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GPUs offer significant potential for accelerating diffraction data processing leading to potential speed-ups of up to 10,000-fold. However, achieving this potential is substantially limited by the extensive effort required to redesign algorithms so that they are optimized for GPU architectures. Diffraction data analysis inherently lends itself to vectorization, yet existing data structures frequently introduce serial dependencies incompatible with the parallel architecture of GPUs, severely diminishing computational efficiency. Historically, diffraction data algorithms development was heavily influenced by memory constraints, as memory-efficient algorithms often performed better and were faster on CPUs. Transitioning to GPUs introduces distinct challenges, notably designing computations around two- or three-dimensional grids, wherein each grid point's calculation is handled independently by dedicated GPU threads. This grid-based parallelism enables GPUs to manage hundreds of thousands of simultaneous threads effectively. I will describe these challenges and opportunities with specific examples of both legacy and newly developed GPU-oriented algorithms. These include (a) transforming detector-coordinate data into a reciprocal lattice aligned with crystallographic axes, analogous to the traditional XDS algorithm, and (b) using libraries that streamline computational code development, by integration of GPU-based FFTs with intuitive coordinate system representations. Such approaches will allow us to fully leverage GPU capabilities in diffraction data analysis.

The X-ray structure of human ABCG5/ABCG8 heterodimer in a nucleotide-free state, being the first atomic model of an ABC sterol transporter. Human ABCG5/ABCG8 sterol transporter Cholesterol is an essential component of vertebrate cell membranes. Animals maintain sterol balance by limiting dietary sterol uptake from the gut and promoting sterol secretion from hepatocytes into bile. These physiological processes are mediated by ABCG5/ABCG8, a heterodimeric ABC transporter. These authors have solved the X-ray crystal structure of the human ABCG5/ABCG8 heterodimer in a nucleotide-free state. As well as being the first atomic model of an ABC sterol transporter, the structure provides mechanistic insights into sterol transport and establishes a framework for understanding mutations responsible for sitosterolaemia, a human disease characterized by premature atherosclerosis. ATP binding cassette (ABC) transporters play critical roles in maintaining sterol balance in higher eukaryotes. The ABCG5/ABCG8 heterodimer (G5G8) mediates excretion of neutral sterols in liver and intestines 1 , 2 , 3 , 4 , 5 . Mutations disrupting G5G8 cause sitosterolaemia, a disorder characterized by sterol accumulation and premature atherosclerosis. Here we use crystallization in lipid bilayers to determine the X-ray structure of human G5G8 in a nucleotide-free state at 3.9 Å resolution, generating the first atomic model of an ABC sterol transporter. The structure reveals a new transmembrane fold that is present in a large and functionally diverse superfamily of ABC transporters. The transmembrane domains are coupled to the nucleotide-binding sites by networks of interactions that differ between the active and inactive ATPases, reflecting the catalytic asymmetry of the transporter. The G5G8 structure provides a mechanistic framework for understanding sterol transport and the disruptive effects of mutations causing sitosterolaemia.

Members of the Wiskott–Aldrich syndrome protein (WASP) family control cytoskeletal dynamics by promoting actin filament nucleation with the Arp2/3 complex. The WASP relative WAVE regulates lamellipodia formation within a 400-kilodalton, hetero-pentameric WAVE regulatory complex (WRC). The WRC is inactive towards the Arp2/3 complex, but can be stimulated by the Rac GTPase, kinases and phosphatidylinositols. Here we report the 2.3-ångstrom crystal structure of the WRC and complementary mechanistic analyses. The structure shows that the activity-bearing VCA motif of WAVE is sequestered by a combination of intramolecular and intermolecular contacts within the WRC. Rac and kinases appear to destabilize a WRC element that is necessary for VCA sequestration, suggesting the way in which these signals stimulate WRC activity towards the Arp2/3 complex. The spatial proximity of the Rac binding site and the large basic surface of the WRC suggests how the GTPase and phospholipids could cooperatively recruit the complex to membranes. WAVE control of actin polymerization The WAVE protein is a central regulator of actin dynamics during cell motility. WAVE is a member of the Wiskott–Aldrich syndrome protein (WASP) family, which promotes the actin-filament-nucleating activity of the Arp2/3 complex. In cells, WAVE is constitutively incorporated into the 350-kilodalton WAVE regulatory complex (WRC); it is normally present in an inactive state and can be activated by a number of inputs including the RacGTPase. Here, Chen et al . present the structure and mechanistic analysis of the WRC. The combined data reveal how the WAVE protein is inhibited within the WRC complex and provide mechanisms for WRC activation at the plasma membrane. In cells, WAVE protein, a central regulator of actin dynamics during cell motility, is constitutively incorporated into WAVE regulatory complex (WRC), is normally present in an inactive state and can be activated by a number of inputs. These authors present the structure and mechanistic analysis of WRC. The combined data reveal how the WAVE protein is inhibited within the WRC complex and provide mechanisms for WRC activation at the plasma membrane.

