MX2: a high-flux undulator microfocus beamline serving both the chemical and macromolecular crystallography communities at the Australian Synchrotron

MX1 is a bending-magnet crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X-rays in the energy range from 8 to 18 keV to a focal spot at the sample position of 120 µm FWHM. The beamline endstation and ancillary equipment facilitate local and remote access for both chemical and biological macromolecular crystallography. Here, the design of the beamline and endstation are discussed. The beamline has enjoyed a full user program for the last seven years and scientific highlights from the user program are also presented.

BL19U2: Small-angle X-ray scattering beamline for biological macromolecules in solution at SSRF

BackgroundThe RCSB Protein Data Bank (PDB) provides public access to experimentally determined 3D-structures of biological macromolecules (proteins, peptides and nucleic acids). While various tools are available to explore the PDB, options to access the global structural diversity of the entire PDB and to perceive relationships between PDB structures remain very limited.MethodsA 136-dimensional atom pair 3D-fingerprint for proteins (3DP) counting categorized atom pairs at increasing through-space distances was designed to represent the molecular shape of PDB-entries. Nearest neighbor searches examples were reported exemplifying the ability of 3DP-similarity to identify closely related biomolecules from small peptides to enzyme and large multiprotein complexes such as virus particles. The principle component analysis was used to obtain the visualization of PDB in 3DP-space.ResultsThe 3DP property space groups proteins and protein assemblies according to their 3D-shape similarity, yet shows exquisite ability to distinguish between closely related structures. An interactive website called PDB-Explorer is presented featuring a color-coded interactive map of PDB in 3DP-space. Each pixel of the map contains one or more PDB-entries which are directly visualized as ribbon diagrams when the pixel is selected. The PDB-Explorer website allows performing 3DP-nearest neighbor searches of any PDB-entry or of any structure uploaded as protein-type PDB file. All functionalities on the website are implemented in JavaScript in a platform-independent manner and draw data from a server that is updated daily with the latest PDB additions, ensuring complete and up-to-date coverage. The essentially instantaneous 3DP-similarity search with the PDB-Explorer provides results comparable to those of much slower 3D-alignment algorithms, and automatically clusters proteins from the same superfamilies in tight groups.ConclusionA chemical space classification of PDB based on molecular shape was obtained using a new atom-pair 3D-fingerprint for proteins and implemented in a web-based database exploration tool comprising an interactive color-coded map of the PDB chemical space and a nearest neighbor search tool. The PDB-Explorer website is freely available at www.cheminfo.org/pdbexplorer and represents an unprecedented opportunity to interactively visualize and explore the structural diversity of the PDB.ᅟGraphical abstractᅟMaps of PDB in 3DP-space color-coded by heavy atom count and shape. Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-015-0776-9) contains supplementary material, which is available to authorized users.

Chapter 6 - Characterization of analytes and matrices

The Macromolecular Crystallography (MX) Beamlines at the Australian Synchrotron collect data on protein samples (PX) and chemical samples (CX). This broad range of sample types requires us to consider a number of experimental and data processing considerations. Protein samples have very large unit cells but diffract weakly, the chemical samples on the other hand have very small unit cell and diffract comparatively strongly. From an experimental point of view, this requires substantially different geometrical considerations which can be handled by changing the energy of the monochromated X-rays and detector distance. Another consideration is detector type, the detectors at the MX beamlines are from the Area Detector Systems Corporation (ADSC) and they have generally been used for PX data collections. This has lead to investigation regarding high incidence angle phosphor thickness corrections. As the detectors have mainly been used for PX work, the software for sample handling has also been developed with PX considerations rather than CX. For example, the software for space group determinations is set my default to assumes that your space group is only ever one of the 65 space groups that don't contain mirror, inversion or glide operations. Another area of interest is the way data scaling is handled. The data is often scaled with PX data for a number of reasons, with the most common scaling of data is due t the prevalence of radiation damage to the samples. By contrast the most common form of scaling for CX data is for sample anisotropy in strong absorbers. A discussion of the challenges faced for the chemical crystallography experiments at the Australian Synchrotron will be presented.

A bacterium can be harnessed to do the job, but can that process be scaled up?

