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9Å structure of soluble gp140 HIV envelope trimers bound to 17b neutralizing FAB fragment. Courtesy of S. Subramaniam, Laboratory of Cell Biology, Center for Cancer Research, National Cancer Institute, NIH, Bethesda.

Cryo-TEM Advantages

How Transmission Electron Microscopy complements protein X-ray crystallography and NMR

Protein molecules carry out the majority of functions within cells and most of these activities involve the interaction of multiple proteins and other macromolecules.  The determination of the structure of a single protein using protein X-ray crystallography or NMR has become easier over the past twenty years.  However, solving the structure of a protein complex is still very challenging.  Getting a protein complex to crystallize is not always possible and the large size of many complexes makes them difficult to study with NMR. 

TEM has the ability to determine the structures of these macromolecular complexes.  TEM works best for complexes that are 250kDa or larger which complements protein X-ray crystallography and NMR studies of individual proteins or domains. In recent years, through significant technical advances in electron microscope design and electron detection technology, the resolutions achievable using cryo-TEM have improved and several protein structures have been solved at better than 3.5Å resolution. The combination of protein X-ray crystallography, NMR and TEM offer the ability to not only resolve smaller proteins at high resolution but to also examine entire proteins complexes as one large macromolecular structure with structural details of some components and molecular modeling enabling the creation a complete atomic model.  Cryo-TEM can also study heterogeneous samples and provide structural details about dynamic and complexes that are difficult to examine with other structural biology techniques.

How TEM can help with challenges in structural biology

TEM is a powerful tool that provides direct images of macromolecules and can help with many structural biology projects.  TEM can provide a big picture view of larger complexes and provide information on the stability and dynamics of a macromolecular complex.  When a complex or protein cannot be crystallized, TEM can often provide a structure, sometimes at near atomic resolution.  Even when a protein complex may be able to be crystallized, TEM can provide an initial 3D model of the structure that may help in determining higher resolution structures.  TEM provides valuable structural information that complements data from other structural biology techniques to help create a more complete picture of macromolecular complexes and help in the generation or mechanistic models to describe their functions.

 

Main advantages of TEM:

  • No need for crystals
  • No upper limit to the size or complexity of the macromolecular complex
  • Near atomic resolutions are possible with the latest microscopes and detectors
  • Heterogeneous samples can be analyzed allowing dynamic and unstable complexes to be studied
  • Only micrograms of proteins are required for analysis
  • Cryo-TEM visualizes macromolecules in a fully hydrated, close to native state

 

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How TEM is used in Structural Biology

In structural biology, TEM is a technique where two dimensional images of individual macromolecular complexes are taken with a transmission electron microscope.  These two dimensional images can be mathematically aligned, through image processing techniques, to generate a 3D volume of the macromolecules.  The samples are typically either encased in a heavy metal stain (negative stain), such as uranyl acetate or imaged at cryogenic temperatures with the sample embedded in vitreous ice, free of strain (Cryo-TEM) .  In order to prepare proteins for cryo-TEM, the specimen is applied to a carbon coated EM grid with a series of small holes (µm size range).  The sample is blotted away, leaving a thin film with the specimen residing within the small holes.  The grid is then rapidly plunged into liquid ethane or propane, cooled to liquid nitrogen temperatures.  The specimen must then be maintained at cryogenic temperatures in order to prevent a phase transition, which would result in formation of crystalline ice and damage to the specimen.  The resulting sample is subsequently imaged in an electron microscope, and reconstruction software is used to create the 3D structure from the individual particle images. Although individual particles are imaged at low contrast, the resulting structure can be extremely high resolution due to averaging of hundreds or thousands of particles.

A field of maturing capsids of herpes simplex virus imaged on a FEI Titan Krios using the Falcon 2 Direct Electron Detector. The capsids are 1250 Å in diameter and have a mass of > 200 megadaltons. Courtesy of Anastasia Aksyuk, William Newcomb, and Alasdair Steven, Laboratory of Structural Biology Research, National Institute of Arthritis and Musculoskeletal and Skin Diseases, USA.

Main application in Cryo-TEM: Single Particle Analysis and Tomography

Single particle analysis (SPA) and tomography can both be performed on the same microscope and they provide complementary information. SPA can deliver high-resolution images of a molecular complex, but lacks biological context. Electron tomography can provide contextual information but has limited resolution of individual particles.

Electron tomography is a well-established technique for recording images at various tilts and subsequent back-projection into a 3D reconstruction (Video 1).  The sample can be a purified macromolecular complex, a whole virus, or even an entire cell.

Single particle analysis (SPA) is another way to create 3D images. With this technique data from a large number of two dimensional projection images, featuring identical copies of a macromolecule in different orientations, are combined to generate a3D reconstruction of the structure (Video 2). 

 

Animations courtesy of Max Planck Institute of Biochemistry, Martinsried, Germany

Video 1: Single Particle Analysis.  A solution of purified protein is vitrified and subsequently imaged in the electron microscope.   Data from 1000s of particles are collected and then classified and averaged resulting in 3D reconstruction of the macromolecule. 
Video 2: In a tomography experiment, back projections of individual 2D images taken at different tilt angles are reconstructed and averaged to provide a 3D structure of the sample. 

Documents

Towards an integrative structural biology approach: combining Cryo-TEM, X-ray crystallography, and NMR

Cryo-transmission electron microscopy (CryoTEM) and particularly single particle analysis is rapidly becoming the premier method for determining the three dimensional structure of protein complexes, and viruses. In the last several years there have been dramatic technological improvements in Cryo-TEM, such as advancements in automation and use of improved detectors, as well as improved image processing techniques. While Cryo-TEM was once thought of as a low resolution structural technique, the method is currently capable of generating nearly atomic resolution structures on a routine basis. Moreover, the combination of Cryo-TEM and other methods such as X-ray crystallography, nuclear magnetic resonance spectroscopy, and molecular dynamics modeling are allowing researchers to address scientific questions previously thought intractable. Future technological developments are widely believed to further enhance the method and it is not inconceivable that Cryo-TEM could become as routine as X-ray crystallography for protein structure determination.

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Structural Mechanism of Trimeric HIV-1 Envelope Glycoprotein Activation

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Titan Krios - Published Article List

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Subnanometre-resolution structure of the intact Thermus thermophilus H1-driven ATP synthase

Products for Cryo-TEM Advantages

Quanta SEM for Materials Science
The Quanta™ 50 SEM series is the third generation Quanta system built on the success of previous generations of ESEM™ Schottky FEG. This series has an easy-to-use and flexible user interface with functions to maximize productivity and allow all the data to be collected. Designed by microscopists for microscopists, this instrument series is truly above and beyond 'easy to use'.
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 F200C TEM for Life Sciences
The Thermo Scientific™ Talos™ is a 200kV S/TEM designed for fast, precise and quantitative characterization of nanomaterials in multiple dimensions. It accelerates materials nanoanalysis based on higher data quality, faster acquisition, simplified, easy and automated operation.
Helios G4 CX DualBeam for Materials Science
The latest technological innovations of the Thermo Scientific™ Helios G4 CX DualBeam™ microscope, in combination with the easiest to use, most comprehensive software and our application expertise, allow Helios G4 CX with optional AS&V4 software for the highest-quality, fully automated acquisition of multi-modal 3D datasets.
Helios G4 UX DualBeam for Materials Science
The latest technological innovations of the Thermo Scientific™ Helios G4 DualBeam™ microscope, in combination with the easiest to use, most comprehensive software and our application expertise, allow for the fastest and easiest preparation of site-specific, ultra-thin HR-S/TEM samples for a wide range of materials.

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