In cryogenic electron microscopy single particle analysis (cryo-EM SPA), the sample-containing solution exists in an extremely high surface-to- volume ratio during the transitional period between post-blotting and plunge-freezing steps of sample preparation. The instability of ice presents an impediment to high-resolution structure determination because samples used for cryo-EM must be prepared as a thin-layer of sample- containing solution embedded in vitreous ice. The primary hazard a thin specimen is exposed to is diffusion- mediated adsorption to the air-water interfaces (AWIs). Adsorption has effects that undermine structure determination such as: 1) denaturation, 2) aggregation, and 3) preferred orientation. Severe preferred orientation will result in an anisotropic 3D reconstruction, which will result in a distorted map. Our objective is to investigate the use of human apoferritin to stabilize thin-layer cryo-EM specimens. Apoferritin is a very stable molecule with a well-known structure that can be efficiently reconstructed. We hypothesize that the use of human apoferritin: (1) will stabilize the ice layer, (2) will not add additional noise, and (3) will provide contrast for motion correction. Adding apoferritin mixed with a target molecule may result in stabilization of the ice layer and modulation of preferred orientation. Coproheme decarboxylase (ChdCs/HemQ) and glutamine synthetase (GS) were chosen as the target molecules because of their preferred orientation in cryo-EM experiments. We prepared grids of apoferritin mixed with ChdCs and GS in several molar ratios for cryo-EM SPA, and for cryogenic electron tomography (cryo-ET) using a 200 kV microscope. We analyzed the impact of apoferritin on the reconstruction of each target molecule, and the position of particles in the ice.

Stu2p/XMAP215 proteins are essential microtubule polymerases that use multiple αβ-tubulin-interacting TOG domains to bind microtubule plus ends and catalyze fast microtubule growth. We report here the structure of the TOG2 domain from Stu2p bound to yeast αβ-tubulin. Like TOG1, TOG2 binds selectively to a fully ‘curved’ conformation of αβ-tubulin, incompatible with a microtubule lattice. We also show that TOG1-TOG2 binds non-cooperatively to two αβ-tubulins. Preferential interactions between TOGs and fully curved αβ-tubulin that cannot exist elsewhere in the microtubule explain how these polymerases localize to the extreme microtubule end. We propose that these polymerases promote elongation because their linked TOG domains concentrate unpolymerized αβ-tubulin near curved subunits already bound at the microtubule end. This tethering model can explain catalyst-like behavior and also predicts that the polymerase action changes the configuration of the microtubule end. Dynamic filaments of proteins, called microtubules, have several important roles inside cells. Microtubules provide structural support for the cell; they help to pull chromosomes apart during cell division; and they guide the trafficking of proteins and molecules across the cell. The building blocks of microtubules are proteins called αβ-tubulin, which are continually added to and removed from the ends of a microtubule, causing it to grow and shrink. Other proteins that interact with the microtubules can help to speed up these construction and deconstruction processes. Ayaz et al. took a closer look at the structure of one particular family of proteins that make it easier for the microtubules to grow, using a technique called X-ray crystallography. The resulting images show two sites—called TOG1 and TOG2—on the enzymes that attach to the αβ-tubulin proteins. Ayaz et al. found that this binding can only occur when αβ-tubulin has a curved shape, which only happens when the tubulins are not included in, or are only bound weakly to the end of, a microtubule. Previous research suggested that the two binding sites might work together to provide ‘scaffolding’ that stabilizes the microtubule. However, genetic experiments by Ayaz et al. show that microtubules will grow even if one of the binding sites is missing. Both TOG1 and TOG2 bind to αβ-tubulin in the same way, and by using computer simulations Ayaz et al. found that this helps to speed up the growth of microtubules. This is because the enzyme's two sites concentrate the individual tubulin building blocks at the ends of the filament. For example, TOG2 could bind to the end of the microtubule, while TOG1 holds an αβ-tubulin protein nearby and ready to bind to the filament's end. This tethering allows the microtubules to be assembled more efficiently.