The Gag polyprotein of retroviruses drives immature virus assembly by forming hexameric protein lattices. The assembly is primarily mediated by protein-protein interactions between capsid (CA) domains and by interactions between nucleocapsid (NC) domains and RNA. Specific interactions between NC and the viral RNA are required for genome packaging. Previously reported cryoelectron microscopy analysis of immature Mason-Pfizer monkey virus (M-PMV) particles suggested that a basic region (residues RKK) in CA may serve as an additional binding site for nucleic acids. Here, we have introduced mutations into the RKK region in both bacterial and proviral M-PMV vectors and have assessed their impact on M-PMV assembly, structure, RNA binding, budding/release, nuclear trafficking, and infectivity using in vitro and in vivo systems. Our data indicate that the RKK region binds and structures nucleic acid that serves to promote virus particle assembly in the cytoplasm. Moreover, the RKK region appears to be important for recruitment of viral genomic RNA into Gag particles, and this function could be linked to changes in nuclear trafficking. Together these observations suggest that in M-PMV, direct interactions between CA and nucleic acid play important functions in the late stages of the viral life cycle. Assembly of retrovirus particles is driven by the Gag polyprotein, which can self-assemble to form virus particles and interact with RNA to recruit the viral genome into the particles. Generally, the capsid domains of Gag contribute to essential protein-protein interactions during assembly, while the nucleocapsid domain interacts with RNA. The interactions between the nucleocapsid domain and RNA are important both for identifying the genome and for self-assembly of Gag molecules. Here, we show that a region of basic residues in the capsid protein of the betaretrovirus Mason-Pfizer monkey virus (M-PMV) contributes to interaction of Gag with nucleic acid. This interaction appears to provide a critical scaffolding function that promotes assembly of virus particles in the cytoplasm. It is also crucial for packaging the viral genome and thus for infectivity. These data indicate that, surprisingly, interactions between the capsid domain and RNA play an important role in the assembly of M-PMV.

Fluorescence Applications in Molecular Neurobiology

Our desire to understand how the individual molecules that make up cells organize, interact, and communicate to form living systems has lead to the burgeoning field of chemical biology, an interfacial area of science that combines aspects of chemistry — the study of matter and its transformations, and biology — the study of living things and their interactions with the environment. The defining feature of chemical biology is the use of chemical approaches and small molecules to interrogate or manipulate biology.1,2 These small molecules are synthetic or naturally occurring ones that, for example, bind to DNA to affect protein expression levels, bind to proteins to inhibit their function, interact with lipids to alter membrane integrity, or become fluorescent in response to a metabolic event. Because small molecules can affect biochemical function, there is a clear link between chemical biology and pharmacology and medicine.3 While small molecules are usually implied as being organic compounds,4 inorganic small molecules also have a long history in both biology and medicine. Ancient civilizations used gold and copper for healing purposes, and the modern era of drug discovery was ushered in when arsenic-containing salvarsan was discovered as an anti-syphilis agent to become the world’s first blockbuster drug.5 Inorganic compounds should therefore not be overlooked in the realm of chemical biology, since their distinctive electronic, chemical, and photophysical properties render them particularly useful for a variety of applications.6-8 What are the properties of metal ions that impart utility to biology? Because inorganic elements comprise the bulk of the periodic table, the diversity of these properties is likewise broad and has been thoroughly covered by several books in the field of bioinorganic chemistry.9-11 A brief summary of the general chemical properties of metals is given below. Charge. Metal ions are positively charged in aqueous solution, but that charge can be manipulated depending on the coordination environment so that a metal complexed by ligands can be cationic, anionic, or neutral. Interactions with ligands. Metal ions bind to ligands (both organic and inorganic) via interactions that are often strong and selective. The ligands impart their own functionality and can tune properties of the overall complex that are unique from those of the individual ligand or metal. The thermodynamic and kinetic properties of metal—ligand interactions influence ligand exchange reactions. Structure and bonding. Metal—ligand complexes span a range of coordination geometries that give them unique shapes compared to organic molecules. The bond lengths, bond angles, and number of coordination sites can vary depending on the metal and its oxidation state. Lewis acid character. Metal ions with high electron affinity can significantly polarize groups that are coordinated to them, facilitating hydrolysis reactions. Partially filled d-shell. For the transition metals, the variable number of electrons in the d-shell orbitals (or f-shell for lanthanides) imparts interesting electronic and magnetic properties to transition metal complexes. Redox activity. Coupled with the variability of electrons in the d-shell is the ability for many transition metals to undergo 1-electron oxidation and reduction reactions. Biology has taken advantage of these chemical properties of metals to perform several functional roles, which are summarized in Table 1. This is by no means an exhaustive list, but rather a primer to highlight important themes. Some metal ions, particularly the alkali and alkaline earth metals, are stable in aqueous solution as cations, making Na+, K+ and Ca2+ ideal for maintaining charge balance and electrical conductivity.10 On the other hand, the distinct architectures accessible via metal—ligand bonding interactions impart important structural roles to metal ions that encompass both macroscopic structural stabilization, as in biomineralized tissues,12 as well as molecular structural stabilization, as in proteins and nucleic acids that are stabilized in a preferred fold by metal ions.13-16 Metal—ligand bonding is also significant in its reversibility. For example, Nature takes advantage of reversible binding of metal ions like Ca2+ and Zn2+ to proteins or other storage repositories in order to propagate various biochemical signals.13,17 Metal ions themselves can be their own signal to adjust DNA transcription, as in the case of metalloregulatory proteins.18,19 Reversible metal—ligand coordination is also exploited to bind and release molecules to and from a metal center, a prime example being O2 binding and release from hemoglobin. Table 1 Functional roles of inorganic elements found in biology, with selected representative examples.

