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Electron Microscopy Solutions
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单颗粒分析

蛋白质复合物分析面临的挑战

要想以当今社会所要求的速度进行药物开发,就需要尽可能接近 活体 以高分辨率研究分子机制。直到最近,用于实现生物分子的高分辨率结构的主要技术是晶体学。

冷冻透射电镜是 《Nature Methods》 评选出的“2015 年度方法”,现在能够以大体积和清晰度解析近乎原子级的分辨率结构。使用冷冻透射电镜了解分子结构,如蛋白质、蛋白质复合物和蛋白核糖核酸缔合物(生命的基本构件),从而大规模开发制药解决方案来治疗疾病和病变。

揭示三维大分子蛋白质复合物面临的挑战

直到最近,为了通过 X 射线晶体分析法重建和呈现蛋白质,他们才设计了一套方法对蛋白质进行结晶处理。结晶是一个非常耗时的过程,而且并不总是能取得成功,因为某些蛋白质没有结晶。这项技术主要用于对单分子和二聚结构进行结晶。尽管大型蛋白质复合物对于研究本体机能更有意义,但目前稳定和结晶这些结构存在一定难度。

 冷冻透射电镜的优势:

  • 分解样品中难以结晶的蛋白质结构
  • 分析极具挑战性的异质样品和抗体

冷冻透射电镜如何与 X 射线晶体分析法和 NMR 互补

Single Particle Analysis Workflow

A step-by-step solution for resolving 3D protein complexes

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Step one: Sample Preparation

The first step in the cryo-TEM workflow is key in order to produce the highest quality 3D protein complex structures.

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Step two: Single Particle Imaging

Maintaining sample integrity while transferring to an imaging platform for screening and analysis is the next step towards obtaining structural data.

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Step three: Data Acquistion

We take a series of 2D projection images using very low electron dose and computationally extract them. Software then orients them relative to one another to generate a three dimensional structure.

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Step four: Single Particle Reconstruction

For single particle reconstructions, the data produced can immediately be imported into various reconstruction packages.

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Step one: Sample Preparation

Sample Vitrification

This is the step which prepares the sample for cryo-TEM imaging. Vitrification cools the sample so rapidly that water molecules do not have time to crystallize, forming instead an amorphous solid that does little or no damage to the sample structure. Vitrification can be applied to protein solutions, cell suspensions or thin tissue slices.

To enable optimal results, the vitrification step needs to be standardized and reproducible. FEI achieves exactly this by offering an automated, programmable approach to vitrification

Products & Solutions

Vitrification with Vitrobot

Create high quality vitrified samples for single particle analysis or cryo-tomography research applications with Vitrobot. Vitrobot offers fully automated vitrification, fast and easy. It performs the cryo-fixation process at constant physical and mechanical conditions like temperature, relative humidity, blotting conditions and freezing velocity. This ensures high quality cryo-fixation results and a high sample preparation throughput prior to cryo-TEM observation.

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Step two: Single Particle Imaging

Fixed in an ultra-thin vitrified ice layer, most biological materials are highly beam sensitive. As a result, only low-dose beams can be used in imaging. In the past, this has presented a challenge to acquiring sufficient image resolution and contrast. This is no longer the case.

Products & Solutions

Imaging Platforms

Glacios Cryo-TEM

The new Thermo Scientific™ Glacios™ Cryo Transmission Electron Microscope (Cryo-TEM) delivers a complete and affordable Cryo-EM solution to a broad range of scientists. It features 200 kV XFEG optics, the industry-leading Autoloader (cryogenic sample manipulation robot) and the same innovative automation for ease of use as on the Krios G3i Cryo-TEM. The Glacios Cryo-TEM bundles all this into a small footprint that simplifies installation.

Krios G3i Cryo-TEM

The new Thermo Scientific™ Krios™ G3i Cryo Transmission Electron Microscope (Cryo-TEM) enables life science researchers to unravel life at the molecular level—easier, faster, and more reliably than ever before. Its highly stable 300 kV TEM platform and industry-leading Autoloader (cryogenic sample manipulation robot) are designed for automated applications, such as single particle analysis (SPA) and cryo-tomography. Designed-in connectivity ensures a robust and risk-free pathway throughout the entire workflow, from sample preparation and optimization to image acquisition and data processing.

