Validation of the orthogonal tilt reconstruction method with a biological test sample

Electron microscopy of frozen-hydrated samples (cryo-EM) can yield high resolution structures of macromolecular complexes by accurately determining the orientation of large numbers of experimental views of the sample relative to an existing 3D model. The "initial model problem", the challenge of obtaining these orientations ab initio, remains a major bottleneck in determining the structure of novel macromolecules, chiefly those lacking internal symmetry. We previously proposed a method for the generation of initial models--orthogonal tilt reconstruction (OTR)--that bypasses limitations inherent to the other two existing methods, random conical tilt (RCT) and angular reconstitution (AR). Here we present a validation of OTR with a biological test sample whose structure was previously solved by RCT: the complex between the yeast exosome and the subunit Rrp44. We show that, as originally demonstrated with synthetic data, OTR generates initial models that do not exhibit the "missing cone" artifacts associated with RCT and show an isotropic distribution of information when compared with the known structure. This eliminates the need for further user intervention to solve these artifacts and makes OTR ideal for automation and the analysis of heterogeneous samples. With the former in mind, we propose a set of simple quantitative criteria that can be used, in combination, to select from a large set of initial reconstructions a subset that can be used as reliable references for refinement to higher resolution.     

The orthogonal tilt reconstruction method: an approach to generating single-class volumes with no missing cone for ab initio reconstruction of asymmetric particles

Generating reliable initial models is a critical step in the reconstruction of asymmetric single-particles by 3D electron microscopy. This is particularly difficult to do if heterogeneity is present in the sample. The Random Conical Tilt (RCT) method, arguably the most robust presently to accomplish this task, requires significant user intervention to solve the "missing cone" problem. We present here a novel approach, termed the orthogonal tilt reconstruction method, that eliminates the missing cone altogether, making it possible for single-class volumes to be used directly as initial references in refinement without further processing. The method involves collecting data at +45 degrees and -45 degrees tilts and only requires that particles adopt a relatively large number of orientations on the grid. One tilted data set is used for alignment and classification and the other set--which provides views orthogonal to those in the first--is used for reconstruction, resulting in the absence of a missing cone. We have tested this method with synthetic data and compared its performance to that of the RCT method. We also propose a way of increasing the level of homogeneity in individual 2D classes (and volumes) in a heterogeneous data set and identifying the most homogeneous volumes.     

Structure of functional single AQP0 channels in phospholipid membranes

Aquaporin-0 (AQP0) is the most prevalent intrinsic protein in the plasma membrane of lens fiber cells where it functions as a water selective channel and also participates in fiber-fiber adhesion. We report the 3D envelope of purified AQP0 reconstituted with random orientation in phospholipid bilayers as single particles. The envelope was obtained by combining freeze-fracture, shadowing and random conical tilt electron microscopy followed by single particle image processing. Two-dimensional analysis of 2547 untilted images produced eight class averages exhibiting "square" and "octagonal" shapes with a continuum of variation. We reconstructed in 3D five class averages that best described the data set. The reconstructions ("molds") appeared as metal cups exhibiting external and internal surfaces. We used the internal surface of the mold to calculate the "imprints" that represent the AQP0 particles protruding from the hydrophobic core of the phospholipid bilayer. The complete envelope of the channel, formed by joining the square and octagonal imprints, described accurately the size, shape, oligomeric state, orientation, and molecular weight of the AQP0 channel inserted in the phospholipid bilayer. Rigid body docking of the atomic model of the aquaporin-1 (AQP1) tetramer showed that the freeze-fracture envelope accounted for the conserved transmembrane domain (approximately 73% similarity between AQP0 and AQP1) but not for the amino and carboxyl termini. We suggest that the discrepancy might reflect differences in the location of the amino and carboxyl termini in the crystal and in the phospholipid bilayer.     

