Laboratory cryo soft X-ray microscopy

Lens-based water-window X-ray microscopy allows two- and three-dimensional (2D and 3D) imaging of intact unstained cells in their near-native state with unprecedented contrast and resolution. Cryofixation is essential to avoid radiation damage to the sample. Present cryo X-ray microscopes rely on synchrotron radiation sources, thereby limiting the accessibility for a wider community of biologists. In the present paper we demonstrate water-window cryo X-ray microscopy with a laboratory-source-based arrangement. The microscope relies on a λ=2.48-nm liquid-jet high-brightness laser-plasma source, normal-incidence multilayer condenser optics, 30-nm zone-plate optics, and a cryo sample chamber. We demonstrate 2D imaging of test patterns, and intact unstained yeast, protozoan parasites and mammalian cells. Overview 3D information is obtained by stereo imaging while complete 3D microscopy is provided by full tomographic reconstruction. The laboratory microscope image quality approaches that of the synchrotron microscopes, but with longer exposure times. The experimental image quality is analyzed from a numerical wave-propagation model of the imaging system and a path to reach synchrotron-like exposure times in laboratory microscopy is outlined.     

Dynamics in cryo EM reconstructions visualized with maximum-likelihood derived variance maps

CryoEM data capture the dynamic character associated with biological macromolecular assemblies by preserving the various conformations of the individual specimens at the moment of flash freezing. Regions of high variation in the data set are apparent in the image reconstruction due to the poor density that results from the lack of superposition of these regions. These observations are qualitative and, to date, only preliminary efforts have been made to quantitate the heterogeneity in the ensemble of particles that are individually imaged. We developed and tested a quantitative method for simultaneously computing a reconstruction of the particle and a map of the space-varying heterogeneity of the particle based on an entire data set. The method uses a maximum likelihood algorithm that explicitly takes into account the continuous variability from one instance to another instance of the particle. The result describes the heterogeneity of the particle as a variance to be plotted at every voxel of the reconstructed density. The test, employing time resolved data sets of virus maturation, not only recapitulated local variations obtained with difference map analysis, but revealed a remarkable time dependent reduction in the overall particle dynamics that was unobservable with classical methods of analysis.     

A binary segmentation approach for boxing ribosome particles in cryo EM micrographs

Three-dimensional reconstruction of ribosome particles from electron micrographs requires selection of many single-particle images. Roughly 100,000 particles are required to achieve approximately 10 A resolution. Manual selection of particles, by visual observation of the micrographs on a computer screen, is recognized as a bottleneck in automated single-particle reconstruction. This paper describes an efficient approach for automated boxing of ribosome particles in micrographs. Use of a fast, anisotropic non-linear reaction-diffusion method to pre-process micrographs and rank-leveling to enhance the contrast between particles and the background, followed by binary and morphological segmentation constitute the core of this technique. Modifying the shape of the particles to facilitate segmentation of individual particles within clusters and boxing the isolated particles is successfully attempted. Tests on a limited number of micrographs have shown that over 80% success is achieved in automatic particle picking.     

Limiting factors in atomic resolution cryo electron microscopy: no simple tricks

To bring cryo electron microscopy (cryoEM) of large biological complexes to atomic resolution, several factors--in both cryoEM image acquisition and 3D reconstruction--that may be neglected at low resolution become significantly limiting. Here we present thorough analyses of four limiting factors: (a) electron-beam tilt, (b) inaccurate determination of defocus values, (c) focus gradient through particles, and (d) particularly for large particles, dynamic (multiple) scattering of electrons. We also propose strategies to cope with these factors: (a) the divergence and direction tilt components of electron-beam tilt could be reduced by maintaining parallel illumination and by using a coma-free alignment procedure, respectively. Moreover, the effect of all beam tilt components, including spiral tilt, could be eliminated by use of a spherical aberration corrector. (b) More accurate measurement of defocus value could be obtained by imaging areas adjacent to the target area at high electron dose and by measuring the image shift induced by tilting the electron beam. (c) Each known Fourier coefficient in the Fourier transform of a cryoEM image is the sum of two Fourier coefficients of the 3D structure, one on each of two curved 'characteristic surfaces' in 3D Fourier space. We describe a simple model-based iterative method that could recover these two Fourier coefficients on the two characteristic surfaces. (d) The effect of dynamic scattering could be corrected by deconvolution of a transfer function. These analyses and our proposed strategies offer useful guidance for future experimental designs targeting atomic resolution cryoEM reconstruction.     

Imaging biological structures with the cryo atomic force microscope.

