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Through an expansive international effort that involved data collection on 12 small-angle X-ray scattering (SAXS) and four small-angle neutron scattering (SANS) instruments, 171 SAXS and 76 SANS measurements for five proteins (ribonuclease A, lysozyme, xylanase, urate oxidase and xylose isomerase) were acquired. From these data, the solvent-subtracted protein scattering profiles were shown to be reproducible, with the caveat that an additive constant adjustment was required to account for small errors in solvent subtraction. Further, the major features of the obtained consensus SAXS data over the q measurement range 0-1 Å -1 are consistent with theoretical prediction. The inherently lower statistical precision for SANS limited the reliably measured q-range to <0.5 Å -1 , but within the limits of experimental uncertainties the major features of the consensus SANS data were also consistent with prediction for all five proteins measured in H 2 O and in D 2 O. Thus, a foundation set of consensus SAS profiles has been obtained for benchmarking scattering-profile prediction from atomic coordinates. Additionally, two sets of SAXS data measured at different facilities to q > 2.2 Å -1 showed good mutual agreement, affirming that this region has interpretable features for structural modelling. SAS measurements with inline size-exclusion chromatography (SEC) proved to be generally superior for eliminating sample heterogeneity, but with unavoidable sample dilution during column elution, while batch SAS data collected at higher concentrations and for longer times provided superior statistical precision. Careful merging of data measured using inline SEC and batch modes, or low- and high-concentration data from batch measurements, was successful in eliminating small amounts of aggregate or interparticle interference from the scattering while providing improved statistical precision overall for the benchmarking data set., (open access.)

Crystallography is commonly used for studying the structures of protein-protein complexes. However, a crystal structure does not define a unique protein-protein interface, and distinguishing a 'biological interface' from 'crystal contacts' is often not straightforward. A number of computational approaches exist for distinguishing them, but their error rate is high, emphasizing the need to obtain further data on the biological interface using complementary structural and functional approaches. In addition to reviewing the computational and experimental approaches for addressing this problem, we highlight two relevant examples. The first example from our laboratory involves the structure of acyl-CoA thioesterase 7, where each domain of this two-domain protein was crystallized separately, but both yielded a non-functional assembly. The structure of the full-length protein was uncovered using a combination of complementary approaches including chemical cross-linking, analytical ultracentrifugation and mutagenesis. The second example involves the platelet glycoprotein Ibalpha-thrombin complex. Two groups reported the crystal structures of this complex, but all the interacting interfaces differed between the two structures. Our computational analysis did not fully resolve the reasons for the discrepancies, but provided interesting insights into the system. This review highlights the need to complement crystallographic studies with complementary experimental and computational approaches.

High-resolution techniques are the mainstay of structural biologists; however, to address challenging biological systems many are now turning to hybrid approaches that use complementary structural data. In this review we outline the types of structural problems that benefit from combining results of many methods, we summarise the types of data that can be generated by complementary approaches, and we highlight the application of combined methods in structural biology with recent structural studies of membrane proteins, mega-complexes and inherently flexible proteins.

Increasing the success in obtaining structures and maximizing the value of the structures determined are the two major goals of target selection in structural proteomics. This chapter presents an efficient and flexible target selection procedure supplemented with a Web-based resource that is suitable for small- to large-scale structural genomics projects that use crystallography as the major means of structure determination. Based on three criteria, biological significance, structural novelty, and "crystallizability," the approach first removes (filters) targets that do not meet minimal criteria and then ranks the remaining targets based on their "crystallizability" estimates. This novel procedure was designed to maximize selection efficiency, and its prevailing criteria categories make it suitable for a broad range of structural proteomics projects.

Expression of insoluble protein in E. coli is a major bottleneck of high throughput structural biology projects. Refolding proteins into native conformations from inclusion bodies could significantly increase the number of protein targets that can be taken on to structural studies. This chapter presents a simple assay for screening insoluble protein targets and identifying those that are most amenable to refolding. The assay is based on the observation that when proteins are refolded while bound to metal affinity resin, misfolded proteins are generally not eluted by imidazole. This difference is exploited here to distinguish between folded and misfolded proteins. Two implementations of the assay are described. The assay fits well into a standard high throughput structural biology pipeline, because it begins with the inclusion body preparations that are a byproduct of small-scale, automated expression and purification trials and does not require additional facilities. Two formats of the assay are described, a manual assay that is useful for screening small numbers of targets, and an automated implementation that is useful for large numbers of targets.

This chapter describes the methodology adopted in a project aimed at structural and functional characterization of proteins that potentially play an important role in mammalian macrophages. The methodology that underpins this project is applicable to both small research groups and larger structural genomics consortia. Gene products with putative roles in macrophage function are identified using gene expression information obtained via DNA microarray technology. Specific targets for structural and functional characterization are then selected based on a set of criteria aimed at maximizing insight into function. The target proteins are cloned using a modification of Gateway cloning technology, expressed with hexa-histidine tags in E. coli, and purified to homogeneity using a combination of affinity and size exclusion chromatography. Purified proteins are finally subjected to crystallization trials and/or NMR-based screening to identify candidates for structure determination. Where crystallography and NMR approaches are unsuccessful, chemical cross-linking is employed to obtain structural information. This resulting structural information is used to guide cell biology experiments to further investigate the cellular and molecular function of the targets in macrophage biology. Jointly, the data sheds light on the molecular and cellular functions of macrophage proteins.

