Cryo-EM

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

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Overview: 

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 the K2 direct detection cameras.

“At present only the Gatan K2, when operated in counting mode, can produce a DQE(0) as high as 80% in conjunction with reasonably small exposure times.  The K2 detector frame rate is about 10 times higher than that available with the two other detector brands.”– Subramaniam, S., Kuhlbrandt, W. & Henderson, R. (2016). IUCrJ 3, 3-7.

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 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 150 kDa. Structural biologists frequently use cryo-EM to study viruses, small organelles, and macromolecular biological complexes, as well as molecular interactions in supramolecular assemblies or machines.

During cryo-EM, a transmission electron microscope (TEM) is used to record high-resolution images of thousands to hundreds of thousands 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 2 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 face plate 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 K2 Summit 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 the K2 Summit camera, the detective quantum efficiency (DQE) performance at half Nyquist is unparalleled at 52%; more than a six-fold improvement over conventional cameras.

 

Efficient frontier of resolution

The K2 Summit 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. The K2 Summit or Quantum LS 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 and buffer concentrations
Allows study of larger assemblies Useful to characterize molecules larger than 150 kDa that include multiple subunits, 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 water 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 blotting paper is briefly used to remove excess liquid. The TEM grid is then plunged into liquid ethane 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 ethane.

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 transfer from the workstation to the TEM. As seen here, the model 914 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 TEM before acquiring images.

The high DQE associated with the K2 Summit 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 the K2 camera, which saves full frames at up to 40 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 single-particle reconstruction of 20S proteasome at 2.8 Å resolution.

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