Energy filter with direct electron detector for electron cryo tomography

用于电子冷冻断层扫描的带有直接电子探测器的能量滤波器

基本信息

批准号：
BB/L014211/1
负责人：
Helen Saibil
金额：
$ 83.33万
依托单位：
Birkbeck, University of London
依托单位国家：
英国
项目类别：
Research Grant
财政年份：
2013
资助国家：
英国
起止时间：
2013 至无数据
项目状态：
已结题

来源：
https://gtr.ukri.org/projects?ref=BB%2FL014211%2F1
关键词：
Energy filter direct electron detector

项目摘要

The machinery of life operates in biological cells and tissues at the molecular and atomic level. A detailed description of these molecular machines, their internal organization, and their interactions within cells is fundamental to understanding living organisms in health and disease. At Birkbeck we study the three-dimensional structure of these machines with the aim of understanding how they work. Electron microscopy (EM) has become a major technique for observing biological machines both in isolation and in their cellular context. Electron microscopy can provide highly detailed information on the structure of biological molecules as well as overall perspectives on the organization of cells and tissues. The EM approach that allows us to determine the three-dimensional structures of cellular machinery is electron tomography. In principle, this is very similar to computed tomography in medicine, except that it works at very high magnification and shows us cells and molecules rather than bones, blood vessels and organs.The samples are rapidly frozen using cryogenic liquids and examined at very low temperature. With this type of preparation they can be imaged in their natural, hydrated state. Advances in electron microscopy methods in the last decade have enabled us to obtain snapshots of biological molecules at different stages of their normal operation, revealing details of their mechanisms of action. However, the unstained biological structures trapped by rapid freezing in their native environment scatter electrons very weakly and are highly sensitive to the electron beam. The resulting images have poor contrast and a high background, limiting the detectable structural features in the images. In this equipment grant, we are requesting funding for a high-resolution electron energy filter and electron detector to help us record fine structural details in tomography images of cells. The filter removes unwanted, inelastic scattering that arises when electrons pass through samples as thick as cells. Filtering out this type of scattering makes the images clearer, so that more structural details can be discerned and interpreted. This energy filter will enable us to extract more information about how the cellular machinery functions.Specific projects that will benefit from this new equipment include: 1) studies of pathogen-infected cells (including Malaria, Chlamydia and Toxoplasma) that will reveal how these intracellular invaders sabotage host cell systems; 2) studies of bacterial secretion systems that will reveal how bacteria interact with their environment to promote their own survival and replication; 3) studies of the process by which viruses infect bacteria; 4) studies to reveal how our immune cells protect our bodies from infection or cancer; 5) studies of the architecture and microtubule cytoskeleton of neuronal cells that will reveal how they adapt their shape during brain development and how this is disrupted in human diseases; 6) studies of amyloid protein deposits in living cells that will reveal how the cell's quality control machinery handles protein misfolding that causes neurodegenerative disease.

生命机器在分子和原子水平上在生物细胞和组织中运转。对这些分子机器、它们的内部组织以及它们在细胞内的相互作用的详细描述对于理解健康和疾病中的生物体至关重要。在伯克贝克学院，我们研究这些机器的三维结构，目的是了解它们的工作原理。电子显微镜 (EM) 已成为观察隔离和细胞环境中生物机器的主要技术。电子显微镜可以提供有关生物分子结构的高度详细信息以及细胞和组织组织的整体视角。使我们能够确定细胞机械的三维结构的电磁方法是电子断层扫描。原则上，这与医学中的计算机断层扫描非常相似，不同之处在于它以非常高的放大倍数工作，并向我们显示细胞和分子，而不是骨骼、血管和器官。使用低温液体快速冷冻样本，并在非常低的温度下进行检查。通过这种类型的制备，它们可以在自然、水合的状态下成像。过去十年电子显微镜方法的进步使我们能够获得生物分子在正常运行的不同阶段的快照，揭示其作用机制的细节。然而，在原生环境中通过快速冷冻捕获的未染色的生物结构散射电子非常微弱，并且对电子束高度敏感。生成的图像对比度差且背景高，限制了图像中可检测的结构特征。在这笔设备拨款中，我们请求资助高分辨率电子能量过滤器和电子探测器，以帮助我们记录细胞断层扫描图像中的精细结构细节。该过滤器可以消除电子穿过细胞一样厚的样品时产生的不必要的非弹性散射。滤除这种类型的散射可以使图像更清晰，从而可以辨别和解释更多的结构细节。这种能量过滤器将使我们能够提取有关细胞机器如何运作的更多信息。将从这种新设备中受益的具体项目包括：1）对病原体感染细胞（包括疟疾、衣原体和弓形虫）的研究，这将揭示这些细胞内如何入侵者破坏宿主细胞系统； 2）细菌分泌系统的研究，将揭示细菌如何与其环境相互作用以促进自身的生存和复制； 3）研究病毒感染细菌的过程； 4）研究揭示我们的免疫细胞如何保护我们的身体免受感染或癌症； 5）对神经元细胞的结构和微管细胞骨架的研究，将揭示它们在大脑发育过程中如何调整其形状，以及在人类疾病中这种结构如何受到破坏； 6) 对活细胞中淀粉样蛋白沉积物的研究，将揭示细胞的质量控制机制如何处理导致神经退行性疾病的蛋白质错误折叠。

项目成果

期刊论文数量（10）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Visualising the cytoskeletal machinery in neuronal growth cones using cryo-electron tomography.

DOI：
10.1242/jcs.259234
发表时间：
2022-04-01
期刊：
Journal of cell science
影响因子：
4
作者：
通讯作者：

Mitotic phosphorylation by NEK6 and NEK7 reduces the microtubule affinity of EML4 to promote chromosome congression

DOI：
10.1126/scisignal.aaw2939
发表时间：
2019-08-13
期刊：
SCIENCE SIGNALING
影响因子：
7.3
作者：
Adib, Rozita;Montgomery, Jessica M.;Fry, Andrew M.
通讯作者：
Fry, Andrew M.

Cryo-EM structure of a microtubule-bound parasite kinesin motor and implications for its mechanism and inhibition.

DOI：
10.1016/j.jbc.2021.101063
发表时间：
2021-11
期刊：
The Journal of biological chemistry
影响因子：
0
作者：
Cook AD;Roberts AJ;Atherton J;Tewari R;Topf M;Moores CA
通讯作者：
Moores CA

Structure of the Bacterial Sex F Pilus Reveals an Assembly of a Stoichiometric Protein-Phospholipid Complex.

DOI：
10.1016/j.cell.2016.08.025
发表时间：
2016-09-08
期刊：
Cell
影响因子：
64.5
作者：
Costa TRD;Ilangovan A;Ukleja M;Redzej A;Santini JM;Smith TK;Egelman EH;Waksman G
通讯作者：
Waksman G

Cryo-EM of kinesin-binding protein: challenges and opportunities from protein-surface interactions.