) domain is inactive because of active site substitutions. We further demonstrate that in C. albicans, the type II DHQase Dqd1 can compensate for the inactive DHQase domain of Aro1, suggesting an unrecognized essential role for this enzyme in shikimate biosynthesis. In contrast, in Candida glabrata and Candida parapsilosis, which do not encode a Dqd1 homolog, Aro1 DHQase domains are enzymatically active, highlighting diversity across Candida species.

Optimal structure determination from sub-optimal diffraction data.

Herein we present the newest version of the HKL-3000 system that integrates data collection, data reduction, phasing, model building, refinement, and validation. The system significantly accelerates the process of structure determination and has proven its high value for the determination of very high-quality structures. The heuristic for choosing the best approach for every step of structure determination for various quality samples and diffraction data has been optimized. The latest modifications increase the likelihood of a successful structure determination with challenging data. The HKL-3000 is a successor of HKL and HKL-2000 programs. The use of the HKL family of programs has been reported for over 73,000 PDB deposits, that is, almost 50% of macromolecular structures determined with X-ray diffraction.

A trimethoprim derivative impedes antibiotic resistance evolution.

The antibiotic trimethoprim (TMP) is used to treat a variety of Escherichia coli infections, but its efficacy is limited by the rapid emergence of TMP-resistant bacteria. Previous laboratory evolution experiments have identified resistance-conferring mutations in the gene encoding the TMP target, bacterial dihydrofolate reductase (DHFR), in particular mutation L28R. Here, we show that 4'-desmethyltrimethoprim (4'-DTMP) inhibits both DHFR and its L28R variant, and selects against the emergence of TMP-resistant bacteria that carry the L28R mutation in laboratory experiments. Furthermore, antibiotic-sensitive E. coli populations acquire antibiotic resistance at a substantially slower rate when grown in the presence of 4'-DTMP than in the presence of TMP. We find that 4'-DTMP impedes evolution of resistance by selecting against resistant genotypes with the L28R mutation and diverting genetic trajectories to other resistance-conferring DHFR mutations with catalytic deficiencies. Our results demonstrate how a detailed characterization of resistance-conferring mutations in a target enzyme can help identify potential drugs against antibiotic-resistant bacteria, which may ultimately increase long-term efficacy of antimicrobial therapies by modulating evolutionary trajectories that lead to resistance.

Structure and gating mechanism of the α7 nicotinic acetylcholine receptor.

The α7 nicotinic acetylcholine receptor plays critical roles in the central nervous system and in the cholinergic inflammatory pathway. This ligand-gated ion channel assembles as a homopentamer, is exceptionally permeable to Ca2+, and desensitizes faster than any other Cys-loop receptor. The α7 receptor has served as a prototype for the Cys-loop superfamily yet has proven refractory to structural analysis. We present cryo-EM structures of the human α7 nicotinic receptor in a lipidic environment in resting, activated, and desensitized states, illuminating the principal steps in the gating cycle. The structures also reveal elements that contribute to its function, including a C-terminal latch that is permissive for channel opening, and an anionic ring in the extracellular vestibule that contributes to its high conductance and calcium permeability. Comparisons among the α7 structures provide a foundation for mapping the gating cycle and reveal divergence in gating mechanisms in the Cys-loop receptor superfamily.

Structural basis for IFN antagonism by human respiratory syncytial virus nonstructural protein 2.

Human respiratory syncytial virus (RSV) nonstructural protein 2 (NS2) inhibits host interferon (IFN) responses stimulated by RSV infection by targeting early steps in the IFN-signaling pathway. But the molecular mechanisms related to how NS2 regulates these processes remain incompletely understood. To address this gap, here we solved the X-ray crystal structure of NS2. This structure revealed a unique fold that is distinct from other known viral IFN antagonists, including RSV NS1. We also show that NS2 directly interacts with an inactive conformation of the RIG-I-like receptors (RLRs) RIG-I and MDA5. NS2 binding prevents RLR ubiquitination, a process critical for prolonged activation of downstream signaling. Structural analysis, including by hydrogen-deuterium exchange coupled to mass spectrometry, revealed that the N terminus of NS2 is essential for binding to the RIG-I caspase activation and recruitment domains. N-terminal mutations significantly diminish RIG-I interactions and result in increased IFNß messenger RNA levels. Collectively, our studies uncover a previously unappreciated regulatory mechanism by which NS2 further modulates host responses and define an approach for targeting host responses.

High-resolution cryo-EM reconstructions in the presence of substantial aberrations.