Electrochemical oxidation of organic compounds is a widely used approach for wastewater purification, energy production, and synthesis of value added products. In all cases listed noble metals are used as catalysts due to their high activity and lack of selectivity. Unfortunately these catalysts are expensive, which hinders their large-scale application for wastewater purification. At the same time Nature not only found the right answer for pollutants removal, but uses these electrochemical oxidation processes to gain energy. Numerous redox enzymes (mainly oxidases and dehydrogenases) are capable of fast, efficient, and selective oxidation of complex and simple organic compounds. The enzyme high redox activity is a result of years of evolutionary improvements of enzymes’ structure and function. A key moment is the specific design of their active center supported by the surrounding amino acid residues and enzyme tertiary structure. The main problem with utilization of enzymatic systems is their short life and limited range of environmental conditions they are able to operate in. Thus, the development of more robust inorganic catalysts by mimicking the enzyme active center provides a new avenue in the development of electrochemical catalysis for oxidation of organic compounds. An inorganic catalyst mimicking the Mn-binding site of oxalate oxidase and oxalate decarboxylase has been developed under this study. Oxalate oxidase and oxalate decarboxylase were chosen as enzyme models since they are capable of oxidation of small organic acids, more specifically oxalic acid. The importance of oxalic acid, especially for clinical diagnosis and in food and water technology, is now widely recognized. Small carboxylic or dicarboxylic acids (such as formic, maleic, and acetic acids) are intermediate products in catalytic oxidation of aromatic compounds present in wastewater. At the same time oxalic acid is a compound that is toxic to almost all organisms. The accumulation of oxalic acid cause hyperoxaluria, formation of calcium oxalate stones in the kidney, renal failure, cardiomyopathy, and cardiac conductance disorders. The developed catalyst, abbreviated here as MnAAPyr, is a non-platinum based catalyst. It has been synthesized using sacrificial support method [1], which was developed in our group mainly to synthesize materials with high activity towards oxygen reduction reaction. Along with its low cost, the main advantage of this catalyst is its high catalytic surface area i.e. the active sites of the material are situated within its own structure, turning it into a highly porous framework incorporating plenty of accessible active sites. The performed XPS analyses of the synthesized MnAAPyr show the presence of Mn-Nx coordination. Furthermore, DFT calculations were used to study the structure and the formation energy of the various Mn-Nxactive sites in which Mn is assumed to be coordinated with two, three, or four nitrogen atoms [2]. The activity of the MnAAPyr catalyst towards oxalate oxidation was studied using cyclic voltammetry at pH 4 and pH 7.5 (Fig. 1). MnAAPyr demonstrated similar catalytic activity at both pHs. The influence of the nature of the possible active sites was further evaluated using DFT approach by looking into the adsorption energy of oxalic acid and oxalate anion since this step was found to be the rate-limiting step in oxalate oxidation (Table 1 and Figure 2). As predicted by DFT oxalic acid and oxalate anion have the highest adsorption energy on Mn-N3, which implies higher activity of this active site in comparison to the other Mn-Nxcomposites. The mechanism of oxalate oxidation was experimentally studied through FTIR and RRDE measurements. We were interested in CO2 production as well as in the possibility of H2O2 generation as a by-product in a similar fashion to the mechanism occurring when oxalate oxidase is used. The activity of the MnAAPyr catalyst towards other small organic compounds was also tested. Along with the oxidation of small organics, it was established that this material shows electrocatalytic activity even towards oxidation of complex sugars such as glucose and sucrose. The broad range of organic compounds the catalyst can covert confirms its applicability in wastewater treatment and energy harvesting, where non-selectivity of the catalyst is beneficial. 1. Pylypenko, S., et al., Non-platinum oxygen reduction electrocatalysts based on pyrolyzed transition metal macrocycles. Electrochimica Acta, 2008. 53: p. 7875–7883. 2. Svedruzic, D., et al., The enzymes of oxalate metabolism: unexpected structures and mechanisms. Archives of Biochemistry and Biophysics, 2005. 433: p. 176–192. Figure 1

Experimental and theoretical approaches to interactions between DNA and purine metabolism products

A biomimetic and bioactive scaffold with intelligently pulsatile teriparatide delivery for local and systemic osteoporosis regeneration

Molecules of organic compounds may have the same atomic composition but differ from each other based on either the way in which the atoms are connected or their special arrangement in space. Such variations are collectively called isomerism and include structural isomerism such as carbon chain, positional, and functional isomerism, and tautomerism. Molecular variation based on spatial arrangement is called stereoisomerism and includes geometrical (cis–trans or E–Z) and R–S (or optical) isomerism. In organic compounds and drug molecules, isomerism means variation not only in physical and chemical properties but also in biological effects. By using practical examples and problem-solving exercises, this chapter outlines the chemical basis and biological significance of isomerism.

Characteristics of virus-cell complexes

The effectiveness of modified sperm washes in severely oligoasthenozoospermic men infected with human immunodeficiency and hepatitis C viruses