Low-Resolution Density Maps from Atomic Models: How Stepping “Back” Can Be a Step “Forward”

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15–40 Å), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

Low-resolution density maps from atomic models: how stepping "back" can be a step "forward"

Atomic-resolution structures have had a tremendous impact on modern biological science. Much useful information also has been gleaned by merging and correlating atomic-resolution structural details with lower-resolution (15-40 A), three-dimensional (3D) reconstructions computed from images recorded with cryo-transmission electron microscopy (cryoTEM) procedures. One way to merge these structures involves reducing the resolution of an atomic model to a level comparable to a cryoTEM reconstruction. A low-resolution density map can be derived from an atomic-resolution structure by retrieving a set of atomic coordinates editing the coordinate file, computing structure factors from the model coordinates, and computing the inverse Fourier transform of the structure factors. This method is a useful tool for structural studies primarily in combination with 3D cryoTEM reconstructions. It has been used to assess the quality of 3D reconstructions, to determine corrections for the phase-contrast transfer function of the transmission electron microscope, to calibrate the dimensions and handedness of 3D reconstructions, to produce difference maps, to model features in macromolecules or macromolecular complexes, and to generate models to initiate model-based determination of particle orientation and origin parameters for 3D reconstruction.     

Subdiffraction‐resolution live‐cell imaging for visualizing thylakoid membranes

The chloroplast is the chlorophyll‐containing organelle that produces energy through photosynthesis. Within the chloroplast is an intricate network of thylakoid membranes containing photosynthetic membrane proteins that mediate electron transport and generate chemical energy. Historically, electron microscopy (EM) has been a powerful tool for visualizing the macromolecular structure and organization of thylakoid membranes. However, an understanding of thylakoid membrane dynamics remains elusive because EM requires fixation and sectioning. To improve our knowledge of thylakoid membrane dynamics we need to consider at least two issues: (i) the live‐cell imaging conditions needed to visualize active processes in vivo; and (ii) the spatial resolution required to differentiate the characteristics of thylakoid membranes. Here, we utilize three‐dimensional structured illumination microscopy (3D‐SIM) to explore the optimal imaging conditions for investigating the dynamics of thylakoid membranes in living plant and algal cells. We show that 3D‐SIM is capable of examining broad characteristics of thylakoid structures in chloroplasts of the vascular plant Arabidopsis thaliana and distinguishing the structural differences between wild‐type and mutant strains. Using 3D‐SIM, we also visualize thylakoid organization in whole cells of the green alga Chlamydomonas reinhardtii. These data reveal that high light intensity changes thylakoid membrane structure in C. reinhardtii. Moreover, we observed the green alga Chromochloris zofingiensis and the moss Physcomitrella patens to show the applicability of 3D‐SIM. This study demonstrates that 3D‐SIM is a promising approach for studying the dynamics of thylakoid membranes in photoautotrophic organisms during photoacclimation processes.     

Microscopy in 3D: a biologist's toolbox

The power of fluorescence microscopy to study cellular structures and macromolecular complexes spans a wide range of size scales, from studies of cell behavior and function in physiological 3D environments to understanding the molecular architecture of organelles. At each length scale, the challenge in 3D imaging is to extract the most spatial and temporal resolution possible while limiting photodamage/bleaching to living cells. Several advances in 3D fluorescence microscopy now offer higher resolution, improved speed, and reduced photobleaching relative to traditional point-scanning microscopy methods. We discuss a few specific microscopy modalities that we believe will be particularly advantageous in imaging cells and subcellular structures in physiologically relevant 3D environments.     

Visualization of the nucleus and nuclear envelope in situ by SEM in tissue culture cells

Our previous work characterizing the biogenesis and structural integrity of the nuclear envelope and nuclear pore complexes (NPCs) has been based on amphibian material but has recently progressed into the analysis of tissue-culture cells. This protocol describes methods for the high resolution visualization, by field-emission scanning electron microscopy (FESEM), of the nucleus and associated structures in tissue culture cells. Imaging by fluorescence light microscopy shows general nuclear and NPC information at a resolution of approximately 200 nm, in contrast to the 3-5 nm resolution provided by FESEM or transmission electron microscopy (TEM), which generates detail at the macromolecular level. The protocols described here are applicable to all tissue culture cell lines tested to date (HeLa, A6, DLD, XTC and NIH 3T3). The processed cells can be stored long term under vacuum. The protocol can be completed in 5 d, including 3 d for cell growth, 1 d for processing and 1 d for imaging.     

