Courtesy: Robert L. Duda (a), Matthijn Vos (b) and James F. Conway (a); a. University of Pittsburgh; b. FEI Company

Structural Biology

Revealing 3D macromolecular complexes

Structural biology explores the functional mechanistics of molecular structures such as proteins, protein complexes and protein-ribonucleic acid associations - the fundamental building blocks of life. 

Scientists seek to understand how these structures are designed and how structural variations affect functionality. Structural biology research is essential to understanding the origins of events such as pathogen-induced and age related diseases, allergic hypersensitivities, and cell growth and differentiation.

Today, many structural biologists are interested in finding innovative ways to intervene in disease processes and find new preventive measures, treatments, and pharmaceutical agents. This requires the in vivo exploration of the molecular mechanisms of diseases, to unambiguously determine the conformation of flexible protein structures in their natural biological context. Understanding these activities will unravel the fundamentals of life and may consequently lead towards wide scale pharmaceutical solutions for treating fundamental diseases and disorders.

Challenges in revealing 3D macromolecular protein complexes

Until recently, scientists have had to engineer and crystalize proteins in order to reconstruct and visualize them via X-ray crystallography. Besides the fact that crystallization is a time-consuming process - which may not always be successful as some proteins do not crystallize - the technology is also mainly applicable to crystallization of monomeric and dimeric structures only. Although large protein complexes provide more insight to native function, it has been difficult to stabilize and crystallize these structures.

 Cryo-TEM advantages:

  • Resolves protein structures from samples that are difficult to crystallize
  • Analyze challenging, heterogeneous samples and antibodies

How cryo-TEM complements X-ray crystallography and NMR

Courtesy: Sriram  Subramaniam, Lab of Cell Biology, National Cancer Institute, National Institutes of Health

Better understanding of molecular functions, faster and reliably

FEI's Cryo-TEM workflow methodology provides solutions to the scientific challenges of today's scientific community. This highly automated workflow delivers the highest throughput at the best possible data quality. The FEI workflow is unique as it connects the best optical platform and detector technology with dedicated software solutions and newly developed sample preparation methods.

In summary, a tiny droplet of purified protein solution or cell suspension is placed on an EM grid and vitrified with the FEI Vitrobot. Subsequently, this sample is imaged using a transmission electron microscope like the Titan Krios, Tecnai Arctica or Talos and the resulting signal is detected using Falcon direct electron detection.

In Single Particle Analysis (SPA), automated image acquisition software EPU acquires the data which is processed using dedicated reconstruction software. The result is a high resolution 3D image of the viral particle or protein (complex).

Tomography is used to obtain ultra-structural 3D information from cells and cell organelles. The vitrified sample is imaged under various alpha tilts and the resulting data is subsequently processed to obtain the original 3D information.

By optimally linking high resolution SPA data into their natural biological context by using template matching software ARGOS, it is possible to localize the object of interest in its 3D context and to define the real orientation of the high resolution object. This adds an additional dimension which provides a better understanding of the functioning of molecular complexes, a cell or cell component. 

Achieving breakthrough results using cryo electron microscopy

Prof. Holger Stark, a leading scientist within the field of structural biology has adopted the FEI workflow to resolve macromolecular structures. Watch this video to explore the capabilities of cryo electron microscopy today and how the study proteins and macromolecular complexes in their native state has led to breakthrough results.


Article - Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation

HIV-1 infection begins with the binding of trimeric viral envelope glycoproteins (Env) to CD4 and a co-receptor on target Tcells. Understanding how these ligands influence the structure of Env is of fundamental interest for HIV vaccine development. Using cryo-electron microscopy, we describe the contrasting structural outcomes of trimeric Env binding to soluble CD4, to the broadly neutralizing, CD4-binding site antibodies VRC01, VRC03 and b12, or to the monoclonal antibody 17b, a co-receptor mimic.

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Products for Structural Biology

Titan Krios
The Titan Krios is the most powerful and flexible high resolution electron microscope for 2D and 3D characterization of protein structures and protein complexes. With automated sample loading and reliable unattended operation, Titan Krios achieves a level of productivity that is unmatched.
Tecnai Arctica
The next generation automated, high resolution imaging TEM platform - Tecnai Arctica provides a platform which combines excellent optical performance and thermal and mechanical stability with high sample throughput.
Falcon II Direct Electron Detector
The Falcon is based upon direct electron detection that enables the acquisition of low-noise images of delicate biological samples and other beam-sensitive materials that require low electron dose interactions to prevent radiation damage of the material. Serving applications like cryo-tomography and single particle analysis, Falcon is designed for optimized image detection of those beam-sensitive specimens that require extreme low-dose conditions. Improved spatial resolution and superior image quality are now available at almost no electron dose.
Vitrobot completely automates the vitrification process to provide fast, easy, reproducible sample preparation - the first step in obtaining high quality images and repeatable experimental results.
EPU - Automated Single Particles Acquisition Software
Effective cryo-TEM workflows require automation in order to achieve optimal results and high thoughput. EPU enables effective workflows as it facilitates the process of optimal area selection – the first crucial step of the total single particle analysis workflow.

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