Computational techniques for single-particle averaging and reconstruction of molecules imaged in the electron microscope have been developed in my lab since 1976. The advent of the frozen-hydrated sample preparation method (cryogenic electron microscopy, or cryo-EM) in the 1980s made it possible, with the help of these computational techniques, to obtain 3D images of asymmetric molecules in their native state without the need for crystallization. This is done by combining tens of thousands of images showing different views of the macromolecule that exists in as many “copies” in the sample. In 1998 such a map, obtained by my group from a sample of the Haloarcula marismortui ribosome, was used by Thomas Steitz (HHMI, Yale University) in the phasing of his X-ray data, which two years later resulted in the first atomic-level structure of a bacterial 50S subunit.
The only hurdle preventing achievement of near-atomic resolution in single-particle cryo-EM has been the inferior quality of the recording media, film or CCD cameras. Due to this problem, the best resolution of molecules lacking symmetry, obtained for data recorded on film, was in the range of 5Å (ribosome of Trypanosoma brucei; Hashem et al., Nature 2013). This resolution hurdle was recently overcome with the introduction, around 2012, of the first commercial direct electron detectors. Since then several groups, including ours, have obtained density maps in the 2.2 - 3.5 Å range which allow atomic structures to be built by tracing the chains and identifying signatures of residues along the way.
Through the use of maximum-likelihood classification, single-particle cryo-EM makes it possible to analyze the motions and dynamic changes of "molecular machines" (assemblies of dynamically interacting molecules that perform one of the cell's functions, such as transcription or translation) during their functional cycle. All functionally relevant states of the molecule equilibrating in the sample can be imaged at the same time, provided they are present with sufficient occupancy.
The main focus of research in my group is translation, the process by which the genetic message carried by mRNA is translated into a polypeptide chain, which then folds into a working protein. In these studies we address the structural basis of translation in both eubacteria and eukaryotes. In the past we have characterized numerous functionally relevant states during the elongation cycle, allowing domain motions and binding states of elongation factors and tRNA to be described. We have also studied key processes during translation initiation, termination and recycling. The recent aforementioned advances in recording media have now enabled us to look at mechanisms of translation that are not fully understood with near-atomic resolution. An example is provided by the interaction between an EF-G mutant (H94A) and the 70S E. coli ribosome immediately after peptidyl-transfer (Figure 1), a study in collaboration with Dr. Suparna Sanyal at Uppsala University. We discovered that EF-G is able to engage the ribosome in its two “macrostates”, related to each other by intersubunit rotation. Since only one of these states leads to GTPase activation on EF-G, the modeling of the density maps in the two states enabled us to formulate the necessary and sufficient conditions for GTPase activation and translocation in structural terms (Li et al., Science Advances 2015).
The best resolution we have obtained thus far has been 2.5Å, for the 60S subunit of the ribosome from T. cruzi (3.4Å for its small subunit, on account of high conformational variability). At this resolution, we are able to model the atomic structure directly from the density map and characterize features unique to trypanosomes, such as the way the 28S rRNA is re-joint from its 6 individually processed pieces.
The standard cryo-EM experiment involves blotting of excess sample off the grid, to its required thickness of ~1000Å. The necessary time (several seconds) for this sample preparation step prohibits investigation of reaction intermediates in a pre-equilibrium system. We have now implemented and further advanced a mixing-spraying device, initially developed at the Wadsworth Center and Rensselaer Polytechnic Institute, which facilitates time-resolved studies (10 – 1000 ms range) of a reaction started by mixing two components. Following a successful proof-of-concept study of ribosomal subunit association (Chen et al., Structure 2015), we have just completed a study of short-lived intermediates during ribosome recycling, in collaboration with Dr. Mans Ehrenberg of Uppsala University. Planned experiments with this device will focus on the mechanism of translation initiation (in collaboration with Dr. Ruben Gonzalez) and decoding (with Dr. Mans Ehrenberg).
Finally, in collaboration with Drs. Andrew Marks and Wayne Hendrickson at Columbia University, our lab has recently been successful in visualizing the calcium release channel (Ryanodine receptor) bound with different agonists. The resolution of these maps (up to 3.6Å) has been sufficient to build atomic models for closed and open states, and to develop a model for the mechanism of channel activation and gating. Incidentally, the first reconstruction of the Ryanodine receptor was done in my lab in 1989, in collaboration with Dr. Sidney Fleischer of Vanderbilt University, at a resolution of 37Å (Wagenknecht et al., Nature 1989). Comparison of these two maps with ten-fold difference in resolution gives us a sense of how far this field has advanced in the intervening time.
This work is being supported, in part, by grants from the National Institutes of Health.
As of March 11, 2016