Computational approaches for automatic structural analysis of large biomolecular complexes

We present computational solutions to two problemsof macromolecular structure interpretation from reconstructedthree-dimensional electron microscopy (3D-EM) maps of largebio-molecular complexes at intermediate resolution (5A-15A). Thetwo problems addressed are: (a) 3D structural alignment (matching)between identified and segmented 3D maps of structure units(e.g. trimeric configuration of proteins), and (b) the secondarystructure identification of a segmented protein 3D map (i.e.locations of a-helices, b -sheets). For problem (a), we presentan efficient algorithm to correlate spatially (and structurally)two 3D maps of structure units. Besides providing a similarityscore between structure units, the algorithm yields an effectivetechnique for resolution refinement of repeated structure units,by 3D alignment and averaging. For problem (b), we present anefficient algorithm to compute eigenvalues and link eigenvectorsof a Gaussian convoluted structure tensor derived from theprotein 3D Map, thereby identifying and locating secondarystructural motifs of proteins. The efficiency and performanceof our approach is demonstrated on several experimentallyreconstructed 3D maps of virus capsid shells from single-particlecryo-EM, as well as computationally simulated protein structuredensity 3D maps generated from protein model entries in theProtein Data Bank.     

Defining and solving the essential protein-protein interactions in HIV infection

The structure determination of macromolecular complexes is entering a new era. The methods of optical microscopy, electron microscopy, X-ray crystallography, and nuclear magnetic resonance increasingly are being combined in hybrid method approaches to achieve an integrated view of macromolecular complexes that span from cellular context to atomic detail. A particularly important application of these hybrid method approaches is the structural analysis of the Human Immunodeficiency Virus (HIV) proteins with their cellular binding partners. High resolution structure determination of essential HIV - host cell protein complexes and correlative analysis of these complexes in the live cell can serve as critical guides in the design of a broad, new class of therapeutics that function by disrupting such complexes. Here, with the hope of stimulating some discussion, we will briefly review some of the literature in the context of what could be done to further apply structural methods to HIV research. We have chosen to focus our attention on certain aspects of the HIV replication cycle where we think that structural information would contribute substantially to the development of new therapeutic and vaccine targets for HIV.     

A Highlights from MBoC Selection: Three-dimensional superresolution fluorescence microscopy maps the variable molecular architecture of the nuclear pore complex

Nuclear pore complexes (NPCs) are large macromolecular machines that mediate the traffic between the nucleus and the cytoplasm. In vertebrates, each NPC consists of ∼1000 proteins, termed nucleoporins, and has a mass of more than 100 MDa. While a pseudo-atomic static model of the central scaffold of the NPC has recently been assembled by integrating data from isolated proteins and complexes, many structural components still remain elusive due to the enormous size and flexibility of the NPC. Here, we explored the power of three-dimensional (3D) superresolution microscopy combined with computational classification and averaging to explore the 3D structure of the NPC in single human cells. We show that this approach can build the first integrated 3D structural map containing both central as well as peripheral NPC subunits with molecular specificity and nanoscale resolution. Our unbiased classification of more than 10,000 individual NPCs indicates that the nuclear ring and the nuclear basket can adopt different conformations. Our approach opens up the exciting possibility to relate different structural states of the NPC to function in situ.     

DynBench3D,a Web-Resource to Dynamically Generate Benchmark Sets of Large Heteromeric Protein Complexes

Multi-protein machines are responsible for most cellular tasks, and many efforts have been invested in the systematic identification and characterization of thousands of these macromolecular assemblies. However, unfortunately, the (quasi) atomic details necessary to understand their function are available only for a tiny fraction of the known complexes. The computational biology community is developing strategies to integrate structural data of different nature, from electron microscopy to X-ray crystallography, to model large molecular machines, as it has been done for individual proteins and interactions with remarkable success. However, unlike for binary interactions, there is no reliable gold-standard set of three-dimensional (3D) complexes to benchmark the performance of these methodologies and detect their limitations. Here, we present a strategy to dynamically generate non-redundant sets of 3D heteromeric complexes with three or more components. By changing the values of sequence identity and component overlap between assemblies required to define complex redundancy, we can create sets of representative complexes with known 3D structure (i.e., target complexes). Using an identity threshold of 20% and imposing a fraction of component overlap of < 0.5, we identify 495 unique target complexes, which represent a real non-redundant set of heteromeric assemblies with known 3D structure. Moreover, for each target complex, we also identify a set of assemblies, of varying degrees of identity and component overlap, that can be readily used as input in a complex modeling exercise (i.e., template subcomplexes). We hope that resources like this will significantly help the development and progress assessment of novel methodologies, as docking benchmarks and blind prediction contests did. The interactive resource is accessible at https://DynBench3D.irbbarcelona.org.     