It has long been recognized that one of the major limitations in biological atomic force microscopy (AFM) is the softness of most biological samples, which are easily deformed or damaged by the AFM tip, because of the high pressure in the contact area, especially from the very sharp tips required for high resolution. Another is the molecular motion present at room temperature due to thermal fluctuation. Using an AFM operated in liquid nitrogen vapor (cryo-AFM), we demonstrate that cryo-AFM can be applied to a large variety of biological samples, from immunoglobulins to DNA to cell surfaces. The resolution achieved with cryo-AFM is much improved when compared with AFM at room temperature with similar specimens, and is comparable to that of cryo-electron microscopy on randomly oriented macromolecules. We will also discuss the technical problems that remain to be solved for achieving even higher resolution with cryo-AFM and other possible applications of this novel technique.     

A streamlined workflow for automated cryo focused ion beam milling

Cryo-electron tomography (cryo-ET) is an emerging technique to study the cellular architecture and the structure of proteins at high resolution in situ. Most biological specimens are too thick to be directly investigated and are therefore thinned by milling with a focused ion beam under cryogenic conditions (cryo-FIB). This procedure is prone to contaminations, which makes it a tedious process, often leading to suboptimal results. Here, we present new hardware that overcomes the current limitations. We developed a new glove box and a high vacuum cryo transfer system and installed a stage heater, a cryo-shield and a cryo-shutter in the FIB milling microscope. This reduces the ice contamination during the transfer and milling process and simplifies the handling of the sample. In addition, we tested a new software application that automates the key milling steps. Together, these improvements allow for high-quality, high-throughput cryo-FIB milling. This paves the way for new types of experiments, which have been previously considered infeasible.     

Fine structure of granal thylakoid membrane organization using cryo electron tomography

The architecture of grana membranes from spinach chloroplasts was studied by cryo electron tomography. Tomographic reconstructions of ice-embedded isolated grana stacks enabled to resolve features of photosystem II (PSII) in the native membrane and to assign the absolute orientation of individual membranes of granal thylakoid discs. Averaging of 3D sub-volumes containing PSII complexes provided a 3D structure of the PSII complex at 40 ? resolution. Comparison with a recently proposed pseudo-atomic model of the PSII supercomplex revealed the presence of unknown protein densities right on top of 4 light harvesting complex II (LHCII) trimers at the lumenal side of the membrane. The positions of individual dimeric PSII cores within an entire membrane layer indicates that about 23% supercomplexes must be of smaller size than full C(2)S(2)M(2) supercomplexes, to avoid overlap.     

The integrative role of cryo electron microscopy in molecular and cellular structural biology

After gradually moving away from preparation methods prone to artefacts such as plastic embedding and negative staining for cell sections and single particles, the field of cryo electron microscopy (cryo‐EM) is now heading off at unprecedented speed towards high‐resolution analysis of biological objects of various sizes. This ‘revolution in resolution’ is happening largely thanks to new developments of new‐generation cameras used for recording the images in the cryo electron microscope which have much increased sensitivity being based on complementary metal oxide semiconductor devices. Combined with advanced image processing and 3D reconstruction, the cryo‐EM analysis of nucleoprotein complexes can provide unprecedented insights at molecular and atomic levels and address regulatory mechanisms in the cell. These advances reinforce the integrative role of cryo‐EM in synergy with other methods such as X‐ray crystallography, fluorescence imaging or focussed‐ion beam milling as exemplified here by some recent studies from our laboratory on ribosomes, viruses, chromatin and nuclear receptors. Such multi‐scale and multi‐resolution approaches allow integrating molecular and cellular levels when applied to purified or in situ macromolecular complexes, thus illustrating the trend of the field towards cellular structural biology.     

Structure of complex viruses and virus-infected cells by electron cryo tomography

In microbiology, and in particular in virus research, electron microscopy (EM) is an important tool, offering a broad approach for investigating viral structure throughout their intracellular and extracellular life cycles. Currently, molecular tools and rapid developments in advanced light microscopy dominate the field and supply an enormous amount of information concerning virus biology. In recent years, numerous fascinating high-resolution EM structures obtained by single-particle electron cryo microscopy (cryo-EM) were revealed for viral particles that possess icosahedral symmetry. However, no comprehensive three-dimensional analysis of complex viruses or viruses within cells has yet been achieved using EM. Recent developments in electron cryo-tomography render this a proficient tool for the analysis of complex viruses and viruses within cells in greater detail.     

