Technical Highlight - May 2012
Short description: A new strategy using Fabs allows visualization of small protein molecules by cryoEM.
While single particle electron cryomicrocopy (cryoEM) has had enormous success in recent years in the structure determination of large, highly symmetric protein assemblies (at near atomic resolution) and large, non-symmetric macromolecular complexes (at 4-5 Å resolution), imaging small molecules (<100 kDa) by cryoEM is nearly impossible. Indeed, it is very difficult to obtain resolutions higher than 20 Å for reconstructions from proteins smaller than 200 kDa.
In the past, small proteins embedded in vitreous ice were difficult to visualize in the noisy images created from limited electron doses. Technical improvements, including the use of a field emission electron source and phase plate technology, may overcome this. However, even when the signal-to-noise issue is addressed, there remains the problem of obtaining accurate image alignments from small particles that often lack well-defined features. In addition, model-induced bias is a frequent issue, making a 3D reconstruction difficult to validate.
To tackle these problems, Cheng and colleagues present a new technique that involves the use of monoclonal fragments antigen binding (Fabs). The Fabs increase the effective size of the protein and also provide a distinct, recognizable feature that aids in image alignment. In addition, the Fab could serve as a validation marker for the 3D reconstruction.
Using phage display and hybridoma technologies, the authors isolate several candidate Fabs to complex with five protein examples: HIV-1 integrase (IN), human proprotein convertase subtilisin/kexin type 9 (PCSK9), a bacterial homolog of the mammalian vesicular glutamate transporter (EcoliVGLUT3), and an ATP-binding cassette (ABC) transporter. Key to the approach is the requirement that the Fab bind not only with high-affinity, but also with rigidity, which is why the use of Fabs that recognize a conformational rather than a linear epitope is preferred. 2D class averages were obtained for each Fab-protein complex, revealing that a stable and rigid complex was formed.
The authors first used random conical tilt procedure and projection matching to obtain single particle negative stain EM reconstructions of the Fab-protein complexes to 25–26 Å resolution. 3D reconstructions of HIV-1 IN and PCSK9 were obtained and compared against their known crystal structures. Both reconstructions produced densities that matched well with the shapes of the individual components. They then obtained a 13.3 Å- resolution cryoEM reconstruction of HIV-IN, using the negative stain reconstruction as a reference model. This technique shows the promise of being applicable to many small proteins, including integral membrane proteins.
S. Wu et al. Fabs Enable Single Particle cryoEM Studies of Small Proteins.
Structure. 20, 582-592 (2012). doi:10.1016/j.str.2012.02.017