Improving Resolution and Resolvability of Single Particle CryoEM using Gaussian Mixture Models

Cryogenic electron microscopy is widely used in structural biology, but the resolution it achieves is often limited by the dynamics of the macromolecule. Here, we developed a refinement protocol based on Gaussian mixture models that integrate particle orientation and conformation estimation, and improves the alignment for flexible domains of protein structures. We demonstrated this protocol on multiple datasets, resulting in improved resolution and resolvability by visual and quantitative measures

Identifying Components in 3D Density Maps of Protein Nanomachines by Multi-scale Segmentation

Segmentation of density maps obtained using cryo-electron microscopy (cryo-EM) is a challenging task, and is typically accomplished by time-intensive interactive methods. The goal of segmentation is to identify the regions inside the density map that correspond to individual components. We present a multi-scale segmentation method for accomplishing this task that requires very little user interaction. The method uses the concept of scale space, which is created by convolution of the input density map with a Gaussian filter. The latter process smoothes the density map. The standard deviation of the Gaussian filter is varied, with smaller values corresponding to finer scales and larger values to coarser scales. Each of the maps at different scales is segmented using the watershed method, which is very efficient, completely automatic, and does not require the specification of seed points. Some detail is lost in the smoothing process. A sharpening process reintroduces detail into the segmentation at the coarsest scale by using the segmentations at the finer scales. We apply the method to simulated density maps, where the exact segmentation (or ground truth) is known, and rigorously evaluate the accuracy of the resulting segmentations

A tail-like assembly at the portal vertex in intact herpes simplex type-1 virions

Herpes viruses are prevalent and well characterized human pathogens. Despite extensive study, much remains to be learned about the structure of the genome packaging and release machinery in the capsids of these large and complex double-stranded DNA viruses. However, such machinery is well characterized in tailed bacteriophage, which share a common evolutionary origin with herpesvirus. In tailed bacteriophage, the genome exits from the virus particle through a portal and is transferred into the host cell by a complex apparatus (i.e. the tail) located at the portal vertex. Here we use electron cryo-tomography of human herpes simplex type-1 (HSV-1) virions to reveal a previously unsuspected feature at the portal vertex, which extends across the HSV-1 tegument layer to form a connection between the capsid and the viral membrane. The location of this assembly suggests that it plays a role in genome release into the nucleus and is also important for virion architecture

The roles of withdrawal in the negotiator personality-tactic relationship

The personality of a negotiator shall affect his choice of tactics. Moreover, mixed predictions of the personality-tactic relationship have been derived from prior studies. One possible explanation is the influence of other intervening factors. In this regard, this study examines the role of withdrawal, as an intervening variable, in the negotiator personality-tactic relationship. State of withdrawal refers to the level of interest to continue with a negotiation. In a state of complete withdrawal, the interest to continue no longer exists and breakdown of the negotiation is inevitable. With the participation of practicing professionals, an experiment was used to collect data for the study. It was found that competitors are prone to withdraw and use more distributive tactics. However, this pattern changes with the composition of the dyad. If the negotiating counterpart is a cooperator, a competitor will adopt a more integrative approach. This finding reminds the importance of the personality factor in selecting members of a negotiating team

Electron Cryomicroscopy of Biological Machines at Subnanometer Resolution

Advances in electron cryomicroscopy (cryo-EM) have made possible the structural determination of large biological machines in the resolution range of 6–9 Å. Rice dwarf virus and the acrosomal bundle represent two distinct types of machines amenable to cryo-EM investigations at subnanometer resolutions. However, calculating the density map is only the first step, and much analysis remains to extract structural insights and the mechanism of action in these machines. This paper will review the computational and visualization methodologies necessary for analysis (structure mining) of the computed cryo-EM maps of these machines. These steps include component segmentation, averaging based on local symmetry among components, density connectivity trace, incorporation of bioinformatics analysis, and fitting of high-resolution component data, if available. The consequences of these analyses can not only identify accurately some of the secondary structure elements of the molecular components in machines but also suggest structural mechanisms related to their biological functions

Surveillance cultures of samples obtained from biopsy channels and automated endoscope reprocessors after high-level disinfection of gastrointestinal endoscopes

