In-house X-ray Source Research Articles

Kilohertz Macromolecular Crystallography Using an EIGER Detector at Low X-ray Fluxes

Time-resolved in-house macromolecular crystallography is primarily limited by the capabilities of the in-house X-ray sources. These sources can only provide a time-averaged structure of the macromolecules. A significant effort has been made in the development of in-house laser-driven ultrafast X-ray sources, with one of the goals as realizing the visualization of the structural dynamics of macromolecules at a very short timescale within the laboratory-scale infrastructure. Most of such in-house ultrafast X-ray sources are operated at high repetition rates and usually deliver very low flux. Therefore, the necessity of a detector that can operate at the repetition rate of the laser and perform extremely well under low flux conditions is essential. Here, we present experimental results demonstrating the usability of the hybrid-pixel detectors, such as Eiger X 1M, and provide experimental proof that they can be successfully operated to collect macromolecular crystallographic data up to a detector frame rate of 3 kHz from synchrotron sources. Our results also show that the data reduction and structural analysis are successful at such high frame rates and fluxes as low as 108 photons/s, which is comparable to the values expected from a typical laser-driven X-ray source.

Small-Angle X-Ray Scattering for the Study of Proteins in the Ubiquitin Pathway.

Small-angle X-ray scattering (SAXS) is an invaluable complement to other biophysical methods used to interrogate the structure and dynamics of proteins. Here, we describe the standard experimental protocol used in our laboratory to analyze proteins in the ubiquitin pathway. The method addresses buffer selection, data collection using an in-house X-ray source, diagnostic tests to assess data quality, and computational approaches to interpret SAXS data.

Optimizing data collection with the latest-generation in-house X-ray sources

Optimizing data collection with the latest-generation in-house X-ray sources

Cloning, expression, purification, characterization, crystallization and X-ray crystallographic analysis of recombinant Der f 21 (rDer f 21) from Dermatophagoides farinae.

Dermatophagoides farinae is one of the major house dust mite (HDM) species that cause allergic diseases. N-terminally His-tagged recombinant Der f 21 (rDer f 21), a group 21 allergen, with the signal peptide truncated was successfully overexpressed in an Escherichia coli expression system. The purified rDer f 21 protein was initially crystallized using the sitting-drop vapour-diffusion method. Well diffracting protein crystals were obtained after optimization of the crystallization conditions using the hanging-drop vapour-diffusion method with a reservoir solution consisting of 0.19 M Tris-HCl pH 8.0, 32% PEG 400 at 293 K. X-ray diffraction data were collected to 1.49 Å resolution using an in-house X-ray source. The crystal belonged to the C-centered monoclinic space group C2, with unit-cell parameters a = 123.46, b = 27.71, c = 90.25 Å, β = 125.84°. The calculated Matthews coefficient (VM) of 2.06 Å(3) Da(-1) suggests that there are two molecules per asymmetric unit, with a solvent content of 40.3%. Despite sharing high sequence identity with Blo t 5 (45%) and Blo t 21 (41%), both of which were determined to be monomeric in solution, size-exclusion chromatography, static light scattering and self-rotation function analysis indicate that rDer f 21 is likely to be a dimeric protein.

Stabilization of porcine pancreatic elastase crystals by glutaraldehyde cross-linking.

Elastase is a serine protease from the chymotrypsin family of enzymes with the ability to degrade elastin, an important component of connective tissues. Excessive elastin proteolysis leads to a number of pathological diseases. Porcine pancreatic elastase (PPE) is often used for drug development as a model for human leukocyte elastase (HLE), with which it shares high sequence identity. Crystals of PPE were grown overnight using sodium sulfate and sodium acetate at acidic pH. Cross-linking the crystals with glutaraldehyde was needed to resist the soaking procedure with a diethyl N-(methyl)pyridinyl-substituted oxo-β-lactam inhibitor. Crystals of PPE bound to the inhibitor belonged to the orthorhombic space group P2₁2₁2₁, with unit-cell parameters a = 51.0, b = 58.3, c = 74.9 Å, and diffracted to 1.8 Å resolution using an in-house X-ray source.

Alternative phasing method in macromolecular crystallography

Selenium is the most widely used heavy atom for experimental phasing, either by single anomalous scattering (SAD) or multiple-wavelength anomalous diffraction (MAD) procedures. The use of the single isomorphous replacement (SIR) or single isomorphous replacement with anomalous scattering (SIRAS) phasing procedure with selenomethionine (Mse) containing proteins is not so commonly used, as it requires isomorphous native data. Several non-redundant X-ray diffraction data sets from various Mse derivatised protein crystals were collected at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation with 266 nm lasers. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation damage-induced phasing (RIP) technique [1]. These can be coupled with the anomalous signal from the dataset collected at the selenium absorption edge to obtain SIRAS phases in a UV-RIPAS phasing experiment [2]. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing at synchrotron [1,2] and at in-house X-ray source [3].

