Current outcomes when optimizing 'standard' sample preparation for single-particle cryo-EM.

Although high-resolution single-particle cryo-electron microscopy (cryo-EM) is now producing a rapid stream of breakthroughs in structural biology, it nevertheless remains the case that the preparation of suitable frozen-hydrated samples on electron microscopy grids is often quite challenging. Purified samples that are intact and structurally homogeneous - while still in the test tube - may not necessarily survive the standard methods of making extremely thin, aqueous films on grids. As a result, it is often necessary to try a variety of experimental conditions before finally finding an approach that is optimal for the specimen at hand. Here, we summarize some of our collective experiences to date in optimizing sample preparation, in the hope that doing so will be useful to others, especially those new to the field. We also hope that an open discussion of these common challenges will encourage the development of more generally applicable methodology. Our collective experiences span a diverse range of biochemical samples and most of the commonly used variations in how grids are currently prepared. Unfortunately, none of the currently used optimization methods can be said, in advance, to be the one that ultimately will work when a project first begins. Nevertheless, there are some preferred first steps to explore when facing specific problems that can be more generally recommended, based on our experience and that of many others in the cryo-EM field.

Near-concentric Fabry-Pérot cavity for continuous-wave laser control of electron waves.

Manipulating free-space electron wave functions with laser fields can bring about new electron-optical elements for transmission electron microscopy (TEM). In particular, a Zernike phase plate would enable high-contrast TEM imaging of soft matter, leading to new opportunities in structural biology and materials science. A Zernike phase plate can be implemented using a tight, intense continuous laser focus that shifts the phase of the electron wave by the ponderomotive potential. Here, we use a near-concentric cavity to focus 7.5 kW of continuous-wave circulating laser power at 1064 nm into a 7 µm mode waist, achieving a record continuous laser intensity of 40 GW/cm2. Such parameters are sufficient to impart a phase shift of 1 rad to a 10 keV electron beam, or 0.16 rad to a 300 keV beam. Our numerical simulations confirm that the standing-wave phase shift profile imprinted on the electron wave by the intra-cavity field can serve as a nearly ideal Zernike phase plate.

Accurate modeling of single-particle cryo-EM images quantitates the benefits expected from using Zernike phase contrast.

The use of a Zernike-type phase plate in biologic cryo-electron microscopy allows the imaging, without using defocus, of what are predominantly phase objects. It is thought that such phase-plate implementations might result in higher quality images, free from the problems of CTF correction that occur when images must be recorded at extremely high values of defocus. In single-particle cryo-electron microscopy it is hoped that these improvements in image quality will facilitate work on structures that have proved difficult to study, either because of their relatively small size or because the structures are not completely homogeneous. There is still a need, however, to quantitate how much improvement can be gained by using a phase plate for single-particle cryo-electron microscopy. We present a method for quantitatively modeling the images recorded with 200keV electrons, for single particles embedded in vitreous ice. We then investigate what difference the use of a phase-plate device could have on the processing of single-particle data. We confirm that using a phase plate results in single-particle datasets in which smaller molecules can be detected, particles can be more accurately aligned and problems of heterogeneity can be more easily addressed.

Design of an electron microscope phase plate using a focused continuous-wave laser.

We propose a Zernike phase contrast electron microscope that uses an intense laser focus to convert a phase image into a visible image. We present the relativistic quantum theory of the phase shift caused by the laser-electron interaction, study resonant cavities for enhancing the laser intensity and discuss applications in biology, soft-materials science and atomic and molecular physics.

Ultrathin carbon support films for electron microscopy.

Carbon support films only 4 to 6 angstroms thick have been made for use in electron microscopy. The determination of their thickness is based on geometrical calculation, electron scattering measurements, and elemental microanalysis.

Electron diffraction of frozen, hydrated protein crystals.

High-resolution electron diffraction patterns have been obtained from frozen, hydrated catalase crystals to demonstrate the feasibility of using a frozen specimen hydration technique. The use of frozen specimens to maintain the hydration of complex biological structures has certain advantages over previously developed liquid hydration techniques.

Specimen Behavior in the Electron Beam.

