Tundra Cryo TEM Applications

With the Thermo Scientific Tundra Cryo-TEM, configured with the Thermo Scientific Falcon C Direct Electron Detector, you can visually resolve protein structures and produce 3D reconstructions down to 2.1 Å. The Tundra Cryo TEM can also be utilized for negative-stain electron microscopy, an easy and cost-effective method for the quality assessment of purified biological specimens at room temperature. In addition, the Tundra Cryo-TEM can visualize sections of resin-embedded cells and tissues or isolated particles of protein complexes and viral assemblies.

Single particle cryo-EM analysis

The 100 keV Tundra Cryo-TEM can efficiently validate ligand and protein binding when in fast mode, taking over 10,000 images in as little as a few days’ acquisition time. With the Tundra Cryo-TEM, you can take a closer look at receptors that affect the central nervous system or investigate prokaryotic ribosomes. From Nobel –prize-winning discoveries, like TRPV5, to small potential drug targets, like GABAA receptors, the Tundra Cryo-TEM can help expand your scientific discovery by visually resolving structures and producing 3D reconstructions at ~2–5 Å resolution. When the Tundra Cryo-TEM is configured with a Falcon C Direct Electron Detector, the resolution can be improved, particularly for small and asymmetric protein complexes such as transthyretin (55 kDa). 

 

Structure of transthyretin (55 kDa) at 3.5 Å, highlighting the ligand binding pocket reconstructed with data from Tundra Cryo-TEM with Falcon C Direct Electron Detector.

Molecular structures determined using the Tundra Cryo-TEM

2.1 Å, Apoferritin (~500 kDa)

  • Number of images: 5,418
  • Number of particles: 249,808 (octahedral)
  • Acquisition time: 18 hrs
  • Detector: Falcon C

2.7 Å, T20S Proteasome (~700 kDa)

  • Number of images: 11,819
  • Number of particles: 184,727 (D7)
  • Acquisition time: 38 hrs
  • Detector: Falcon C

3.5 Å, Transthyretin (55 kDa)

  • Number of images: 10,137
  • Number of particles: 131,895
  • Acquisition time: 38hrs
  • Detector: Falcon C

5 Å, Hemoglobin (64 kDa)

  • Number of images: 5,399
  • Number of particles: 60,381 (D2)
  • Acquisition time: 42 hrs
  • Detector: Falcon C

4.3 Å, CAK complex (84 kDa)

  • Number of images: 5,034
  • Number of particles: 67,846 (C1)
  • Acquisition time: 40 hrs
  • Detector: Falcon C

2.9 Å, Aldolase (160 kDA)

  • Number of images: 10,400
  • Number of particles: 143,939 (D2)
  • Acquisition time: 38 hrs
  • Detector: Falcon C

     

3.5 Å, 70S Ribosome (~2.5 MDa)

  • Number of images: 21,552
  • Number of particles: 150,000 (C1)
  • Acquisition time: 37 hrs
  • Detector: Ceta-F CMOS

 

3.0 Å, AAV6 (~3.7 MDa)

  • Number of images: 5,058
  • Number of particles: 46,646 (Icosahedral)
  • Acquisition time: 17 hrs
  • Detector: Ceta-F CMOS
     

4.2 Å, TRPV5 (~330 kDa)

  • Number of images: 10,876
  • Number of particles: 184,800 (C4)
  • Acquisition time: 30 hrs
  • Detector: Ceta-F CMOS

Drug discovery with the Tundra Cryo-TEM

Cryo-electron microscopy (cryo-EM) is rapidly drawing interest in structure-based drug discovery and design, since it can accurately and rapidly visualize to high resolutions the interactions between drug and receptor of a multitude of samples. Here we show an example of a human CDK-activating kinase (CAK), a challenging protein for traditional structural biology methods. Structures determined on the Tundra Cryo-TEM provide insight into inhibitor interactions and the basis for rational design of next-generation therapeutics.

Reconstruction map of human CDK-activating kinase

The reconstruction map of the human CDK-activating kinase (CAK) was obtained at 4.3 Å resolution using the Tundra Cryo-TEM and Falcon C Direct Electron Detector. The structural insights at this resolution allowed researchers to determine how large ligands bind to the protein: a promising target for cancer therapeutics. The Tundra Cryo-TEM generated structure shows distinct CAK subunits: Cyclin H brown, MAT1 orange, CDK7 grey, AMP-PNP (an inhibitor of CAK) purple.

 

Sample courtesy of Basil Greber, Institute of Cancer Research, London

Cryo-TEM sample optimization and connectivity

The Tundra Cryo-TEM can be used to optimize samples, offering efficient sample iteration and optimization. It serves as a valuable screening tool, providing biochemical sample optimization in less time than higher-level cryo-EM instruments. In the example of CAK complex, when using Krios G4 Cryo-TEM, higher resolution reconstruction can be achieved to reveal more structural information.

Rapid in-house sample-optimization of cryo-EM workflow shows how the Tundra Cryo-TEM is used to determine the right criteria for sample preparation to generate a high-resolution structure.

Room temperature TEM applications

The Tundra Cryo-TEM can be utilized for negative-stain electron microscopy, an easy and cost-effective method for the quality assessment of purified biological specimens at room temperature to quickly assess the stability, homogeneity, and concentration of purified samples. In addition, room temperature sections of resin-embedded cells and tissues or isolated particles of protein complexes and viral assemblies can also be visualized with the Tundra Cryo-TEM.

T20S proteasome stained with uranyl formate (2%) and visualized with the Tundra Cryo-TEM.

Sample courtesy of New York Structural Biology Institute. 

Resin-embedded tobacco leaf visualized with the Tundra Cryo-TEM.

Sample courtesy of Sarah Powers, Doug Allen Lab, Janithri Wickramanayake, and Kirk Czymmek, Advanced BioImaging Laboratory, Donald Danforth Plant Science Center.

For Research Use Only. Not for use in diagnostic procedures.