Acquisition of 200keV FEG Cryo-Electron Microscope

电子显微镜（EM）是研究蛋白质聚合物、蛋白质- dna复合物和病毒等大分子复合物结构的一种极其强大的技术。电子显微镜在样品制备、仪器和成像方面取得了重大进展。这些进步使得利用电子显微镜来确定蛋白质的三维结构成为可能。此外，它们还允许进行低分辨率的研究，这是对x射线晶体学和核磁共振等技术获得的研究的补充。一台由飞利浦/FEI制造的200 KeV场发射枪低温电子显微镜将大大提高弗吉尼亚大学医学院六名研究人员的研究计划。这种显微镜将有一个高度相干的电子源，将允许分辨率比传统仪器高得多。该显微镜将配备用于冷冻水合标本的成像，在液氮温度下保持在显微镜中。它还将具有图像采集能力，使用CCD相机，这将允许在线图像分析。将利用该仪器提供的更高分辨率的项目有：1)研究f -肌动蛋白，以及f -肌动蛋白与肌动蛋白结合蛋白的复合物；2)蛋白质- dna复合物的研究，特别是螺旋重组细丝，如RecA/Rad51/UvsX，环状解旋酶，如rho， DnaB和T7 gp4；3)单纯疱疹病毒二十面体衣壳的研究；4)利用脂质单层上形成的二维晶体研究Ca2+依赖性膜结合蛋白（如膜联蛋白）；5)利用脂质单层上形成的二维晶体研究蛋白激酶C同工酶；6)离子通道的研究，如VacA六聚体，使用二维晶体的冷冻电镜来补充正在进行的原子力显微镜研究。许多项目已经产生的结果是在负染色标本的常规透射电镜可获得的可靠分辨率的极限。新仪器不仅将加强这些个人研究项目，而且将大大加强弗吉尼亚大学结构生物学的环境以及研究生和博士后的培训。

ABSTRACT0002805Edward H. EgelmanUniversity of VirginiaAcquisition of 200 KeV FEG Cryo-Electron Microscope Electron microscopy (EM) is an extremely powerful technique for studying the structure of macromolecular complexes, such as protein polymers, protein-DNA complexes and viruses. Significant advances in electron microscopy have been made in specimen preparation, instrumentation and image. These advances make it possible to use an electron microscopic to determine the three-dimensional structure of a protein. In addition, they also allow for lower-resolution studies that are complementary to those obtained by such techniques as x-ray crystallography and nuclear magnetic resonance. A 200 KeV Field Emission Gun cryo-electron microscope, manufactured by Philips/FEI, will significantly enhance the research programs of a group of six investigators at the University of Virginia Medical School. This microscope will have a highly coherent electron source that will allow for resolutions much higher than obtainable with conventional instruments. The microscope will be equipped for the imaging of frozen-hydrated specimens, maintained in the microscope at liquid nitrogen temperatures. It will also have image acquisition capabilities, using a CCD camera, that will allow for on-line image analysis. The projects that will take advantage of the increased resolution provided by this instrument are: 1) studies of F-actin, and complexes of F-actin with actin-binding proteins; 2) studies of protein-DNA complexes, particularly helical recombination filaments such as RecA/Rad51/UvsX, and ring helicases such as rho, DnaB and T7 gp4; 3) studies of the icosahedral Herpes Simplex virus capsid; 4) studies of Ca2+-dependent membrane binding proteins such as annexin, using two dimensional crystals formed on lipid monolayers; 5) studies of Protein Kinase C isozymes using two dimensional crystals formed on lipid monolayers; and 6) studies of ion channels, such as VacA hexamers, using cryo-EM of two-dimensional crystals to complement ongoing studies with Atomic Force Microscopy. Many of the projects have already produced results that are at the limit of reliable resolution obtainable by conventional TEM of negatively stained specimens. The new instrument will not only enhance these individual research programs, but will greatly strengthen the environment for structural biology at the University of Virginia and the training of graduate students and postdoctoral fellows.