The structural determination of biological macromolecules has been transformative for understanding biochemical mechanisms and developing therapeutics. However, the ultimate goal of characterizing ...how structural dynamics underpin biochemical processes has been difficult. This is largely due to significant technical challenges that hinder data collection and analysis on the native timescales of macromolecular dynamics. Single‐particle cryo‐EM provides a powerful platform to approach this challenge, since samples can be frozen faster than the single‐turnover timescales of most biochemical reactions. In order to enable time‐resolved analysis, significant innovations in the handling and preparation of cryo‐EM samples have been implemented, bringing us closer to the goal of the direct observation of protein dynamics in the milliseconds to seconds range. Here, the current state of time‐resolved cryo‐EM is reviewed and the most promising future research directions are discussed.
Time‐resolved cryo‐EM is an emerging technique in structural biology that allows the user to capture structural states which would otherwise be too transient for standard methods. There has been a resurgence in technical advancements in this field in the last five years and this review provides a summary of the technical highlights.
Recent developments in the CCP‐EM software suite Burnley, Tom; Palmer, Colin M.; Winn, Martyn
Acta crystallographica. Section D, Structural biology,
June 2017, Volume:
73, Issue:
6
Journal Article
Peer reviewed
Open access
As part of its remit to provide computational support to the cryo‐EM community, the Collaborative Computational Project for Electron cryo‐Microscopy (CCP‐EM) has produced a software framework which ...enables easy access to a range of programs and utilities. The resulting software suite incorporates contributions from different collaborators by encapsulating them in Python task wrappers, which are then made accessible via a user‐friendly graphical user interface as well as a command‐line interface suitable for scripting. The framework includes tools for project and data management. An overview of the design of the framework is given, together with a survey of the functionality at different levels. The current CCP‐EM suite has particular strength in the building and refinement of atomic models into cryo‐EM reconstructions, which is described in detail.
An overview is given of the new CCP‐EM software suite, including the underpinning framework, the current functionality and the distribution procedure. This first version of the suite has a focus on building and refining atomic models.
Partially recorded data are frequently encountered in many applications and usually clustered by first removing incomplete cases or features with missing values, or by imputing missing values, ...followed by application of a clustering algorithm to the resulting altered dataset. Here, we develop clustering methodology through a model‐based approach using the marginal density for the observed values, assuming a finite mixture model of multivariate t distributions. We compare our approximate algorithm to the corresponding full expectation–maximization (EM) approach that considers missing values in the incomplete dataset and makes a missing at random (MAR) assumption, as well as case deletion and imputation methods. Since only the observed values are utilized, our approach is computationally more efficient than imputation or full EM. Simulation studies demonstrate that our approach has favourable recovery of the true cluster partition compared to case deletion and imputation under various missingness mechanisms and is at least competitive with the full EM approach, even when MAR assumptions are violated. Our methodology is demonstrated on a problem of clustering gamma‐ray bursts and is implemented at https://github.com/emilygoren/MixtClust.
When traditional marketing practices are unsuitable for small and medium enterprises, entrepreneurs have to unlearn traditional principles and replace them with new innovative thoughts and actions, ...such as entrepreneurial marketing (EM). This paper examines the impact of EM dimensions on small and medium-sized enterprises (SMEs) in Kosovo. Findings reveal that respondents tend to be highly opportunity focused and understand the importance of resource leveraging. While value creation is seen as a very important entrepreneurial marketing dimension, respondents are reserved with respect to taking risks; furthermore, they do not tend to be proactive, innovative nor customer oriented. Finally, the limitations of the study and the suggestions for future research are provided.