In-house zinc SAD phasing at Cu Kα edge.

De novo zinc single-wavelength anomalous dispersion (Zn-SAD) phasing has been demonstrated with the 1.9 Å resolution data of glucose isomerase and 2.6 Å resolution data of Staphylococcus aureus Fur (SaFur) collected using in-house Cu Kα X-ray source. The successful in-house Zn-SAD phasing of glucose isomerase, based on the anomalous signals of both zinc ions introduced to crystals by soaking and native sulfur atoms, drove us to determine the structure of SaFur, a zinc-containing transcription factor, by Zn-SAD phasing using in-house X-ray source. The abundance of zinc-containing proteins in nature, the easy zinc derivatization of the protein surface, no need of synchrotron access, and the successful experimental phasing with the modest 2.6 Å resolution SAD data indicate that inhouse Zn-SAD phasing can be widely applicable to structure determination.

Purification, crystallization and preliminary X-ray diffraction analysis of effector protein MoHrip2 fromMagnaporthe oryzae

MoHrip2, a novel effector protein from the pathogenic fungus Magnaporthe oryzae, was purified and crystallized using the sitting-drop vapour-diffusion method. Native crystals and selenomethionine-labelled crystals were obtained using 2.2 M ammonium sulfate as a precipitant. A native data set was collected to 2.0 Å resolution at 100 K using an in-house X-ray source and a selenomethionine-labelled data set containing anomalous signal was collected to 1.8 Å resolution at 100 K using a synchrotron source. Based on the anomalous signal generated from the Se atom, the MoHrip2 structure was successfully solved using the single-wavelength anomalous dispersion (SAD) method.

Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein MoHrip1 from Magnaporthe oryzae.

The effector protein MoHrip1 from the pathogenic fungus Magnaporthe oryzae was purified and crystallized using the sitting-drop vapour-diffusion method. Native crystals appeared in a solution composed of 0.005 M cobalt(II) chloride hexahydrate, 0.005 M nickel(II) chloride hexahydrate, 0.005 M cadmium chloride hydrate, 0.005 M magnesium chloride hexahydrate, 0.1 M HEPES pH 7.5, 12%(w/v) polyethylene glycol 3350. A native data set was collected to 1.9 Å resolution at 100 K using an in-house X-ray source. The structure of MoHrip1 was successfully determined by molecular replacement using a homologous structure.

Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the N-terminal domain of serine glutamate repeat A (SgrA) protein fromEnterococcus faecium

Serine glutamate repeat A (SgrA) protein is an LPxTG surface adhesin of Enterococcus faecium and is the first bacterial nidogen-binding protein identified to date. It has been suggested that it binds to human nidogen, the extracellular matrix molecule of basal lamina, and plays a key role in the invasion and colonization of eukaryotic host cells. SgrA(28-288), having both a putative ligand-binding A domain and repetitive B domain, was expressed in Escherichia coli and purified using Ni-affinity and hydrophobic interaction chromatography. Further, the putative ligand-binding region, rSgrA(28-153), was subcloned, overexpressed and purified in both native and selenomethionine-derivative forms. The native rSgrA(28-153) protein crystallized in the monoclinic space group P2(1) and diffracted to 3.3 Å resolution using an in-house X-ray source, with unit-cell parameters a = 35.84, b = 56.35, c = 60.20 Å, β = 106.5°.

Crystallization and preliminary diffraction analysis of the catalytic domain of major extracellular endoglucanase from Xanthomonas campestris pv. campestris.

Cellulases, such as endoglucanases, exoglucanases and β-glucosidases, are important enzymes used in the process of enzymatic hydrolysis of plant biomass. The bacteria Xanthomonas campestris pv. campestris expresses a large number of hydrolases and the major endoglucanase (XccEG), a member of glycoside hydrolase family 5 (GH5), is the most strongly secreted extracellularly. In this work, the native XccEG was purified from the extracellular extract and crystallization assays were performed on its catalytic domain. A complete data set was collected on an in-house X-ray source. The crystal diffracted to 2.7 Å resolution and belonged to space group C2, with unit-cell parameters a = 174.66, b = 141.53, c = 108.00 Å, β = 110.49°. The Matthews coefficient suggests a solvent content of 70.1% and the presence of four protein subunits in the asymmetric unit.

Purification, crystallization and preliminary X-ray diffraction analysis of the effector protein PevD1 from Verticillium dahliae.