Here, an analysis is performed of how uncorrected antisymmetric aberrations, such as coma and trefoil, affect cryo-EM single-particle reconstruction (SPR) results, and an analytical formula quantifying information loss owing to their presence is inferred that explains why Fourier-shell coefficient-based statistics may report significantly overestimated resolution if these aberrations are not fully corrected. The analysis is validated with reference-based aberration refinement for two cryo-EM SPR data sets acquired with a 200 kV microscope in the presence of coma exceeding 40 µm, and 2.3 and 2.7 Šreconstructions for 144 and 173 kDa particles, respectively, were obtained. The results provide a description of an efficient approach for assessing information loss in cryo-EM SPR data acquired in the presence of higher order aberrations, and address inconsistent guidelines regarding the level of aberrations that is acceptable in cryo-EM SPR experiments.

Structures of teixobactin-producing nonribosomal peptide synthetase condensation and adenylation domains.

The recently discovered antibiotic teixobactin is produced by uncultured soil bacteria. The antibiotic inhibits cell wall synthesis of Gram-positive bacteria by binding to precursors of cell wall building blocks, and therefore it is thought to be less vulnerable to development of resistance. Teixobactin is synthesized by two nonribosomal peptide synthetases (NRPSs), encoded by txo1 and txo2 genes. Like other NRPSs, the Txo1 and Txo2 synthetases are large, multifunctional, and comprised of several modules. Each module is responsible for catalysis of a distinct step of teixobactin synthesis and contains specific functional units, commonly including a condensation (C) domain, an adenylation (A) domain, and a peptidyl carrier protein (PCP) domain. Here we report the structures of the C-A bidomains of the two L-Ser condensing modules, from Txo1 and Txo2, respectively. In the structure of the C domain of the L-Ser subunit of Txo1, a large conformational change is observed, featuring an outward swing of its N-terminal α-helix. This repositioning, if functionally validated, provides the necessary conformational change for the condensation reaction in C domain, and likely represents a regulatory mechanism. In an Acore subdomain, a well-coordinated Mg2+ cation is observed, which is required in the adenylation reaction. The Mg2+-binding site is defined by a largely conserved amino acid sequence motif and is coordinated by the α-phosphate group of AMP (or ATP) when present, providing some structural evidence for the role of the metal cation in the catalysis of A domain.

NMR and crystallographic structural studies of the extremely stable monomeric variant of human cystatin C with single amino acid substitution.

Human cystatin C (hCC), a member of the superfamily of papain-like cysteine protease inhibitors, is the most widespread cystatin in human body fluids. This small protein, in addition to its physiological function, is involved in various diseases, including cerebral amyloid angiopathy, cerebral hemorrhage, stroke, and dementia. Physiologically active hCC is a monomer. However, all structural studies based on crystallization led to the dimeric structure formed as a result of a three-dimensional exchange of the protein domains (3D domain swapping). The monomeric structure was obtained only for hCC variant V57N and for the protein stabilized by an additional disulfide bridge. With this study, we extend the number of models of monomeric hCC by an additional hCC variant with a single amino acid substitution in the flexible loop L1. The V57G variant was chosen for the X-ray and NMR structural analysis due to its exceptional conformational stability in solution. In this work, we show for the first time the structural and dynamics studies of human cystatin C variant in solution. We were also able to compare these data with the crystal structure of the hCC V57G and with other cystatins. The overall cystatin fold is retained in the solute form. Additionally, structural information concerning the N terminus was obtained during our studies and presented for the first time. DATABASE: Crystallographic structure: structural data are available in PDB databases under the accession number 6ROA. NMR structure: structural data are available in PDB and BMRB databases under the accession numbers 6RPV and 34399, respectively.

Inhibition of Marburg Virus RNA Synthesis by a Synthetic Anti-VP35 Antibody.

Marburg virus causes sporadic outbreaks of severe hemorrhagic fever with high case fatality rates. Approved, effective, and safe therapeutic or prophylactic countermeasures are lacking. To address this, we used phage display to engineer a synthetic antibody, sFab H3, which binds the Marburg virus VP35 protein (mVP35). mVP35 is a critical cofactor of the viral replication complex and a viral immune antagonist. sFab H3 displayed high specificity for mVP35 and not for the closely related Ebola virus VP35. sFab H3 inhibited viral-RNA synthesis in a minigenome assay, suggesting its potential use as an antiviral. We characterized sFab H3 by a combination of biophysical and biochemical methods, and a crystal structure of the complex solved to 1.7 Å resolution defined the molecular interface between the sFab H3 and mVP35 interferon inhibitory domain. Our study identifies mVP35 as a therapeutic target using an approach that provides a framework for generating engineered Fabs targeting other viral proteins.

Divergent kinase regulates membrane ultrastructure of the Toxoplasma parasitophorous vacuole.

