3D Protein Structure Determination by CryoEM

3D protein structure determination by cryoEM takes advantage of being able to trap a large number of protein particles in many different orientations in a thin layer of vitreous ice. Images are recorded and a method called single particle reconstruction is used to compute a 3D map of the particles. The particles are first identified in the images, aligned relative to each other and averaged. The averages represent 2D projection views of the particles and many different views are combined computationally to generate a 3D structure. Recent improvements in electron microscopes, detectors and computational algorithms now allow determination of protein structures at high resolution.

 

Titan Krios

At NanoImaging Services, a Titan Krios electron microscope, equipped with an energy filter, phase plate and K2 direct detector, allows determination of protein structures at near atomic to atomic resolution.

 


3D structure of Apoferritin reconstructed to a resolution of 2.4.

Detail of the 2.4 Apoferritin map.

 

These analyses help answer questions like:

  • Protein Function
    How does the arrangement of atoms in the protein explain its function?
  • Morphology
    What is the shape and relative size of the protein? Does it have symmetry?
  • Conformational States
    Does the protein exhibit different conformations or does it undergo conformational changes in different environments? Does the protein have flexible domains?
  • Particle Interactions
    How does the protein interact with ligands, e.g. a small drug or Fab fragment? Are these interactions affecting its structure?