Miniaturizing EM Sample Preparation: Opportunities, Challenges, and "Visual Proteomics".

This review compares and discusses conventional versus miniaturized specimen preparation methods for transmission electron microscopy (TEM). The progress brought by direct electron detector cameras, software developments and automation have transformed transmission cryo-electron microscopy (cryo-EM) and made it an invaluable high-resolution structural analysis tool. In contrast, EM specimen preparation has seen very little progress in the last decades and is now one of the main bottlenecks in cryo-EM. Here, we discuss the challenges faced by specimen preparation for single particle EM, highlight current developments, and show the opportunities resulting from the advanced miniaturized and microfluidic sample grid preparation methods described, such as visual proteomics and time-resolved cryo-EM studies.

Principles Governing the Self-Assembly of Coiled-Coil Protein Nanoparticles.

Self-assembly refers to the spontaneous organization of individual building blocks into higher order structures. It occurs in biological systems such as spherical viruses, which utilize icosahedral symmetry as a guiding principle for the assembly of coat proteins into a capsid shell. In this study, we characterize the self-assembling protein nanoparticle (SAPN) system, which was inspired by such viruses. To facilitate self-assembly, monomeric building blocks have been designed to contain two oligomerization domains. An N-terminal pentameric coiled-coil domain is linked to a C-terminal coiled-coil trimer by two glycine residues. By combining monomers with inherent propensity to form five- and threefold symmetries in higher order agglomerates, the supposition is that nanoparticles will form that exhibit local and global symmetry axes of order 3 and 5. This article explores the principles that govern the assembly of such a system. Specifically, we show that the system predominantly forms according to a spherical core-shell morphology using a combination of scanning transmission electron microscopy and small angle neutron scattering. We introduce a mathematical toolkit to provide a specific description of the possible SAPN morphologies, and we apply it to characterize all particles with maximal symmetry. In particular, we present schematics that define the relative positions of all individual chains in the symmetric SAPN particles, and provide a guide of how this approach can be generalized to nonspherical morphologies, hence providing unprecedented insights into their geometries that can be exploited in future applications.

Contributions of the lower dimer to supramolecular actin patterning revealed by TIRF microscopy.

Two distinct dimers are formed during the initial steps of actin polymerization. The first one, referred to as the 'lower dimer' (LD) was discovered many years ago by means of chemical crosslinking. Owing to its transient nature, a biological relevance had long been precluded when, using LD-specific antibodies, we detected LD-like contacts in actin assemblies that are associated with the endolysosomal compartment in a number of different cell lines. Moreover, immunofluorescence showed the presence of LD-related structures at the cell periphery of migrating fibroblasts, in the nucleus, and in association with the centrosome of interphase cells. Here, we explore contributions of the LD to the assembly of supramolecular actin structures in real time by total internal reflection fluorescence (TIRF) microscopy. Our data shows that while LD on its own cannot polymerize under filament forming conditions, it is able to incorporate into growing F-actin filaments. This incorporation of LD triggers the formation of X-shaped filament assemblies with barbed ends that are pointing in the same direction in the majority of cases. Similarly, an increased frequency of junction sites was observed when filaments were assembled in the presence of oxidized actin. This data suggests that a disulfide bridge between Cys374 residues might stabilize LD-contacts. Based on our findings, we propose two possible models for the molecular mechanism underlying the supramolecular actin patterning in LD-related structures.

Cholesteryl ester transfer between lipoproteins does not require a ternary tunnel complex with CETP.

The cholesteryl ester transfer protein (CETP) enables the transfer of cholesteryl ester (CE) from high-density lipoproteins (HDL) to low-density lipoproteins (LDL) in the plasma compartment. CETP inhibition raises plasma levels of HDL cholesterol; a ternary tunnel complex with CETP bridging HDL and LDL was suggested as a mechanism. Here, we test whether the inhibition of CETP tunnel complex formation is a promising approach to suppress CE transfer from HDL to LDL, for potential treatment of cardio-vascular disease (CVD). Three monoclonal antibodies against different epitopes of CETP are assayed for their potential to interfere with CE transfer between HDL and/or LDL. Surprisingly, antibodies that target the tips of the elongated CETP molecule, interaction sites sterically required to form the suggested transfer complexes, do not interfere with CETP activity, but an antibody binding to the central region does. We show that CETP interacts with HDL, but not with LDL. Our findings demonstrate that a ternary tunnel complex is not the mechanistic prerequisite to transfer CE among lipoproteins.