Apicomplexan parasites replicate within a protective organelle, called the parasitophorous vacuole (PV). The Toxoplasma gondii PV is filled with a network of tubulated membranes, which are thought to facilitate trafficking of effectors and nutrients. Despite being critical to parasite virulence, there is scant mechanistic understanding of the network's functions. Here, we identify the parasite-secreted kinase WNG1 (With-No-Gly-loop) as a critical regulator of tubular membrane biogenesis. WNG1 family members adopt an atypical protein kinase fold lacking the glycine rich ATP-binding loop that is required for catalysis in canonical kinases. Unexpectedly, we find that WNG1 is an active protein kinase that localizes to the PV lumen and phosphorylates PV-resident proteins, several of which are essential for the formation of a functional intravacuolar network. Moreover, we show that WNG1-dependent phosphorylation of these proteins is required for their membrane association, and thus their ability to tubulate membranes. Consequently, WNG1 knockout parasites have an aberrant PV membrane ultrastructure. Collectively, our results describe a unique family of Toxoplasma kinases and implicate phosphorylation of secreted proteins as a mechanism of regulating PV development during parasite infection.

The Bear Giant-Skipper genome suggests genetic adaptations to living inside yucca roots.

Giant-Skippers (Megathymini) are unusual thick-bodied, moth-like butterflies whose caterpillars feed inside Yucca roots and Agave leaves. Giant-Skippers are attributed to the subfamily Hesperiinae and they are endemic to southern and mostly desert regions of the North American continent. To shed light on the genotypic determinants of their unusual phenotypic traits, we sequenced and annotated a draft genome of the largest Giant-Skipper species, the Bear (Megathymus ursus violae). The Bear skipper genome is the least heterozygous among sequenced Lepidoptera genomes, possibly due to much smaller population size and extensive inbreeding. Their lower heterozygosity helped us to obtain a high-quality genome with an N50 of 4.2 Mbp. The ~ 430 Mb genome encodes about 14000 proteins. Phylogenetic analysis supports placement of Giant-Skippers with Grass-Skippers (Hesperiinae). We find that proteins involved in odorant and taste sensing as well as in oxidative reactions have diverged significantly in Megathymus as compared to Lerema, another Grass-Skipper. In addition, the Giant-Skipper has lost several odorant and gustatory receptors and possesses many fewer (1/3-1/2 of other skippers) anti-oxidative enzymes. Such differences may be related to the unusual life style of Giant-Skippers: they do not feed as adults, and their caterpillars feed inside Yuccas and Agaves, which provide a source of antioxidants such as polyphenols.

Crystal structure of the human NK1 tachykinin receptor.

The NK1 tachykinin G-protein-coupled receptor (GPCR) binds substance P, the first neuropeptide to be discovered in mammals. Through activation of NK1R, substance P modulates a wide variety of physiological and disease processes including nociception, inflammation, and depression. Human NK1R (hNK1R) modulators have shown promise in clinical trials for migraine, depression, and emesis. However, the only currently approved drugs targeting hNK1R are inhibitors for chemotherapy-induced nausea and vomiting (CINV). To better understand the molecular basis of ligand recognition and selectivity, we solved the crystal structure of hNK1R bound to the inhibitor L760735, a close analog of the drug aprepitant. Our crystal structure reveals the basis for antagonist interaction in the deep and narrow orthosteric pocket of the receptor. We used our structure as a template for computational docking and molecular-dynamics simulations to dissect the energetic importance of binding pocket interactions and model the binding of aprepitant. The structure of hNK1R is a valuable tool in the further development of tachykinin receptor modulators for multiple clinical applications.

Functional diversification of the NleG effector family in enterohemorrhagic Escherichia coli.

The pathogenic strategy of Escherichia coli and many other gram-negative pathogens relies on the translocation of a specific set of proteins, called effectors, into the eukaryotic host cell during infection. These effectors act in concert to modulate host cell processes in favor of the invading pathogen. Injected by the type III secretion system (T3SS), the effector arsenal of enterohemorrhagic E. coli (EHEC) O157:H7 features at least eight individual NleG effectors, which are also found across diverse attaching and effacing pathogens. NleG effectors share a conserved C-terminal U-box E3 ubiquitin ligase domain that engages with host ubiquitination machinery. However, their specific functions and ubiquitination targets have remained uncharacterized. Here, we identify host proteins targeted for ubiquitination-mediated degradation by two EHEC NleG family members, NleG5-1 and NleG2-3. NleG5-1 localizes to the host cell nucleus and targets the MED15 subunit of the Mediator complex, while NleG2-3 resides in the host cytosol and triggers degradation of Hexokinase-2 and SNAP29. Our structural studies of NleG5-1 reveal a distinct N-terminal α/ß domain that is responsible for interacting with host protein targets. The core of this domain is conserved across the NleG family, suggesting this domain is present in functionally distinct NleG effectors, which evolved diversified surface residues to interact with specific host proteins. This is a demonstration of the functional diversification and the range of host proteins targeted by the most expanded effector family in the pathogenic arsenal of E. coli.