Water structure around a left-handed Z-DNA fragment analyzed by cryo neutron crystallography

Even in high-quality X-ray crystal structures of oligonucleotides determined at a resolution of 1 Å or higher, the orientations of first-shell water molecules remain unclear. We used cryo neutron crystallography to gain insight into the H-bonding patterns of water molecules around the left-handed Z-DNA duplex [d(CGCGCG)]₂. The neutron density visualized at 1.5 Å resolution for the first time allows us to pinpoint the orientations of most of the water molecules directly contacting the DNA and of many second-shell waters. In particular, H-bond acceptor and donor patterns for water participating in prominent hydration motifs inside the minor groove, on the convex surface or bridging nucleobase and phosphate oxygen atoms are finally revealed. Several water molecules display entirely unexpected orientations. For example, a water molecule located at H-bonding distance from O6 keto oxygen atoms of two adjacent guanines directs both its deuterium atoms away from the keto groups. Exocyclic amino groups of guanine (N2) and cytosine (N4) unexpectedly stabilize waters H-bonded to O2 keto oxygens from adjacent cytosines and O6 keto oxygens from adjacent guanines, respectively. Our structure offers the most detailed view to date of DNA solvation in the solid-state undistorted by metal ions or polyamines.     

Towards an atlas of mammalian cell ultrastructure by cryo soft X-ray tomography

We provide a catalog of 3D cryo soft X-ray tomography (cryo-SXT) images obtained from ～6 to 12μm thick mouse adenocarcinoma cells. Included are multiple representative images of nuclei, nucleoli, nuclear membrane, nuclear membrane channels, mitochondria, lysosomes, endoplasmic reticulum, filaments and plasma membrane, plus three structures not previously described by cryo-SXT, namely Golgi, microvilli and nuclear-membrane blebs. Sections from the 3D cryo-SXT tomograms for all the preceding structures closely resemble those seen by thin-section transmission electron microscopy (TEM). Some structures such as nuclear-membrane channels and nuclear-membrane blebs are more easily detected by cryo-SXT than TEM most likely due to their better contrast and cellular preservation in cryo-SXT combined with the ability to rapidly locate these structures within a full 3D image. We identify and discuss two current limitations in cryo-SXT: variability in image quality and difficulties in detecting weaker contrast structures such as chromatin and various nuclear bodies. Progress on these points is likely to come from the solution of several technical problems in image acquisition, plus the implementation of advanced cryo soft X-ray microscopy approaches such as phase contrast or optical sectioning.     

Practical workflow for cryo focused-ion-beam milling of tissues and cells for cryo-TEM tomography

Vitreous freezing offers a way to study cells and tissue in a near-native state by cryo-transmission electron microscopy (cryo-TEM), which is important when structural information at the macromolecular level is required. Many cells – especially those in tissue – are too thick to study intact in the cryo-TEM. Cryo focused-ion-beam (cryo-FIB) milling is being used in a few laboratories to thin vitreously frozen specimens, thus avoiding the artifacts and difficulties of cryo-ultramicrotomy. However, the technique is challenging because of the need to avoid devitrification and frost accumulation during the entire process, from the initial step of freezing to the final step of loading the specimen into the cryo-TEM. We present a robust workflow that makes use of custom fixtures and devices that can be used for high-pressure-frozen bulk tissue samples as well as for samples frozen on TEM grids.     

High-resolution cryo-EM performance comparison of two latest-generation cryo electron microscopes on the human ribosome

Recent technological advances in cryo electron microscopy (cryo-EM) have led to new opportunities in the structural biology field. Here we benchmark the performance of two 300 kV latest-generation cryo electron microscopes, Titan Krios G4 from Thermofisher Scientific and CRYO ARM 300 from Jeol, with regards to achieving high resolution single particle reconstructions on a real case sample. We compare potentially limiting factors such as drift rates, astigmatism & coma aberrations and performance during image processing and show that both microscopes, while comprising rather different technical setups & parameter settings and equipped with different types of energy filters & cameras, achieve a resolution of around 2 Å on the human ribosome, a non-symmetric object which constitutes a key drug target. Astigmatism correction, CTF refinement and correction of higher order aberrations through refinement in separate optics groups helped to account for astigmatism/coma caused by beam tilting during multi-spot and multi-hole acquisition in neighbouring holes without stage movement. The obtained maps resolve Mg²⁺ ions, water molecules, inhibitors and side-chains including chemical modifications. The fact that both instruments can resolve such detailed features will greatly facilitate understanding molecular mechanisms of various targets and helps in cryo-EM structure based drug design. The methods and analysis tools used here will be useful also to characterize existing instruments and optimize data acquisition settings and are applicable broadly to other drug targets in structural biology.     