It has long been known that cryo-EM specimens are severely damaged by a level of electron exposure that is much lower than what is needed to obtain high-resolution images from single macromolecules. Perhaps less well appreciated in the cryo-EM literature, the vitreous ice in which samples are suspended is equally sensitivity to radiation damage. This chapter provides a review of several fundamental topics such as inelastic scattering of electrons, radiation chemistry, and radiation biology, which-together-can help one to understand why radiation damage occurs so "easily." This chapter also addresses the issue of beam-induced motion that occurs at even lower levels of electron exposure. While specimen charging may be a contributor to this motion, it is argued that both radiation-induced relief of preexisting stress and damage-induced generation of additional stress may be the dominant causes of radiation-induced movement.

Chemical and physical evidence for multiple functional steps comprising the M state of the bacteriorhodopsin photocycle.

In the photocycle of bacteriorhodopsin (bR), light-induced transfer of a proton from the Schiff base to an acceptor group located in the extracellular half of the protein, followed by reprotonation from the cytoplasmic side, are key steps in vectorial proton pumping. Between the deprotonation and reprotonation events, bR is in the M state. Diverse experiments undertaken to characterize the M state support a model in which the M state is not a static entity, but rather a progression of two or more functional substates. Structural changes occurring in the M state and in the entire photocycle of wild-type bR can be understood in the context of a model which reconciles the chloride ion-pumping phenotype of mutants D85S and D85T with the fact that bR creates a transmembrane proton-motive force.

Crystal structure of the D85S mutant of bacteriorhodopsin: model of an O-like photocycle intermediate.

Crystal structures are reported for the D85S and D85S/F219L mutants of the light-driven proton/hydroxyl-pump bacteriorhodopsin. These mutants crystallize in the orthorhombic C222(1) spacegroup, and provide the first demonstration that monoolein-based cubic lipid phase crystallization can support the growth of well-diffracting crystals in non-hexagonal spacegroups. Both structures exhibit similar and substantial differences relative to wild-type bacteriorhodopsin, suggesting that they represent inherent features resulting from neutralization of the Schiff base counterion Asp85. We argue that these structures provide a model for the last photocycle intermediate (O) of bacteriorhodopsin, in which Asp85 is protonated, the proton release group is deprotonated, and the retinal has reisomerized to all-trans. Unlike for the M and N photointermediates, where structural changes occur mainly on the cytoplasmic side, here the large-scale changes are confined to the extracellular side. As in the M intermediate, the side-chain of Arg82 is in a downward configuration, and in addition, a pi-cloud hydrogen bond forms between Trp189 NE1 and Trp138. On the cytoplasmic side, there is increased hydration near the surface, suggesting how Asp96 might communicate with the bulk during the rise of the O intermediate.

High-voltage electron diffraction from bacteriorhodopsin (purple membrane) is measurably dynamical.

Electron diffraction patterns of 45 A thick two-dimensional crystalline arrays of a cell membrane protein, bacteriorhodopsin, have been recorded at two electron voltages, namely 20 and 120 kV. Significant intensity differences are observed for Friedel mates at 20 kV, but deviations from Friedel symmetry are quite small at 120 kV. It does not seem likely that the measured Friedel differences can be accounted for by complex atomic structure factors, by curvature of the Ewald sphere, or by effects that might occur as a result of inelastic scattering (absorption). It is therefore concluded that dynamical diffraction within the single molecular layer of these crystals is responsible for the observed Friedel differences. The results are useful in estimating the maximum specimen thickness for which the kinematic approximation may be safely used in electron crystallography of biological macromolecules at the usual electron voltage of 100 kV, or even at higher voltages. The results show that the Friedel differences are independent of resolution and this opens up the possibility that dynamical effects occurring at lower voltages might be used to phase higher-voltage kinematic diffraction intensities.

Validity domain of the weak-phase-object approximation for electron diffraction of thin protein crystals.

The domain of validity of the weak-phase-object (WPO) approximation is evaluated for high-energy electrons (100 keV, 500 keV and 1 MeV) scattered by crystalline biological macromolecules. Cytochrome b5 is used as an example in which calculated dynamical diffraction intensities are used to simulate observed diffraction intensities which are then compared with intensities calculated by the weak-phase-object approximation. Three criteria of validity are used, namely the crystallographic residual (R value), the interpretability of difference Patterson maps, and the results of phasing by the heavy-atom isomorphous replacement method. The present calculations indicate that the error associated with the WPO approximation is quite acceptable up to a specimen thickness of 200 A for 100 keV electrons, which is two to four times the thickness limit for crystalline organic structures with much smaller unit-cell dimensions. An equally acceptable thickness limit at 500 keV and 1MeV is about 300-350 A.

