Summary
Although high‐resolution single‐particle cryo‐electron microscopy (cryo‐EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the ...preparation of suitable frozen‐hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous – while still in the test tube – may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo‐EM field.
Abstract
Magnetars, isolated neutron stars with magnetic-field strengths typically ≳10
14
G, exhibit distinctive months-long outburst epochs during which strong evolution of soft X-ray pulse ...profiles, along with nonthermal magnetospheric emission components, is often observed. Using near-daily NICER observations of the magnetar SGR 1830-0645 during the first 37 days of a recent outburst decay, a pulse peak migration in phase is clearly observed, transforming the pulse shape from an initially triple-peaked to a single-peaked profile. Such peak merging has not been seen before for a magnetar. Our high-resolution phase-resolved spectroscopic analysis reveals no significant evolution of temperature despite the complex initial pulse shape, yet the inferred surface hot spots shrink during peak migration and outburst decay. We suggest two possible origins for this evolution. For internal heating of the surface, tectonic motion of the crust may be its underlying cause. The inferred speed of this crustal motion is ≲100 m day
−1
, constraining the density of the driving region to
ρ
∼ 10
10
g cm
−3
, at a depth of ∼200 m. Alternatively, the hot spots could be heated by particle bombardment from a twisted magnetosphere possessing flux tubes or ropes, somewhat resembling solar coronal loops, that untwist and dissipate on the 30–40 day timescale. The peak migration may then be due to a combination of field-line footpoint motion (necessarily driven by crustal motion) and evolving surface radiation beaming. This novel data set paints a vivid picture of the dynamics associated with magnetar outbursts, yet it also highlights the need for a more generic theoretical picture where magnetosphere and crust are considered in tandem.
Calcium transport plays an important role in regulating mitochondrial physiology and pathophysiology. The mitochondrial calcium uniporter (MCU) is a calcium-selective ion channel that is the primary ...mediator for calcium uptake into the mitochondrial matrix. Here, we present the cryo-electron microscopy structure of the full-length MCU from
to an overall resolution of ~3.7 angstroms. Our structure reveals a tetrameric architecture, with the soluble and transmembrane domains adopting different symmetric arrangements within the channel. The conserved W-D-Φ-Φ-E-P-V-T-Y sequence motif of MCU pore forms a selectivity filter comprising two acidic rings separated by one helical turn along the central axis of the channel pore. The structure combined with mutagenesis gives insight into the basis of calcium recognition.
Summary
We report on a genomic and functional analysis of a novel marine siphovirus, the Vibrio phage SIO‐2. This phage is lytic for related Vibrio species of great ecological interest including the ...broadly antagonistic bacterium Vibrio sp. SWAT3 as well as notable members of the Harveyi clade (V. harveyi ATTC BAA‐1116 and V. campbellii ATCC 25920). Vibrio phage SIO‐2 has a circularly permuted genome of 80 598 bp, which displays unusual features. This genome is larger than that of most known siphoviruses and only 38 of the 116 predicted proteins had homologues in databases. Another divergence is manifest by the origin of core genes, most of which share robust similarities with unrelated viruses and bacteria spanning a wide range of phyla. These core genes are arranged in the same order as in most bacteriophages but they are unusually interspaced at two places with insertions of DNA comprising a high density of uncharacterized genes. The acquisition of these DNA inserts is associated with morphological variation of SIO‐2 capsid, which assembles as a large (80 nm) shell with a novel T = 12 symmetry. These atypical structural features confer on SIO‐2 a remarkable stability to a variety of physical, chemical and environmental factors. Given this high level of functional and genomic novelty, SIO‐2 emerges as a model of considerable interest in ecological and evolutionary studies.
Conservation management is expected to increase local biodiversity, but uniform management may lead to biotic homogenization and diversity losses at the regional scale. We evaluated the effects of ...renewed grazing and cutting management carried out across a whole region, on the diversity of plants and seven arthropod groups. Changes in occurrence over 17 years of intensive calcareous grassland management were analysed at the species level, which gave insight into the exact species contributing to regional homogenization or differentiation. Reponses were compared between species differing in habitat affinity, dispersal ability, food specialisation and trophic level. Local species richness increased over the sampling period for true bugs and millipedes, while carabid beetles and weevils declined in local species richness. Species richness remained unchanged for plants, woodlice, ants and spiders. Regional diversity and compositional variation generally followed local patterns. Diversity shifts were only to a limited extent explained by species’ habitat affinity, dispersal ability, trophic level and food specialisation. We conclude that implementation of relatively uniform conservation management across a region did not lead to uniform changes in local species composition. This is an encouraging result for conservation managers, as it shows that there is not necessarily a conflict of interest between local and regional conservation goals. Our study also demonstrates that shifts in diversity patterns differ markedly between taxonomic groups. Single traits provide only limited understanding of these differences. This highlights the need for a wide taxonomic scope when evaluating conservation management and demonstrates the need to understand the mechanisms underlying occurrence shifts.
