Multiwavelength Anomalous Dispersion Experiment Research Articles

Fast, automated, continuous energy scans for experimental phasing at the BioMAX beamline.

In X-ray macromolecular crystallography (MX), single-wavelength anomalous dispersion (SAD) and multi-wavelength anomalous dispersion (MAD) techniques are commonly used for obtaining experimental phases. For an MX synchrotron beamline to support SAD and MAD techniques it is a prerequisite to have a reliable, fast and well automated energy scan routine. This work reports on a continuous energy scan procedure newly implemented at the BioMAX MX beamline at MAX IV Laboratory. The continuous energy scan is fully automated, capable of measuring accurate fluorescence counts over the absorption edge of interest while minimizing the sample exposure to X-rays, and is about a factor of five faster compared with a conventional step scan previously operational at BioMAX. The implementation of the continuous energy scan facilitates the prompt access to the anomalous scattering data, required for the SAD and MAD experiments.

BL-11C Micro-MX: a high-flux microfocus macromolecular-crystallography beamline for micrometre-sized protein crystals at Pohang Light Source II.

BL-11C, a new protein crystallography beamline, is an in-vacuum undulator-based microfocus beamline used for macromolecular crystallography at the Pohang Accelerator Laboratory and it was made available to users in June 2017. The beamline is energy tunable in the range 5.0-20 keV to support conventional single- and multi-wavelength anomalous-dispersion experiments against a wide range of heavy metals. At the standard working energy of 12.659 keV, the monochromated beam is focused to 4.1 µm (V) × 8.5 µm (H) full width at half-maximum at the sample position and the measured photon flux is 1.3 × 1012 photons s-1. The experimental station is equipped with a Pilatus3 6M detector, a micro-diffractometer (MD2S) incorporating a multi-axis goniometer, and a robotic sample exchanger (CATS) with a dewar capacity of 90 samples. This beamline is suitable for structural determination of weakly diffracting crystalline substances, such as biomaterials, including protein, nucleic acids and their complexes. In addition, serial crystallography experiments for determining crystal structures at room temperature are possible. Herein, the current beamline characteristics, technical information for users and some recent scientific highlights are described.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of the L,D-transpeptidase LdtMt1 from Mycobacterium tuberculosis.

Mycobacterium tuberculosis is capable of adapting to prolonged periods of dormancy, a state which is resistant to killing by antimycobacterial agents. The L,D-transpeptidation reaction catalysed by the L,D-transpeptidase LdtMt1 is likely to play an essential role in the adaptation of M. tuberculosis to its dormant state. LdtMt1 has been successfully crystallized using vapour-diffusion methods. The crystals of this protein belonged to space group P6₅22, with unit-cell parameters a=57.25, b=57.25, c=257.96 Å, α=90, β=90, γ=120°. Diffraction data have also been collected from a selenomethionine derivative to 2.9 Å resolution. Model building using the phases derived from the multiwavelength anomalous dispersion experiment is in progress.

Expression, purification, crystallization and preliminary X-ray crystallographic analysis of a resuscitation-promoting factor from Mycobacterium tuberculosis.

The resuscitation-promoting factor RpfB, the most complex of the five resuscitation-promoting factors produced by M. tuberculosis, is devoted to bacterial reactivation from the dormant state. RpfB consists of 362 residues predicted to form five domains. An RpfB fragment containing the protein catalytic domain and a G5 domain has been successfully crystallized using vapour-diffusion methods. This is the first crystallographic study of a resuscitation-promoting factor. Crystals of this protein belong to space group I422, with unit-cell parameters a = 97.63, b = 97.63, c = 114.87 A. Diffraction data have also been collected from a selenomethionine derivative at 2.9 A resolution. Model building using the phases derived from the multiwavelength anomalous dispersion experiment is in progress.

