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It was summer … I was freezing as I entered the Civil Engineering building of the Polytechnique School of Engineering where the entrance exam for the University of Sao Paulo (USP) Schools and Institutes was being held … Following my BSc degree in Physics, I got an MSc degree in Condensed Matter and Material Science and continued to obtain a PhD in Experimental Physics and Crystallography from the Physics Institute, USP. It was as a graduate student that I started to spin the thread that has been defining my career … The love for crystals and X-rays, and an obsession for light sources … Today Vivian Stojanoff leads the User, Training and Outreach program of the Center for Biomolecular Structure (CBMS) at the National Synchrotron Light Source II. Her research focus on the application and development of X-ray characterization and crystallization methods of biomolecules.

Electric fields have been employed to promote macromolecular crystallization for several decades. Although crystals grown in electric fields seem to present higher diffraction quality, these methods are not widespread. For most configurations, electrodes are in direct contact with the protein solution. Here, we propose a configuration that can be easily extended to standard crystallization methods for which the electrodes are not in direct contact with the protein solution. Furthermore, the proposed electrode configuration supplies an external DC electric field. Glucose Isomerase from Streptomyces rubiginosus crystals were grown at room temperature using the microbatch method in the presence of 1, 2, 4, and 6 kV. Several crystallization trials were carried out for reproducibility and statistical analysis purposes. The comparison with crystals grown in the absence of electric fields showed that crystallization in the presence of electric fields increases the size of crystals, while decreasing the number of nucleations. X-ray diffraction analysis of the crystals showed that those grown in the presence of electric fields are of higher crystal quality.

A gel can be considered to be a two-phase (liquid and solid) system, which lacks flow once it reaches a stationary state. The solid phase is usually a tridimensional polymeric mesh, while the liquid phase is usually found in three forms: contained in great cavities, retained in the capillary pores between micelles, or adsorbed on the surface of a micelle. The influence of the use of gels in crystal growth is diverse and depends on the type of gel being used. A decrease in solubility of any solute in the liquid may occur if the solvent interacts extensively with the polymeric section, hence, the nucleation in gels in these cases apparently occurs at relatively low supersaturations. However, if the pore size is small enough, there is a possibility that a higher supersaturation is needed, due to the compartmentalization of solvents. Finally, this may also represent an effect in the diffusion of substances. This review is divided into three main parts; the first evaluates the theory and practice used for the obtainment of polymorphs. The second part describes the use of gels into crystallogenesis of different substances. The last part is related to the particularities of protein crystal polymorphism, as well as modern trends in gel growth for high-resolution X-ray crystallography.

Energy buffering systems are key for homeostasis during variations in energy supply. Spiders are the most important predators for insects and therefore key in terrestrial ecosystems. From biomedical interest, spiders are important for their venoms and as a source of potent allergens, such as arginine kinase (AK, EC 2.7.3.3). AK is an enzyme crucial for energy metabolism, keeping the pool of phosphagens in invertebrates, and also an allergen for humans. In this work, we studied AK from the Argentininan spider Polybetes pythagoricus ( Pp AK), from its complementary DNA to the crystal structure. The Pp AK cDNA from muscle was cloned, and it is comprised of 1068 nucleotides that encode a 384-amino acids protein, similar to other invertebrate AKs. The apparent Michaelis-Menten kinetic constant ( K m ) was 1.7 mM with a k cat of 75 s −1 . Two crystal structures are presented, the apo Pv AK and Pp AK bound to arginine, both in the open conformation with the active site lid (residues 310–320) completely disordered. The guanidino group binding site in the apo structure appears to be organized to accept the arginine substrate. Finally, these results contribute to knowledge of mechanistic details of the function of arginine kinase.

Micrometeorites (MMs) are small particles that account for most of the extraterrestrial material deposited on Earth. Synchrotron X-ray fluorescence and diffraction allowed for chemical and mineral characterization to distinguish MM from atmospheric particulate. The relative components of iron, nickel, and other elements were considered in the identification of ferrous MM while high amounts of titanium were considered an indication that the particles were of atmospheric origin. Out of 100 samples collected by high school students and teachers, eight were taken to a synchrotron for analysis. Of those eight, three exhibited extraterrestrial compositions. X-ray absorption near-edge structure analysis revealed that the same three samples contained sulfide, the main sulfur form constituent in MM. X-ray microdiffraction analysis showed the presence of the minerals pentlandite and forsterite. Collectively, these results support the extraterrestrial nature of the three particles.

