Single-particle cryo-electron microscopy pdf download

Due to the low contrast, the object of choice cannot be discriminated from all kinds of contaminants and breakdown products. The low contrast is, however, likely to be improved in the near future by instrumental improvements, such as implementing phase plates in the microscopes, such as the Zernike phase plate Yamaguchi et al.

There are several advantages of cryo-EM of vitrified specimens: specimen flattening and other drying artifacts are circumvented. Moreover, cryo-images better reflect the true density of a protein, because the contrast directly originates from scattering by the protein rather than from the surrounding stain.

Also, the interaction of negative stain with the protein is often quite complex if the object is not fully embedded. In thinner stain layers, the upper part of the protein could easily be less well embedded in the stain layer, as pointed out in Fig.

This means that the contributions of the upper- and lower half of a protein in the final recorded image do not have the same weighting. In contrast, the embedding in a full ice layer gives a more straightforward signal. Cryo-negative staining represents a complementary method for the conventional negative stain EM and a valuable alternative in particular for situations where cryo-EM reaches its limits in terms of visibility of the protein complexes De Carlo et al.

In cryo-negative staining, particles become embedded in a rather thick layer of stain which is not fully dehydrated, which may prevent flattening and preferential staining. An example of the footprint effect of negative staining. The crystal is composed of rows of monomers. Within the rows, the monomers are either up- or down-oriented, and there is a substantial difference in overall contrast between individual rows of monomers in the upper layer with respect to the lower layer.

The purpose of single particle EM is to determine the structure of macromolecules from images of individual particles or single particle projections. Because isolated macromolecules, prepared on a carbon support film or in a thin layer of ice over a holey carbon film, usually exhibit a full range of orientations, resulting projections will differ as well, and substantial processing is needed before averaging can take place.

Basically, the method of single particle analysis consists of only a few crucial steps, of which two are illustrated in Fig. If projections result from one type of orientation on the support film, averaging is possible after alignment. The alignment step brings projections in equivalent positions by computing rotational and translational shifts.

In the case of the example, a supercomplex of trimeric photosystem I PSI surrounded by a ring of 18 copies of the antenna protein IsiA, a set of projections has been brought in register. It can be seen that by increasing the number of summed projections the noise is gradually reduced Fig. It is very obvious that from individual, noisy projections the number of IsiA copies cannot be retrieved and that processing is indispensable. The basics of single particle EM, explained from an analysis of the photosystem I—IsiA supercomplex from the cyanobacterium Synechococcus , extracted from negatively stained EM specimens Boekema et al.

After translational and rotational alignment of a data set of about single particle projections showing the complex in a position as in the membrane plane, sums with increasing numbers of copies in equivalent positions show the gradual improvement in the signal-to-noise ratio upper part of the picture.

However, these particle projections may not all be identical, because small tilt variations on the membrane plane may lead to different positions. Indeed, after multivariate statistical analysis and classification, it became clear that only a small number of projections show threefold rotational symmetry which is indicative for a position parallel to the membrane lower row , left. The other two classes middle and right show the supercomplex in tilted positions. Just summing of projections, however, is meaningless when the projections arise from particles in different orientations toward the plane.

In order to deal with this, data sets have to be treated with multivariate statistical analysis together with automated classification see Van Heel et al. After statistical analysis and classification, those images that are most similar can be grouped together.

In the case of the data set of PSI—IsiA, it turned out that further processing of projections after the alignment step improved the final sums. The particle projections were not all identical, because small tilt variations on the support film led to different positions. The statistical analysis and classification showed that only a small number of projections had threefold rotational symmetry, indicative for a position parallel to the membrane Fig.

In favorable cases, the molecules show random orientation in the ice layer or on the support film. If not, specimens can be tilted in the microscope in order to obtain 2D projection maps of the molecules viewed from different angles. However, in general, 3D information is much more valuable especially for spherical objects as ribosomes and virus molecules. In the s and s, single particle analysis was still a matter of hard labor, including the recording on photographic emulsion, scanning the images by densitometers and processing, which was less sophisticated Fig.

In recent years, single particle method has been developed much in a direction of automation of all steps, i. The use of scanning slow-scan CCD cameras, which can be programmed to record hundreds of images in a semi-automated way, helped tremendously Fig. In the near future, it is expected that direct electron counters with superior recording qualities will replace the CCD cameras Faruqi and Henderson and that further automation will provide structures within hours after sample insertion in the microscope.

This is similar to the phase contrast light microscope, for which Frits Zernike was awarded the Nobel prize for physics in Implementation in commercial electron microscopes will be a logical next step in improving EM methods. Grigorieff N. Penczek PA. Walz T. Refereed Designation Refereed. The dawn of direct detectors.

Heterogeneity: a blessing and a curse. Acknowledgements We thank R. Glaeser for his help in preparing this Primer. Authors Allison Doerr View author publications.

Rights and permissions Reprints and Permissions. About this article. Cite this article Doerr, A. Copy to clipboard. Nobrega , Marnix Vlot , Patrick A. Dreesens , Hubertus J. Beaumont , Rob Lavigne , Bas E. Ascher , William L. Search Search articles by subject, keyword or author. Show results from All journals This journal.

In his review Dubochet et al. As a demonstration, we obtain reconstructions of apoferritin using an FEI Arctica microscope to 2. An all-LN 2 cold chain can simplifiy sample preparation workflows and design of automated instruments that eliminate manual sample handling after sample deposition.