Non-insulin dependent diabetes mellitus (NIDDM) is a major public health problem, but its aetiology remains poorly understood. We have performed a comprehensive study of the genetic basis of diabetes ...in the Goto-Kakizaki (GK) rat, the most widely used animal model of non-obese NIDDM. The genetic dissection of NIDDM using this model has allowed us to map three independent loci involved in the disease. In addition, we identify a major factor affecting body weight, but not glucose tolerance, on chromosome 7 and map a further 10 regions that are suggestive for linkage. We conclude that NIDDM is polygenic and fasting hyperglycaemia and postprandial hyperglycaemia clearly have distinct genetic bases.
We describe a map of 1.42 million single nucleotide polymorphisms (SNPs) distributed throughout the human genome, providing an average density on available sequence of one SNP every 1.9 kilobases. ...These SNPs were primarily discovered by two projects: The SNP Consortium and the analysis of clone overlaps by the International Human Genome Sequencing Consortium. The map integrates all publicly available SNPs with described genes and other genomic features. We estimate that 60,000 SNPs fall within exon (coding and untranslated regions), and 85% of exons are within 5 kb of the nearest SNP. Nucleotide diversity varies greatly across the genome, in a manner broadly consistent with a standard population genetic model of human history. This high-density SNP map provides a public resource for defining haplotype variation across the genome, and should help to identify biomedically important genes for diagnosis and therapy.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Our electron photoemission experiments demonstrate that the magnetization of the ferromagnetic state of UTe is proportional to the binding energy of the hybridized band centered around 50 meV below ...EF. This proportionality is direct evidence that the ferromagnetism of UTe is itinerant; i.e., the 5f electrons are not fully localized close to the atomic core. This mechanism of itinerant ferromagnetism differs from the traditional picture for 5f-electron magnetism in an essential and a novel way. We propose a simple model for the observed proportionality between the temperature dependence of the magnetization and the binding energy of the hybridized band near EF. This model allows us to estimate the effective magnetic interaction and to identify signatures of itinerant ferromagnetism in other materials.
Morphogenesis of bacteriophage P22 involves the packaging of double-stranded DNA into a preassembled procapsid. DNA is translocated by a powerful virally encoded molecular motor called terminase, ...which comprises large (gp2, 499 residues) and small (gp3, 162 residues) subunits. While gp2 contains the phosphohydrolase and endonuclease activities of terminase, the function of gp3 may be to regulate specific and nonspecific modes of DNA recognition as well as the enzymatic activities of gp2. Electron microscopy shows that wild-type gp3 self-assembles into a stable and monodisperse nonameric ring. A three-dimensional reconstruction at 18 Å resolution provides the first glimpse of P22 terminase architecture and implies two distinct modes of interaction with DNA—involving a central channel of 20 Å diameter and radial spikes separated by 34 Å. Electromobility shift assays indicate that the gp3 ring binds double-stranded DNA nonspecifically
in vitro via electrostatic interactions between the positively charged C-terminus of gp3 (residues 143–152) and phosphates of the DNA backbone. Raman spectra show that nonameric rings formed by subunits truncated at residue 142 retain the subunit fold despite the loss of DNA-binding activity. Difference density maps between gp3 rings containing full-length and C-terminally truncated subunits are consistent with localization of residues 143–152 along the central channel of the nonameric ring. The results suggest a plausible molecular mechanism for gp3 function in DNA recognition and translocation.
Crohn's disease and ulcerative colitis (the inflammatory bowel diseases) have a strong genetic component. Although over 20 putative susceptibility loci have been identified by individual genome ...scans, the majority of these loci have not been replicated. Many individual studies are at the lower limit of acceptable power for complex disease linkage analysis. Genome scan meta-analysis (GSMA), by use of sample sizes an order of magnitude greater than individual linkage studies, has increased power to detect novel loci, may confirm or refute regions detected in smaller individual studies, and enables regions to be prioritized for further gene identification efforts. Genome scan data (markers, significance scores) were obtained from 10 separate studies and meta-analysis was performed using the GSMA method. These studies comprised 1952 inflammatory bowel disease, 1068 Crohn's disease and 457 ulcerative colitis affected relative pairs. Study results were divided into 34 cM chromosomal bins, ranked, weighted by study size, summed across studies and bin-by-bin significance obtained by simulation. A region on chromosome 6p (containing the HLA) met genome wide significance for inflammatory bowel disease. Loci meeting suggestive significance for inflammatory bowel disease were 2q, 3q, 5q, 7q and 16 (NOD2/CARD15 region); Crohn's disease, 2q, 3q, 6p, 16 (NOD2/CARD15 region), 17q, 19p; and ulcerative colitis, 2q. Clustering of adjacent bins was observed for chromosomes 6p, 16, 19p. The meta-analysis has identified novel loci and prioritized genomic regions for further gene identification studies.
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