Crystallographic legacy of Ned Seeman.

We trace the career path of Nadrian Seeman, the inventor of DNA nanotechnology. The influence of his early training in crystallography and how this led to his success in creating self-assembled crystals are highlighted.

Lessons from high-throughput protein crystallization screening: 10 years of practical experience.

X-ray crystallography provides the majority of our structural biological knowledge at a molecular level and, in terms of pharmaceutical design, is a valuable tool to accelerate discovery. It is the premier technique in the field, but its usefulness is significantly limited by the need to grow well-diffracting crystals. It is for this reason that high-throughput crystallization has become a key technology that has matured over the past 10 years through the field of structural genomics.

The crystal structure and activity of a putative trypanosomal nucleoside phosphorylase reveal it to be a homodimeric uridine phosphorylase.

Purine nucleoside phosphorylases (PNPs) and uridine phosphorylases (UPs) are closely related enzymes involved in purine and pyrimidine salvage, respectively, which catalyze the removal of the ribosyl moiety from nucleosides so that the nucleotide base may be recycled. Parasitic protozoa generally are incapable of de novo purine biosynthesis; hence, the purine salvage pathway is of potential therapeutic interest. Information about pyrimidine biosynthesis in these organisms is much more limited.

Purification of transmembrane proteins from Saccharomyces cerevisiae for X-ray crystallography.

To enhance the quantity and quality of eukaryotic transmembrane proteins (TMPs) available for structure determination by X-ray crystallography, we have optimized protocols for purification of TMPs expressed in the yeast Saccharomyces cerevisiae. We focused on a set of the highest-expressing endogenous yeast TMPs for which there are established biochemical assays. Genes encoding the target TMPs are transferred via ligation-independent cloning to a series of vectors that allow expression of reading frames fused to C-terminal His10 and ZZ (IgG-binding) domains that are separated from the reading frame by a cleavage site for rhinovirus 3C protease.

Heterologous expression of L. major proteins in S. cerevisiae: a test of solubility, purity, and gene recoding.

High level expression of many eukaryotic proteins for structural analysis is likely to require a eukaryotic host since many proteins are either insoluble or lack essential post-translational modifications when expressed in E. coli. The well-studied eukaryote Saccharomyces cerevisiae possesses several attributes of a good expression host: it is simple and inexpensive to culture, has proven genetic tractability, and has excellent recombinant DNA tools.

Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins.

Elucidating the structures of membrane proteins is essential to our understanding of disease states and a critical component in the rational design of drugs. Structural characterization of a membrane protein begins with its detergent solubilization from the lipid bilayer and its purification within a functionally stable protein-detergent complex (PDC). Crystallization of the PDC typically occurs by changing the solution environment to decrease solubility and promote interactions between exposed hydrophilic surface residues.

Understanding the physical properties that control protein crystallization by analysis of large-scale experimental data.

Crystallization is the most serious bottleneck in high-throughput protein-structure determination by diffraction methods. We have used data mining of the large-scale experimental results of the Northeast Structural Genomics Consortium and experimental folding studies to characterize the biophysical properties that control protein crystallization. This analysis leads to the conclusion that crystallization propensity depends primarily on the prevalence of well-ordered surface epitopes capable of mediating interprotein interactions and is not strongly influenced by overall thermodynamic stability.

Establishing a training set through the visual analysis of crystallization trials. Part II: crystal examples.

In the automated image analysis of crystallization experiments, representative examples of outcomes can be obtained rapidly. However, while the outcomes appear to be diverse, the number of crystalline outcomes can be small. To complement a training set from the visual observation of 147 456 crystallization outcomes, a set of crystal images was produced from 106 and 163 macromolecules under study for the North East Structural Genomics Consortium (NESG) and Structural Genomics of Pathogenic Protozoa (SGPP) groups, respectively.

Establishing a training set through the visual analysis of crystallization trials. Part I: approximately 150,000 images.

Structural crystallography aims to provide a three-dimensional representation of macromolecules. Many parts of the multistep process to produce the three-dimensional structural model have been automated, especially through various structural genomics projects. A key step is the production of crystals for diffraction.

Structures of substrate- and inhibitor-bound adenosine deaminase from a human malaria parasite show a dramatic conformational change and shed light on drug selectivity.

Plasmodium and other apicomplexan parasites are deficient in purine biosynthesis, relying instead on the salvage of purines from their host environment. Therefore, interference with the purine salvage pathway is an attractive therapeutic target. The plasmodial enzyme adenosine deaminase (ADA) plays a central role in purine salvage and, unlike mammalian ADA homologs, has a further secondary role in methylthiopurine recycling.

Structural genomics of pathogenic protozoa: an overview.

The Structural Genomics of Pathogenic Protozoa (SGPP) Consortium aimed to determine crystal structures of proteins from trypanosomatid and malaria parasites in a high throughput manner. The pipeline of target selection, protein production, crystallization, and structure determination, is sketched. Special emphasis is given to a number of technology developments including domain prediction, the use of "co-crystallants," and capillary crystallization.

Structure of a Trypanosoma brucei alpha/beta-hydrolase fold protein with unknown function.

The structure of a structural genomics target protein, Tbru020260AAA from Trypanosoma brucei, has been determined to a resolution of 2.2 A using multiple-wavelength anomalous diffraction at the Se K edge. This protein belongs to Pfam sequence family PF08538 and is only distantly related to previously studied members of the alpha/beta-hydrolase fold family.