Cooling in LN 2 may also reduce stresses that contribute to beam-induced sample motion. Critical cooling rates CCRs — the minimum cooling rates required for sample vitrification — depend on the maximum tolerable or detectable ice fraction in otherwise vitrified samples Berejnov et al.

Ice formation in aqueous solutions at large cooling rates occurs at large supercoolings and is homogeneous-nucleation limited Warkentin et al. Fortunately, this is not necessary Wieferig et al. Crudely, high-resolution particle imaging is possible as long as the volume fraction of ice relative to the biomolecule is small within the sample so that, for most particles, there is no visible or strongly diffracting ice within the volume around a particle that contributes to the particle image; similarly, proximity of neighbors in the dense arrays of randomly oriented biomolecules often observed in cryo-EM images does not prevent high-resolution reconstructions.

CCRs decrease exponentially with solute concentration Warkentin et al. Despite using liquid ethane, one of the most effective liquid cryogens, and despite modest cooling rates required to vitrify water, samples for single-particle cryo-EM can develop substantial areas of crystalline ice during cooling.

Why might cooling rates be so low? Cold gas above a liquid cryogen precools samples as they are plunged through it Ryan, Both commercial and home-built cryo-EM plunge-cooling instruments plunge the sample into a small ethane-filled cup surrounded by a larger LN 2 -filled container Fig. S1 of the supporting information. The ethane level is typically at a millimetre or more below the top of its cup. The top of the ethane cup may be just above or well below the top of the LN 2 container.

Consequently, cooling of the sample and foil between grid bars may largely occur in the cold gas, before the sample reaches the ethane. As discussed in Section 5. Confusion about the importance of this precooling when thermocouples are plunged into LN 2 , and when samples much thinner than available thermocouples are cooled, appears to have caused the cooling potential of LN 2 relative to ethane for thin samples to have been underestimated. Three different types of grids i.

Details of grid and foil fabrication are given in the supporting information. S2 and S3 , which is based on insights into the physics of cryocooling described by Kriminski et al. The gas management manifold Fig. This ensures that nearly all sample cooling occurs once the sample enters the LN 2.

The gas management manifold also isolates all cold surfaces from ambient air to minimize or eliminate frost accumulation. Crystals are looped or scooped out of solution onto the loop, and then the wand, base, loop and crystals are loaded on the NANUQ vertical translation stage. When the access door to the vertical stage is closed, the cold gas above the LN 2 within the plunge bore is removed and replaced with dry ambient-temperature gas, the sample is plunged into the LN 2 , and then the sample is translated and released into the storage puck.

To use this instrument for cryocooling cryo-EM grids, two generations of prototype grid holders consisting of custom forceps attached to standard crystallography goniometer bases were fabricated.

Initial experiments in summer used a protein-free 0. When sample plunging was complete, the UniPuck was removed from NANUQ and transferred to an LN 2 -filled insulated container, and samples were removed from the puck one by one and released into standard cryo-EM grid boxes. Second-generation prototype grid holders improved ease of gripping and grid perpendicularlity to the LN 2 surface during the plunge. The absolute thickness was determined in two holes on a type B grid sample 2 using tilt-series tomography Fig.

At each tilt, a five-frame movie was collected for 0. Data acquisition was automated using the SerialEM software Mastronarde, A dataset for sample 1 on an UltrAuFoil grid consisted of micrographs, with a measured defocus range from 0. After patch-based motion correction and CTF estimation using Patch CTF, micrographs were manually inspected and were selected for use.

Particles were extracted at 0. This class was subjected to homogeneous refinement with octahedral symmetry enforced and both global and local CTF refinement, resulting in a 2.

A dataset collected for sample 2 on a prototype grid consisted of total micrographs. Measured defocus ranged from 0. Micrographs were patch-motion corrected and patch-CTF corrected. After manual inspection, micrographs were selected for continued processing. Homogeneous refinement of this class with octahedral symmetry enforced and global CTF refinement produced a 2. Model building and refinement were performed using each map individually. This model was placed in the map using phenix.

The final refinement used one half map, and validation was carried out against the other half map. ChimeraX Goddard et al.

Statistics for the two models are given in Tables 1 and S1 of the supporting information. Often this assessment is done on a simple side-entry microscope e. Single-particle analysis with cryo-EM depends on the computational averaging of thousands of images of identical particles, and therefore structural heterogeneity should be minimized in order to simplify structure determination.

Although the single particle analysis workflow can alleviate partial heterogeneity in the specimen with 3D classification procedures, biochemical purification of the sample to isolate the target proteins is required. The biological specimen should remain active in the in vivo optimized conditions buffer composition, etc.

A suitable biochemical or functional assay might also be exploited to test the activity of the protein. For compatibility with the electron microscope vacuum, and to lock the individual particles in their native states, the solution containing the sample material must be frozen.

In order to preserve the macromolecular structures, freezing has to happen rapidly enough to avoid crystalline ice formation; during vitrification an amorphous solid forms instead that does little or no damage to the sample structure.

Afterwards, the sample must be kept at liquid nitrogen temperatures at all times to preserve the amorphous nature of the embedding ice layer and to avoid damage to the biological particles. This operation produces a frozen hydrated sample, where the individual molecules of the specimen are well distributed and embedded in a very thin layer of amorphous vitreous ice. The whole procedure can be simplified using semi-automated plungers such as the Thermo Scientific Vitrobot System.

A set of key parameters, such as specimen blotting time, blotting force, relative humidity and temperature, allows for reproducible preparation of high-quality vitrified specimens.