BACKGROUND: The instrument channels of gastrointestinal (GI) endoscopes may be heavily contaminated with bacteria even after high-level disinfection (HLD). The British Society of Gastroenterology guidelines emphasize the benefits of manually brushing endoscope channels and using automated endoscope reprocessors (AERs) for disinfecting endoscopes. In this study, we aimed to assess the effectiveness of decontamination using reprocessors after HLD by comparing the cultured samples obtained from biopsy channels (BCs) of GI endoscopes and the internal surfaces of AERs. METHODS: We conducted a 5-year prospective study. Every month random consecutive sampling was carried out after a complete reprocessing cycle; 420 rinse and swabs samples were collected from BCs and internal surface of AERs, respectively. Of the 420 rinse samples collected from the BC of the GI endoscopes, 300 were obtained from the BCs of gastroscopes and 120 from BCs of colonoscopes. Samples were collected by flushing the BCs with sterile distilled water, and swabbing the residual water from the AERs after reprocessing. These samples were cultured to detect the presence of aerobic and anaerobic bacteria and mycobacteria. RESULTS: The number of culture-positive samples obtained from BCs (13.6%, 57/420) was significantly higher than that obtained from AERs (1.7%, 7/420). In addition, the number of culture-positive samples obtained from the BCs of gastroscopes (10.7%, 32/300) and colonoscopes (20.8%, 25/120) were significantly higher than that obtained from AER reprocess to gastroscopes (2.0%, 6/300) and AER reprocess to colonoscopes (0.8%, 1/120). CONCLUSIONS: Culturing rinse samples obtained from BCs provides a better indication of the effectiveness of the decontamination of GI endoscopes after HLD than culturing the swab samples obtained from the inner surfaces of AERs as the swab samples only indicate whether the AERs are free from microbial contamination or not

Quantitative analysis of cryo-EM density map segmentation by watershed and scale-space filtering, and fitting of structures by alignment to regions

Cryo-electron microscopy produces 3D density maps of molecular machines, which consist of various molecular components such as proteins and RNA. Segmentation of individual components in such maps is a challenging task, and is mostly accomplished interactively. We present an approach based on the immersive watershed method and grouping of the resulting regions using progressively smoothed maps. The method requires only three parameters: the segmentation threshold, a smoothing step size, and the number of smoothing steps. We first apply the method to maps generated from molecular structures and use a quantitative metric to measure the segmentation accuracy. The method does not attain perfect accuracy, however it produces single or small groups of regions that roughly match individual proteins or subunits. We also present two methods for fitting of structures into density maps, based on aligning the structures with single regions or small groups of regions. The first method aligns centers and principal axes, whereas the second aligns centers and then rotates the structure to find the best fit. We describe both interactive and automated ways of using these two methods. Finally, we show segmentation and fitting results for several experimentally-obtained density maps.National Institutes of Health (U.S.) (Grant PN2EY016525)National Institutes of Health (U.S.) (Grant R01GM079429)National Institutes of Health (U.S.) (Grant P41RR02250)National Science Foundation (U.S.) (IIS-0705644

GENFIRE: A generalized Fourier iterative reconstruction algorithm for high-resolution 3D imaging

Tomography has made a radical impact on diverse fields ranging from the study of 3D atomic arrangements in matter to the study of human health in medicine. Despite its very diverse applications, the core of tomography remains the same, that is, a mathematical method must be implemented to reconstruct the 3D structure of an object from a number of 2D projections. In many scientific applications, however, the number of projections that can be measured is limited due to geometric constraints, tolerable radiation dose and/or acquisition speed. Thus it becomes an important problem to obtain the best-possible reconstruction from a limited number of projections. Here, we present the mathematical implementation of a tomographic algorithm, termed GENeralized Fourier Iterative REconstruction (GENFIRE). By iterating between real and reciprocal space, GENFIRE searches for a global solution that is concurrently consistent with the measured data and general physical constraints. The algorithm requires minimal human intervention and also incorporates angular refinement to reduce the tilt angle error. We demonstrate that GENFIRE can produce superior results relative to several other popular tomographic reconstruction techniques by numerical simulations, and by experimentally by reconstructing the 3D structure of a porous material and a frozen-hydrated marine cyanobacterium. Equipped with a graphical user interface, GENFIRE is freely available from our website and is expected to find broad applications across different disciplines.Comment: 18 pages, 6 figure