一个结构性病毒学的集成工作流程,使用100keV电子显微镜
Rasangi Pathirage1, Moumita Dutta1, Ruth J Parsons1,2
1Duke University, Duke Human Vaccine Institute, Durham NC 27710, USA.
bioRxiv : the preprint server for biology
|December 22, 2025
概括
研究人员现在可以在自己的实验室中使用100keV显微镜实现高质量的冷电子显微镜 (cryo-EM) 结构生物学. 这使得冷电磁器的使用变得民主化,使其能够独立运行,并为结构确定提供培训.
科学领域:
- 结构生物学 结构生物学
- 生物物理学的生物物理.
- 生物化学 生物化学
背景情况:
- 低温电子显微镜 (cryo-EM) 对于研究灵活和异质的生物样本至关重要.
- 高端300keV冷电磁显微镜提供高分辨率数据,但价格昂贵,可访问性有限.
- 对先进的冷电磁设备的有限访问阻碍了研究进步和培训.
研究的目的:
- 介绍一个结构生物学实验室内100keV冷电磁器的成功用户管理的操作.
- 详细介绍安装,维护和运行具有成本效益的冷电磁系统的实际方面.
- 为了证明独立,高质量的冷EM数据收集和处理在核心设施之外的可行性.
主要方法:
- 100千伏电子显微镜的安装和日常维护.
- 集成的工作流包括网格选,数据收集和数据处理.
- 使用Ceta CMOS摄像头进行低分辨率和猎C直接探测器进行高分辨率重建.
主要成果:
- 演示了适合原子模型构建的高质量低分辨率重建和高分辨率重建的例行高质量重建.
- 展示了使用病毒表面糖蛋白作为案例研究的完整工作流.
- 在一个月的定期使用和培训内,初学者在独立的显微镜操作中获得了熟练程度.
结论:
- 用户管理的100 keV冷EM可实现综合结构生物学研究,将蛋白质生产与结构确定联系起来.
- 该模型提供了一个可访问的培训平台,促进了对冷电磁技术的更广泛的专业知识.
- 代表了现代研究小组首次成功演示,独立管理冷EM操作,并利用其结构生物学能力.
更多相关视频
13:43A Robust Single-Particle Cryo-Electron Microscopy cryo-EM Processing Workflow with cryoSPARC, RELION, and Scipion
Published on: January 31, 2022
14.9K
07:56User-friendly, High-throughput, and Fully Automated Data Acquisition Software for Single-particle Cryo-electron Microscopy
Published on: July 29, 2021
3.9K
相关概念视频
01:28Cryo-electron Microscopy
4.1K
Conventional electron microscopy (EM) involves dehydration, fixation, and staining of biological samples, which distorts the native state of biological molecules and results in several artifacts. Also, the high-energy electron beam damages the sample and makes it difficult to obtain high-resolution images. These issues can be addressed using cryo-EM, which uses frozen samples and gentler electron beams. The technique was developed by Jacques Dubochet, Joachim Frank, and Richard Henderson, for...
4.1K
01:25Overview of Electron Microscopy
12.9K
The wavelengths of visible light ultimately limit the maximum theoretical resolution of images created by light microscopes. Most light microscopes can only magnify 1000X, and a few can magnify up to 1500X. Electrons, like electromagnetic radiation, can behave like waves, but with wavelengths of 0.005 nm, they produce significantly greater resolution up to 0.05 nm as compared to 500 nm for visible light. An electron microscope (EM) can create a sharp image that is magnified up to 2,000,000X.
12.9K
01:07Electron Microscope Tomography and Single-particle Reconstruction
2.8K
Transmission electron microscopy (TEM) can be used to determine the 3D structure of biological samples with the help of techniques such as electron microscope tomography and single-particle reconstruction. While single-particle reconstruction can examine macromolecules and macromolecular complexes in vitro conditions only, tomography permits the study of cell components or small cells in vivo.
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission...
Electron Tomography
Electron tomography can be performed either in TEM or STEM (scanning transmission...
2.8K
01:28Three-Dimensional Microscopy in Microbiology
732
Three-dimensional imaging techniques are essential in cell biology, allowing researchers to visualize intricate cellular structures with high resolution. Two prominent methods, Differential Interference Contrast Microscopy (DIC) and Confocal Scanning Laser Microscopy (CSLM), provide distinct advantages for imaging live and thick specimens, respectively.Differential Interference Contrast MicroscopyDIC microscopy enhances contrast in transparent, unstained samples by converting phase...
732
01:20Preparation of Samples for Electron Microscopy
6.7K
To be visualized by an electron microscope, either transmission or scanning, biological samples need to be fixed (stabilized) so the electron beam does not destroy them and dried thoroughly (desiccated/dehydrated) so the vacuum does not affect them. Fixation needs to be done as quickly as possible because the sample properties will start changing as soon as it is removed from its natural environment. For example, in a tissue sample, the oxygen levels begin decreasing, causing an altered...
6.7K