Real-space analysis of radiation-induced specific changes with independent component analysis.

A method of analysis is presented that allows for the separation of specific radiation-induced changes into distinct components in real space. The method relies on independent component analysis (ICA) and can be effectively applied to electron density maps and other types of maps, provided that they can be represented as sets of numbers on a grid. Here, for glucose isomerase crystals, ICA was used in a proof-of-concept analysis to separate temperature-dependent and temperature-independent components of specific radiation-induced changes for data sets acquired from multiple crystals across multiple temperatures. ICA identified two components, with the temperature-independent component being responsible for the majority of specific radiation-induced changes at temperatures below 130 K. The patterns of specific temperature-independent radiation-induced changes suggest a contribution from the tunnelling of electron holes as a possible explanation. In the second case, where a group of 22 data sets was collected on a single thaumatin crystal, ICA was used in another type of analysis to separate specific radiation-induced effects happening on different exposure-level scales. Here, ICA identified two components of specific radiation-induced changes that likely result from radiation-induced chemical reactions progressing with different rates at different locations in the structure. In addition, ICA unexpectedly identified the radiation-damage state corresponding to reduced disulfide bridges rather than the zero-dose extrapolated state as the highest contrast structure. The application of ICA to the analysis of specific radiation-induced changes in real space and the data pre-processing for ICA that relies on singular value decomposition, which was used previously in data space to validate a two-component physical model of X-ray radiation-induced changes, are discussed in detail. This work lays a foundation for a better understanding of protein-specific radiation chemistries and provides a framework for analysing effects of specific radiation damage in crystallographic and cryo-EM experiments.

Complete Genome of Achalarus lyciades, The First Representative of the Eudaminae Subfamily of Skippers.

BACKGROUND: The Hoary Edge Skipper (Achalarus lyciades) is an eastern North America endemic butterfly from the Eudaminae subfamily of skippers named for an underside whitish patch near the hindwing edge. Its caterpillars feed on legumes, in contrast to Grass skippers (subfamily Hesperiinae) which feed exclusively on monocots. RESULTS: To better understand the evolution and phenotypic diversification of Skippers (family Hesperiidae), we sequenced, assembled and annotated a complete genome draft and transcriptome of a wild-caught specimen of A. lyciades and compared it with the available genome of the Clouded Skipper (Lerema accius) from the Grass skipper subfamily. The genome of A. lyciades is nearly twice the size of L. accius (567 Mbp vs. 298 Mbp), however it encodes a smaller number of proteins (15881 vs. 17411). Gene expansions we identified previously in L. accius apparently did not occur in the genome of A. lyciades. For instance, a family of hypothetical cellulases that diverged from an endochitinase (possibly associated with feeding of L. accius caterpillars on nutrient-poor grasses) is absent in A. lyciades. While L. accius underwent gene expansion in pheromone binding proteins, A. lyciades has more opsins. This difference may be related to the mate recognition mechanisms of the two species: visual cues might be more important for the Eudaminae skippers (which have more variable wing patterns), whereas odor might be more important for Grass skippers (that are hardly distinguishable by their wings). Phylogenetically, A. lyciades is a sister species of L. accius, the only other Hesperiidae with a complete genome. CONCLUSIONS: A new reference genome of a dicot-feeding skippers, the first from the Eudaminae subfamily, reveals its larger size and suggests hypotheses about phenotypic traits and differences from monocot-feeding skippers.

The first complete genomes of Metalmarks and the classification of butterfly families.

Sequencing complete genomes of all major phylogenetic groups of organisms opens unprecedented opportunities to study evolution and genetics. We report draft genomes of Calephelis nemesis and Calephelis virginiensis, representatives of the family Riodinidae. They complete the genomic coverage of butterflies at the family level. At 809 and 855 Mbp, respectively, they become the largest available Lepidoptera genomes. Comparison of butterfly genomes shows that the divergence between Riodinidae and Lycaenidae dates to the time when other families started to diverge into subfamilies. Thus, Riodinidae may be considered a subfamily of Lycaenidae. Calephelis species exhibit unique gene expansions in actin-disassembling factor, cofilin, and chitinase. The functional implications of these gene expansions are not clear, but they may aid molting of caterpillars covered in extensive setae. The two Calephelis species diverged about 5 million years ago and they differ in proteins involved in metabolism, circadian clock, regulation of development, and immune responses.