Atomic structure and hierarchical assembly of a cross-ß amyloid fibril.

The cross-ß amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of ß-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-ß amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale--including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy--we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent ß-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

Optimizing the design of protein nanoparticles as carriers for vaccine applications.

Successful vaccine development remains a huge challenge for infectious diseases such as malaria, HIV and influenza. As a novel way to present antigenic epitopes to the immune system, we have developed icosahedral self-assembling protein nanoparticles (SAPNs) to serve as a prototypical vaccine platform for infectious diseases. Here we examine some biophysical factors that affect the self-assembly of these nanoparticles, which have as basic building blocks coiled-coil oligomerization domains joined by a short linker region. Relying on in silico computer modeling predictions, we selected five different linker regions from the RCSB protein database that connect oligomerization domains, and then further studied the self-assembly and stability of in vitro produced nanoparticles through biophysical characterization of formed particles. One design in particular, T2i88, revealed excellent self-assembly and homogeneity thus paving the way toward a more optimized nanoparticle for vaccine applications. FROM THE CLINICAL EDITOR: Despite the widespread use of vaccines worldwide, successful development of vaccines against some diseases remains a challenge still. In this article, the authors investigated the physic-chemical and biological properties of icosahedral self-assembling protein nanoparticles (SAPNs), which mimic viral particles, in order to utilize this technology as potential platform for future design of vaccines.

openBEB: open biological experiment browser for correlative measurements.

BACKGROUND: New experimental methods must be developed to study interaction networks in systems biology. To reduce biological noise, individual subjects, such as single cells, should be analyzed using high throughput approaches. The measurement of several correlative physical properties would further improve data consistency. Accordingly, a considerable quantity of data must be acquired, correlated, catalogued and stored in a database for subsequent analysis. RESULTS: We have developed openBEB (open Biological Experiment Browser), a software framework for data acquisition, coordination, annotation and synchronization with database solutions such as openBIS. OpenBEB consists of two main parts: A core program and a plug-in manager. Whereas the data-type independent core of openBEB maintains a local container of raw-data and metadata and provides annotation and data management tools, all data-specific tasks are performed by plug-ins. The open architecture of openBEB enables the fast integration of plug-ins, e.g., for data acquisition or visualization. A macro-interpreter allows the automation and coordination of the different modules. An update and deployment mechanism keeps the core program, the plug-ins and the metadata definition files in sync with a central repository. CONCLUSIONS: The versatility, the simple deployment and update mechanism, and the scalability in terms of module integration offered by openBEB make this software interesting for a large scientific community. OpenBEB targets three types of researcher, ideally working closely together: (i) Engineers and scientists developing new methods and instruments, e.g., for systems-biology, (ii) scientists performing biological experiments, (iii) theoreticians and mathematicians analyzing data. The design of openBEB enables the rapid development of plug-ins, which will inherently benefit from the "house keeping" abilities of the core program. We report the use of openBEB to combine live cell microscopy, microfluidic control and visual proteomics. In this example, measurements from diverse complementary techniques are combined and correlated.

Molecular architecture of Streptococcus pneumoniae TIGR4 pili.

Although the pili of Gram-positive bacteria are putative virulence factors, little is known about their structure. Here we describe the molecular architecture of pilus-1 of Streptococcus pneumoniae, which is a major cause of morbidity and mortality worldwide. One major (RrgB) and two minor components (RrgA and RrgC) assemble into the pilus. Results from TEM and scanning transmission EM show that the native pili are approximately 6 nm wide, flexible filaments that can be over 1 microm long. They are formed by a single string of RrgB monomers and have a polarity defined by nose-like protrusions. These protrusions correlate to the shape of monomeric RrgB-His, which like RrgA-His and RrgC-His has an elongated, multi-domain structure. RrgA and RrgC are only present at the opposite ends of the pilus shaft, compatible with their putative roles as adhesin and anchor to the cell wall surface, respectively. Our structural analyses provide the first direct experimental evidence that the native S. pneumoniae pilus shaft is composed exclusively of covalently linked monomeric RrgB subunits oriented head-to-tail.