Symmetry-adapted spherical harmonics method for high-resolution 3D single-particle reconstructions

Three-dimensional (3D) reconstruction is the last and an essential step toward high-resolution structural determination in single-particle cryo-electron microscopy (cryoEM). We have implemented a new algorithm for reconstructing 3D structures of macromolecular complexes with icosahedral symmetry from cryoEM images. Icosahedral symmetry-adapted functions (ISAFs) are used to interpolate structural factors in the reciprocal space to generate a 3D reconstruction in spherical coordinates. In our implementation, we introduced a recursive method for deriving higher order ISAFs from three lower order seed functions. We demonstrate improvements of our new method in both the noise suppression and the effective resolution in 3D reconstruction over the commonly used Fourier-Bessel synthesis method introduced by Crowther et al. three decades ago. Our 3D reconstruction method can be extended to macromolecular complexes with other symmetry types and is thus likely to impact future high-resolution cryoEM single-particle reconstruction efforts in general.     

Visualizing Proteins and Macromolecular Complexes by Negative Stain EM: from Grid Preparation to Image Acquisition

Single particle electron microscopy (EM), of both negative stained or frozen hydrated biological samples, has become a versatile tool in structural biology ¹. In recent years, this method has achieved great success in studying structures of proteins and macromolecular complexes ^{2, 3}. Compared with electron cryomicroscopy (cryoEM), in which frozen hydrated protein samples are embedded in a thin layer of vitreous ice ⁴, negative staining is a simpler sample preparation method in which protein samples are embedded in a thin layer of dried heavy metal salt to increase specimen contrast ⁵. The enhanced contrast of negative stain EM allows examination of relatively small biological samples. In addition to determining three-dimensional (3D) structure of purified proteins or protein complexes ⁶, this method can be used for much broader purposes. For example, negative stain EM can be easily used to visualize purified protein samples, obtaining information such as homogeneity/heterogeneity of the sample, formation of protein complexes or large assemblies, or simply to evaluate the quality of a protein preparation.In this video article, we present a complete protocol for using an EM to observe negatively stained protein sample, from preparing carbon coated grids for negative stain EM to acquiring images of negatively stained sample in an electron microscope operated at 120kV accelerating voltage. These protocols have been used in our laboratory routinely and can be easily followed by novice users.     

Distribution and movement of membrane-associated platelet glycoproteins: use of colloidal gold with correlative video-enhanced light microscopy, low-voltage high-resolution scanning electron microscopy, and high-voltage transmission electron microscopy

Scanning electron microscopy (SEM), especially low-voltage (1 KeV) high-resolution SEM, can be used in conjunction with stereo pair high-voltage (1 MeV) transmission electron microscopy (HVEM) of whole spread cells or thick sections effectively to correlate surface structure with internal structure. Surface features such as microvilli, pits, pseudopodia, ruffles, attached virus, and other surface-related morphologic characteristics can be identified using SEM, while underlying cytoskeletal structure and organelle organization can be viewed by HVEM of the same preparation. However, the need to "prepare" cells for electron microscopy precludes observation in the living state. The use of several types of video-enhanced light microscopy (VLM) permits observation of living cells such that certain surface and internal features can be observed at a relatively high level of resolution or detection. Thus, changes in living cells can be followed, and at appropriate times the cells may be chemically fixed or rapidly frozen and prepared for ultrastructural examination by electron microscopy. We have utilized VLM in conjunction with SEM and HVEM to correlate changes in shape and surface structure with changes in the internal structure of platelets. In addition, we have found it advantageous to use colloidal gold-labeling procedures, because these markers are detectable by all three forms of microscopy. Using this approach we have labeled platelet membrane GPIIb/IIIa, a receptor for RGD-containing adhesive proteins, with gold-fibrinogen or gold-anti-IIb/IIIa. The initial binding and subsequent movement of gold-fibrinogen-IIb/IIIa complexes in living platelets was followed by VLM. The movement of individual labels could be mapped. Subsequent observation by low-voltage (1 KeV) high-resolution SEM and HVEM permits visualization of the same individual receptors tracked by LM. The final position on the membrane or the position-in-transit when fixative was added was determined relative to surface ultrastructure (SEM) and internal, particularly cytoskeletal, ultrastructure (HVEM).     

1D2DSimScore: A novel method for comparing contacts in biomacromolecules and their complexes

The biologically relevant structures of proteins and nucleic acids and their complexes are dynamic. They include a combination of regions ranging from rigid structural segments to structural switches to regions that are almost always disordered, which interact with each other in various ways. Comparing conformational changes and variation in contacts between different conformational states is essential to understand the biological functions of proteins, nucleic acids, and their complexes. Here, we describe a new computational tool, 1D2DSimScore, for comparing contacts and contact interfaces in all kinds of macromolecules and macromolecular complexes, including proteins, nucleic acids, and other molecules. 1D2DSimScore can be used to compare structural features of macromolecular models between alternative structures obtained in a particular experiment or to score various predictions against a defined “ideal” reference structure. Comparisons at the level of contacts are particularly useful for flexible molecules, for which comparisons in 3D that require rigid-body superpositions are difficult, and in biological systems where the formation of specific inter-residue contacts is more relevant for the biological function than the maintenance of a specific global 3D structure. Similarity/dissimilarity scores calculated by 1D2DSimScore can be used to complement scores describing 3D structural similarity measures calculated by the existing tools.     