Elemental distribution in striated muscle and the effects of hypertonicity: Electron probe analysis of cryo sections

A method of rapid freezing in supercooled Freon 22 (monochlorodifluoromethane) followed by cryoultramicrotomy is described and shown to yield ultrathin sections in which both the cellular ultrastructure and the distribution of diffusible ions across the cell membrane are preserved and intracellular compartmentalization of diffusabler ions can be quantitated. Quantitative electron probe analysis (Shuman, H., A.V. Somlyo, and A.P. Somlyo. 1976. Ultramicros. 1:317-339.) of freeze-dried ultrathin cryto sections was found to provide a valid measure of the composition of cells and cellular organelles and was used to determine the ionic composition of the in situ terminal cisternae of the sarcoplasmic reticulum (SR), the distribution of CI in skeletal muscle, and the effects of hypertonic solutions on the subcellular composition if striated muscle. There was no evidence of sequestered CI in the terminal cisternae of resting muscles, although calcium (66mmol/kg dry wt +/- 4.6 SE) was detected. The values of [C1](i) determined with small (50-100 nm) diameter probes over cytoplasm excluding organelles over nuclei or terminal cisternae were not significantly different. Mitochondria partially excluded C1, with a cytoplasmic/ mitochondrial Ci ratio of 2.4 +/- 0.88 SD. The elemental concentrations (mmol/kg dry wt +/- SD) of muscle fibers measured with 0.5-9-μm diameter electron probes in normal frog striated muscle were: P, 302 +/- 4.3; S, 189 +/- 2.9;C1, 24 +/- 1.1;K, 404 +/- 4.3, and Mg, 39 +/- 2.1. It is concluded that: (a) in normal muscle the "excess CI" measured with previous bulk chemical analyses and flux studies is not compartmentalized in the SR or in other cellular organelles, and (b) the cytoplasmic C1 in low [K](0) solutions exceeds that predicted by a passive electrochemical distribution. Hypertonic 2.2 X NaCl, 2.5 X sucrose, or 2.2 X Na isethionate produced: (a) swollen vacuoles, frequently paired, adjacent to the Z lines and containing significantly higher than cytoplasmic concentrations of Na and Cl or S (isethionate), but no detectable Ca, and (b) granules of Ca, Mg, and P = approximately (6 Ca + 1 Mg)/6P in the longitudinal SR. It is concluded that hypertonicity produces compartmentalized domains of extracellular solutes within the muscle fibers and translocates Ca into the longitudinal tubules.     

ContinuousFlex: Software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy and tomography data

ContinuousFlex is a user-friendly open-source software package for analyzing continuous conformational variability of macromolecules in cryo electron microscopy (cryo-EM) and cryo electron tomography (cryo-ET) data. In 2019, ContinuousFlex became available as a plugin for Scipion, an image processing software package extensively used in the cryo-EM field. Currently, ContinuousFlex contains software for running (1) recently published methods HEMNMA-3D, TomoFlow, and NMMD; (2) earlier published methods HEMNMA and StructMap; and (3) methods for simulating cryo-EM and cryo-ET data with conformational variability and methods for data preprocessing. It also includes external software for molecular dynamics simulation (GENESIS) and normal mode analysis (ElNemo), used in some of the mentioned methods. The HEMNMA software has been presented in the past, but not the software of other methods. Besides, ContinuousFlex currently also offers a deep learning extension of HEMNMA, named DeepHEMNMA. In this article, we review these methods in the context of the ContinuousFlex package, developed to facilitate their use by the community.     

Biased coarse-grained molecular dynamics simulation approach for flexible fitting of X-ray structure into cryo electron microscopy maps

Several approaches have been introduced to interpret, in terms of high-resolution structure, low-resolution structural data as obtained from cryo-EM. As conformational changes are often observed in biological molecules, these techniques need to take into account the flexibility of proteins. Flexibility has been described in terms of movement between rigid domains and between rigid secondary structure elements, which present some limitations for studying dynamical properties. Normal mode analysis has also been used, but is limited to medium resolution data. All-atom molecular dynamics fitting techniques are more appropriate to fit structures into higher-resolution data as full protein flexibility is considered, but are cumbersome in terms of computational time. Here, we introduce a coarse-grained approach; a Go-model was used to represent biological molecules, combined with biased molecular dynamics to reproduce accurately conformational transitions. Illustrative examples on simulated data are shown. Accurate fittings can be obtained for resolution ranging from 5 to 20 Å. The approach was also tested on experimental data of Elongation Factor G and Escherichia coli RNA polymerase, where its validity is compared to previous models obtained from different techniques. This comparison demonstrates that quantitative flexible techniques, as opposed to manual docking, need to be considered to interpret low-resolution data.