Observe biological specimens in their native environment at cryo-temperatures.

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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®).

The efficient frontier of resolution     Advantages     Workflow for cryo-EM

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, and 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 an 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 which 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


Workflow for single-particle cryo-EM

Step 1: Purify

To study molecules by single-particle cryo-EM, a specimen must be purified and structurally intact to produce high-quality 3D reconstructions. Ideally, you need to maintain the specimen in a buffer solution that keeps it biochemically active. Molecules within the specimen should be in a high enough concentration that you can study them under a microscope, but not so high that they aggregate together. Finally, experimental conditions should be optimized to promote a uniform conformational state for your molecule of interest.

Step 2: Plunge freeze

Each specimen is frozen to prevent it from freeze-drying within the vacuum of the microscope. Nearly instantaneous freezing prevents the formation of ice crystals that will disrupt the structure of the specimen.

To start, you apply a small amount of the specimen in solution to a TEM grid before the blotting paper is briefly used to remove excess liquid. The TEM grid is then plunged into liquid ethane or a mixture of ethane/propane to quickly wet the specimen, remove heat, and create non-crystalline or vitreous ice. The image shows the Cryoplunge® 3 system just prior to blotting and submersion of the specimen into the liquid cryogen.

Step 3: Transfer to TEM

Once frozen, you transfer the specimen to a specialized TEM holder that keeps it at liquid nitrogen temperature. To prevent specimen contamination, a cryo-workstation protects the specimen when you load it into the holder, then a cryo-shield encapsulates it during the transfer from the workstation to the TEM. As seen here, the cryo-transfer holder is being retracted from the workstation prior to insertion into the TEM.

Step 4: Image specimen

A specimen’s structural integrity is damaged when exposed to electrons, and typically a total dose of 10 – 30 e-2 can be used before high-resolution structural information is lost. To prevent specimen damage, low-dose imaging procedures are used to navigate to the desired areas and focus the electron beam before acquiring images.

The high DQE associated with Gatan's direct detector electron counting and super-resolution modes enables you to acquire the highest quality images of delicate biological samples. This high image signal-to-noise ratio allows you to discern water molecules, ions, and ligand structures in the 3D particle reconstruction. You can further improve image quality by utilizing the dose fractionation feature on Gatan's direct detection cameras, which saves full frames at up to 75 frames per second, to later correct for specimen motion and minimize drift.

Step 5: Analyze and reconstruct

Once imaged, the Gatan Microscopy Suite® software supports your analysis and export of data into multiple formats. Data from Gatan cameras is imported into a wide variety of 3rd party software tools for 3D reconstruction and visualization, including EMAN, Frealign, Relion, and many others. The image shows a 3D cryo-EM density of 20S proteasome at 2.8 Å resolution.

Research Spotlight

Diamond Light Source - eBIC for Industry

eBIC for Industry is a recently inaugurated center offering professional cryo-EM services to the global pharmaceutical and biotech industry.

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