Characterization of Trypanosoma brucei dihydroorotate dehydrogenase as a possible drug target; structural, kinetic and RNAi studies.

Nucleotide biosynthesis pathways have been reported to be essential in some protozoan pathogens. Hence, we evaluated the essentiality of one enzyme in the pyrimidine biosynthetic pathway, dihydroorotate dehydrogenase (DHODH) from the eukaryotic parasite Trypanosoma brucei through gene knockdown studies. RNAi knockdown of DHODH expression in bloodstream form T.

Blocking S-adenosylmethionine synthesis in yeast allows selenomethionine incorporation and multiwavelength anomalous dispersion phasing.

Saccharomyces cerevisiae is an ideal host from which to obtain high levels of posttranslationally modified eukaryotic proteins for x-ray crystallography. However, extensive replacement of methionine by selenomethionine for anomalous dispersion phasing has proven intractable in yeast. We report a general method to incorporate selenomethionine into proteins expressed in yeast based on manipulation of the appropriate metabolic pathways.

The structure of Plasmodium vivax phosphatidylethanolamine-binding protein suggests a functional motif containing a left-handed helix.

The structure of a putative Raf kinase inhibitor protein (RKIP) homolog from the eukaryotic parasite Plasmodium vivax has been studied to a resolution of 1.3 A using multiple-wavelength anomalous diffraction at the Se K edge. This protozoan protein is topologically similar to previously studied members of the phosphatidylethanolamine-binding protein (PEBP) sequence family, but exhibits a distinctive left-handed alpha-helical region at one side of the canonical phospholipid-binding site.

Efficient optimization of crystallization conditions by manipulation of drop volume ratio and temperature.

An efficient optimization method for the crystallization of biological macromolecules has been developed and tested. This builds on a successful high-throughput technique for the determination of initial crystallization conditions. The optimization method takes an initial condition identified through screening and then varies the concentration of the macromolecule, precipitant, and the growth temperature in a systematic manner.

Using fragment cocktail crystallography to assist inhibitor design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase.

The 1.8 A resolution de novo structure of nucleoside 2-deoxyribosyltransferase (EC 2.4.

Thermofluor-based high-throughput stability optimization of proteins for structural studies.

Production of proteins well suited for structural studies is inherently difficult and time-consuming. Protein sample homogeneity, stability, and solubility are strongly correlated with the proteins' probability of yielding crystals, and optimization of these properties will improve success rates of crystallization. In the current study, we applied the thermofluor method as a high-throughput approach for identifying optimal protein formulation for crystallization.

Structure of ribose 5-phosphate isomerase from Plasmodium falciparum.

The structure of ribose 5-phosphate isomerase from Plasmodium falciparum, PFE0730c, has been determined by molecular replacement at 2.09 angstroms resolution. The enzyme, which catalyzes the isomerization reaction that interconverts ribose 5-phosphate and ribulose 5-phosphate, is a member of the pentose phosphate pathway.

Structure of the conserved hypothetical protein MAL13P1.257 from Plasmodium falciparum.

The structure of a conserved hypothetical protein, PlasmoDB sequence MAL13P1.257 from Plasmodium falciparum, Pfam sequence family PF05907, has been determined as part of the structural genomics effort of the Structural Genomics of Pathogenic Protozoa consortium. The structure was determined by multiple-wavelength anomalous dispersion at 2.

Structure of Lmaj006129AAA, a hypothetical protein from Leishmania major.

The gene product of structural genomics target Lmaj006129 from Leishmania major codes for a 164-residue protein of unknown function. When SeMet expression of the full-length gene product failed, several truncation variants were created with the aid of Ginzu, a domain-prediction method. 11 truncations were selected for expression, purification and crystallization based upon secondary-structure elements and disorder.

Crystal structure of glyceraldehyde-3-phosphate dehydrogenase from Plasmodium falciparum at 2.25 A resolution reveals intriguing extra electron density in the active site.

The crystal structure of D-glyceraldehyde-3-phosphate dehydrogenase (PfGAPDH) from the major malaria parasite Plasmodium falciparum is solved at 2.25 A resolution. The structure of PfGAPDH is of interest due to the dependence of the malaria parasite in infected human erythrocytes on the glycolytic pathway for its energy generation.

Automatic classification of sub-microlitre protein-crystallization trials in 1536-well plates.

A technique for automatically evaluating microbatch (400 nl) protein-crystallization trials is described. This method addresses analysis problems introduced at the sub-microlitre scale, including non-uniform lighting and irregular droplet boundaries. The droplet is segmented from the well using a loopy probabilistic graphical model with a two-layered grid topology.

A deliberate approach to screening for initial crystallization conditions of biological macromolecules.

A method to rationally predict crystallization conditions for a previously uncrystallized macromolecule has not yet been developed. One way around this problem is to determine initial crystallization conditions by casting a wide net, surveying a large number of chemical and physical conditions to locate crystallization leads. A facility that executes the rapid survey of crystallization lead conditions is described in detail.

The crystal structure of MT0146/CbiT suggests that the putative precorrin-8w decarboxylase is a methyltransferase.

The CbiT and CbiE enzymes participate in the biosynthesis of vitamin B12. They are fused together in some organisms to form a protein called CobL, which catalyzes two methylations and one decarboxylation on a precorrin intermediate. Because CbiE has sequence homology to canonical precorrin methyltransferases, CbiT was hypothesized to catalyze the decarboxylation.