Automated acquisition of electron microscopic random conical tilt sets

Single particle reconstruction using the random conical tilt data collection geometry is a robust method for the initial determination of macromolecular structures by electron microscopy. Unfortunately, the broad adoption of this powerful approach has been limited by the practical challenges inherent in manual data collection of the required pairs of matching high and low tilt images (typically 60 degrees and 0 degrees). The microscopist is obliged to keep the imaging area centered during tilting as well as to maintain accurate focus in the tilted image while minimizing the overall electron dose, a challenging and time consuming process. To help solve these problems, we have developed an automated system for the rapid acquisition of accurately aligned and focused tilt pairs. The system has been designed to minimize the dose incurred during alignment and focusing, making it useful in both negative stain and cryo-electron microscopy. The system includes a feature for montaging untilted images to ensure that all of the particles in the tilted image may be used in the reconstruction.     

virtualspecies,an R package to generate virtual species distributions

virtualspecies is a freely available package for R designed to generate virtual species distributions, a procedure increasingly used in ecology to improve species distribution models. This package combines the existing methodological approaches with the objective of generating virtual species distributions with increased ecological realism. The package includes 1) generating the probability of occurrence of a virtual species from a spatial set of environmental conditions (i.e. environmental suitability), with two different approaches; 2) converting the environmental suitability into presence–absence with a probabilistic approach; 3) introducing dispersal limitations in the realised virtual species distributions and 4) sampling occurrences with different biases in the sampling procedure. The package was designed to be extremely flexible, to allow users to simulate their own defined species–environment relationships, as well as to provide a fine control over every simulation parameter. The package also includes a function to generate random virtual species distributions. We provide a simple example in this paper showing how increasing ecological realism of the virtual species impacts the predictive performance of species distribution models. We expect that this new package will be valuable to researchers willing to test techniques and protocols of species distribution models as well as various biogeographical hypotheses.     

XMIPP: a new generation of an open-source image processing package for electron microscopy

X-windows based microscopy image processing package (Xmipp) is a specialized suit of image processing programs, primarily aimed at obtaining the 3D reconstruction of biological specimens from large sets of projection images acquired by transmission electron microscopy. This public-domain software package was introduced to the electron microscopy field eight years ago, and since then it has changed drastically. New methodologies for the analysis of single-particle projection images have been added to classification, contrast transfer function correction, angular assignment, 3D reconstruction, reconstruction of crystals, etc. In addition, the package has been extended with functionalities for 2D crystal and electron tomography data. Furthermore, its current implementation in C++, with a highly modular design of well-documented data structures and functions, offers a convenient environment for the development of novel algorithms. In this paper, we present a general overview of a new generation of Xmipp that has been re-engineered to maximize flexibility and modularity, potentially facilitating its integration in future standardization efforts in the field. Moreover, by focusing on those developments that distinguish Xmipp from other packages available, we illustrate its added value to the electron microscopy community.     

Dynactin 3D Structure: Implications for Assembly and Dynein Binding

The multisubunit protein complex, dynactin, is an essential component of the cytoplasmic dynein motor. High-resolution structural work on dynactin and the dynein/dynactin supercomplex has been limited to small subunits and recombinant fragments that do not report fully on either ≈ 1 MDa assembly. In the present study, we used negative-stain electron microscopy and image analysis based on random conical tilt reconstruction to obtain a three-dimensional (3D) structure of native vertebrate dynactin. The 35-nm-long dynactin molecule has a V-shaped shoulder at one end and a flattened tip at the other end, both offset relative to the long axis of the actin-related protein (Arp) backbone. The shoulder projects dramatically away from the Arp filament core in a way that cannot be appreciated in two-dimensional images, which has implications for the mechanism of dynein binding. The 3D structure allows the helical parameters of the entire Arp filament core, which includes the actin capping protein, CP, to be determined for the first time. This structure exhibits near identity to F-actin and can be well fitted into the dynactin envelope. Molecular fitting of modeled CP-Arp polymers into the envelope shows that the filament contains between 7 and 9 Arp protomers and is capped at both ends. In the 7 Arp model, which agrees best with measured Arp stoichiometry and other structural information, actin capping protein (CP) is not present at the distal tip of the structure, unlike what is seen in the other models. The 3D structure suggests a mechanism for dynactin assembly and length specification.     

