Services

Services

Tecnai Spirit user

The core is committed to transmission electron microscopy of samples larger than 250 KDa, with the goal of image reconstruction at better than 1nm resolution. The facility's three electron microscopes provide a progression of capability, from the Tecnai Spirit for initial screening of negative-stained samples, to the Tecnai F20 for preliminary electron cryomicroscopy, to the advanced Titan Krios, which is dedicated to automated data collection for high-resolution analysis.

The primary techniques used here are:

Electron Cryo-Microscopy (Cryo-EM)

  • unstained, unfixed samples
  • single particles from solutions with potential resolution < 1nm
  • helical and two-dimensional crystals
  • some atomic models rival X-ray crystallography and NMR

Electron Cryo-Tomography (CET)

  • 3-D maps of non-regular structures, larger cellular complexes and cell sections
  • uses tilt series to generate 3-D structure
  • resolution 3-4 nm for irregular structures, higher for regular structures

Micro Electron Diffraction (MicroED)

  • full diffraction data on three dimensional micro crystals
  • may get data from 2 µm crystals too small for X-ray crystallography
  • potential for atomic resolution

 

At this time, the MEMC is not providing data analysis services. However, some guidance and suggestions are available.

Freezing sampleUsing the facilities

Trained researchers can use microscopes themselves. Alternatively, MEMC personnel can assist researchers in collecting images.

Please contact Kelly Dryden (kdryden@virginia.edu) to discuss how best to use the facility to aid your project. A project application will be required, at which time access to the scheduling system will be provided.

Clients will need to provide a 1Tb-4Tb USB storage drive for multi-day data collection on the Titan Krios. Images collected on the Spirit or F20 will accessible by download from a remote server.

Sample preparation

Researchers can freeze samples manually in liquid ethane or using an FEI Vitrobot. Several options are also available for negative-staining.

Typical sample requirements are 3 µL of at concentrations of 0.1-1.0 mg/ml.

References

American Laboratory recently published an introduction to cryo electron microscopy for structure determination. Some use this technique to study proteins in membranes.

University of Virginia work

Unified polymerization mechanism for the assembly of ASC-dependent inflammasomes. Lu A, Magupalli VG, Ruan J, Yin Q, Atianand MK, Vos MR, Schröder GF, Fitzgerald KA, Wu H, Egelman EH. Cell. 2014 Mar 13;156(6):1193-206. doi: 10.1016/j.cell.2014.02.008