Implementation of a flash-photolysis system for time-resolved cryo-electron microscopy

We describe here the implementation of a flash-photolysis system for time-resolved cryo-electron microscopy. A previously designed computer-controlled cryo-plunging apparatus [White, H.D., Thirumurugan, K., Walker, M.L., Trinick, J., 2003. A second generation apparatus for time-resolved electron cryo-microscopy using stepper motors and electrospray. J. Struct. Biol. 144, 246–252] was used as a hardware platform, onto which a xenon flash lamp and liquid light pipe were mounted. The irradiation initiates a reaction through cleavage of the photolabile blocking group from a biologically active compound. The timespan between flashing and freezing in cryogen is on the order of milliseconds, and defines the fastest observable reaction. Blotting of excess fluid, which takes on the order of 1 s, is done before irradiation and thus does not represent a rate-limiting step. A specimen-heating problem, identified by measurements with a thermocouple, was alleviated with the use of thick, aluminum-coated grids.     

Structure determination of clathrin coats to subnanometer resolution by single particle cryo-electron microscopy

Clathrin triskelions can assemble into lattices of different shapes, sizes and symmetries. For many years, the structures of clathrin lattices have been studied by single particle cryo-electron microscopy, which probed the architecture of the D6 hexagonal barrel clathrin coat at the molecular level. By introducing additional image processing steps we have recently produced a density map for the D6 barrel clathrin coat at subnanometer resolution, enabling us to generate an atomic model for this lattice [Fotin, A., Cheng, Y., Sliz, P., Grigorieff, N., Harrison, S.C., Kirchhausen, T., Walz, T., 2004. Molecular model for a complete clathrin lattice from electron cryomicroscopy. Nature 432, 573-579]. We describe in detail here the image processing steps that we have added to produce a density map at this high resolution. These procedures should be generally applicable and may thus help determine the structures of other large protein assemblies to higher resolution by single particle cryo-electron microscopy.     

Three-dimensional reconstruction of bovine brain V-ATPase by cryo-electron microscopy and single particle analysis

Bovine V-ATPase from brain clathrin-coated vesicles was investigated by cryo-electron microscopy and single particle analysis. Our studies revealed great flexibility of the central linker region connecting V₁ and V₀. As a consequence, the two sub-complexes were processed separately and the resulting volumes were merged computationally. We present the first three-dimensional (3D) map of a V-ATPase obtained from cryo-electron micrographs. The overall resolution was estimated 34 Å by Fourier shell correlation (0.5 cutoff). Our 3D reconstruction shows a large peripheral stalk and a smaller, isolated peripheral density, suggesting a second, less well-resolved peripheral connection. The 3D map reveals new features of the large peripheral stator and of the collar-like density attached to the membrane domain. Our analyses of the membrane domain indicate the presence of six proteolipid subunits. In addition, we could localize the V₀ subunit a flanking the large peripheral stalk.     

Tools for correlative cryo-fluorescence microscopy and cryo-electron tomography applied to whole mitochondria in human endothelial cells

Cryo-electron tomography (cryo-ET) allows for the visualization of biological material in a close-to-native state, in three dimensions and with nanometer scale resolution. However, due to the low signal-to-noise ratio inherent to imaging of the radiation-sensitive frozen-hydrated samples, it appears oftentimes impossible to localize structures within heterogeneous samples. Because a major potential for cryo-ET is thereby left unused, we set out to combine cryo-ET with cryo-fluorescence microscopy (cryo-FM), in order to facilitate the search for structures of interest. We describe a cryo-FM setup and workflow for correlative cryo-fluorescence and cryo-electron microscopy (cryo-CLEM) that can be easily implemented. Cells are grown on finder grids, vitally labeled with one or two fluorescent dyes, and vitrified. After a structure is located by cryo-FM (with 0.4 μm resolution), its image coordinates are translated to cryo-ET stage coordinates via a home-built software routine. We tested our workflow on whole mount primary human umbilical vein endothelial cells. The correlative routine enabled us to investigate mitochondrial ultrastructure for the first time on intact human mitochondria, and led us to find mitochondrial cristae that were connected to the intermembrane space via large slits, which challenges the current view that such connections are established exclusively via small circular pores. Taken together, this study emphasizes that cryo-CLEM can be a routinely used technique that opens up exciting new possibilities for cryo-ET.     

Initial evaluation of a direct detection device detector for single particle cryo-electron microscopy

We report on initial results of using a new direct detection device (DDD) for single particle reconstruction of vitreous ice embedded specimens. Images were acquired on a Tecnai F20 at 200 keV and a nominal magnification of 29,000×. This camera has a significantly improved signal to noise ratio and modulation transfer function (MTF) at 200 keV compared to a standard CCD camera installed on the same microscope. Control of the DDD has been integrated into Leginon, an automated data collection system. Using GroEL as a test specimen, we obtained images of ∼30 K particles with the CCD and the DDD from the same specimen sample using essentially identical imaging conditions. Comparison of the maps reconstructed from the CCD images and the DDD images demonstrates the improved performance of the DDD. We also obtained a 3D reconstruction from ∼70 K GroEL particles acquired using the DDD; the quality of the density map demonstrates the potential of this new recording device for cryoEM data acquisition.     

