Cryo-Electron Microscopy: Bridging the Micro-Nano Gap

Titan Krios Suite
Titan Krios Suite

To understand human physiology and the pathology of diseases, it is important to investigate the underlying biological processes on all spatial scales. These scales range from the patient, to organs, to tissues within an organ, to individual cells within the tissue, to molecular machines within the cells, down to the atomic level. The detailed knowledge of macromolecular interactions within and in between cells in a given tissue, combined with an integrated view of an organism, significantly increases the chances for finding new cures.

From the days of the invention in the 1930s by Ernst Ruska and Max Knoll, electron microscopes have been used to capture images of biological specimens. The technological advances of the 1940s allowed the observation of cellular organelles embedded in heavy metal stains by transmission electron microscopy (TEM). The 1960s brought the ability to generate 3D structures from individual macromolecular assemblies. Further developments in the 1970s provided the means to image these macromolecules while retaining them hydrated in biochemically functional buffer conditions by instantaneously freezing them at -180°C. However, the resolution attainable for these samples through electron microscopy (cryoEM) was limited. X-ray crystallography continued to be the method of choice for resolving molecular structures; as a robust and mature technology, X-ray crystallography routinely provided de novo structural models. This technology, however, is not without limitations: large quantities of the molecules have to be isolated and crystallized; these processes are often difficult and time-consuming. The resulting crystals may capture the molecules in a conformation that does not occur naturally in biologically relevant environments. Lastly, molecules of high medical importance are often very hard or practically impossible to crystallize.

Recently, advances of hardware and image analysis capabilities revolutionized the field of cryoEM, enabling the generation of high-resolution structural data from vitrified samples at resolutions that rival X-ray crystallography, without the need for crystallization. In addition, cryoEM is parsimonious in its material requirements, also handling fully hydrated pleiomorphic macromolecular assemblies. Of currently available equipment, the most powerful cryoEM instruments that operate at these low temperatures is the FEI Titan Krios. Today, the Titan Krios, equipped with a direct detector and contrast-enhancing (phase plate) technology, can generate large volumes of high-resolution, high-contrast data from automated data collection runs. The analysis of these data yields molecular structures with the resolution of a fraction of a nanometer (2-3Å); individual residues within proteins or nucleic acids are clearly visible at that scale (see Figure 1).

Figure 1
Figure 1. Scheme of hybrid methods technology integrating live-cell light microscopy and 3D electron cryo-tomography. The approach allows generating and linking data that can span six orders of magnitude in length scale (Ångstroms to millimeters) and five orders of magnitude in time scale (milliseconds to minutes). Here, a series of correlated fluorescence and electron cryo-microscopy images of a phalloidin-stained cell is shown, ranging from an overview of the fluorescence image in (A) to individual actin filaments traced in red in a 3.8-nm slice of a cryo-tomogram in (E) (in collaboration with Drs. Pollard, Li and Volkmann).


The NIH’s Office of Research Infrastructure Programs (ORIP), through its Shared Instrumentation Program (S10), supports grant awards for the acquisition of the state-of-the art instruments that enable and enhance research of NIH-supported investigators. Dr. Dorit Hanein, the Principal Investigator at the Sanford Burnham Prebys Medical Discovery Institute (SBP), was awarded an S10 grant to support the purchase of the Titan Krios (see Figure 2). She commissioned the microscope in 2015. A separate NIH shared instrumentation award in 2008 funded ancillary instruments required for the preparation of samples for cryoEM experiments. Also necessary are dedicated high-end computers, adequate data storage capacity, and sophisticated software for on-the-fly processing and real-time feedback for data quality to optimize the microscope usage. Setting up the cryoEM research program also required additional investments such as the allocation of space, maintenance, and support for the operation of the instruments, including highly trained personnel. Investments at SBP created the environment benefiting scientific discovery.


Figure 2
Figure 2. Titan Krios Suite at SBP


The availability of the Titan Krios and ancillary equipment through the S10 mechanism already has enormously increased the imaging capabilities of SBP investigators and collaborators for determining high-resolution macromolecular structures. For Dr. Hanein’s research, it provided a paradigm shift as it offers an opportunity for high-impact, rigorous, and robust discoveries in basic biological research and medicine in situ by investigating macromolecules inside mammalian cells at high resolution.


