The Royal Swedish Academy of Sciences has decided to award Jacques Dubochet, Joachim Frank, and Richard Henderson the Nobel Prize for Chemistry 2017 for "developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution". Scientific Background on the Nobel Prize in Chemistry 2017: The Development of Cryo-Electron Microscopy
Cryo-EM is being led by the technology and innovation behind Gatan's direct detection cameras (K3® and K2®).
What is single-particle cryo-EM?
Single-particle cryo-electron microscopy (cryo-EM), is an increasingly popular technique used by structural biologists to solve structures at atomic resolution. This technique complements x-ray crystallography because it reveals structural details without the need for a crystalline specimen. Through the examination of a frozen-hydrated specimen in vitreous (non-crystalline) ice, the specimen ultrastructure, buffer composition, and ligand distribution from its native state are maintained. Cryo-EM also complements structural studies using nuclear magnetic resonance (NMR) in that it enables the study of specimens larger than 90 kDa. Structural biologists frequently use cryo-EM to study viruses, small organelles, macromolecular biological complexes, purified proteins as well as molecular interactions in supramolecular assemblies or machines.
During single-particle cryo-EM, a transmission electron microscope (TEM) is used to record high-resolution images of thousands to hundreds of thousands of identical but randomly oriented particles (molecules) from each specimen. These images are then grouped, aligned, and averaged with image classification algorithms to distinguish between multiple orientations of the 3D molecule. With a well-behaved sample, cryo-EM can solve molecular structures with a resolution below 1.5 Å; a resolution level that was inconceivable only a few years ago.
This rapid improvement of cryo-EM resolution has been called the “resolution revolution” and is the direct result of direct detection cameras. Conventional cameras for electron microscopy use a scintillator to convert the electron image to a light image, and a fiber optic faceplate to transfer the image to a CCD or CMOS image sensor for analog recording. When the results are converted and recorded, there is a loss of high-resolution detail that has long-held cryo-EM from achieving its true potential.
Direct detection cameras now directly measure the electron image to avoid the loss of detail from the conventional camera conversion steps. The Gatan K3 camera is a unique direct detection camera that uses electron counting with super-resolution technology to record the image. This technology recognizes and counts individual image electrons in real-time, while it rejects the analog read noise. As shown with Gatan's direct detection cameras, the detective quantum efficiency (DQE) performance at half Nyquist is unparalleled.
The efficient frontier of resolution
Gatan's direct detection camera, often in combination with the GIF Quantum LS imaging filter, continues to yield breakthrough results that define the efficient frontier of resolution. Cutting-edge publications show these products, along with improved data acquisition and image processing strategies, remain at the forefront of higher resolution reconstructions for smaller molecules, such as the 2.2 Å structure of β-galactosidase.
The graph above is a comparison between the resolution and molecule size for published single-particle cryo-EM structures. Gatan's direct detectors have been employed in all the structures that define the high-resolution frontier across a range of molecular sizes.
Advantages of single-particle cryo-EM
Now that single-particle cryo-EM provides structures with comparable resolution to x-ray crystallography, there are a number of unique advantages that are helping the technique gain traction amongst structural biologists:
| Capability | Advantage |
|---|---|
| Examines structures in their native, hydrated state | Maintains your specimen in a biologically relevant environment, including sample concentration and buffer composition |
| Allows study of larger assemblies | Useful to characterize molecules larger than 150 kDa that include multiple subunits that are heterogeneous, metastable or extremely difficult to crystallize |
| Elucidates atomic resolution structures | Enables observation of asymmetric side chains, hydrogen bonds, and water molecules in addition to alpha helices and beta sheets |
| Controls the chemical environment | Allows you to vary experimental conditions to examine molecules in different functional states |
| Eliminates crystallization steps | Avoids long, uncertain preparation steps; shortens your time to publish |
Related materials
Nyquist frequency
Dose fractionation and motion correction
Improving DQE with counting and super-resolution
Modulation transfer function (MTF) curves
| Modulation transfer function (MTF) curves | ||
|---|---|---|
| 200 kV | 300 kV | |
| K3 | CDS | CDS |
| Standard | Standard | |
| K2 | Standard | Standard |