Talos™ Arctica

The Talos Arctica is a 200kV FEG Transmission and Scanning Electron Microscope (STEM). It is a powerful, stable, and versatile system for delivering high-resolution 3D characterization of biological and biomaterials samples in cell biology, structural biology, and nanotechnology research. Talos enables scientists to quickly obtain better insight and understanding of macromolecular structures, cellular components, cells, and tissues in three dimensions.

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Step three: Data Acquistion

FEI offers a complete range of detection cameras and software to support efficient data recording as part of the single particle analysis workflow. With the ability to efficiently detect low-contrast signals, scientists can now capture image information with high sensitivity for higher resolution images.

Products & Solutions

FEI EPU Automated SPA Software

As part of single particle analysis workflow, FEI offers powerful EPU automated acquisition software for data collection. FEI EPU software streamlines the acquisition of large data sets, using thousands or tens of thousands of nominally identical particles. After conformational classification and particle averaging, the result is a high-resolution 3D representation.

FEI Phase Plate

Vitrified samples typically exhibit low intrinsic contrast and require low-dose imaging techniques. FEI Phase Plate achieves a significantly improved contrast at low spatial frequencies, revealing greater levels of detail, as shown in the example to the right. With higher image contrast, each tilt image can be recorded at a lower electron dose with less damage to the specimen.

FEI Falcon Direct Electron Detector

The Falcon 3EC is the first direct electron detector to benefit from our next-generation image processing pipeline. It is seamlessly connected to a dedicated fast storage server, which makes waiting for frames to be stored a thing of the past. Images, including individual frames or dose fractions, are processed on-the-fly and stored on the storage server. The increased frame rate, further reduced noise levels, and powerful imaging pipeline enable electron counting and even on-the-fly drift correction, providing the highest quality data in the shortest amount of time.

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Step four: Single Particle Reconstruction

Our goal is to improve the rate of data collection and through ease of use, get better structures leading to better publications-ultimately leading to breakthrough discoveries. Publications are key here. They are essentially the scientific currency. Accurate, detailed, three-dimensional models of intricate biological structures at the sub cellular and molecular scale are key to developing these publications.

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三聚体 HIV-1 包膜糖蛋白活化的结构机理

HIV-1 感染首先从三聚体病毒包膜糖蛋白 (Env) 与 CD4 和目标 T 细胞的共受体相结合开始。了解这些配合基如何影响 Env 的结构是研制 HIV 疫苗的根本。利用冷冻电子显微镜,我们描述了三聚体 Env 与下列物质相结合时产生的迥然不同的结构:可溶的 CD4;具有广泛中和作用、结合了 CD4 的部位抗体 VRC01、VRC03 和 b12;或模仿共受体的单克隆抗体17b。

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Protein Data Bank Structures