Looking back at a quarter-century of research at the Maurice E. Müller Institute for Structural Biology.

The Maurice E. Müller Institute, embedded in the infrastructure of the Biozentrum, University of Basel, was founded in 1985 and financed by the Maurice E. Müller Foundation of Switzerland. For 26 years its two founders, Ueli Aebi and Andreas Engel, pursued the vision of integrated structural biology. This paper reviews selected publications issuing from the Maurice E. Müller Institute for Structural Biology and marks the end of this era.

Optimizing the refolding conditions of self-assembling polypeptide nanoparticles that serve as repetitive antigen display systems.

Nanoparticles show great promise as potent vaccine candidates since they are readily taken up by the antigen presenting cells of the immune system. The particle size and the density of the B cell epitopes on the surface of the particles greatly influences the strength of the humoral immune response. We have developed a novel type of nanoparticle composed of peptide building blocks (Raman et al., 2006) and have used such particles to design vaccines against malaria and SARS (Kaba et al., 2009; Pimentel et al., 2009). Here we investigate the biophysical properties and the refolding conditions of a prototype of these self-assembling polypeptide nanoparticles (SAPNs). SAPNs are formed from a peptide containing a pentameric and a trimeric coiled-coil domain. At near physiological conditions the peptide self-assembles into about 27 nm, roughly spherical SAPNs. The average size of the SAPNs increases with the salt concentration. The optimal pH for their formation is between 7.5 and 8.5, while aggregation occurs at lower and higher values. A glycerol concentration of about 5% v/v is required for the formation of SAPNs with regular spherical shapes. These studies will help to optimize the immunological properties of SAPNs.

Connecting µ-fluidics to electron microscopy.

A versatile methodology for electron microscopy (EM) grid preparation enabling total content sample analysis is presented. A microfluidic-dialysis conditioning module to desalt or mix samples with negative stain solution is used, combined with a robotic writing table to micro-pattern the EM grids. The method allows heterogeneous samples of minute volumes to be processed at physiological pH for structure and mass analysis, and allows the preparation characteristics to be finely tuned.

The chlorosome of Chlorobaculum tepidum: size, mass and protein composition revealed by electron microscopy, dynamic light scattering and mass spectrometry-driven proteomics.

Chlorosomes, the antenna complexes of green bacteria, are unique antenna systems in which pigments are organized in aggregates. Studies on isolated chlorosomes from Chlorobaculum tepidum based on SDS-PAGE, immunoblotting and molecular biology have revealed that they contain ten chlorosomal proteins, but no comprehensive information is available about the protein composition of the entire organelle. To extend these studies, chlorosomes were isolated from C. tepidum using three related and one independent isolation protocol and characterized by absorption spectroscopy, tricine SDS-PAGE, dynamic light scattering (DLS) and electron microscopy. Tricine SDS-PAGE showed the presence of more than 20 proteins with molecular weights ranging between 6 and 70 kDa. The chlorosomes varied in size. Their hydrodynamic radius (R(h) ) ranged from 51 to 75 nm and electron microscopy indicated that they were on average 140 nm wide and 170 nm long. Furthermore, the mass of 184 whole chlorosome organelles determined by scanning transmission electron microscopy ranged from 27 to 237 MDa being on average 88 (±28) MDa. In contrast their mass-per-area was independent of their size, indicating that there is a strict limit to chlorosome thickness. The average protein composition of the C. tepidum chlorosome organelles was obtained by MS/MS-driven proteomics and for the first time a detailed protein catalogue of the isolated chlorosomal proteome is presented. Based on the proteomics results for chlorosomes isolated by different protocols, four proteins that are involved in the electron or ion transport are proposed to be tightly associated with or incorporated into C. tepidum chlorosomes as well as the ten Csm proteins known to date.

Amyloid structure and assembly: insights from scanning transmission electron microscopy.

