EXPLORING THE DYNAMICS OF SUPRAMOLECULAR MACHINES
WITH CRYO-ELECTRON MICROSCOPY
JOACHIM FRANK
Howard Hughes Medical Institute, Department of Biochemistry and Molecular Biophysics,
650 W. 168th Street, New York, NY 10032, USA
Present state of research
Supramolecular machines perform their work in the cell by going through many different
states, distinguished by different conformations and free-energy levels. Ideally, in order
to find out how these machines work, we would create a suitable in vitro environment
containing all components including energy supply that allows the machine to function.
We would then aim to take a “movie,” capturing their structure at highest resolution in a
continuous fashion. Keeping within that film analogy, we might consider taking a large
number of “snapshots” in equal small time intervals, each short enough, as in the
macroscopic world, to eliminate jarring transitions. However, we would find out that this
project has flaws both on the conceptual and the practical level. Conceptually, it is
incorrect to equate a molecular machine’s progress to the workings of a macroscopic
machine in motion since the states are not ordered in sequence of time but are visited in a
stochastic manner, with occasional irreversible events such as NTP hydrolysis as the only
mark of progress. In practical terms, there are in fact two problems, one affecting the
way data for any given state can be captured, the other affecting the ability to obtain
coverage of states in a continuum.
First of all, the visualization of a structure requires some form of radiation which
imparts energy on the molecule and changes it in the very process. Minimization of these
damaging interactions calls for a radiation dose so low that averaging over a sufficiently
large population is required for visualization. This requirement, in turn, translates into a
complicated way each snapshot must be taken: the structural information in every state
has to be gathered from many different copies of the molecule. While in forming such an
average, crystallographic approaches -- X-ray and electron crystallography -- are able to
take advantage of the regular arrangement of molecules in a crystal, single-particle
electron microscopy must first determine the precise orientation of each molecule from
its projection image [1]. The second hurdle interfering with the idea of making a movie
is that only a limited number of states are sufficiently populated to allow a threedimensional structure to be determined. Thus the “movie frames” in between these states
remain empty, undetermined.
The difference between crystallographic and single-molecule approaches has
opposite consequences for resolution and functional relevance: the high order achievable
in a crystal makes it possible to obtain very high resolution, but the conformational state
that the molecule is trapped in may not be relevant to its function. When, on the other
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hand, the structure is obtained from multiple images of free-standing “single” molecules
as they are engaged in their work, functional relevance is guaranteed (e.g. [2]), yet until
recently atomic resolution has not been achieved, except for viruses and other molecules
with high symmetry [3,4].
However, the introduction, in the past year, of direct-detection cameras [5,6], some
of which have single-electron counting and super-resolution capabilities has radically
changed this situation. Recent accomplishments obtained with the help of such cameras
portray a field in rapid transition, with the ultimate claim to occupy a position in
structural biology matching the one held until now by X-ray crystallography. Threedimensional density maps (reflecting the reconstructed Coulomb potential in a 3D array)
obtained in the 3Å range for particles with high symmetry [7,8] and now even entirely
asymmetric assemblies [9] enable de novo chain tracing and the construction of accurate
atomic models without the aid of fitting existing structures. This of course pertains not
only to the protein parts of a molecular machine, but to nucleic acid components, as well.
Another aspect to be emphasized in single-particle electron microscopy is the ability
to obtain an entire inventory of co-existing states of a macromolecule from a single
sample. Recent development of powerful software using maximum-likelihood methods
has made it possible to extract multiple structures, one for each state, from a
heterogeneous mixture [10]. This capability means that to some extent, with help from
other techniques such as single-molecule FRET, inferences can be drawn on the
dynamics of the supramolecular machine (“Story in a Sample” [11]).
So far I have spoken of a reductionist, in vitro approach by electron microscopy
toward study of supramolecular machines, whose aim is a portrayal of their structure and
dynamics at atomic resolution. Another approach, electron tomography, is the attempt to
visualize the machine within the context of the cell. The progress with this approach in
recent years, with automated tilt data collection having become routine, has been quite
impressive [12]. In rare cases the interesting part of a cell is thin enough to be penetrated
by the electron beam. For thicker samples, high-pressure freezing must be used followed
by sectioning with diamond knife or Focused Ion Beam (FIB) milling. While FIB
milling of a frozen sample requires very specialized equipment, it is becoming the
preferred method of sectioning of biological material as it is virtually free of artifacts
associated with cutting [13, 14].
