Software tools for molecular microscopy

In our role as the editors of a special edition of the Journal of Structural Biology published in 1996 and devoted to the development of software tools, we offer our view of past developments and future prospects in this area. The astonishing progress in computer hardware over the past decade has fueled a significant increase in computational power available for the solution of macromolecular structures. At the same time the relatively slow growth and development of the accompanying software reflects the difficulties of developing large, complex and very specialized analytical methods.     

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.     

Atomic force microscopy of macromolecular interactions.

The introduction of functional imaging tools and techniques that operate at molecular-length scales has provided investigators with unique approaches to characterizing biomolecular structure and function relationships. Recent advances in the field of scanning probe techniques and, in particular, atomic force microscopy have yielded tantalizing insights into the dynamics of protein self-assembly and the mechanics of protein unfolding.     

Gorgon and pathwalking: macromolecular modeling tools for subnanometer resolution density maps

The complex interplay of proteins and other molecules, often in the form of large transitory assemblies, are critical to cellular function. Today, X-ray crystallography and electron cryo-microscopy (cryo-EM) are routinely used to image these macromolecular complexes, though often at limited resolutions. Despite the rapidly growing number of macromolecular structures, few tools exist for modeling and annotating structures in the range of 3-10 ? resolution. To address this need, we have developed a number of utilities specifically targeting subnanometer resolution density maps. As part of the 2010 Cryo-EM Modeling Challenge, we demonstrated two of our latest de novo modeling tools, Pathwalking and Gorgon, as well as a tool for secondary structure identification (SSEHunter) and a new rigid-body/flexible fitting tool in Gorgon. In total, we submitted 30 structural models from ten different subnanometer resolution data sets in four of the six challenge categories. Each of our utlities produced accurate structural models and annotations across the various density maps. In the end, the utilities that we present here offer users a robust toolkit for analyzing and modeling protein structure in macromolecular assemblies at non-atomic resolutions.     

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.     

Software identification tools for biodiversity and biosecurity monitoring

The taxonomic crisis, associated with declining taxonomic expertise and resources, poses serious problems for biodiversity and biosecurity monitoring and management. While there has been a dramatic increase in the amount of taxonomic information available on the Web, resulting from such global initiatives as Species 2000, All Species and the Global Biodiversity Information Facility (GBIF), the lack of identification services has meant this information is often not accessible to many potential users. Apart from the immediate problems raised by the inability of ecologists, conservation managers, plant health inspectors and other practitioners to identify specimens of concern, this in turn means they are also unable to access the increasing wealth of information about particular organisms that is potentially available to them.     

Graphical method for force analysis: macromolecular mechanics with atomic force microscopy

We present a graphical method for a unifying, quantitative analysis of molecular bonding-force measurements by atomic force microscopy (AFM). The method is applied to interpreting a range of phenomena commonly observed in the experimental AFM measurements of noncovalent, weak bonds between biological macromolecules. The analysis suggests an energy landscape underlying the intermolecular force and demonstrates that many observations, such as "snaps-on," "jumps-off," and hysteresis loops, are different manifestations of a double-well energy landscape. The analysis gives concrete definitions for the operationally defined "attractive" and "adhesive" forces in terms of molecular parameters. It is shown that these operationally defined quantities are usually functions of the experimental setup, such as the stiffness of the force probe and the rate of its movement. The analysis reveals a mechanical instability due to the multistate nature of molecular interactions and provides new insight into macromolecular viscosity. The graphical method can equally be applied to a quantitative analysis of multiple unfolding of subunits of the giant muscle protein titin under AFM.     