Exploring vitreous cryo-section-induced compression at the macromolecular level using electron cryo-tomography; 80S yeast ribosomes appear unaffected

Vitreous cryo-section-induced compression influences the interpretation and the reliability of electron microscopy images and tomographic reconstructions. Previous studies of this deformation have been focused at the cellular level where considerable compression occurs, yet the degree of possible intracellular macromolecular deformation has remained unclear. Here, electron cryo-tomographic reconstructions of vitreous cryo-sections show that 80S ribosomes, both intracellular and in an isolated state, appear able to resist section-induced compression. Our observations indicate that vitreous section-induced compression is non-uniform between whole cells that have been sectioned and their intracellular macromolecular complexes. We conclude that electron cryo-tomography of vitreous cryo-sections, in spite of section-induced compression, is a suitable technique for charting the structural organization of cellular nanomachines, such as ribosomes, in a cellular environment.     

Single particle macromolecular structure determination via electron microscopy

Three-dimensional structure determination of macromolecules and macromolecular complexes is an integral part of understanding biological functions. For large protein and macromolecular complexes structure determination is often performed using electron cryomicroscopy where projection images of individual macromolecular complexes are combined to produce a three-dimensional reconstruction. Single particle methods have been devised to perform this structure determination for macromolecular complexes with little or no underlying symmetry. These computational methods generally involve an iterative process of aligning unique views of the macromolecular images followed by determination of the angular components that define those views. In this review, this structure determination process is described with the aim of clarifying a seemingly complex structural method.     

Structural studies of the human PBAF chromatin-remodeling complex

ATP-dependent chromatin remodeling is one of the central processes responsible for imparting fluidity to chromatin and thus regulating DNA transactions. Although knowledge on this process is accumulating rapidly, the basic mechanism (or mechanisms) by which the remodeling complexes alter the structure of a nucleosome is not yet understood. Structural information on these macromolecular machines should aid in interpreting the biochemical and genetic data; to this end, we have determined the structure of the human PBAF ATP-dependent chromatin-remodeling complex preserved in negative stain by electron microscopy and have mapped the nucleosome binding site using two-dimensional (2D) image analysis. PBAF has an overall C-shaped architecture--with a larger density to which two smaller knobs are attached--surrounding a central cavity; one of these knobs appears to be flexible and occupies different positions in each of the structures determined. The 2D analysis of PBAF:nucleosome complexes indicates that the nucleosome binds in the central cavity.     

3‐D analysis of bacterial cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using complementary microscopy tomography approaches

The formation of cell‐(iron)mineral aggregates as a consequence of bacterial iron oxidation is an environmentally widespread process with a number of implications for processes such as sorption and coprecipitation of contaminants and nutrients. Whereas the overall appearance of such aggregates is easily accessible using 2‐D microscopy techniques, the 3‐D and internal structure remain obscure. In this study, we examined the 3‐D structure of cell‐(iron)mineral aggregates formed during Fe(II) oxidation by the nitrate‐reducing Acidovorax sp. strain BoFeN1 using a combination of advanced 3‐D microscopy techniques. We obtained 3‐D structural and chemical information on different cellular encrustation patterns at high spatial resolution (4–200 nm, depending on the method): more specifically, (1) cells free of iron minerals, (2) periplasm filled with iron minerals, (3) spike‐ or platelet‐shaped iron mineral structures, (4) bulky structures on the cell surface, (5) extracellular iron mineral shell structures, (6) cells with iron mineral filled cytoplasm, and (7) agglomerations of extracellular globular structures. In addition to structural information, chemical nanotomography suggests a dominant role of extracellular polymeric substances (EPS) in controlling the formation of cell‐(iron)mineral aggregates. Furthermore, samples in their hydrated state showed cell‐(iron)mineral aggregates in pristine conditions free of preparation (i.e., drying/dehydration) artifacts. All these results were obtained using 3‐D microscopy techniques such as focused ion beam (FIB)/scanning electron microscopy (SEM) tomography, transmission electron microscopy (TEM) tomography, scanning transmission (soft) X‐ray microscopy (STXM) tomography, and confocal laser scanning microscopy (CLSM). It turned out that, due to the various different contrast mechanisms of the individual approaches, and due to the required sample preparation steps, only the combination of these techniques was able to provide a comprehensive understanding of structure and composition of the various Fe‐precipitates and their association with bacterial cells and EPS.