DNA replication requires processivity factors that allow replicative DNA polymerases to extend long stretches of DNA. Some DNA viruses encode their own replicative DNA polymerase, such as the white spot syndrome virus (WSSV) that infects decapod crustaceans but still require host replication accessory factors. We have determined by X-ray diffraction the three-dimensional structure of the Pacific white leg shrimp Litopenaeus vannamei Proliferating Cell Nuclear Antigen (LvPCNA). This protein is a member of the sliding clamp family of proteins, that binds DNA replication and DNA repair proteins through a motif called PIP-box (PCNA-Interacting Protein). The crystal structure of LvPCNA was refined to a resolution of 3 Å, and allowed us to determine the trimeric protein assembly and details of the interactions between PCNA and the DNA. To address the possible interaction between LvPCNA and the viral DNA polymerase, we docked a theoretical model of a PIP-box peptide from the WSSV DNA polymerase within LvPCNA crystal structure. The theoretical model depicts a feasible model of interaction between both proteins. The crystal structure of shrimp PCNA allows us to further understand the mechanisms of DNA replication processivity factors in non-model systems.

Here we investigate the catalytic site of tRNA methyltransferase enzyme (TrmD) in Mycobacterium abscessus as a potential antibiotic target. Previously, molecular dynamic simulation has been employed to predict the relationship between the natural ligand, S-adenosyl-L-methionine, and a divalent metallic ion. However no experimental crystallographic evidence has been obtained thus far. Three protein structures of this TrmD with S-adenosyl-L-homocysteine have been determined by X-ray crystallography. Here we report the first structure to be found with the cationic magnesium bound in the catalytic site. The models and analysis provided in this study offer insight into the complex methylation reaction that occurs in the catalytic site and overall means of survival for certain infectious bacteria. This work directly contributes to pharmaceutical efforts in creating antimicrobial treatments with TrmD acting as a potential drug target.

Abstract Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli , a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E . coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum . The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E . coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.

Acyl carrier proteins (ACPs) are essential to the production of fatty acids. In some species of marine bacteria, ACPs are arranged into tandem repeats joined by peptide linkers, an arrangement that results in high fatty acid yields. By contrast, Escherichia coli , a relatively low producer of fatty acids, uses a single-domain ACP. In this work, we have engineered the native E . coli ACP into tandem di- and tri-domain constructs joined by a naturally occurring peptide linker from the PUFA synthase of Photobacterium profundum . The size of these tandem fused ACPs was determined by size exclusion chromatography to be higher (21 kDa, 36 kDa and 141 kDa) than expected based on the amino acid sequence (12 kDa, 24 kDa and 37 kDa, respectively) suggesting the formation of a flexible extended conformation. Structural studies using small-angle X-ray scattering (SAXS), confirmed this conformational flexibility. The thermal stability for the di- and tri-domain constructs was similar to that of the unfused ACP, indicating a lack of interaction between domains. Lastly, E . coli cultures harboring tandem ACPs produced up to 1.6 times more fatty acids than wild-type ACP, demonstrating the viability of ACP fusion as a method to enhance fatty acid yield in bacteria.

Type 1 pili-adhesive fibers expressed in most members of the Enterobacteriaceae family-mediate binding to mannose receptors on host cells through the FimH adhesin. Pilus biogenesis proceeds by way of the chaperone/usher pathway. The x-ray structure of the FimC-FimH chaperone-adhesin complex from uropathogenic Escherichia coli at 2.5 angstrom resolution reveals the basis for carbohydrate recognition and for pilus assembly. The carboxyl-terminal pilin domain of FimH has an immunoglobulin-like fold, except that the seventh strand is missing, leaving part of the hydrophobic core exposed. A donor strand complementation mechanism in which the chaperone donates a strand to complete the pilin domain explains the basis for both chaperone function and pilus biogenesis.