Amyloid fibrils are filamentous protein aggregates implicated in several common diseases such as Alzheimer's disease and type II diabetes. Similar structures are also the molecular principle of the infectious spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and of the so-called yeast prions, inherited non-chromosomal elements found in yeast and fungi. Scanning transmission electron microscopy (STEM) is often used to delineate the assembly mechanism and structural properties of amyloid aggregates. In this review we consider specifically contributions and limitations of STEM for the investigation of amyloid assembly pathways, fibril polymorphisms and structural models of amyloid fibrils. This type of microscopy provides the only method to directly measure the mass-per-length (MPL) of individual filaments. Made on both in vitro assembled and ex vivo samples, STEM mass measurements have illuminated the hierarchical relationships between amyloid fibrils and revealed that polymorphic fibrils and various globular oligomers can assemble simultaneously from a single polypeptide. The MPLs also impose strong constraints on possible packing schemes, assisting in molecular model building when combined with high-resolution methods like solid-state nuclear magnetic resonance (NMR) and electron paramagnetic resonance (EPR).

Rhodopsin-transducin heteropentamer: three-dimensional structure and biochemical characterization.

The process of vision is initiated when the G protein-coupled receptor, rhodopsin (Rho), absorbs a photon and transitions to its activated Rho(∗) form. Rho(∗) binds the heterotrimeric G protein, transducin (G(t)) inducing GDP to GTP exchange and G(t) dissociation. Using nucleotide depletion and affinity chromatography, we trapped and purified the resulting nucleotide-free Rho(∗)·G(t) complex. Quantitative SDS-PAGE suggested a 2:1 molar ratio of Rho(∗) to G(t) in the complex and its mass determined by scanning transmission electron microscopy was 221±12kDa. A 21.6Å structure was calculated from projections of negatively stained Rho(∗)·G(t) complexes. The molecular envelope thus determined accommodated two Rho molecules together with one G(t) heterotrimer, corroborating the heteropentameric structure of the Rho(∗)·G(t) complex.

Bipartite design of a self-fibrillating protein copolymer with nanopatterned peptide display capabilities.

The development of biomatrices for technological and biomedical applications employs self-assembled scaffolds built from short peptidic motifs. However, biopolymers composed of protein domains would offer more varied molecular frames to introduce finer and more complex functionalities in bioreactive scaffolds using bottom-up approaches. Yet, the rules governing the three-dimensional organization of protein architectures in nature are complex and poorly understood. As a result, the synthetic fabrication of ordered protein association into polymers poses major challenges to bioengineering. We have now fabricated a self-assembling protein nanofiber with predictable morphologies and amenable to bottom-up customization, where features supporting function and assembly are spatially segregated. The design was inspired by the cross-linking of titin filaments by telethonin in the muscle sarcomere. The resulting fiber is a two-protein system that has nanopatterned peptide display capabilities as shown by the recruitment of functionalized gold nanoparticles at regular intervals of ∼ 5 nm, yielding a semiregular linear array over micrometers. This polymer promises the uncomplicated display of biologically active motifs to selectively bind and organize matter in the fine nanoscale. Further, its conceptual design has high potential for controlled plurifunctionalization.

MASDET-A fast and user-friendly multiplatform software for mass determination by dark-field electron microscopy.

Electron microscopy has been used to measure the mass of biological nanoparticles since the early 60s, and is the only way to obtain the mass of large structures or parameters such as the mass-per-length of filaments. The ability of this method to sort heterogeneous samples both in terms of mass and shape promises to make it a key tool for proteomics down to the single cell level. A new multiplatform software package, MASDET, that can be run under MATLAB or as a standalone program is described. Based on a user-friendly graphical interface MASDET streamlines mass evaluation and greatly increases the speed of required optimisation procedures. Importantly, the immediate application of Monte-Carlo simulations to describe multiple scattering is possible, allowing the mass analysis of thicker samples and the generation of mass thickness maps.

Assembly of Kch, a putative potassium channel from Escherichia coli.