Chemical bonding effects in the determination of protein structures by electron crystallography.

Scattering of electrons is affected by the distribution of valence electrons that participate in chemical bonding and thus change the electrostatic shielding of the nucleus. This effect is particularly significant for low-angle scattering. Thus, while chemical bonding effects are difficult to measure with small-unit cell materials, they can be substantial in the study of proteins by electron crystallography. This work investigates the magnitude of chemical bonding effects for a representative collection of protein fragments and a model ligand for nucleotide-binding proteins within the resolution range generally used in determining protein structures by electron crystallography. Electrostatic potentials were calculated by ab initio methods for both the test molecules and for superpositions of their free atoms. Differences in scattering amplitudes can be well over 10% in the resolution range below 5 A and are especially large in the case of ionized side chains and ligands. We conclude that the use of molecule-based scattering factors can provide a much more accurate representation of the low-resolution data obtained in electron crystallographic studies. The comparison of neutral and ionic structure factors at resolutions below 5 A can also provide a sensitive determination of charge states, important for biological function, that is not accessible from X-ray crystallographic measurements.

Specimen flatness of thin crystalline arrays: influence of the substrate.

The extreme degree of specimen flatness (i.e. planarity) required for high-resolution electron diffraction and electron microscopy at high tilt angles cannot be realized with thin, sheet-like crystals of biological macromolecules, just on the basis of the intrinsic stiffness of the specimen itself. In an effort to improve the rate of success at which suitably flat specimens are prepared, this paper analyzes several different factors that can either limit or enhance the specimen flatness. If specimens are adsorbed (by attractive forces) to a support film, such as evaporated carbon, which itself is not flat to atomic dimensions, quantitative calculations show that it is quite likely that the specimen will be too wrinkled to be used for high-resolution studies. Adsorption to an air-water interface is more likely to result in the necessary degree of flatness. Repulsive interactions, which might be used to "sandwich" a specimen between two interfaces, are estimated to be too "soft", i.e. too long-range in character, to be effective. Finally, if only one edge of a specimen sticks firmly to a substrate, then surface tension forces can pull the specimen taut over the surface of the substrate, so that the specimen itself can be more flat than the surface of the substrate upon which it is deposited. A second, important consideration in many studies is the fact that cooling the specimen to low temperature can result in specimen wrinkling, because of the fact that the biological crystal has a much larger coefficient of thermal expansion than that of the evaporated carbon film. In this case one expects that cooling-induced wrinkling might be reduced by using a metal support grid which has a smaller thermal coefficient than that of the carbon film. The validity of this qualitative idea is supported by experiments which show that cooling-induced wrinkling of glucose-embedded purple membrane can be prevented if molybdenum grids are used rather than copper.

Difference Fourier analysis of "surface features" of bacteriorhodopsin using glucose-embedded and frozen-hydrated purple membrane.

A difference Fourier map of the projected structure of bacteriorhodopsin has been synthesized from electron diffraction amplitudes collected from membranes prepared in the glucose-embedded state and the frozen-hydrated state. Phases of a recently published data set for glucose-embedded specimens were used for the difference Fourier map. Moderate resolution (9 A) and high resolution (4.25 A) maps both indicate that glucose is exchangeable for water in the region of the map corresponding to the lipid regions. We interpret this as indicating that there is a small surface depression in this region of the structure. The depth of this feature is estimated to be 1/6 the thickness of the protein region in the membrane. The data obtained in this study rules out the existence of an aqueous transmembrane channel, the dimensions of which are large enough to allow free exchange of glucose for water. Several new features are also observed in the protein region of the membrane. These features are probably due to segments of the polypeptide at the aqueous interface that are well ordered in frozen-hydrated specimens but not in glucose-embedded specimens. Candidate structures for the origin of these features are extensions of the helices, or linker regions connecting the helices.

Preparation of frozen-hydrated specimens for high resolution electron microscopy.

