Setting up and operating a cryo-EM laboratory

Abstract

Cryo-electron microscopy (cryo-EM) has become the technique of choice for structural biology of macromolecular assemblies, after the ‘resolution revolution’ that has occurred in this field since 2012. With a suitable instrument, an appropriate electron detector and, last but not least, a cooperative sample it is now possible to collect images from which macromolecular structures can be determined to better than 2 Å resolution, where reliable atomic models can be built. By electron tomography and sub-tomogram averaging of cryo-samples, it is also possible to reconstruct subcellular structures to sub-nanometre resolution. This review describes the infrastructure that is needed to achieve this goal. Ideally, a cryo-EM lab will have a dedicated 300 kV electron microscope for data recording and a 200 kV instrument for screening cryo-samples, both with direct electron detectors, and at least one 120 kV EM for negative-stain screening at room temperature. Added to this should be ancillary equipment for specimen preparation, including a light microscope, carbon coater, plasma cleaner, glow discharge unit, a device for fast, robotic sample freezing, liquid nitrogen storage Dewars and a ready supply of clean liquid nitrogen. In practice, of course, the available budget will determine the number and types of microscopes and how elaborate the lab can be. The cryo-EM lab should be designed with adequate space for the electron microscopes and ancillary equipment, and should allow for sufficient storage space. Each electron microscope room should be connected to the image-processing computers by fibre-optic cables for the rapid transfer of large datasets. The cryo-EM lab should be overseen by a facility manager whose responsibilities include the day-to-day tasks to ensure that all microscopes are operating perfectly, organising service and repairs to minimise downtime, and controlling the budget. Large facilities will require additional support staff who help to oversee the operation of the facility and instruct new users.

Broadening access to cryoEM through centralized facilities

“…CryoEM, similar to other structural biology methods, including NMR, X-ray crystallography, and solution X-ray scattering, employs sophisticated instrumentation that requires a high level of expertise for operation and maintenance. The infrastructure requirements to site and support science that makes use of high-end instrumentation are not trivial, and pooling resources leads to an economy of scale [23]. The centralization of resources is well established in structural biology with access to regional NMR centers and biologically focused X-ray beamlines at synchrotrons.…”

“…Large facilities have a greater return from the resources invested because they can increase efficiency and quality through establishment of common standards and protocols [23]. A primary example of how standardization has assisted the adoption of structural biology is X-ray beamlines I).…”

High-speed high-resolution data collection on a 200 keV cryo-TEM

“…As of September 2021, there are 297 EM maps deposited in the Electron Microscopy Data Bank (EMDB), collected with a Talos Arctica which are <4 A ˚resolution (Patwardhan, 2017). Since 200 keV cryo-TEMs cost significantly less than their 300 keV counterparts and are also capable of obtaining EM maps at near-atomic resolutions, we aim to further expand their utility as high-resolution data collection instruments (Mills, 2021;Sader et al, 2020). Importantly, the methods for acceleration of data collection described herein will also aid in more rapid data collection for other 200 and 300 keV cryo-TEMs equipped with DEDs.…”

Cryo-electron microscopy of the f1 filamentous phage reveals a new paradigm in viral infection and assembly

“…Whilst the latter study provided the first direct view of the pVIII filament, atomic model building was not possible. With improvements in cryoEM methodology 29 , a high-resolution cryoEM structure of a related pVIII protein filament from phage Ike was determined 30 . The four minor cap proteins have not however been amenable to structural characterisation, with the exception of N- terminal fragments of pIII 14, 15, 31 .…”