Attempts to explore the structure and function of Kch, a putative potassium channel of Escherichia coli have yielded varying results; potassium-associated functions have been found in vivo but not in vitro. Here the kch gene is shown to produce two proteins, full-length Kch and the large C-terminal cytosolic domain (the RCK domain). Further, these two proteins are associated at the initial stages of purification. Previous structural studies of full-length Kch claim that the isolated protein forms large aggregates that are not suitable for analysis. The results presented here show that the purified protein sample, although heterogeneous, has one major population with a mass of about 400kDa, implying the presence of two Kch tetramers in a complex form. A three dimensional reconstruction at 25A based on electron microscopy data from negatively stained particles, revealed a 210A long and 95A wide complex in which the two tetrameric Kch units are linked by their RCK domains, giving rise to a large central ring of density. The formation of this dimer of tetramers on expression or during purification, may explain why attempts to reconstitute Kch into liposomes for activity measurements have failed.

Omp85(Tt) from Thermus thermophilus HB27: an ancestral type of the Omp85 protein family.

Proteins belonging to the Omp85 family are involved in the assembly of beta-barrel outer membrane proteins or in the translocation of proteins across the outer membrane in bacteria, mitochondria, and chloroplasts. The cell envelope of the thermophilic bacterium Thermus thermophilus HB27 is multilayered, including an outer membrane that is not well characterized. Neither the precise lipid composition nor much about integral membrane proteins is known. The genome of HB27 encodes one Omp85-like protein, Omp85(Tt), representing an ancestral type of this family. We overexpressed Omp85(Tt) in T. thermophilus and purified it from the native outer membranes. In the presence of detergent, purified Omp85(Tt) existed mainly as a monomer, composed of two stable protease-resistant modules. Circular dichroism spectroscopy indicated predominantly beta-sheet secondary structure. Electron microscopy of negatively stained lipid-embedded Omp85(Tt) revealed ring-like structures with a central cavity of approximately 1.5 nm in diameter. Single-channel conductance recordings indicated that Omp85(Tt) forms ion channels with two different conducting states, characterized by conductances of approximately 0.4 nS and approximately 0.65 nS, respectively.

What transmission electron microscopes can visualize now and in the future.

Our review concentrates on the progress made in high-resolution transmission electron microscopy (TEM) in the past decade. This includes significant improvements in sample preparation by quick-freezing aimed at preserving the specimen in a close-to-native state in the high vacuum of the microscope. Following advances in cold stage and TEM vacuum technology systems, the observation of native, frozen hydrated specimens has become a widely used approach. It fostered the development of computer guided, fully automated low-dose data acquisition systems allowing matched pairs of images and diffraction patterns to be recorded for electron crystallography, and the collection of entire tilt-series for electron tomography. To achieve optimal information transfer to atomic resolution, field emission electron guns combined with acceleration voltages of 200-300 kV are now routinely used. The outcome of these advances is illustrated by the atomic structure of mammalian aquaporin-O and by the pore-forming bacterial cytotoxin ClyA resolved to 12 A. Further, the Yersinia injectisome needle, a bacterial pseudopilus and the binding of phalloidin to muscle actin filaments were chosen to document the advantage of the high contrast offered by dedicated scanning transmission electron microscopy (STEM) and/or the STEM's ability to measure the mass of protein complexes and directly link this to their shape. Continued progress emerging from leading research laboratories and microscope manufacturers will eventually enable us to determine the proteome of a single cell by electron tomography, and to more routinely solve the atomic structure of membrane proteins by electron crystallography.

The Moraxella IgD-binding protein MID/Hag is an oligomeric autotransporter.

The immunoglobulin D (IgD)-binding protein MID/Hag of the human respiratory pathogen Moraxella catarrhalis is an outer membrane protein of approximately 200kDa belonging to the autotransporter family. MID also functions as an adhesin and hemagglutinin. In the present paper, the ultrastructure of MID was mapped. Using a series of Escherichia coli transformants, the last 210 aa of the C-terminal region were shown to translocate protein MID through the outer membrane suggesting that MID has a beta-barrel structure comprising of 10 transmembrane beta-sheets. Electron microscopy mapping with gold-labelled specific antibodies, and partial unravelling using guanidine hydrochloride showed that the rest of the MID protein forms an approximately 120nm long, fibrillar structure in which the individual monomers fold back on themselves to expose a globular distal domain at their tips comprising both the IgD-binding (MID962-1200) and adhesive (MID764-913) regions. This positions their N-termini close to the C-terminal membrane spanning domains. Mass measurements by scanning transmission electron microscopy (STEM) verified that the MID molecule is an oligomer.