High-resolution cryo-electron microscopy on macromolecular complexes and cell organelles

Cryo-electron microscopy techniques and computational 3-D reconstruction of macromolecular assemblies are tightly linked tools in modern structural biology. This symbiosis has produced vast amounts of detailed information on the structure and function of biological macromolecules. Typically, one of two fundamentally different strategies is used depending on the specimens and their environment. A: 3-D reconstruction based on repetitive and structurally identical unit cells that allow for averaging, and B: tomographic 3-D reconstructions where tilt-series between approximately ±60 and ±70° at small angular increments are collected from highly complex and flexible structures that are beyond averaging procedures, at least during the first round of 3-D reconstruction. Strategies of group A are averaging-based procedures and collect large number of 2-D projections at different angles that are computationally aligned, averaged together, and back-projected in 3-D space to reach a most complete 3-D dataset with high resolution, today often down to atomic detail. Evidently, success relies on structurally repetitive particles and an aligning procedure that unambiguously determines the angular relationship of all 2-D projections with respect to each other. The alignment procedure of small particles may rely on their packing into a regular array such as a 2-D crystal, an icosahedral (viral) particle, or a helical assembly. Critically important for cryo-methods, each particle will only be exposed once to the electron beam, making these procedures optimal for highest-resolution studies where beam-induced damage is a significant concern. In contrast, tomographic 3-D reconstruction procedures (group B) do not rely on averaging, but collect an entire dataset from the very same structure of interest. Data acquisition requires collecting a large series of tilted projections at angular increments of 1–2° or less and a tilt range of ±60° or more. Accordingly, tomographic data collection exposes its specimens to a large electron dose, which is particularly problematic for frozen-hydrated samples. Currently, cryo-electron tomography is a rapidly emerging technology, on one end driven by the newest developments of hardware such as super-stabile microscopy stages as well as the latest generation of direct electron detectors and cameras. On the other end, success also strongly depends on new software developments on all kinds of fronts such as tilt-series alignment and back-projection procedures that are all adapted to the very low-dose and therefore very noisy primary data. Here, we will review the status quo of cryo-electron microscopy and discuss the future of cellular cryo-electron tomography from data collection to data analysis, CTF-correction of tilt-series, post-tomographic sub-volume averaging, and 3-D particle classification. We will also discuss the pros and cons of plunge freezing of cellular specimens to vitrified sectioning procedures and their suitability for post-tomographic volume averaging despite multiple artifacts that may distort specimens to some degree.     

Computational methods for analyzing conformational variability of macromolecular complexes from cryo-electron microscopy images

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Software tools for analyzing pairwise alignments of long sequences.

Pairwise comparison of long stretches of genomic DNA sequence can identify regions conserved across species, which often indicate functional significance. However, the novel insights frequently must be windowed from a flood of information; for instance, running an alignment program on two 50-kilobase sequences might yield over a hundred pages of alignments. Direct inspection of such a volume of printed output is infeasible, or at best highly undesirable, and computer tools are needed to summarize the information, to assist in its analysis, and to report the findings. This paper describes two such software tools. One tool prepares publication-quality pictorial representations of alignments, while another facilitates interactive browsing of pairwise alignment data. Their effectiveness is illustrated by comparing the beta-like globin gene clusters between humans and rabbits. A second example compares the chloroplast genomes of tobacco and liverwort.     

Software for systems biology: from tools to integrated platforms

Understanding complex biological systems requires extensive support from software tools. Such tools are needed at each step of a systems biology computational workflow, which typically consists of data handling, network inference, deep curation, dynamical simulation and model analysis. In addition, there are now efforts to develop integrated software platforms, so that tools that are used at different stages of the workflow and by different researchers can easily be used together. This Review describes the types of software tools that are required at different stages of systems biology research and the current options that are available for systems biology researchers. We also discuss the challenges and prospects for modelling the effects of genetic changes on physiology and the concept of an integrated platform.     

