Phoebe Stewart

Structure of macromolecular assemblies by cryo-electron microscopy and three-dimensional image processing

Cryo-electron microscopy (cryo-EM) combined with computer image reconstruction is a powerful way to visualize the structure of large macromolecular assemblies. The technique involves freezing a droplet of sample in a cryogen, which preserves the sample in an amorphous ice layer on an EM sample grid. The grid is kept frozen (at liquid nitrogen or helium temperatures) while it is imaged in an electron microscope and digital images are collected. Image analysis software is then used to synthesize images of thousands of particles into a detailed three-dimensional structure. Atomic resolution structures of component molecules or domains can often be mapped into the cryo-EM density. This approach has been successfully applied to icosahedral viruses as well as symmetric and asymmetric protein assemblies (>200 kDa). Recent advances in liquid helium cryo-microscopy, automation of data acquisition, and image processing on multi-processor computers, offer the potential of reaching near atomic resolution (3-5 Angstroms) by cryo-EM methods.

Ongoing and new projects include:

  1. Adenovirus, adenovirus-receptor and antibody complexes, engineered adenovirus vectors
  2. The DNA-dependent protein kinase, DNA-PK, an enzyme involved in DNA double-strand break repair and immunoglobulin gene rearrangement
  3. The vault particle, a ubiquitous cytoplasmic RNA-protein complex implicated in cancer cell multi-drug resistance

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