Structural studies of T4S systems by electron microscopy

Abstract: Type IV secretion (T4S) systems are large dynamic nanomachines that transport DNA and/or proteins through the membranes of bacteria. Analysis of T4S system architecture is an extremely challenging task taking into account their multi protein organisation and lack of overall global symmetry. Nonetheless the last decade demonstrated an amazing progress achieved by X-ray crystallography and cryo-electron microscopy. In this review we present a structural analysis of this dynamic complex based on recent advances in biochemical, biophysical and structural studies

李郝体制与国民党派系斗争

第一章分析了郝伯村与李登辉的权力斗争与利益交换，二月政争后， 李登辉为了达到打击李焕等目的，提名郝柏村组阁，李郝体制于是形成，李登辉这 种为求得与党内反对派达成暂时权力平衡的做法，并没有从根本上消除党内斗争 的根源，加上李郝两人完全不同的政治理念与历史渊源，使李郝体制存在许多先天性 矛盾，两人“肝胆相照”关系存在许多变数。 第二章分析了郝内阁两次人事改组中李郝的互相妥协，郝柏村上台后，力主“行 政院”工作，处处显现强势作风，二李登辉表面上不动声色，暗中却在筹划主控大 利益，各守分际，尽量压制冲突性利益，形成了政治生态上的暂时平衡．第三章分析了李郝在“新大陆政策”及“务实外交”上的...学位：历史学硕士院系专业：台湾研究所_专门史学号：1990110

Electron imaging with Medipix2 hybrid pixel detector

The electron imaging performance of Medipix2 is described. Medipix2 is a hybrid pixel detector composed of two layers. It has a sensor layer and a layer of readout electronics, in which each 55 μm×55 μm pixel has upper and lower energy discrimination and MHz rate counting. The sensor layer consists of a 300 μm slab of pixellated monolithic silicon and this is bonded to the readout chip. Experimental measurement of the detective quantum efficiency, DQE(0) at 120 keV shows that it can reach 85% independent of electron exposure, since the detector has zero noise, and the DQE(Nyquist) can reach 35% of that expected for a perfect detector (4/π2). Experimental measurement of the modulation transfer function (MTF) at Nyquist resolution for 120 keV electrons using a 60 keV lower energy threshold, yields a value that is 50% of that expected for a perfect detector (2/π). Finally, Monte Carlo simulations of electron tracks and energy deposited in adjacent pixels have been performed and used to calculate expected values for the MTF and DQE as a function of the threshold energy. The good agreement between theory and experiment allows suggestions for further improvements to be made with confidence. The present detector is already very useful for experiments that require a high DQE at very low doses

Structural analysis of protein complexes by cryo electron microscopy

Structural studies of biocomplexes using single-particle cryo-electron microscopy (cryo-EM) is now a well-established technique in structural biology and has become competitive with X-ray crystallography. The latest advances in EM enable us to determine structures of protein complexes at 3–5 Å resolution for an extremely broad range of sizes from ~200 kDa up to hundreds of megadaltons (Bartesaghi et al., Science 348(6239):1147–1151, 2051; Bai et al., Nature 525(7568):212–217, 2015; Vinothkumar et al., Nature 515(7525):80–84, 2014; Grigorieff and Harrison, Curr Opin Struct Biol 21(2):265–273, 2011). The majority of biocomplexes comprise a number of different components and are not amenable to crystallisation. Secretion systems are typical examples of such multi-protein complexes, and structural studies of them are extremely challenging. The only feasible approach to revealing their spatial organisation and functional modification is cryo-EM. The development of systems for digital registration of images and algorithms for the fast and efficient processing of recorded images and subsequent analysis facilitated the determination of structures at near-atomic resolution. In this review we will describe sample preparation for cryo-EM, how data are collected by new detectors, and the logistics of image analysis through the basic steps required for reconstructions of both small and large biological complexes and their refinement to nearly atomic resolution. The processing workflow is illustrated using examples of EM analysis of a Type IV Secretion System