My lab’s recent research contributions
My lab studies the mechanism of protein biosynthesis in both eubacteria and eukaryotes,
using single-particle cryo-electron microscopy of ribosomes that are in various states of
translation. These states are characterized by different conformations of the ribosome
itself and different binding configurations of mRNA, tRNA, as well as a variety of
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translation factors (in the case of bacteria, EF-G, EF-Tu, etc.). From density maps
reconstructed, such as in ref. [2], atomic models are built by docking and flexible fitting
of structures in the Protein Data Bank. Of special interest to us has been translocation, or
the mechanism by which the ribosome transports mRNA and tRNAs bound to it to the
next codon in each cycle of the polypeptide elongation. We early recognized that this
movement is facilitated by a ratchet-like reorganization of the ribosome [15]. More
recently we discovered [16] that the ribosome at the point after peptide bond formation
exists in multiple conformational states characterized by different intersubunit rotations
and tRNA binding configurations. Thanks to the above-mentioned development of novel
classification software these states could be extracted and independently reconstructed
[17].
Most recent contributions by my group to the field of biosynthesis include the 5-Å
resolution reconstruction of the ribosome from Trypanosoma brucei, a eukaryotic
parasite causing Sleeping Sickness [18], and the determination of the structure of the
mammalian translation pre-initiation complex [19]. At the date of preparation of this
manuscript, the T. brucei ribosome reconstruction represented one of the highestresolution structures of an asymmetric molecule, though still employing the old
technology of recording on film (Fig. 1). Compared to the ribosomes of other
eukaryotes, the T. brucei ribosome possesses unusual features, such as its large RNA
expansion segments which may be docking platforms for T. brucei-specific protein
factors, possibly associated with the need of the parasite to rapidly adapt to hosts with
widely different body temperatures.
Fig. 1. Cryo-EM density map of the ribosome from Trypanosoma brucei at 5Å resolution. Left: the ribosome
(grey) viewed from the solvent side of the small subunit; right: viewed from the solvent side of the large
subunit. Ribosomal RNA expansion segments are rendered in different colors.
The visualization of the mammalian 43S pre-initiation complex presents an example
for a highly heterogeneous specimen that could nevertheless be characterized by a series
of reconstructions which depict different combinations of factors attached to the small
ribosomal subunit. Of these, only one representing 4.4% of the whole dataset contained
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the initiator tRNA and most factors that are important in setting up the 43S initiation
complex and the staging for the scanning of the mRNA for the start codon (Fig. 2).
Fig. 2. 43S translation pre-initiation complex at 11.6Å resolution, reconstructed from a subpopulation of 29,000
cryo-EM particle images. The components of this supramolecular complex are: 40S ribosomal subunit (yellow),
eIF3 (red), DHX29 (green), initiator tRNA (yellow-orange), and eIF2 (orange, attached to initiator tRNA).
Outlook to future developments of research
As noted in the introduction, the new generation of detectors employed in the
transmission electron microscope is currently revolutionizing cryo-electron microscopy
as a tool in structural research, in particular in the study of supramolecular complexes.
Due the gain in contrast and resolution achievable, it will soon be possible to reconstruct
supramolecular machines in their entirety – proteins along with the nucleic acid
components -- in multiple, functionally relevant states. The novel technology has a
bearing on both different experimental approaches outlined in the beginning: singleparticle reconstruction and electron tomography of cell sections. Both techniques will be
transformed because of the gains in resolution. Determination of atomic structures by
single-particle reconstruction will become routine for “well-behaved” supramolecular
complexes, i.e. those that occur only in a few well-populated states. High-throughput
methods will be available similar to those now found in X-ray crystallography. At the
same time, electron tomography will reach the range of resolutions that allows the
signatures of conformational states of a molecule to be recognized within the context of a
cell, enabling the tracking of information relevant for the description of its functional
state in situ. In this way, it will be possible to directly link localized processes in the cell
to the dynamical behavior of atomic structures determined by a combination of singleparticle cryo-EM, X-ray crystallography, and single-molecule FRET.
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Acknowledgments
Funding has been provided by Howard Hughes Medical Institute and grants NIH R01
GM29169 and GM55440. I thank Yaser Hashem for the preparation of the figures and
for a critical reading of the manuscript.
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