Electron Cryomicroscopy of Biological Machines at Subnanometer Resolution

Advances in electron cryomicroscopy (cryo-EM) have made possible the structural determination of large biological machines in the resolution range of 6–9 Å. Rice dwarf virus and the acrosomal bundle represent two distinct types of machines amenable to cryo-EM investigations at subnanometer resolutions. However, calculating the density map is only the first step, and much analysis remains to extract structural insights and the mechanism of action in these machines. This paper will review the computational and visualization methodologies necessary for analysis (structure mining) of the computed cryo-EM maps of these machines. These steps include component segmentation, averaging based on local symmetry among components, density connectivity trace, incorporation of bioinformatics analysis, and fitting of high-resolution component data, if available. The consequences of these analyses can not only identify accurately some of the secondary structure elements of the molecular components in machines but also suggest structural mechanisms related to their biological functions

The role of the coherence in the cross-correlation analysis of diffraction patterns from two-dimensional dense mono-disperse systems

The investigation of the static and dynamic structural properties of colloidal systems relies on techniques capable of atomic resolution in real space and femtosecond resolution in time. Recently, the cross-correlation function (CCF) analysis of both X-rays and electron diffraction patterns from dilute and dense aggregates has demonstrated the ability to retrieve information on the sample's local order and symmetry. Open questions remain regarding the role of the beam coherence in the formation of the diffraction pattern and the properties of the CCF, especially in dense systems. Here, we simulate the diffraction patterns of dense two-dimensional monodisperse systems of different symmetries, varying the transverse coherence of the probing wave, and analyze their CCF. We study samples with different symmetries at different size scale, as for example, pentamers arranged into a four-fold lattice where each pentamer is surrounded by triangular lattices, both ordered and disordered. In such systems, different symmetry modulations are arising in the CCF at specific scattering vectors. We demonstrate that the amplitude of the CCF is a fingerprint of the degree of the ordering in the sample and that at partial transverse coherence, the CCF of a dense sample corresponds to that of an individual scattering object.Comment: 22 pages, 7 figure

Structural and biophysical analysis of important biomedical enzymes and nano-architectures

Dopa decarboxylase (DDC) is an important enzyme in the catecholamine biosynthesis pathways. Catecholamines, e.g., dopamine, serotonin, etc. often are the major neuromodulators or neurotransmitters. Hence, DDC plays a key role in regulation of neurodegenerative diseases like Parkinson’s disease (PD). In order to achieve a medicine for PD, a successful inhibitor for DDC, that could reduce the activity of DDC in the blood while making it more effective in brain, is required. An effective design of an inhibitor requires a detailed structural study of human DDC. It was aimed to solve the DDC structure by X-ray crystallography. In order to have enough protein the DDC encoding gene has been cloned in the pET21d vector which was later termed as pET-DDC-His. However, it required numerous trials and errors until a suitable condition for soluble DDC expression was found. Addition of additives like PLP, ethanol, a complex of sorbitol and betaine in the growth medium of the bacteria did not help bring the protein in the soluble part as it formed inclusion bodies. Several soluble protein fusions with DDC, like Thioredoxin and Glutathione-S-transferase were also not quite helpful towards achieving soluble expression of DDC. Finally, a coexpression of DDC along with bacterial chaperone proteins, e.g., GroEL and GroES (after cotransforming both the DDC and Chaperone protein encoding plasmid in the same E.coli cell, used for expression) lead to solubilization of recombinant human DDC. This enzyme was then purified to homogeneity by successively passing the crude bacterial proteins through Ni-chelate-affinity chromatography and Size Exclusion Chromatography. The purified protein (>90 % purity) did not produce a good yield (4mg/ 8L culture), but this was enough to start the initial crystallization trial. Using a scale up to a 50 L culture, quite a good amount of protein was achieved. The homogeneity of DDC was further confirmed by using Multi-Angle Light Scattering and Blue Native PAGE. The dimeric enzyme preparation was then utilized for crystallization using the Hanging Drop Vapor Diffusion method. In a particular condition of the crystal screens trigonal bipyramidal crystals formed. However, these crystals did not show good diffraction when bombarded with X-ray beams. Later, this particular crystallization condition remained irreproducible. The peptide nanoparticle, designed and produced in our lab, could possibly be a very valuable tool in biomedical applications, e.g., in designing vaccines, delivering drugs, bioimaging, serodiagnosis, etc. The design of the peptide nanoparticles is based on the application of the symmetry elements of virus icosahedral capsid on a specially designed building block peptide. The designed peptide building block contains two oligomerization motifs, i.e., a trimeric coiled coil and a pentameric coiled coil joined by a linker region. Sixty such peptide units, upon self-assembly, would produce peptide nanoparticle mimicking a small icosahedral virus particle. The peptide chains in the building block provide flexibility in the design so that an additional peptide could be attached to it at the C-terminus in order to functionalize the peptide nanoparticle for various biomedical applications. First of all, the functional peptide at the C-terminus could be an epitope for the antibody of a life threatening disease like HIV. These peptide nanoparticles can then function as the potent vaccine candidate for that particular disease. In this thesis work, I have attached the two epitopes against the two broadly neutralizing classes of antibody for HIV infection, 2F5 and 4E10, to the peptide nanoparticle. Secondly, another sequence of peptide, which proved to have the capacity of seeding gold on its surface, was attached to the building block peptide unit. The nanoparticle, functionalized with such a peptide, can decorate a gold layer surrounding it. Gold coating on the peptide nanoparticle scaffold can provide a nanostructure, called ‘nanoshells’, which could be very important in the field of therapeutics because of its ability in easy detection and quick treatment of cancer cells. Lastly, I added three peptides; those are recognized in the culture filtrates of M.tuberculosis isolated from TB patients, separately, to the basic peptide construct to form three different nanoparticles. Also, I tried to make a single nanoparticle that displays all the three peptides on its surface. Such a nanoparticle could be a very useful tool in the serodiagnosis or the antibody-based rapid detection of the deadly disease- Tuberculosis. The nanoparticle formation in each of the above-mentioned cases was more or less successful. One of the constructs could successfully even produce gold shells on the peptide nanoparticle

