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Marabini R.,Autonomous University of Madrid | Carragher B.,Scripps Research Institute | Chen S.,MRC LMB | Chen J.,Massachusetts Institute of Technology | And 18 more authors.
Journal of Structural Biology | Year: 2015

Image formation in bright field electron microscopy can be described with the help of the contrast transfer function (CTF). In this work the authors describe the "CTF Estimation Challenge", called by the Madrid Instruct Image Processing Center (I2PC) in collaboration with the National Center for Macromolecular Imaging (NCMI) at Houston. Correcting for the effects of the CTF requires accurate knowledge of the CTF parameters, but these have often been difficult to determine. In this challenge, researchers have had the opportunity to test their ability in estimating some of the key parameters of the electron microscope CTF on a large micrograph data set produced by well-known laboratories on a wide set of experimental conditions. This work presents the first analysis of the results of the CTF Estimation Challenge, including an assessment of the performance of the different software packages under different conditions, so as to identify those areas of research where further developments would be desirable in order to achieve high-resolution structural information. © 2015 Elsevier Inc.

Hansen C.G.,MRC LMB | Nichols B.J.,MRC LMB
Trends in Cell Biology | Year: 2010

Caveolae are ampullate (flask-shaped) invaginations that are abundant in the plasma membrane of many mammalian cell types. Although caveolae are implicated in a wide range of processes including endothelial transcytosis, lipid homeostasis and cellular signalling, a detailed molecular picture of many aspects of their function has been elusive. Until recently, the only extensively characterised protein components of caveolae were the caveolins. Recently, data from several laboratories have demonstrated that a family of four related proteins, termed cavins 1-4, plays key roles in caveolar biogenesis and function. Salient properties of the cavin family include their propensity to form complexes with each other and their different but overlapping tissue distribution. This review summarises recent data on the cavins, and sets them in the context of open questions on the construction and function of caveolae. The discovery of cavins implies that caveolae might have unexpectedly diverse structural properties, in accord with the wide range of functions attributed to these 'little caves'. © 2010 Elsevier Ltd.

Wayland M.T.,University of Cambridge | Defaye A.,Aix - Marseille University | Rocha J.,MRC LMB | Jayaram S.A.,MRC LMB | And 6 more authors.
Journal of Insect Physiology | Year: 2014

The intestinal physiology of Drosophila melanogaster can be monitored in an integrative, non-invasive manner by analysing graphical features of the excreta produced by flies fed on a dye-supplemented diet. This assay has been used by various labs to explore gut function and its regulation. To facilitate its use, we present here a free, stand-alone dedicated software tool for the analysis of fly excreta. The Ultimate Reader of Dung (T.U.R.D.) is designed to offer a flexible environment for a wide range of experimental designs, with special attention to automation and high-throughput processing. This software detects the distinctive changes in acid-base and water balance previously reported to occur in response to dietary challenges and mating. We have used T.U.R.D. to test the contribution of the bacterial environment of the flies to various intestinal parameters including the established diet- and mating-triggered responses. To this end, we have analysed the faecal patterns of flies reared in germ-free conditions, upon re-association with controlled microbiota and subjected to food-borne or systemic, non-lethal bacterial infections. We find that the tested faecal outputs are unchanged in all these conditions, suggesting that the impact of the bacterial environment on the intestinal features highlighted by faecal deposit analysis is minimal. © 2014 The Authors.

Shvets E.,MRC LMB | Ludwig A.,Nanyang Technological University | Nichols B.J.,MRC LMB
Current Opinion in Cell Biology | Year: 2014

Recent data from the study of the cell biology of caveolae have provided insights both into how these flask-shaped invaginations of the plasma membrane are formed and how they may function in different contexts. This review discusses experiments that analyse the composition and ultrastructural distribution of protein complexes responsible for generating caveolae, that suggest functions for caveolae in response to mechanical stress or damage to the plasma membrane, that show that caveolae may have an important role during the signalling events for regulation of metabolism, and that imply that caveolae can act as endocytic vesicles at the plasma membrane. We also highlight unexpected roles for caveolar proteins in regulating circadian rhythms and new insights into the way in which caveolae may be involved in fatty acid uptake in the intestine. Current outstanding questions in the field are emphasised. © 2014 Elsevier Ltd.

To determine the high-resolution structure of some biomolecules, scientists use the well-established technique X-ray crystallography. But for that century-old method to work, they must first grow large crystals of the substances—one-tenth of a millimeter thick and larger, in most cases. Some biomolecules, however, are difficult to work with and form crystals that are too small to analyze this way. A report now shows that a technique called microED (microelectron diffraction) can determine the structures of biomolecules at atomic resolution by probing crystals with only about one-millionth the volume of those needed for X-ray crystallography. In the study, Tamir Gonen of Howard Hughes Medical Institute’s Janelia Research Campus; David S. Eisenberg of the University of California, Los Angeles; and coworkers used microED to determine the structures of aggregates of two peptides from α-synuclein that play key roles in Parkinson’s disease (Nature 2015, DOI: 10.1038/nature15368). The structures they obtained are similar to those uncovered by research teams studying amyloid-forming peptides involved in other neurodegenerative diseases, notes Michel Goedert of the Medical Research Council Laboratory of Molecular Biology (MRC LMB), in Cambridge, England, in a Nature commentary. But they also provide new information that could aid in the development of potential agents to fight Parkinson’s that inhibit α-synuclein aggregate formation. X-ray crystallography requires relatively large crystals because X-rays quickly damage small ones, destroying samples before they can be analyzed effectively. X-ray free-electron laser (XFEL) diffraction instruments use ultrafast pulses that can analyze crystals about one-thousandth the volume of those probed by conventional X-ray crystallography, but XFEL instruments are rare and expensive to use. Single-particle cryoelectron microscopy, which has become increasingly popular in recent years, doesn’t require crystals at all but is limited to analyzing only large biomolecules. In 2013, Gonen’s group at Janelia developed microED, which is in the cryoelectron microscopy family of techniques. The electron beam used by the method interacts with molecules more “softly” than X-rays do but is still damaging. Gonen and coworkers made the technique work by stepping down the power of the electron beam to extremely low levels, permitting sufficient signal to be collected to derive structures. In the new work, the Gonen-Eisenberg team used microED to analyze crystals so small that they can’t be visualized by conventional light microscopy. Gonen’s group has tested microED on enzymes, such as lysozyme, with well-known structures. But the new study is the first to apply the technique to previously unknown structures. MicroED determined the α-synuclein peptide aggregate structures at 1.4-Å resolution—the best resolution ever achieved by any cryoelectron microscopy technique. Cryoelectron microscopy instruments are relatively inexpensive and already widely available in many laboratories, so crystallographers could eventually use microED routinely. Disadvantages of the technique include an inability to analyze large crystals in addition to small ones and problems with phasing, a process needed to calculate structures. This article has been translated into Spanish by Divulgame.org and can be found here.

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