Cryogenic electron microscopy (cryo-EM) is a
cryomicroscopy technique applied on samples cooled to
cryogenic temperatures. For biological specimens, the structure is preserved by embedding in an environment of
vitreous ice. An aqueous sample solution is applied to a grid-mesh and plunge-frozen in liquid
ethane or a mixture of liquid ethane and
propane.[1] While development of the technique began in the 1970s, recent advances in detector technology and software algorithms have allowed for the determination of biomolecular structures at near-atomic resolution.[2] This has attracted wide attention to the approach as an alternative to
X-ray crystallography or
NMR spectroscopy for macromolecular structure determination without the need for crystallization.[3]
In the 1960s, the use of
transmission electron microscopy for structure determination methods was limited because of the radiation damage due to high energy electron beams. Scientists hypothesized that examining specimens at low temperatures would reduce beam-induced radiation damage.[6] Both
liquid helium (−269
°C or 4
K or −452.2
°F) and
liquid nitrogen (−195.79 °C or 77 K or −320 °F) were considered as cryogens. In 1980,
Erwin Knapek and
Jacques Dubochet published comments on beam damage at cryogenic temperatures sharing observations that:
Thin crystals mounted on carbon film were found to be from 30 to 300 times more beam-resistant at 4 K than at room temperature... Most of our results can be explained by assuming that cryoprotection in the region of 4 K is strongly dependent on the temperature.[7]
However, these results were not reproducible and amendments were published in Nature just two years later informing that the beam resistance was less significant than initially anticipated. The protection gained at 4 K was closer to "tenfold for standard samples of L-
valine",[8] than what was previously stated.
The 2010s were marked with drastic advancements of electron cameras. Notably, the improvements made to
direct electron detectors have led to a "resolution revolution"[11] pushing the resolution barrier beneath the crucial ~2-3 Å limit to resolve amino acid position and orientation.[12]
More recently, advancements in the use of protein-based imaging scaffolds are helping to solve the problems of sample orientation bias and size limit.
Proteins smaller than ~50
kDa generally have insufficient
SNR to be able to resolve protein particles in the image, making 3D reconstruction difficult or impossible.[13] Imaging scaffolds boost the SNR of smaller proteins by binding them to a larger object, the scaffold. The
Yeates group at
UCLA were able to create a clearer image of three variants of
KRAS (roughly 19 kDa in size) by utilising a rigidified imaging scaffold, and using
DARPins as modular binding domain between the scaffold and the protein-of-interest.[14]
2017 Nobel Prize in Chemistry
In recognition of the impact cryo-EM has had on biochemistry, three scientists,
Jacques Dubochet,
Joachim Frank and
Richard Henderson, were awarded the
Nobel Prize in Chemistry "for developing cryo-electron microscopy for the high-resolution structure determination of biomolecules in solution."[4]
Traditionally, X-ray crystallography has been the most popular technique for determining the 3D structures of biological molecules.[15] However, the aforementioned improvements in cryo-EM have increased its popularity as a tool for examining the details of biological molecules. Since 2010, yearly cryo-EM structure deposits have outpaced X-ray crystallography.[16] Though X-ray crystallography has drastically more total deposits due to a decades-longer history, total deposits of the two methods are projected to eclipse around 2035.[16]
The resolution of X-ray crystallography is limited by crystal homogeneity,[17] and coaxing biological molecules with unknown ideal crystallization conditions into a crystalline state can be very time-consuming, in extreme cases taking months or even years.[18] To contrast, sample preparation in cryo-EM may require several rounds of screening and optimization to overcome issues such as protein aggregation and preferred orientations,[19][20] but it does not require the sample to form a crystal, rather samples for cryo-EM are flash-frozen and examined in their near-native states.[21]
According to
Proteopedia, the median resolution achieved by X-ray crystallography (as of May 19, 2019) on the
Protein Data Bank is 2.05
Å,[17] and the highest resolution achieved on record (as of September 30, 2022) is 0.48 Å.[22] As of 2020, the majority of the protein structures determined by cryo-EM are at a lower resolution of 3–4 Å.[23] However, as of 2020, the best cryo-EM resolution has been recorded at 1.22 Å,[20] making it a competitor in resolution in some cases.
The
Federal Institute of Technology, the
University of Lausanne and the
University of Geneva opened the Dubochet Center For Imaging (DCI) at the end of November 2021, for the purposes of applying and further developing cryo-EM.[25] Less than a month after the first identification of the
SARS-CoV-2 Omicron variant, researchers at the DCI were able to define its structure, identify the crucial mutations to circumvent individual vaccines and provide insights for new therapeutic approaches.[26]
The Danish National cryo-EM Facility also known as
EMBION was inaugurated on December 1, 2016. EMBION is a cryo-EM consortium between Danish Universities (Aarhus University host and University of Copenhagen co-host).
Advanced methods
Cryogenic electron tomography (cryo-ET), a specialized application where many images are taken of individual samples at various tilt angles, resulting in a 3D reconstruction of a single sample.[27]
^Knapek E, Dubochet J (August 1980). "Beam damage to organic material is considerably reduced in cryo-electron microscopy". Journal of Molecular Biology. 141 (2): 147–161.
doi:
10.1016/0022-2836(80)90382-4.
PMID7441748.