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Selenomethionine labeling is the most common technique used in protein crystallography to derivatize recombinant proteins for experimental phasing using anomalous scattering at tunable synchrotron beamlines. Recently, it has been shown that UV radiation depletes electron density of selenium atoms of selenomethionine residues and that UV radiation-damage-induced phasing (equivalent to single isomorphous replacement) protocol can be applied to calculate experimental phases. Here we present the straightforward integration of a UV source with an in-house diffractometer. We show how this setup can extend the capabilities of a sealed tube X-ray generator and be used for experimental phasing of selenium-labeled proteins., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Two commonly encountered bottlenecks in the structure determination of a protein by X‐ray crystallography are screening for conditions that give high‐quality crystals and, in the case of novel structures, finding derivatization conditions for experimental phasing. In this study, the phasing molecule 5‐amino‐2,4,6‐triiodoisophthalic acid (I3C) was added to a random microseed matrix screen to generate high‐quality crystals derivatized with I3C in a single optimization experiment. I3C, often referred to as the magic triangle, contains an aromatic ring scaffold with three bound I atoms. This approach was applied to efficiently phase the structures of hen egg‐white lysozyme and the N‐terminal domain of the Orf11 protein from Staphylococcus phage P68 (Orf11 NTD) using SAD phasing. The structure of Orf11 NTD suggests that it may play a role as a virion‐associated lysin or endolysin. [ABSTRACT FROM AUTHOR]

MX2 is an in‐vacuum undulator‐based crystallography beamline at the 3 GeV Australian Synchrotron. The beamline delivers hard X‐rays in the energy range 4.8–21 keV to a focal spot of 22 × 12 µm FWHM (H × V). At 13 keV the flux at the sample is 3.4 × 1012 photons s−1. The beamline endstation allows robotic handling of cryogenic samples via an updated SSRL SAM robot. This beamline is ideal for weakly diffracting hard‐to‐crystallize proteins, virus particles, protein assemblies and nucleic acids as well as smaller molecules such as inorganic catalysts and organic drug molecules. The beamline is now mature and has enjoyed a full user program for the last nine years. This paper describes the beamline status, plans for its future and some recent scientific highlights. [ABSTRACT FROM AUTHOR]

Proteins from the GASA/snakin superfamily are common in plant proteomes and have diverse functions, including hormonal crosstalk, development, and defense. One 63-residue member of this family, snakin-1, an antimicrobial protein from potatoes, has previously been chemically synthesized in a fully active form. Herein the 1.5 Å structure of snakin-1, determined by a novel combination of racemic protein crystallization and radiation-damage-induced phasing (RIP), is reported. Racemic crystals of snakin-1 and quasiracemic crystals incorporating an unnatural 4-iodophenylalanine residue were prepared from chemically synthesized D- and L-proteins. Breakage of the C-I bonds in the quasi-racemic crystals facilitated structure determination by RIP. The crystal structure reveals a unique protein fold with six disulfide crosslinks, presenting a distinct electrostatic surface that may target the protein to microbial cell surfaces. [ABSTRACT FROM AUTHOR]

Non-POU domain-containing octamer-binding protein (NONO, a.k.a. p54nrb) is a central player in nuclear gene regulation with rapidly emerging medical significance. NONO is a member of the highly conserved Drosophila behaviour/human splicing (DBHS) protein family, a dynamic family of obligatory dimeric nuclear regulatory mediators. However, work with the NONO homodimer has been limited by rapid irreversible sample aggregation. Here, it is reported that l-proline stabilizes purified NONO homodimers, enabling good-quality solution small-angle X-ray structure determination and crystallization. NONO crystallized in the apparent space group P21 with a unique axis ( b) of 408.9 Å and with evidence of twinning, as indicated by the cumulative intensity distribution L statistic, suggesting the possibility of space group P1. Structure solution by molecular replacement shows a superhelical arrangement of six NONO homodimers (or 12 in P1) oriented parallel to the long axis, resulting in extensive noncrystallographic symmetry. Further analysis revealed that the crystal was not twinned, but the collected data suffered from highly overlapping reflections that obscured the L-test. Optimized data collection on a new crystal using higher energy X-rays, a smaller beam width and an increased sample-to-detector distance produced non-overlapping reflections to 2.6 Å resolution. The steps taken to analyse and overcome this series of practical difficulties and to produce a biologically informative structure are discussed. [ABSTRACT FROM AUTHOR]

Using the UHV experimental endstation on the soft X-ray beamline at the Australian Synchrotron, lysozyme and proteinase K crystals have been exposed to a vacuum of 10−5 mbar, prior to flash-cooling in a bath of liquid nitrogen. Subsequent data collection on the MX2 beamline at the Australian Synchrotron demonstrated that, for lysozyme and proteinase K, it is possible to subject these mounted crystals to a vacuum pressure of 10−5 mbar without destroying the crystal lattice. Despite the lower data quality of the vacuum-pumped crystals compared with control crystals, it is demonstrated that the protein crystals can survive in a vacuum under suitable conditions. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The article discusses the addition to the tutorial for teaching macromolecular crystallography. The updates include experiments on structure determination by single isomorphous replacement including anomalous scattering (SIRAS) using a gold derivative of tetragonal HEWL and a structure determination by ultraviolet radiation damage induced phasing on the protein thaumatin. The update is said to provide a complete description of crystallographic experiment covering all aspects from crystallization to structure determination and interpretation.