Conical tomography II: A method for the study of cellular organelles in thin sections

We have used conical electron tomography in order to reconstruct neuronal organelles in thin sections of plastic embedded rat somato-sensory cortical tissue. The conical tilt series were collected at a 55 degrees tilt and at 5 degrees rotations, aligned using gold particles as fiduciary markers, and reconstructed using the weighted back projection algorithm. After a refinement process based on projection matching, the 3D maps showed the "unit membrane pattern" along the entire reconstructed volume. This pattern is indicative of the bilayer arrangement of phospholipids in biological membranes. Based on Fourier correlation methods as well as the visualization of the "unit membrane" pattern, we estimated resolutions of approximately 4 nm. To illustrate the prospective advantages of conical tomography, we segmented "coated" vesicles in the reconstructed volumes. These vesicles were comprised of a central core enclosing a small lumen, and a protein "coating" extending into the cytoplasm. The "coated" vesicle was attached to the plasma membrane through a complex structure shaped as an arch where the ends are attached to the membrane and the crook is connected to the vesicle. We concluded that conical electron tomography of thin-sectioned specimens provides a powerful experimental approach for studying thin-sectioned neuronal organelles at resolution levels of approximately 4 nm.     

Tomographic subvolume alignment and subvolume classification applied to myosin V and SIV envelope spikes

Electron tomography is a technique for three-dimensional reconstruction, that is widely used for imaging macromolecules, macromolecular assemblies or whole cells. Combined with cryo-electron microscopy, it is capable of visualizing structural detail in a state close to in vivo conditions in the cell. In electron tomography, micrographs are taken while tilting the specimen to different angles about a fixed axis. Due to mechanical constraints, the angular tilt range is limited. As a consequence, the reconstruction of a 3D image is missing data, which for a single axis tilt series is called the “missing wedge”, a region in reciprocal space where Fourier coefficients cannot be obtained experimentally. Tomographic data is analyzed by extracting subvolumes from the raw tomograms, by alignment of the extracted subvolumes, multivariate data analysis, classification, and class-averaging, which results in an increased signal-to-noise ratio and substantial data reduction. Subvolume analysis is a valuable tool to discriminate heterogeneous populations of macromolecules, or conformations of a macromolecule or macromolecular assembly as well as to characterize interactions between macromolecules. However, this analysis is hampered by the lack of data in the original tomograms caused by the missing wedge. Here, we report enhancements of our subvolume processing protocols in which the problem of the missing data in reciprocal space is addressed by using constrained correlation and weighted averaging in reciprocal space. These procedures are applied to the analysis of myosin V and simian immunodeficiency virus (SIV) envelope spikes. We also investigate the effect of the missing wedge on image classification and establish limits of reliability by model calculations with generated phantoms.     

The first three-dimensional structure of phosphofructokinase from Saccharomyces cerevisiae determined by electron microscopy of single particles

Phosphofructokinaseis a key regulatory enzyme of the glycolytic pathway. We have determined the structure of this enzyme from Saccharomyces cerevisiae to a resolution of 2.0 nm. This is the first structure available for this family of enzymes in eukaryotic organisms. Phosphofructokinase is an octamer composed of 4alpha and 4beta subunits arranged in a dihedral point group symmetry D(2). The enzyme has a very open and elongated structure, with dimensions of 24 nm in length and 17 nm in width. The final structure, calculated from 0 degrees tilt projections of the molecule at random orientations using as reference the volume obtained by the random conical reconstruction technique in ice, has allowed us to discern the shapes of the subunits and their mutual arrangement in the octamer.     

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.     

Why do so many petals have conical epidermal cells?

Background

The conical epidermal cells found on the petals of most Angiosperm species are so widespread that they have been used as markers of petal identity, but their function has only been analysed in recent years. This review brings together diverse data on the role of these cells in pollination biology.

Scope

The published effects of conical cells on petal colour, petal reflexing, scent production, petal wettability and pollinator grip on the flower surface are considered. Of these factors, pollinator grip has been shown to be of most significance in the well-studied Antirrhinum majus/bumble-bee system. Published data on the relationship between epidermal cell morphology and floral temperature were limited, so an analysis of the effects of cell shape on floral temperature in Antirrhinum is presented here. Statistically significant warming by conical cells was not detected, although insignificant trends towards faster warming at dawn were found, and it was also found that flat-celled flowers could be warmer on warm days. The warming observed is less significant than that achieved by varying pigment content. However, the possibility that the effect of conical cells on temperature might be biologically significant in certain specific instances such as marginal habitats or weather conditions cannot be ruled out.

