Expanding capabilities and infrastructure for cryo-EM

Many challenges and considerations must be evaluated when expanding and supporting new cryo-electron microscopy facilities.

The importance of structural biology research has been reinforced during the ongoing COVID-19 pandemic, as newly determined viral protein structures have helped us understand the mode of action of antibodies and drugs against SARS-CoV-2 and its variants. This success is built on decades of work toward the development of various structural biology techniques.

Among these, cryo-electron microscopy (cryo-EM) has recently seen rapid progress, with structures of many previously intractable macromolecules now being determined. This is largely because it requires very little protein and can image proteins as individual molecules that can be averaged to produce high-resolution three-dimensional atomic models. In fact, structures have recently been obtained without a need for extensive purification from lysates¹, and the power of seeing and identifying protein molecule from the images (or class averages) is an added asset (Fig. 1). With better sample preparation methods, next-generation detectors and new image processing software, it will be possible to obtain even better images and structures². Single-particle cryo-EM has become and will continue to grow as a routine method, and in the next few years the time taken from gene to structure is bound to shorten.

Fig. 1: Cryo-EM (or EM in general) can help improve the biochemical quality of the protein for both compositional and conformational heterogeneity.

a, A demonstration of the power of cryo-EM (scale bar, 500 Å). The micrograph reveals a number of different particles; to a trained eye, some of these molecules might be familiar. b, After two-dimensional classification, the identity of the molecule may become clear (box size, 414 Å), and subsequent mass spectrometry can confirm the identification. Image courtesy Shaileshanand Jha, a graduate student at NCBS, Bangalore. Cryo-EM can produce structures of macromolecules from a mixture without purification of a single entity¹. Whether such an approach will become the norm remains to be seen.

A natural extension of imaging with electron microscopy is in situ structure determination. Large molecules at very high resolution can already be studied in the cellular environment³, and this is likely to become even more powerful. The future challenge lies in imaging low-copy-number macromolecules and membrane protein complexes in their native environment. These proteins are often heterogenous in their composition. Integration of different techniques, including light and electron microscopy, mass spectrometry and genetics, will help us realize this goal.

Synchrotron-based models have been very successful across the world, and installation of cryo-EM (and other) facilities within them has been a wise move. Not many countries have such integrated facilities, and structural biology in India has been an uphill task, in particular when it comes to access to brighter synchrotron sources⁴. When working on competitive projects, easy access to a synchrotron facility (preferably at a local site) can make a huge difference for rapid scientific progress.

The advent of cryo-EM is great for countries like India as, in contrast to synchrotron facilities, large infrastructure is not needed. India has a few institutes with functional cryo-EM⁴, and several others are planned to come online within the end of the year. This is extremely encouraging. There are also plans in place for training service engineers and application scientists to augment these facilities. In practical terms, it is often easy to obtain the initial funding for the procurement of the instrument and setting up the facilities. However, subsequent long-term support to cover the costs of maintaining the instrument and providing easy accessibility to researchers without steep charges will ultimately determine the success of such facilities. The inevitable lockdown due to the pandemic has also highlighted our dependence on research consumables delivered from other countries. For instance, an interruption in supply has meant that we have samples and EM time but not enough EM grids. Being able to source consumables locally could alleviate this dependence. Thus, one of the challenges for us in the near future is to build integrated facilities with good user support and long-term sustainability in order to tackle interesting and difficult biological problems across scales.