3D reconstruction of biological structures: automated procedures for alignment and reconstruction of multiple tilt series in electron tomography

Transmission electron microscopy allows the collection of multiple views of specimens and their computerized three-dimensional reconstruction and analysis with electron tomography. Here we describe development of methods for automated multi-tilt data acquisition, tilt-series processing, and alignment which allow assembly of electron tomographic data from a greater number of tilt series, yielding enhanced data quality and increasing contrast associated with weakly stained structures. This scheme facilitates visualization of nanometer scale details of fine structure in volumes taken from plastic-embedded samples of biological specimens in all dimensions. As heavy metal-contrasted plastic-embedded samples are less sensitive to the overall dose rather than the electron dose rate, an optimal resampling of the reconstruction space can be achieved by accumulating lower dose electron micrographs of the same area over a wider range of specimen orientations. The computerized multiple tilt series collection scheme is implemented together with automated advanced procedures making collection, image alignment, and processing of multi-tilt tomography data a seamless process. We demonstrate high-quality reconstructions from samples of well-described biological structures. These include the giant Mimivirus and clathrin-coated vesicles, imaged in situ in their normal intracellular contexts. Examples are provided from samples of cultured cells prepared by high-pressure freezing and freeze-substitution as well as by chemical fixation before epoxy resin embedding

Characterization of the GBoV1 Capsid and Its Antibody Interactions

Human bocavirus 1 (HBoV1) has gained attention as a gene delivery vector with its ability to infect polarized human airway epithelia and 5.5 kb genome packaging capacity. Gorilla bocavirus 1 (GBoV1) VP3 shares 86% amino acid sequence identity with HBoV1 but has better transduction efficiency in several human cell types. Here, we report the capsid structure of GBoV1 determined to 2.76 Å resolution using cryo-electron microscopy (cryo-EM) and its interaction with mouse monoclonal antibodies (mAbs) and human sera. GBoV1 shares capsid surface morphologies with other parvoviruses, with a channel at the 5-fold symmetry axis, protrusions surrounding the 3-fold axis and a depression at the 2-fold axis. A 2/5-fold wall separates the 2-fold and 5-fold axes. Compared to HBoV1, differences are localized to the 3-fold protrusions. Consistently, native dot immunoblots and cryo-EM showed cross-reactivity and binding, respectively, by a 5-fold targeted HBoV1 mAb, 15C6. Surprisingly, recognition was observed for one out of three 3-fold targeted mAbs, 12C1, indicating some structural similarity at this region. In addition, GBoV1, tested against 40 human sera, showed the similar rates of seropositivity as HBoV1. Immunogenic reactivity against parvoviral vectors is a significant barrier to efficient gene delivery. This study is a step towards optimizing bocaparvovirus vectors with antibody escape properties

Structural and Energetic Characterization of the Ankyrin Repeat Protein Family

Ankyrin repeat containing proteins are one of the most abundant solenoid folds. Usually implicated in specific protein-protein interactions, these proteins are readily amenable for design, with promising biotechnological and biomedical applications. Studying repeat protein families presents technical challenges due to the high sequence divergence among the repeating units. We developed and applied a systematic method to consistently identify and annotate the structural repetitions over the members of the complete Ankyrin Repeat Protein Family, with increased sensitivity over previous studies. We statistically characterized the number of repeats, the folding of the repeat-arrays, their structural variations, insertions and deletions. An energetic analysis of the local frustration patterns reveal the basic features underlying fold stability and its relation to the functional binding regions. We found a strong linear correlation between the conservation of the energetic features in the repeat arrays and their sequence variations, and discuss new insights into the organization and function of these ubiquitous proteins.Fil: Parra, Rodrigo Gonzalo. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Espada, Rocío. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Verstraete, Nina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; ArgentinaFil: Ferreiro, Diego Ulises. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Ciudad Universitaria. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Instituto de Química Biológica de la Facultad de Ciencias Exactas y Naturales; Argentin