There was a time in structural biology when solving protein structures using NMR was received with considerable skepticism. In addition to the normal experimental uncertainty, the technique generated structures with additional uncertainty due to the vibrational motions of proteins in solution. That’s part of the reason standard NMR entries in the PDB contain ~20 structures while x-ray structures have just one. However modern NMR methods have advanced to the point that few skeptics are left. The two techniques together were essential in the rapid increase of structural information that’s available today.
According to Dr. Wah Chiu, electron cryomicroscopy today looks a lot like NMR 20 years ago. On his first slide he showed was a definition of Cryo-EM, which looked a lot like a definition of NMR. In bold he emphasized that Cryo-EM is solving structures without crystals. I’ve often heard protein crystallization called ‘black art’ or ‘trying to hold a stack of bowling balls together with tape’. I’m not a practitioner so I’ll assume it’s hard for at least the interesting cases. Getting good crystals is not required but sample preparation rules still apply to Cryo-EM. Wah stresses how diligent and often labor intensive work at the bench yields much better results further along in the pipeline. Once they have a sample though, Wah has a playground full of high-end instruments…
JEOL 3200FSC electron cryomicroscope
1,000-core Linux cluster
images via [http://ncmi.bcm.tmc.edu]
Cryo-EM techniques have been very successful in determining structures of nanomachines (or macromolecular assemblies if you are frustrated with nano-fications) and looks to continue improving over the next 20 years. Large assemblies like capsids, phages, pores, and channels are all possible with Cryo-EM. The resolution is still quite far away from the < 2 Angstroms typical of good x-ray structures. If I remember correctly, Wah said they are currently achieving around 4.7 Angstrom res. and better depending on the system. But resolution isn’t just a number, it’s all about what you can actually see. And what they can actually see now is things like secondary structure and even side-chains. Complete atomic detail is not very far off.
Software and computational techniques influenced by image processing and protein structure prediction efforts are providing atomic details even sooner than expected. Wah’s group has developed SSEHunter to detect secondary structures from the Cryo-EM data and programs like MODELLER are used to characterize each component [paper].
My expectations are quite high. How long before we can see the entire cell and all of it’s components in atomic detail? 5? 10? 20 years?
Futher reading:
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