Structural biology is a branch of molecular biology, biochemistry and biophysics that relates to understanding the structure of macromolecules. In the past, X-ray crystallography was the technique used to produce high-resolution 3D models of macromolecules, but more recently with the technical advances of cryo-TEM the field of single particle analysis (SPA) is now at the forefront of structural biology. This term “particle” is used to refer to the individual biological macromolecules or complexes. Determining structures at 3Å resolution is now almost routine but an increasing number of structures of 2Å or better are reported.

The iterative improvement in electron microscopes and detectors plus more powerful computational tools has led to what has been called the “Resolution Revolution”. In 2017 the Nobel Prize for Chemistry was awarded to Jacques Dubochet, Joachim Frank and Richard Henderson for “Developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution.

Basically, the purified macromolecules are frozen in a thin layer of vitreous ice and then observed in a cryo-TEM. Images of tens of thousands of particles are taken at very low electron dose. The rationale behind this approach is that the particles will lie in different orientations within the vitreous ice. These different orientations will lead to many different shapes being captured on the TEM image as the beam passes through the sample. Via computational analysis, these multiple images are reconstructed into the high-resolution 3D maps of the particle in question.

Rotate the shape to see how the projection changes when illuminated from above. This illustrates how the orientation affects the shape of the particles in the TEM image. Interactive courtesy of Martyn Cook.

Workflow for single particle analysis

Even using ideal imaging parameters, individual electron microscope images do not contain sufficient information to provide an atomic description. To limit radiation damage to the sample the images are collected at low electron doses which are inherently noisy. To work around this, many images are collected and averaged. If identical noisy images can be grouped and averaged, the high-resolution signal in the images adds coherently, while noise is gradually averaged out.

Cryo-EM images of GroEL particles. A. Raw image captured without the use of an energy filter showing how much noise is in the image and how difficult it is to see the actual particles. This is averaged and motion corrected from the original movie. B. A low pass filter has been used on the image in A. to show the particles. Images courtesy of Nick Ariotti, UNSW.

The detail in an image obtained in cryo-TEM is low and the noise is high. By averaging many similar images together the signal is increased and the noise reduced as can be seen here by averaging together 10, 50, 100, 200, 400, 800, 1600 and 3200 images of different particles. Images courtesy of Hari Venugopal, Monash University.

The word ‘resolution’ in cryo-EM refers to how trustworthy various features are in the 3D reconstruction map of the macromolecule, based on the ratio of signal intensity to noise intensity at a particular position.

SPA is based on several initial assumptions.

1. All particles in the specimen have identical structure
2. All particles undergo 3D rigid body transformations (rotations, translations)
3. Particle images are interpreted as the projection of the common structure plus noise.

Structure of apoferritin solved using single particle analysis. Image courtesy of Nick Ariotti, University of New South Wales.