Helical reconstruction in RELION is increasingly being used to determine the atomic structures of amyloid filaments from electron cryo‐microscopy (cryo‐EM) images. However, because the energy ...landscape of amyloid refinements is typically fraught with local optima, amyloid structure determination is often difficult. This paper aims to help RELION users in this process. It discusses aspects of helical reconstruction that are particularly relevant to amyloids, it illustrates the problem of local optima in refinement and how to detect them, and it introduces a new method to calculate 3D initial models from reference‐free 2D class averages. By providing starting models that are closer to the global optimum, this method makes amyloid structure determination easier. All methods described are open‐source and distributed within RELION‐3.1. Their use is illustrated using a publicly available data set on tau filaments from the brain of an individual with Alzheimer's disease.
Amyloid structure determination in RELION is increasingly popular, but may suffer from refinements becoming stuck in local minima. This paper describes a new method for initial model generation that may alleviate this problem.
Central retinal artery occlusion (CRAO) is a rare emergency department presentation with high morbidity and potential for long-term vision loss. Additionally, this finding requires an expeditious ...embolic workup for possible systemic pathology (i.e., stroke). The gold standard for diagnosis is visualization of a pale retina with a "cherry-red spot" on the fovea seen under dilated fundoscopic examination. However, performing a dilated fundoscopic exam is often not practical and technically challenging in the emergency room setting. Alternatively, point of care ultrasound is an inexpensive, non-invasive tool that is already highly utilized in the emergency department and can aid in diagnosis. In the case described in this report, a 66-year-old female presented to the emergency department with painless, monocular vision loss. Ultrasound showed a hyperechoic density on the distal aspect of the optic nerve ("retrobulbar spot sign") and dilated fundoscopic exam showed right eye pale macula with cherry red spot, all consistent with CRAO. Here we present a case that suggests an opportunity for improvement in evaluation of monocular vision loss in the emergency department by adding bedside ocular ultrasound to aid in more rapid diagnosis of CRAO.
Central retinal occlusion, vision loss, point-of-care ultrasound, ocular ultrasound, emboli.
Foreign body ingestions are common complaints in the pediatric emergency department that can be difficult to diagnose in patients with vague symptoms. Magnetic foreign body ingestion can cause ...significant morbidity and mortality in children. Point-of-care ultrasound (POCUS) is an imaging modality that can be used to readily diagnose emergent abdominal pathology in a timely matter at the patient's bedside. In the case described in the report below, a 6-year-old male swallowed multiple round magnets, causing severe abdominal pain. This was detected on POCUS. An abdominal radiograph also confirmed the POCUS findings of multiple hyperechoic round foreign bodies. Key clinical features and sonographic findings are discussed that may help clinicians using POCUS to identify ingested foreign bodies in Pediatric Emergency Departments.
Point-of-care ultrasound, pediatric emergency medicine, foreign body ingestion.
Emphysematous pyelonephritis (EPN) is a rare and life-threatening sequalae of a urinary tract infection marked by a gas-producing necrotizing infection of the renal parenchyma requiring prompt ...treatment. It has been found to be correlated with uncontrolled diabetes mellitus and is more common in females due to their increased susceptibility to urinary tract infections. Early recognition and treatment of EPN are essential to improve the patient's prognosis and prevent further complications. In this case we highlight a progressive case of pyelonephritis complicated by emphysematous abscess formation requiring surgical management. A 49-year-old female with a history of diabetes, hypertension, asthma, and lupus who presented to the emergency department (ED) for evaluation of generalized weakness. Lactate was elevated and urinalysis was suggestive of infection. A computed tomography (CT) scan of the abdomen and pelvis revealed a large subcapsular fluid collection with multiple gas and air-fluid levels along the right kidney, as well as another suspected fluid collection adjacent to the upper pole of the right kidney. The findings suggested complicated pyelonephritis with emphysematous abscess formation. Urology was consulted and they suggested a perinephric drain be placed by interventional radiology (IR). The patient progressed well and was ultimately discharged days later in stable condition. Despite its rarity, healthcare providers should consider infectious processes such as EPN in the differential for generalized weakness and promptly initiate appropriate diagnostic and therapeutic measures.
Weakness, sepsis, urology, CT scan.
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