Failure to express soluble proteins in bacteria is mainly attributed to the properties of the target protein itself, as well as the choice of the vector, the purification tag and the linker between the tag and protein, and codon usage. The expression of proteins with fusion tags to facilitate subsequent purification steps is a widely used procedure in the production of recombinant proteins. However, the additional residues can affect the properties of the protein; therefore, it is often desirable to remove the tag after purification. This is usually done by engineering a cleavage site between the tag and the encoded protein that is recognised by a site-specific protease, such as the one from tobacco etch virus (TEV). In this study, we investigated the effect of four different tags on the bacterial expression and solubility of nine mouse proteins. Two of the four engineered constructs contained hexahistidine tags with either a long or short linker. The other two constructs contained a TEV cleavage site engineered into the linker region. Our data show that inclusion of the TEV recognition site directly downstream of the recombination site of the Invitrogen Gateway vector resulted in a loss of solubility of the nine mouse proteins. Our work suggests that one needs to be very careful when making modifications to expression vectors and combining different affinity and fusion tags and cleavage sites.

Insoluble expression of heterologous proteins in Escherichia coli is a major bottleneck of many structural genomics and high-throughput protein biochemistry projects. Many of these proteins may be amenable to refolding, but their identification is hampered by a lack of high-throughput methods. We have developed a matrix-assisted refolding approach in which correctly folded proteins are distinguished from misfolded proteins by their elution from affinity resin under non-denaturing conditions. Misfolded proteins remain adhered to the resin, presumably via hydrophobic interactions. The assay can be applied to insoluble proteins on an individual basis but is particularly well suited for high-throughput applications because it is rapid, automatable and has no rigorous sample preparation requirements. The efficacy of the screen is demonstrated on small-scale expression samples for 15 proteins. Refolding is then validated by large-scale expressions using SEC and circular dichroism.

B21 is a small‐angle X‐ray scattering (SAXS) beamline with a bending magnet source in the 3 GeV storage ring at the Diamond Light Source Ltd synchrotron in the UK. The beamline utilizes a double multi‐layer monochromator and a toroidal focusing optic to deliver 2 × 1012 photons per second to a 34 × 40 µm (FWHM) focal spot at the in‐vacuum Eiger 4M (Dectris) detector. A high‐performance liquid chromatography system and a liquid‐handling robot make it possible to load solution samples into a temperature‐controlled in‐vacuum sample cell with a high level of automation. Alternatively, a range of viscous or solid materials may be loaded manually using a range of custom sample cells. A default scattering vector range from 0.0026 to 0.34 Å−1 and low instrument background make B21 convenient for measuring a wide range of biological macromolecules. The beamline has run a full user programme since 2013. [ABSTRACT FROM AUTHOR]

Apical membrane antigen 1 (AMA1) of the human malaria parasite Plasmodium falciparum has been implicated in invasion of the host erythrocyte. It interacts with malarial rhoptry neck (RON) proteins in the moving junction that forms between the host cell and the invading parasite. Agents that block this interaction inhibit invasion and may serve as promising leads for anti-malarial drug development. The invasion-inhibitory peptide R1 binds to a hydrophobic cleft on AMA1, which is an attractive target site for small molecules that block parasite invasion. In this work, truncation and mutational analyses show that Phe5-Phe9, Phe12 and Arg15 in R1 are the most important residues for high affinity binding to AMA1. These residues interact with two well-defined binding hot spots on AMA1. Computational solvent mapping reveals that one of these hot spots is suitable for small molecule targeting. We also confirm that R1 in solution binds to AMA1 with 1∶1 stoichiometry and adopts a secondary structure consistent with the major form of R1 observed in the crystal structure of the complex. Our results provide a basis for designing high affinity inhibitors of the AMA1-RON2 interaction. [ABSTRACT FROM AUTHOR]

Abstract: The flood of new genomic sequence information together with technological innovations in protein structure determination have led to worldwide structural genomics (SG) initiatives. The goals of SG initiatives are to accelerate the process of protein structure determination, to fill in protein fold space and to provide information about the function of uncharacterized proteins. In the long-term, these outcomes are likely to impact on medical biotechnology and drug discovery, leading to a better understanding of disease as well as the development of new therapeutics. Here we describe the high throughput pipeline established at the University of Queensland in Australia. In this focused pipeline, the targets for structure determination are proteins that are expressed in mouse macrophage cells and that are inferred to have a role in innate immunity. The aim is to characterize the molecular structure and the biochemical and cellular function of these targets by using a parallel processing pipeline. The pipeline is designed to work with tens to hundreds of target gene products and comprises target selection, cloning, expression, purification, crystallization and structure determination. The structures from this pipeline will provide insights into the function of previously uncharacterized macrophage proteins and could lead to the validation of new drug targets for chronic obstructive pulmonary disease and arthritis. [Copyright &y& Elsevier]

Summary: Latexin, the only known mammalian carboxypeptidase inhibitor, has no detectable sequence similarity with plant and parasite inhibitors, but it is related to a human putative tumor suppressor protein, TIG1. Latexin is expressed in the developing brain, and we find that it plays a role in inflammation, as it is expressed at high levels and is inducible in macrophages in concert with other protease inhibitors and potential protease targets. The crystal structure of mouse latexin, solved at 1.83 Å resolution, shows no structural relationship with other carboxypeptidase inhibitors. Furthermore, despite a lack of detectable sequence duplication, the structure incorporates two topologically analogous domains related by pseudo two-fold symmetry. Surprisingly, these domains share a cystatin fold architecture found in proteins that inhibit cysteine proteases, suggesting an evolutionary and possibly functional relationship. The structure of the tumor suppressor protein TIG1 was modeled, revealing its putative membrane binding surface. [Copyright &y& Elsevier]