The Structure of 2-Oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline Decarboxylase Provides Insights into the Mechanism of Uric Acid Degradation

The complete degradation of uric acid to (S)-allantoin, as recently elucidated, involves three enzymatic reactions. Inactivation by pseudogenization of the genes of the pathway occurred during hominoid evolution, resulting in a high concentration of urate in the blood and susceptibility to gout. Here, we describe the 1.8A resolution crystal structure of the homodimeric 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase, which catalyzes the last step in the urate degradation pathway, for both ligand-free enzyme and enzyme in complex with the substrate analogs (R)-allantoin and guanine. Each monomer comprises ten alpha-helices, grouped into two domains and assembled in a novel fold. The structure and the mutational analysis of the active site have allowed us to identify some residues that are essential for catalysis, among which His-67 and Glu-87 appear to play a particularly significant role. Glu-87 may facilitate the exit of the carboxylate group because of electrostatic repulsion that destabilizes the ground state of the substrate, whereas His-67 is likely to be involved in a protonation step leading to the stereoselective formation of the (S)-allantoin enantiomer as reaction product. The structural and functional characterization of 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline decarboxylase can provide useful information in view of the potential use of this enzyme in the enzymatic therapy of gout.

Crystal structure of phosphoribosylformylglycinamidine synthase II (smPurL) from Thermotoga maritima at 2.15 Å resolution

Crystal structure of phosphoribosylformylglycinamidine synthase II (smPurL) from <i>Thermotoga maritima</i> at 2.15 Å resolution

The macromolecular crystallography station at beamline BM16 at the ESRF

A beamline previously used for powder diffraction (BM16) at the European Synchrotron Radiation Facility has been redesigned and rebuilt to carry out both mcromolecular crystallography (MX) and noncrystalline diffraction/scattering (NCD/S) experiments. The MX station has become available for routine user experiments since September 2003 and the NCD/S station is currently being commissioned. The optics are designed so that both the focal spot and flux are good in the 6–17keV energy range (2.1–0.73Å), while the performance is optimal at the selenium K-edge (12.658keV,0.98Å). The MX facility is fully equipped to carry out multiwavelength anomalous dispersion experiments, in particular at the SeK-edge. Moreover, experiments using lower energies such as those of the Mn and FeK-edges are also possible. Here we describe the optical configuration and the setup for MX experiments, as well as a typical example of the experiments carried out so far.

Crystal structure of an indigoidine synthase A (IndA)‐like protein (TM1464) from Thermotoga maritima at 1.90 Å resolution reveals a new fold

Crystal structure of an indigoidine synthase A (IndA)‐like protein (TM1464) from <i>Thermotoga maritima</i> at 1.90 Å resolution reveals a new fold

The Human TREX2 3′ → 5′-Exonuclease Structure Suggests a Mechanism for Efficient Nonprocessive DNA Catalysis

The 3' --> 5'-exonucleases process DNA ends in many DNA repair pathways of human cells. Determination of the human TREX2 structure is the first of a dimeric 3'-deoxyribonuclease and indicates how this highly efficient nonprocessive enzyme removes nucleotides at DNA 3' termini. Symmetry in the TREX2 dimer positions the active sites at opposite outer edges providing open access for the DNA. Adjacent to each active site is a flexible region containing three arginines positioned appropriately to bind DNA and to control its entry into the active site. Mutation of these three arginines to alanines reduces the DNA binding capacity by approximately 100-fold with no effect on catalysis. The human TREX2 catalytic residues overlay with the bacterial DnaQ family of 3'-exonucleases confirming the structural conservation of the catalytic sites despite limited sequence identity, and mutations of these residues decrease the still measurable activity by approximately 10(5)-fold, confirming their catalytic role.

Crystal structure of a tandem cystathionine‐β‐synthase (CBS) domain protein (TM0935) from Thermotoga maritima at 1.87 Å resolution

Crystal structure of a tandem cystathionine‐β‐synthase (CBS) domain protein (TM0935) from <i>Thermotoga maritima</i> at 1.87 Å resolution

Optimizing data collection for structure determination.