Dr. Hanein and her collaborators develop quantitative approaches for integrating imaging modalities across scales such as correlative fluorescence light microscopy, cellular cryo-tomography, and cryoEM, bridging the micro-nano gap of underlying biological processes.1-8 This correlative approach, when combined with image analysis and data mining, identifies the location of components of macromolecular complexes in a whole cell and informs about their function. Dr. Hanein’s central biological interest is the nanometer-scale structure of the actin cytoskeleton, the girders and cables that control the shapes and movements of cells. The anchoring sites of these girders are mechanosensitive assemblies that transmit force across the cell membrane and regulate biochemical signals in response to changes in the mechanical environment. These macromolecules are building blocks of tissue development and maintenance and also play an important role in cancer progression where loss of these contacts is a defining step of metastasis (see Figure 3).

Figure 3
Figure 3. Electron microscopy of cell-cell junctions probing the transmission of forces between cells. (A) Image of a cell-cell junction between Caco-2 epithelial cells cultured on electron microscopy-amenable substrates. Brackets label actin filament arrays parallel to the junction, and the yellow arrow marks where the actin arrays were in close proximity to cell-cell contacts. (B) Three-dimensional electron tomography reconstruction of the same region shown in (A) rotated 90° clockwise and then tilted 45° around the horizontal axis. The cell-cell junction is highlighted in red. Yellow arrow marks the same region as in (A). Scale bars are 1 μm (in collaboration with Drs. Volkmann, Weis, Nelson and Dunn).


Figure 4
Figure 4. Dr. Hanein and the FEI Titan Krios.



Through the years, Dr. Hanein and her collaborators determined high-resolution structures of molecular assemblies at an increasing level of complexity. For example, they provided the first view of the Arp2/3 complex2-4, initially as an unbound seven-protein assembly and then, in a state when initiating theformation of new actin filaments. With the Titan Krios, they were able to verify the existence of protein complexes in unperturbed cellular environments, reveal new regulatory roles for these complexes, and investigate their function in promoting directed cell migration. Also, they were able to elucidate effects of certain gene mutations and drug-induced changes on protein assemblies in situ, setting foundations for the development of new therapeutic molecules. Such comprehensive observations become feasible because of the throughput imaging capacity of the Titan Krios, combined with the powerful quantitative analysis of the data.



1Page C, Hanein D, Volkmann N. Accurate membrane tracing in three-dimensional reconstructions from electron cryotomography data. Ultramicroscopy 2015;155:20-26. PMC4451430.

2Suraneni P, Rubinstein B, Unruh JR, Durnin M, Hanein D, Li R. The Arp2/3 complex is required for lamellipodia extension and directional fibroblast cell migration. Journal of Cell Biology 2012;197:239-251.

3Suraneni1 P, Fogelson B, Rubinstein B, Noguera P, Volkmann N, Hanein D, Mogilner A, Li R. A mechanism of leading-edge protrusion in the absence of Arp2/3 complex. Molecular Biology of the Cell 2015 26(5):901–912.

4Anderson KL, Page C, Swift MF, Suraneni P, Janssen ME, Pollard TD, Li R, Volkmann N, Hanein D. Nano-scale actin-network characterization of fibroblast cells lacking functional Arp2/3 complex. Journal of Structural Biology 2017;197(3):312–321.

5Xu XP, Kim E, Swift M, Smith JW, Volkmann N, Hanein D. Three-dimensional structures of full-length, membrane-embedded human α(IIb)β(3) integrin complexes. Biophysical Journal 2016;110(4):798-809.

6Chen Z, Sun L, Zhang Z, Fokine A, Padilla-Sanchez V, Hanein D, Jiang W, Rossmann MG, Rao VB. Cryo-EM structure of the bacteriophage T4 isometric head at 3.3-Å resolution and its relevance to the assembly of icosahedral viruses. Proceedings of the National Academies of Science U S A 2017;114(39):E8184-E8193.

7Anderson KL, Page C, Swift MF, Hanein D, Volkmann N. Marker-free method for accurate alignment between correlated light, cryo-light, and electron cryo-microscopy data using sample support features. Journal of Structural Biology 2018;201(1):46-51.

8Buckley CD, Tan J, Anderson KL, Hanein D, Volkmann N, Weis WI, Nelson WJ, Dunn AR. Cell adhesion. The minimal cadherin-catenin complex binds to actin filaments under force. Science 2014;346(6209):1254211.