Robust filtering and particle picking in micrograph images towards 3D reconstruction of purified proteins with cryo-electron microscopy

In order to make a high resolution model of macromolecular structures from cryo-electron microscope (cryo-EM) raw images one has to be precise at every processing step from particle picking to 3D image reconstruction. In this paper we propose a collection of novel methods for filtering cryo-EM images and for automatic picking of particles. These methods have been developed for two cases: (1) when particles can be identified and (2) when particle are not distinguishable. The advantages of these methods are demonstrated in standard purified protein samples and to generalize them we do not use any ad hoc presumption of the geometry of the particle projections. We have also suggested a filtering method to increase the signal-to-noise (S/N) ratio which has proved to be useful for other levels of reconstruction, i.e., finding orientations and 3D model reconstruction.     

Reference-free particle selection enhanced with semi-supervised machine learning for cryo-electron microscopy

Reference-based methods have dominated the approaches to the particle selection problem, proving fast, and accurate on even the most challenging micrographs. A reference volume, however, is not always available and compiling a set of reference projections from the micrographs themselves requires significant effort to attain the same level of accuracy. We propose a reference-free method to quickly extract particles from the micrograph. The method is augmented with a new semi-supervised machine-learning algorithm to accurately discriminate particles from contaminants and noise.     

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.     

Fast automatic particle picking from cryo-electron micrographs using a locally normalized cross-correlation function: a case study

Recent progress in single-particle reconstruction methods and cryo-EM techniques has led to the determination of macromolecular structures with unprecedented resolution. The number of particles that goes into the reconstruction is a key determinant in achieving high resolution. Interactive manual picking of particles from an electron micrograph is a very time-consuming, tedious, and inefficient process. We have implemented a fast automatic particle picking procedure in the SPIDER environment. The procedure makes use of template matching schemes and employs a recently developed locally normalized correlation algorithm based on Fourier techniques. As a test, we have used this procedure to pick 70S Escherichia coli ribosomes from a cryo-electron micrograph. Different search strategies including use of a circular mask and asymmetric masks for different orientations of the particle have been explored, and their relative efficiencies are discussed. The results indicate that the procedure can be optimally used to pick ribosomes in a fully automatic way within the limit of selecting less than 10% false positives while missing about 15% of true positives.     

Preparation of macromolecular complexes for cryo-electron microscopy

This protocol describes the preparation of frozen-hydrated single-particle specimens of macromolecular complexes. First, it describes how to create a grid surface coated with holey carbon by first inducing holes in a Formvar film to act as a template for the holey carbon that is stable under cryo-electron microscopy (cryo-EM) conditions and is sample-friendly. The protocol then describes the steps required to deposit the homogeneous sample on the grid and to plunge-freeze the grid into liquid ethane at the temperature of liquid nitrogen, so that it is suitable for cryo-EM visualization. It takes 4-5 h to make several hundred holey carbon grids and about 1 h to make the frozen-hydrated grids. The time required for sample purification varies from hours to days, depending on the sample and the specific procedure required. A companion protocol details how to collect cryo-EM data using an FEI Tecnai transmission electron microscope that can subsequently be processed to obtain a three-dimensional reconstruction of the macromolecular complex.     

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.     

Development of imaging scaffolds for cryo-electron microscopy

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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.     

Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy

Cryo-EM samples prepared using traditional methods often suffer from too few particles, poor particle distribution, strongly biased orientation, or damage from the air-water interface. Here we report that functionalization of graphene oxide (GO) coated grids with amino groups concentrates samples on the grid with improved distribution and orientation. By introducing a PEG spacer, particles are kept away from both the GO surface and the air-water interface, protecting them from potential denaturation.     

Fast projection matching for cryo-electron microscopy image reconstruction

A new FFT-accelerated projection matching method is presented and tested. The electron microscopy images are represented by their Fourier-Bessel transforms and the 3D model by its expansion in spherical harmonics, or more specifically in terms of symmetry-adapted functions. The rotational and translational properties of these representations are used to quickly access all the possible 2D projections of the 3D model, which allow an exhaustive inspection of the whole five-dimensional domain of parameters associated to each particle.     