Advances in sequencing have generated a large number of complete genomes. Traditionally, phylogenetic analysis relies on alignments of orthologs, but defining orthologs and separating them from paralogs is a complex task that may not always be suited to the large datasets of the future. An alternative to traditional, alignment-based approaches are whole-genome, alignment-free methods. These methods are scalable and require minimal manual intervention. We developed SlopeTree, a new alignment-free method that estimates evolutionary distances by measuring the decay of exact substring matches as a function of match length. SlopeTree corrects for horizontal gene transfer, for composition variation and low complexity sequences, and for branch-length nonlinearity caused by multiple mutations at the same site. We tested SlopeTree on 495 bacteria, 73 archaea, and 72 strains of Escherichia coli and Shigella. We compared our trees to the NCBI taxonomy, to trees based on concatenated alignments, and to trees produced by other alignment-free methods. The results were consistent with current knowledge about prokaryotic evolution. We assessed differences in tree topology over different methods and settings and found that the majority of bacteria and archaea have a core set of proteins that evolves by descent. In trees built from complete genomes rather than sets of core genes, we observed some grouping by phenotype rather than phylogeny, for instance with a cluster of sulfur-reducing thermophilic bacteria coming together irrespective of their phyla. The source-code for SlopeTree is available at: http://prodata.swmed.edu/download/pub/slopetree_v1/slopetree.tar.gz.

Two species of hairstreak butterflies from the genus Calycopis are known in the United States: C. cecrops and C. isobeon. Analysis of mitochondrial COI barcodes of Calycopis revealed cecrops-like specimens from the eastern US with atypical barcodes that were 2.6% different from either USA species, but similar to Central American Calycopis species. To address the possibility that the specimens with atypical barcodes represent an undescribed cryptic species, we sequenced complete genomes of 27 Calycopis specimens of four species: C. cecrops, C. isobeon, C. quintana and C. bactra. Some of these specimens were collected up to 60 years ago and preserved dry in museum collections, but nonetheless produced genomes as complete as fresh samples. Phylogenetic trees reconstructed using the whole mitochondrial and nuclear genomes were incongruent. While USA Calycopis with atypical barcodes grouped with Central American species C. quintana by mitochondria, nuclear genome trees placed them within typical USA C. cecrops in agreement with morphology, suggesting mitochondrial introgression. Nuclear genomes also show introgression, especially between C. cecrops and C. isobeon. About 2.3% of each C. cecrops genome has probably (p-value < 0.01, FDR < 0.1) introgressed from C. isobeon and about 3.4% of each C. isobeon genome may have come from C. cecrops. The introgressed regions are enriched in genes encoding transmembrane proteins, mitochondria-targeting proteins and components of the larval cuticle. This study provides the first example of mitochondrial introgression in Lepidoptera supported by complete genome sequencing. Our results caution about relying solely on COI barcodes and mitochondrial DNA for species identification or discovery.

We recently showed that the Wiskott-Aldrich syndrome protein (WASP) family member, WASH, localizes to endosomal subdomains and regulates endocytic vesicle scission in an Arp2/3-dependent manner. Mechanisms regulating WASH activity are unknown. Here we show that WASH functions in cells within a 500 kDa core complex containing Strumpellin, FAM21, KIAA1033 (SWIP), and CCDC53. Although recombinant WASH is constitutively active toward the Arp2/3 complex, the reconstituted core assembly is inhibited, suggesting that it functions in cells to regulate actin dynamics through WASH. FAM21 interacts directly with CAPZ and inhibits its actin-capping activity. Four of the five core components show distant (approximately 15% amino acid sequence identify) but significant structural homology to components of a complex that negatively regulates the WASP family member, WAVE. Moreover, biochemical and electron microscopic analyses show that the WASH and WAVE complexes are structurally similar. Thus, these two distantly related WASP family members are controlled by analogous structurally related mechanisms. Strumpellin is mutated in the human disease hereditary spastic paraplegia, and its link to WASH suggests that misregulation of actin dynamics on endosomes may play a role in this disorder.

The human genome harbors a variety of genetic variations. Single-nucleotide changes that alter amino acids in protein-coding regions are one of the major causes of human phenotypic variation and diseases. These single-amino acid variations (SAVs) are routinely found in whole genome and exome sequencing. Evaluating the functional impact of such genomic alterations is crucial for diagnosis of genetic disorders. We developed DeepSAV, a deep-learning convolutional neural network to differentiate disease-causing and benign SAVs based on a variety of protein sequence, structural and functional properties. Our method outperforms most stand-alone programs, and the version incorporating population and gene-level information (DeepSAV+PG) has similar predictive power as some of the best available. We transformed DeepSAV scores of rare SAVs in the human population into a quantity termed \"mutation severity measure\" for each human protein-coding gene. It reflects a gene's tolerance to deleterious missense mutations and serves as a useful tool to study gene-disease associations. Genes implicated in cancer, autism, and viral interaction are found by this measure as intolerant to mutations, while genes associated with a number of other diseases are scored as tolerant. Among known disease-associated genes, those that are mutation-intolerant are likely to function in development and signal transduction pathways, while those that are mutation-tolerant tend to encode metabolic and mitochondrial proteins.

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 Å reconstructions 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.