The ultimate purpose of diffraction data collection is to produce a data set which will result in the required structural information about the molecule of interest. This usually entails collecting a complete and accurate set of reflection intensities to as high a resolution as possible. In practice, the characteristics of the crystal and properties of the X-ray source can be limiting factors to the data-set quality that can be achieved and a careful strategy has to be used to extract the maximum amount of information from the data within the experimental constraints. In the particular case of data intended for phasing using anomalous dispersion, the synchrotron beamline properties are relevant to determine how many wavelengths (one or more) should be used for the experiment and what the wavelength values should be. This will in turn affect the detailed strategy for data collection, including decisions about the data-collection sequence and how much data to collect at each wavelength. Collection of multiwavelength anomalous dispersion (MAD) data at three different wavelengths can provide very accurate experimental phases. Two-wavelength MAD experiments may offer the best compromise between phase quality and minimizing the effects of radiation damage to the sample. However, MAD experiments are demanding in terms of beamline wavelength range, easy tunability, stability and reproducibility. When the beamline cannot fulfill these demands, single-wavelength experiments may be a better option.

Generation of noble-gas binding sites for crystallographic phasing using site-directed mutagenesis.

In recent years, the use of noble-gas complexes for the de novo phasing of protein structures has proven to be a useful alternative to selenomethionine and more traditional derivatives, largely owing to improvements in methods for incorporating noble gases within protein crystals. Advantages of noble-gas derivatives include a high degree of isomorphism when using xenon for multiple isomorphous replacement (MIR) and an easily accessible absorption edge when using krypton for multiwavelength anomalous dispersion (MAD) phasing. One problem with this approach is that not all proteins contain cavities which bind noble gases. Even in proteins which do bind noble gases, the resulting derivative may not be sufficient for phasing. Using T4 lysozyme as an example, it is illustrated how this limitation might be overcome by using 'large-to-small' mutations to introduce potential noble-gas binding sites. Wild-type T4 lysozyme contains a single xenon-binding site. By truncating leucine and phenylalanine residues to alanine, it is possible to generate additional noble-gas binding sites within the core of the protein. Combining rotating-anode data from xenon complexes of wild-type and mutant lysozymes yields MIR phases which compare favorably with those determined from a selenomethionine MAD experiment conducted at a synchrotron. Experience with T4 lysozyme suggests that a leucine-to-alanine substitution made at random in a protein of unknown structure has about a 30% chance of providing a useful derivative. This procedure holds promise for the determination of unknown protein structures, especially when selenomethionine-containing protein is not available or when access to a synchrotron is limited.

MAD data collection - current trends.

The multiwavelength anomalous dispersion (MAD) method of protein structure determination is becoming a routine technique in protein crystallography. The increased number of wavelength-tuneable synchrotron beamlines capable of performing challenging MAD experiments, coupled with the widespread availability of charge-coupled device (CCD) based X-ray detectors with fast read-out times have brought MAD structure determination to a new exciting level. Ultrafast MAD data collection is now possible and, with the widespread use of selenium in the form of selenomethionine for phase determination, the method is growing in popularity. Recent developments in crystallographic software are complementing the above advances, paving the way for rapid protein structure determination. An overview of a typical MAD experiment is described, with emphasis on the rates and quality of data acquisition now achievable at third-generation synchrotron sources.

Use of Chemically Modified Nucleotides to Determine a 62-Nucleotide RNA Crystal Structure: A Survey of Phosphorothioates, Br, Pt and Hg

Two important challenges confronting RNA crystallographers are producing crystals and finding isomorphous heavy-atom derivatives. Non-isomorphism can be addressed by determining the phases using the multiwavelength anomalous dispersion (MAD) method. These phases can be greatly improved by combining phases from MAD experiments done on different heavy-atom derivatives. Heavy-atom derivatives can be created by chemically modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH3)3]2+ to N7 of guanine. While phosphorothioates can provide mercury binding sites, disorder can reduce their value for phase determination. The location of these chemical modifications is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crystal packing.