Gold nanoparticle-protein arrays improve resolution for cryo-electron microscopy

Cryo-electron microscopy single particle analysis shows limited resolution due to poor alignment precision of noisy images taken under low electron exposure. Certain advantages can be obtained by assembling proteins into two-dimensional (2D) arrays since protein particles are locked into repetitive orientation, thus improving alignment precision. We present a labeling method to prepare protein 2D arrays using gold nanoparticles (NPs) interconnecting genetic tag sites on proteins. As an example, mycobacterium tuberculosis 20S proteasomes tagged with 6x-histidine were assembled into 2D arrays using 3.9-nm Au NPs functionalized with nickel-nitrilotriacetic acid. The averaged top-view images from the array particles showed higher resolution (by 6-8A) compared to analysis of single particles. The correct 7-fold symmetry was also evident by using array particles whereas it was not clear by analysis of a comparable number of single particles. The applicability of this labeling method for three-dimensional reconstruction of biological macromolecules is discussed.     

Ribosomal dynamics explored by cryo-electron microscopy

Cryo-electron microscopy has furnished direct evidence for conformational changes of the ribosome as it proceeds, in a cyclic manner, through different functional states. Strategies to explore the ribosome dynamics include trapping of particular functionally meaningful states by chemical, genetic, or physical means. The new atomic information obtained by X-ray crystallography should make it possible to track conformational changes observed by cryo-electron microscopy to changes of the underlying structural framework of the ribosome.     

Metallothionein as a clonable high-density marker for cryo-electron microscopy

Cryo-electron microscopy is expanding its scope from macromolecules towards much larger and more complex cellular specimens such as organelles, cells and entire tissues. While isolated macromolecular specimens are typically composed of only very few different components that may be recognized by their shape, size or state of polymerization, cellular specimens combine large numbers of proteinaceous structures as well as nucleic acids and lipid arrays. Consequently, an unambiguous identification of these structures within the context of a whole cell may create a very difficult challenge. On plastic-embedded specimens, or Tokuyasu sections (Tokuyasu, 1980), epitopes that are exposed at the surface can be tagged by antibodies. However, vitrified sections have to be kept at strict cryo-conditions (below -140 °C) and therefore do not allow any post-sectioning treatment of the specimens other than data acquisition in the microscope. Hence, the labels have to be placed into the specimen before freezing. Here we report on the application of a small metal-clustering protein, metallothionein (MTH), as a clonable label capable of clustering metal atoms into a high-density particle with high spatial resolution. We tested MTH as a label for kinesin-decorated microtubules (MTs) as well as the building blocks of desmin intermediate filaments (IFs).     

Golgi apparatus analyzed by cryo-electron microscopy

In 1898, the Golgi apparatus was discovered by light microscopy, and since the 1950s, the ultrastructure composition is known by electron microscopic investigation. The complex three-dimensional morphology fascinated researchers and was sometimes even the driving force to develop novel visualization techniques. However, the highly dynamic membrane systems of Golgi apparatus are delicate and prone to fixation artifacts. Therefore, the understanding of Golgi morphology and its function has been improved significantly with the development of better preparation methods. Nowadays, cryo-fixation is the method of choice to arrest instantly all dynamic and physiological processes inside cells, tissues, and small organisms. Embedded in amorphous ice, such samples can be further processed by freeze substitution or directly analyzed in their fully hydrated state by cryo-electron microscopy and tomography. Even though the overall morphology of vitrified Golgi stacks is comparable to well-prepared and resin-embedded samples, previously unknown structural details can be observed solely based on their native density. At this point, any further improvement of sample preparation would gain novel insights, perhaps not in terms of general morphology, but on fine structural details of this dynamic organelle.     

Focused-ion-beam thinning of frozen-hydrated biological specimens for cryo-electron microscopy

Cryo-electron microscopy can provide high-resolution structural information about cells and organelles in the nearly native, frozen-hydrated state. Applicability, however, is limited by difficulties encountered in preparing suitably thin, vitreously frozen biological specimens. We demonstrate, by cryo-electron tomography of Escherichia coli cells, that a focused ion beam (FIB) can be used to thin whole frozen-hydrated cells in a convenient and essentially artifact-free way.     

Conformational changes studied by cryo-electron microscopy

Biological processes involving movement, such as muscle contraction or the opening of an ion channel through a membrane, are mediated through conformational changes. These changes often occur in large and flexible macromolecular complexes. Cryo-electron microscopy provides a means of capturing different conformational states of such assemblies. Even if the resulting density maps are at low resolution, they can be combined with atomic structures of subcomplexes or isolated components determined by X-ray crystallography or NMR. This review presents a brief summary of the principles and recent advances in macromolecular structure determination by cryo-electron microscopy.     

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.     

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.