A method is presented for preserving the high resolution structure of biological membranes in a frozen-hydrated environment for electron microscopy. The technique consists of sandwiching a specimen between two carbon films and then waiting while some of the solvent evaporates. When the solvent layer is judged to be of an appropriate thickness, the specimen is then frozen in liquid nitrogen. The specimen can then be inserted into the precooled stage of an electron microscope. Electron diffraction studies of the purple membrane of Halobacterium halobium recorded at -120 degrees C have shown that the structure can be preserved to a resolution of 3.5 A. The main advantage of this method over previous techniques is that the hydrating conditions can be accurately controlled.

Assessment of resolution in biological electron crystallography.

The resolution of images or density maps produced by electron microscopy and electron crystallography can be objectively defined in terms of the spatial frequency of the highest resolution diffraction spot, or Fourier coefficient, included in the data processing. In practice, this objective definition of resolution is expected to be too optimistic if the amplitudes of the highest resolution structure factors are too weak, if the population of high resolution reflections is too sparse, or if the signal-to-noise ratio of the high resolution data is too low. Calculated examples are presented here which illustrate how the apparent resolution in images of a membrane protein, bacteriorhodopsin, can be reduced from a nominal value of 3.5 A by weak amplitudes, sparse data or high noise levels. These calculations provide concrete examples which can serve as a guide when estimating whether the objective definition of image resolution is likely to correspond to a practical, structurally useful estimate of image resolution.

Development of methodology for low exposure, high resolution electron microscopy of biological specimens.

Specimen damage resulting from inelastic scattering is one of the factors that limits high-resolution electron microscopy of biological specimens. We have, therefore, sought to develop a method to record images of periodic objects at a reduced electron exposure in order to preserve high-resolution structural detail. The resulting image will tend increasingly to be a statistically noisy one, as the electron exposure is reduced to lower and lower values. Construction of a statistically defined image from such data is possible by spatial averaging of the electron signals from a large number of identical unit cells. In this paper, we have first investigated the theory pertaining to the attainable resolution as a function of the electron exposure, the magnification, and several other relevant parameters. In addition, we report experimental results obtained with a commercial image intensifier and with nuclear track photographic emulsion, both of which are highly sensitive recording devices. Usable images can be recorded and processed at exposures in the image plane as low as 10(-3) electron/micron2 (1.6 x 10(-14) coulomb/cm2).

Factors affecting high resolution fixed-beam transmission electron microscopy.

An experimental and theoretical characterization of a fixed-beam transmission electron microscope with a field emission gun has been made with regard to the factors of electron beam brightness, spatial and temporal coherence of the incident electrons, objective lens current fluctuation, mechanical stability, and specimen contamination. It has been found that mechanical stability and temporal coherence are the primary factors that prevent the contrast transfer function from extending to 2.0 A in our microscope. Different amorphous thin films have also been used in order to compare their suitability for testing the imaging capability of the microscope at atomic resolution.

High resolution cold stage for the JEOL 100B and 100C electron microscopes.

A high resolution, liquid nitrogen-cooled specimen stage has been designed and constructed for use on the JEOL 100B and 100C electron-microscopes. This stage will be useful for imaging biological macromolecular arrays in the frozen-hydrated or glucose-embedded states at low temperature. Images thus obtained should have an increased signal-to-noise ratio due to the radiation damage protection offered by low temperature.

Radiation damage of purple membrane at low temperature.

The intensities of diffracted electron beams for the purple membrane of Halobacterium halobium are found to decay exponentially as a function of the accumulated electron exposure, both at room temperature and at -120 degrees C. This permits us to define the "critical dose" Ne(h,k) for the (h,k) diffracted beam, as being the electron exposure (electrons/A2) at which the diffracteed intensity has fallen to e-1 of its initial value. The critical of purple membrane is found to increase from the room temperature value by at least a factor of four when the specimen is maintained at a temperature of -120 degrees C on a liquid-nitrogen-cooled stage. A relationship derived between the critical dose, Ne, and the dose for optimum imaging, Nopt. Both Ne and Nopt depend, of course, upon the spatial frequency, or resolution. The derivation is valid only for the case in which all sources of noise other than quantum fluctuations are neglected. In this case, Nopt approximately equal to 2.5Ne. Finally, Nuclear Track Emulsion plates have been shown to be advantageous for recording high resolution electron diffraction patterns of small (1 micrometer 2) patches of crystalline biological materials.