(Click on images for more details)
  • CryoEM Helical Reconstruction of TMV
    Author: Ge, P. et al.
    Taken on a FEI TITAN KRIOS at 3.3 Å resolution
  • Life in the extremes: atomic structure of Sulfolobus Turreted Icosahedral Virus
    Author: Veesler, D. et al.
    Taken on a FEI TITAN KRIOS at 4.5 Å resolution
  • Cryo-EM structure of Dengue virus serotype 3 at 28 degrees C
    Author: Fibriansah, G. et al.
    Taken on a FEI TITAN KRIOS at 6.0 Å resolution
  • Structure of beta-galactosidase at 3.2-A resolution obtained by cryo-electron microscopy
    Author: Bartesaghi, A. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • CryoEM single particle reconstruction of anthrax toxin protective antigen pore at 2.9 Angstrom resolution
    Author: Jiang, J. et al.
    Taken on a FEI TITAN KRIOS at 2.9 Å resolution
  • Atomic structure of a non-enveloped virus reveals pH sensors for a coordinated process of cell entry
    Author: Zhang, X. et al.
    Taken on a FEI TITAN KRIOS at 3.3 Å resolution
  • Structure of alpha-1 glycine receptor by single particle electron cryo-microscopy, strychnine-bound state
    Author: Du, J. et al.
    Taken on a FEI TITAN KRIOS at 3.9 Å resolution
  • Electron cryo-microscopy of the IST1-CHMP1B ESCRT-III copolymer
    Author: McCullough, J. et al.
    Taken on a FEI TITAN KRIOS at 4.0 Å resolution
  • Structure of Escherichia coli EF4 in posttranslocational ribosomes (Post EF4)
    Author: Zhang, D. et al.
    Taken on a FEI TITAN KRIOS at 3.700 Å resolution
  • Cryo-EM structure of the magnesium channel CorA in the closed symmetric magnesium-bound state
    Author: Matthies, D. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Structure of the yeast 60S ribosomal subunit in complex with Arx1, Alb1 and C-terminally tagged Rei1
    Author: Greber, B.J. et al.
    Taken on a FEI TITAN KRIOS at 3.410 Å resolution
  • Structure of a Chaperone-Usher pilus reveals the molecular basis of rod uncoilin
    Author: Hospenthal, M.K. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Structure of F-ATPase from Pichia angusta, state1
    Author: Vinothkumar, K.R. et al.
    Taken on a FEI TITAN KRIOS at 7 Å resolution
  • Cryo-EM structure of a BG505 Env-sCD4-17b-8ANC195 complex
    Author: Wang, H. et al.
    Taken on a FEI TITAN KRIOS at 8.9 Å resolution
  • Ca2+ bound aplysia Slo1
    Author: MacKinnon, R. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Volta phase plate cryo-electron microscopy structure of a calcitonin receptor-heterotrimeric Gs protein complex
    Author: Liang, Y.L. et al.
    Taken on a FEI TITAN KRIOS at 4.1 Å resolution
  • CryoEM structure of type II secretion system secretin GspD in E.coli K12
    Author: Yan, Z. et al.
    Taken on a FEI TITAN KRIOS at 3.04 Å resolution
  • Structure of a Pancreatic ATP-sensitive Potassium Channel
    Author: Li, N. et al.
    Taken on a FEI TITAN KRIOS at 5.6 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, three-fold symmetry
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Prefusion structure of MERS-CoV spike glycoprotein, conformation 2
    Author: Yuan, Y. et al.
    Taken on a FEI TITAN KRIOS at 3.2 Å resolution
  • Structure of SARS-CoV spike glycoprotein
    Author: Gui, M. et al.
    Taken on a FEI TITAN KRIOS at 3.8 Å resolution
  • Products for 单颗粒分析

    Titan Krios 生命科学应用
    对于蛋白质结构和蛋白复合物的二维和三维表征来说,Titan Krios 是功能最强大、灵活性最高的高分辨率电子显微镜。凭借自动样品装载和可靠的无人值守操作,Titan Krios 可实现无与伦比的工作效率水平。
    Talos L120C TEM for Life Sciences
    An ideal entry-level solution for imaging and tomography that can be configured as a basic cryo-TEM imaging platform. Fully upgradeable, the Talos L120C TEM will meet your needs, whether those needs are in Cryo or Room Temperature or 2D imaging or 3D imaging and multi-modality imaging experiments.
    Talos Arctica 生命科学应用
    Talos™ Arctica 是一款 200 kV FEG 透射和扫描电子显微镜 (STEM)。它是一款强大、稳定的多功能系统,可在细胞生物学、结构生物学和纳米技术研究中提供生物和生物材料样品的高分辨率三维表征。Talos 使科学家能够快速、更好地洞察和了解三维大分子结构、细胞成分、细胞和组织。
    Talos 的生命科学应用
    TalosTM 是一款 200 kV S/TEM,旨在对纳米材料进行快速、准确、量化的多维表征。它基于更高的数据质量、更快的采集速度以及简化、轻松且自动化的操作,可以加速材料纳米分析。
    用于生命科学的 Vitrobot
    Vitrobot 可完全自动执行玻璃化流程,令样本制备快速、轻松且可重复,样本制备是获得高质量图像和可重复实验结果的第一步。
    EPU 的生命科学应用
    为单颗粒分析带来强大的自动化与可靠性,实现有效的冷冻透射电镜工作流。

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