Five experiments have been designed to be used for teaching macromolecular crystallography. The three proteins used in this tutorial are all commercially available; they can be easily and reproducibly crystallized and mounted for diffraction data collection. For each of the five experiments the raw images and the processed data of a sample diffraction data set as well as the refined coordinates and phases are provided for teaching the steps of data processing and structure determination. [ABSTRACT FROM AUTHOR]

23 different crystal forms of 19 different biological macromolecules were examined with respect to their anomalously scattering substructures using diffraction data collected at a wavelength of 2.0 Å (6.2 keV). In more than 90% of the cases the substructure was found to contain more than just the protein S atoms. The data presented suggest that chloride, sulfate, phosphate or metal ions from the buffer or even from the purification protocol are frequently bound to the protein molecule and that these ions are often overlooked, especially if they are not bound at full occupancy. Thus, in order to fully describe the macromolecule under study, it seems desirable that any structure determination be complemented with a long-wavelength data set. [ABSTRACT FROM AUTHOR]

Complete and highly redundant data sets were collected at different wavelengths between 0.80 and 2.65 Å for a total of ten different protein and DNA model systems. The magnitude of the anomalous signal-to-noise ratio as assessed by the quotient Ranom/Rr.i.m. was found to be influenced by the data-collection wavelength and the nature of the anomalously scattering substructure. By utilizing simple empirical correlations, for instance between the estimated ΔF/F and the expected Ranom or the data-collection wavelength and the expected Rr.i.m., the wavelength at which the highest anomalous signal-to-noise ratio can be expected could be estimated even before the experiment. Almost independent of the nature of the anomalously scattering substructure and provided that no elemental X-ray absorption edge is nearby, this optimal wavelength is 2.1 Å. [ABSTRACT FROM AUTHOR]

The EMBL-Hamburg Automated Crystal Structure Determination Platform is a sys-tem that combines a number of existing macromolecular crystallographic computer programs and several decision-makers into a software pipeline for automated and efficient crystal structure determination. The pipeline can be invoked as soon as X-ray data from derivatized protein crystals have been collected and processed. It is controlled by a web-based graphical user interface for data and parameter input, and for monitoring the progress of structure determination. A large number of possible structure-solution paths are encoded in the system and the optimal path is selected by the decision-makers as the structure solution evolves. The processes have been optimized for speed so that the pipeline can be used effectively for validating the X-ray experiment at a synchrotron beamline. [ABSTRACT FROM AUTHOR]

Complete and highly redundant data sets were collected at nine different wavelengths between 0.80 and 2.65 Å on a xenon derivative of porcine pancreatic elastase in both air and helium atmospheres. The magnitude of the anomalous signal, as assessed by the xenon-peak height in the anomalous difference Patterson synthesis, is affected by the wavelength of data collection as well as by the scaling model used. For data collected at wavelengths longer than 1.7 Å, the use of a three-dimensional scaling protocol is essential in order to obtain the highest possible anomalous signal. Based on the scaling protocols currently available, the optimal wavelength range for data collection appears to be between 2.1 and 2.4 Å. Beyond that, any further increase in signal will be compensated for or even superseded by a concomitant increase in noise, which cannot be fully corrected for. Data collection in a helium atmosphere yields higher I/σ(I) values, but not significantly better anomalous differences, than data collection in air. [ABSTRACT FROM AUTHOR]

Summary Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step in the lysine biosynthesis pathway of bacteria. The pathway can be regulated by feedback inhibition of DHDPS through the allosteric binding of the end product, lysine. The current dogma states that DHDPS from Gram-negative bacteria are inhibited by lysine but orthologs from Gram-positive species are not. The 1.65-Å resolution structure of the Gram-negative Legionella pneumophila DHDPS and the 1.88-Å resolution structure of the Gram-positive Streptococcus pneumoniae DHDPS bound to lysine, together with comprehensive functional analyses, show that this dogma is incorrect. We subsequently employed our crystallographic data with bioinformatics, mutagenesis, enzyme kinetics, and microscale thermophoresis to reveal that lysine-mediated inhibition is not defined by Gram staining, but by the presence of a His or Glu at position 56 ( Escherichia coli numbering). This study has unveiled the molecular determinants defining lysine-mediated allosteric inhibition of bacterial DHDPS. [ABSTRACT FROM AUTHOR]