Atomic force microscopy imaging of DNA under macromolecular crowding conditions

Studying the influence of macromolecular crowding at high ionic strengths on assemblies of biomolecules is of particular interest because these are standard intracellular conditions. However, up to now, no techniques offer the possibility of studying the effect of molecular crowding at the single molecule scale and at high resolution. We present a method to observe double-strand DNA under macromolecular crowding conditions on a flat mica surface by atomic force microscope. By using high concentrations of monovalent salt ([NaCl] > 100 mM), we promote DNA adsorption onto NiCl 2 pretreated muscovite mica. It therefore allows analysis of DNA conformational changes and DNA compaction induced by polyethylene glycol (PEG), a neutral crowding agent, at physiological concentrations of monovalent salt.     

Quantitative microscopy and imaging tools for the mechanical analysis of morphogenesis

The importance of mechanical signals during embryogenesis and development, through both intercellular and extracellular signals, is coming into focus. It is widely hypothesized that physical forces help to guide the shape, cellular differentiation and the patterning of tissues. To test these ideas many classical engineering principles and imaging technologies are being adapted. Recent advances in microscopy, mechanical testing and genetic and pharmacological techniques, alongside computational models are helping to dissect the activity of mechanical signals in development at the cellular and molecular level. These inroads are providing maps of mechanical changes in tissue structure and stiffness, and will permit deeper insights into the role of mechanics in both developmental biology and disease.     

Molecular resolution imaging of macromolecular crystals by atomic force microscopy.

Atomic force microscopy (AFM) images at the molecular level have been obtained for a number of different protein and virus crystals. They can be utilized in some special cases to obtain information useful to crystal structure analyses by x-ray diffraction. In particular, questions of space group enantiomer, the packing of molecules within a unit cell, the number of molecules per asymmetric unit, and the dispositions of multiple molecules within the asymmetric unit may be resolved. In addition, because of the increasing sensitivity and resolution of the AFM technique, some molecular features of very large asymmetric units may be within reach. We describe here high-resolution studies, using AFM, to visualize individual molecules and viruses in their crystal lattices. These investigations included fungal lipase, lysozyme, thaumatin, canavalin, and satellite tobacco mosaic virus (STMV).     

A novel mirror-symmetry analysis approach for the study of macromolecular assemblies imaged by electron microscopy

Multivariate statistical symmetry analysis is widely employed in single-particle electron-microscopy studies for the detection of symmetry components within a set of noisy two-dimensional images. So far, this technique has been used to retrieve information from the analysis of end-on view oriented particles only. Here, we propose a method to detect symmetry components from side- and tilted-view oriented particles. This method is validated using a number of in silico generated as well as real datasets, can be used to analyze stoichiometrically heterogeneous datasets, and is useful for separating particle datasets with respect to their symmetry components. Additionally, translational components in lock-washer ring configurations can be detected. Most relevantly, this method represents a powerful tool for the characterisation of distinct symmetry components within multi-layered protein assemblies, and any putative symmetry mismatch between layers. Such configurations have often been postulated, though rarely observed directly, and are thought to have a crucial role in conferring dynamicity to molecular machineries like nucleic acid packaging motors, ClpAP/ClpXP proteases, flagellar motors and the F1/F0 ATPase.     

Software tools and databases for bacterial systematics and their disseminationvia global networks

The dynamic expansion of the taxonomic knowledge base is fundamental to further developments in biotechnology and sustainable conservation strategies. The vast array of software tools for numerical taxonomy and probabilistic identification, in conjunction with automated systems for data generation are allowing the construction of large computerised strain databases. New techniques available for the generation of chemical and molecular data, associated with new software tools for data analysis, are leading to a quantum leap in bacterial systematics. The easy exchange of data through an interactive and highly distributed global computer network, such as the Internet, is facilitating the dissemination of taxonomic data. Relevant information for comparative sequence analysis, ribotyping, protein and DNA electrophoretic pattern analysis is available on-line through computerised networks. Several software packages are available for the analysis of molecular data. Nomenclatural and taxonomic Authority Files are available from different sources together with strain specific information. The increasing availability of public domain software, is leading to the establishment and integration of public domain databases all over the world, and promoting co-operative research projects on a scale never seen before.