Conclusions

Conical epidermal cells can influence a diverse set of petal properties. The fitness benefits they provide to plants are likely to vary with pollinator and habitat, and models are now required to understand how these different factors interact.     

IMIRS: a high-resolution 3D reconstruction package integrated with a relational image database

Recent advances in electron cryomicroscopy instrumentation and single particle reconstruction have created opportunities for high-throughput and high-resolution three-dimensional (3D) structure determination of macromolecular complexes. However, it has become impractical and inefficient to rely on conventional text file data management and command-line programs to organize and process the increasing numbers of image data required in high-resolution studies. Here, we present a distributed relational database for managing complex datasets and its integration into our high-resolution software package IMIRS (Image Management and Icosahedral Reconstruction System). IMIRS consists of a complete set of modular programs for icosahedral reconstruction organized under a graphical user interface and provides options for user-friendly, step-by-step data processing as well as automatic reconstruction. We show that the integration of data management with processing in IMIRS automates the tedious tasks of data management, enables data coherence, and facilitates information sharing in a distributed computer and user environment without significantly increasing the time of program execution. We demonstrate the applicability of IMIRS in icosahedral reconstruction toward high resolution by using it to obtain an 8-A 3D structure of an intermediate-sized dsRNA virus.     

GGT 2.0: versatile software for visualization and analysis of genetic data

Ever since its first release in 1999, the free software package for visualization of molecular marker data, graphical genotype (GGT), has been constantly adapted and improved. The GGT package was developed in a plant-breeding context and thus focuses on plant genetic data but was not intended to be limited to plants only. The current version has many options for genetic analysis of populations including diversity analyses and simple association studies. A second release of the GGT package, GGT 2.0 (available through http://www.plantbreeding.wur.nl), is therefore presented in this paper. An overview of existing and new features that are available within GGT 2.0, and a case study in which GGT 2.0 is applied to analyze an existing set of plant genetic data, are presented and discussed.     

Localization of the coactivator Cdh1 and the cullin subunit Apc2 in a cryo-electron microscopy model of vertebrate APC/C

The anaphase-promoting complex/cyclosome (APC/C) is a ubiquitin ligase with essential functions in mitosis, meiosis, and G1 phase of the cell cycle. APC/C recognizes substrates via coactivator proteins such as Cdh1, and bound substrates are ubiquitinated by E2 enzymes that interact with a hetero-dimer of the RING subunit Apc11 and the cullin Apc2. We have obtained three-dimensional (3D) models of human and Xenopus APC/C by angular reconstitution and random conical tilt (RCT) analyses of negatively stained cryo-electron microscopy (cryo-EM) preparations, have determined the masses of these particles by scanning transmission electron microscopy (STEM), and have mapped the locations of Cdh1 and Apc2. These proteins are located on the same side of the asymmetric APC/C, implying that this is where substrates are ubiquitinated. We have further identified a large flexible domain in APC/C that adopts a different orientation upon Cdh1 binding. Cdh1 may thus activate APC/C both by recruiting substrates and by inducing conformational changes.     

Three-dimensional structure of the respiratory chain supercomplex I1III2IV1 from bovine heart mitochondria

The respiratory chain complexes can arrange into multienzyme assemblies, so-called supercomplexes. We present the first 3D map of a respiratory chain supercomplex. It was determined by random conical tilt electron microscopy analysis of a bovine supercomplex consisting of complex I, dimeric complex III, and complex IV (I1III2IV1). Within this 3D map the positions and orientations of all the individual complexes in the supercomplex were determined unambiguously. Furthermore, the ubiquinone and cytochrome c binding sites of each complex in the supercomplex could be located. The mobile electron carrier binding site of each complex was found to be in proximity to the binding site of the succeeding complex in the respiratory chain. This provides structural evidence for direct substrate channeling in the supercomplex assembly with short diffusion distances for the mobile electron carriers.