Improved specimen preparation for cryo-electron microscopy using a symmetric carbon sandwich technique

Image shift due to beam-induced specimen charging has become the most severe problem in electron microscopy for imaging two-dimensional (2D) crystals of biological macromolecules, especially in the case of highly tilted specimens. Image shift causes diffraction spots perpendicular to the tilt axis to disappear even at medium or low resolution. The yield of good images from tilted specimens prepared on a single layer of continuous carbon support film is therefore very low. In this paper, we have used 2D crystals of aquaporin-4 to investigate the effect of a carbon sandwich preparation method on specimen charging. We find that a larger number of images show sharp diffraction spots perpendicular to the tilt axis if crystals are placed in between two sheets of carbon film as compared to images taken from specimens prepared by the conventional single carbon support film technique. Our results demonstrate that the reproducible carbon sandwich preparation technique overcomes the severe specimen charging problem and thus has the potential to significantly speed up structure analysis by electron crystallography.     

Cryo-electron microscopy for structural analysis of dynamic biological macromolecules

Background

Since the introduction of what became today's standard for cryo-embedding of biological macromolecules at native conditions more than 30 years ago, techniques and equipment have been drastically improved and the structure of biomolecules can now be studied at near atomic resolution by cryo-electron microscopy (cryo-EM) while capturing multiple dynamic states. Here we review the recent progress in cryo-EM for structural studies of dynamic biological macromolecules.

Deep generative modeling for volume reconstruction in cryo-electron microscopy

Advances in cryo-electron microscopy (cryo-EM) for high-resolution imaging of biomolecules in solution have provided new challenges and opportunities for algorithm development for 3D reconstruction. Next-generation volume reconstruction algorithms that combine generative modelling with end-to-end unsupervised deep learning techniques have shown promise, but many technical and theoretical hurdles remain, especially when applied to experimental cryo-EM images. In light of the proliferation of such methods, we propose here a critical review of recent advances in the field of deep generative modelling for cryo-EM reconstruction. The present review aims to (i) provide a unified statistical framework using terminology familiar to machine learning researchers with no specific background in cryo-EM, (ii) review the current methods in this framework, and (iii) outline outstanding bottlenecks and avenues for improvements in the field.     

Novel organic dyes for multicolor localization‐based super‐resolution microscopy

Precise multicolor single molecule localization‐based microscopy (SMLM) requires bright probes with compatible photo‐chemical and spectral properties to resolve distinct molecular species at the nanoscale. The accuracy of multicolor SMLM is further challenged by color channel crosstalk and chromatic alignment errors. These constrains limit the applicability of known reversibly switchable organic dyes for optimized multicolor SMLM. Here, we tested 28 commercially available dyes for their suitability to super‐resolve a known cellular nanostructure. We identified eight novel dyes in different spectral regimes that enable high quality dSTORM imaging. Among those, the spectrally close dyes CF647 and CF680 comprise an optimal dye pair for spectral demixing‐based, registration free multicolor dSTORM with low crosstalk. Combining this dye pair with the separately excited CF568 we performed 3‐color dSTORM to image the relative nanoscale distribution of components of the endocytic machinery and the cytoskeleton.

A major limitation of multicolor single molecule localization based super‐resolution microscopy (SMLM) is the availability of suitable photo‐switchable fluorescent dyes. By screening 28 commercially available dyes, novel dyes in different spectral regimes were identified that are well suited for dual and triple color SMLM with low crosstalk. These novel dyes are employed to image the relative nanoscale distribution of sub‐cellular components.     

Serial block face‐scanning electron microscopy: A tool for studying embryonic development at the cell–matrix interface

Studies of gene regulation, signaling pathways, and stem cell biology are contributing greatly to our understanding of early embryonic vertebrate development. However, much less is known about the events during the latter half of embryonic development, when tissues comprising mostly extracellular matrix (ECM) are formed. The matrix extends far beyond the boundaries of individual cells and is refractory to study by conventional biochemical and molecular techniques; thus major gaps exist in our knowledge of the formation and three‐dimensional (3D) organization of the dense tissues that form the bulk of adult vertebrates. Serial block face‐scanning electron microscopy (SBF‐SEM) has the ability to image volumes of tissue containing numerous cells at a resolution sufficient to study the organization of the ECM. Furthermore, whereas light microscopy was once relatively straightforward and electron microscopy was performed in specialist laboratories, the tables are turned; SBF‐SEM is relatively straightforward and is becoming routine in high‐end resolution studies of embryonic structures in vivo. In this review, we discuss the emergence of SBF‐SEM as a tool for studying embryonic vertebrate development. Birth Defects Research (Part C) 105:9–18, 2015. © 2015 Wiley Periodicals, Inc.     

Combinatorial microscopy for the study of protein–membrane interactions in supported lipid bilayers: Order parameter measurements by combined polarized TIRFM/AFM

Understanding the mechanisms of peptide-induced membrane disorder is critical to the design of novel antimicrobial and cell-penetrating peptides. One means of quantifying local structure and order/disorder is through the orientational order parameter, typically obtained using various spectroscopic approaches. We report here on the use of an image-based means of tracking the order parameter in supported lipid bilayers during peptide-induced disordering. By coupling polarized total internal reflection fluorescence microscopy with in situ atomic force microscopy, it is now possible to track changes in order parameter associated with peptide binding and insertion, as well as lipid headgroup and acyl chain reordering, while simultaneously resolving molecular-scale topographical changes. Interactions between the model antimicrobial peptide, indolicidin, and its fluorescent analog, TAMRA-indolicidin, with model eukaryotic (DOPC:DSPC:cholesterol) and prokaryotic (DOPE/DOPG) membranes were tracked using the fluorescent lipid reporters, DiI-C₂₀ and BODIPY-PC. Changes in the order parameter upon membrane binding and insertion provided insights into the orientation of the peptide and the role of membrane chemistry and composition on insertion dynamics and membrane restructuring.