The effector protein PevD1 from the pathogenic fungus Verticillium dahliae was purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals appeared in a solution consisting of 4.0 M sodium formate. A native data set was collected at 1.9 Å resolution at 100 K using an in-house X-ray source. Because of the absence of useful methinione in the protein sequence, derivative crystals that contained iodine were obtained by soaking in 1.25 M potassium iodide, and a data set that contained anomalous signal was collected using the same X-ray facility at a wavelength of 1.54 Å. The single-wavelength anomalous dispersion method was used to successfully solve the structure based on the anomalous signal generated from iodine.

Crystallization and preliminary X-ray analysis of a novel halotolerant feruloyl esterase identified from a soil metagenomic library.

Feruloyl esterase cleaves the ester linkage formed between ferulic acid and polysaccharides in plant cell walls and thus has wide potential industrial applications. A novel feruloyl esterase (EstF27) identified from a soil metagenomic library was crystallized and a complete data set was collected from a single cooled crystal using an in-house X-ray source. The crystal diffracted to 2.9 Å resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 94.35, b = 106.19, c = 188.51 Å, α = β = γ = 90.00°. A Matthews coefficient of 2.55 Å(3) Da(-1), with a corresponding solvent content of 51.84%, suggested the presence of ten protein subunits in the asymmetric unit.

Crystallization and preliminary X-ray diffraction analysis of human endoplasmic reticulum aminopeptidase 2.

Endoplasmic reticulum aminopeptidase 2 (ERAP2) is a critical enzyme involved in the final processing of MHC class I antigens. Peptide trimming by ERAP2 and the other members of the oxytocinase subfamily is essential to customize longer precursor peptides in order to fit them to the correct length required for presentation on major histocompatibility complex class I molecules. While recent structures of ERAP1 have provided an understanding of the `molecular-ruler' mechanism of substrate selection, little is known about the complementary activities of its homologue ERAP2 despite their sharing 49% sequence identity. In order to gain insights into the structure-function relationship of the oxytocinase subfamily, and in particular ERAP2, the luminal region of human ERAP2 has been crystallized in the presence of the inhibitor bestatin. The crystals belonged to an orthorhombic space group and diffracted anisotropically to 3.3 Å resolution in the best direction on an in-house X-ray source. A molecular-replacement solution suggested that the enzyme has adopted the closed state as has been observed in other inhibitor-bound aminopeptidase structures.

Crystallization and preliminary X-ray analysis of the RPB5 subunit of human RNA polymerase II.

RPB5 is an essential subunit of eukaryotic RNA polymerase II. It has been proposed to interact with DNA and several key transcription factors during transcription. These interactions are crucial for transcription and its regulation. Here, prior to obtaining complex structures of human RPB5 and its binding partners, recombinant human RPB5 was crystallized alone by vapour diffusion in hanging drops. A complete data set was collected from a single frozen crystal employing an in-house X-ray source. The crystal diffracted to 2.8 Å resolution and belonged to space group P4(3)2(1)2. The likely Matthews coefficient and solvent content of 2.67 Å(3) Da(-1) and 53.92%, respectively, suggested the presence of two protein subunits in the asymmetric unit. The structure was solved using molecular replacement.

Crystallization and preliminary X-ray analysis of a cold-active β-galactosidase from the psychrotrophic and halotolerant Planococcus sp. L4.

β-Galactosidases catalyze the hydrolysis of a galactosyl moiety from the nonreducing termini of oligosaccharides or from glycosides. A novel GH family 42 cold-active β-galactosidase identified from the psychrotrophic and halotolerant Planococcus sp. L4 (BgaP) was crystallized and a complete data set was collected from a single frozen crystal on an in-house X-ray source. The crystal diffracted to 2.8 Å resolution and belonged to space group P1, with unit-cell parameters a = 104.29, b = 118.12, c = 121.12 Å, α = 62.66, β = 69.48, γ=70.74°. Alikely Matthews coefficient of 2.58 Å(3) Da(-1) and solvent content of 52.32% suggested the presence of six protein subunits in the asymmetric unit.

Single isomorphous replacement phasing of selenomethionine-containing proteins using UV-induced radiation damage

The most commonly used heavy-atom derivative, selenium, requires the use of a tunable beamline to access the Se K edge for experimental phasing using anomalous diffraction methods, whereas X-ray diffraction experiments for selenium-specific ultraviolet radiation-damage-induced phasing can be performed on fixed-wavelength beamlines or even using in-house X-ray sources. Several nonredundant X-ray diffraction data sets were collected from three different selenomethionine (Mse) derivatized protein crystals at energies far below the absorption edge before and after exposing the crystal to ultraviolet (UV) radiation using 266 nm lasers of flux density 1.7 × 10¹⁵ photons s⁻¹ mm⁻² for 10-50 min. A detailed analysis revealed that significant changes in diffracted intensities were induced by ultraviolet irradiation whilst retaining crystal isomorphism. These intensity changes allowed the crystal structures to be solved by the radiation-damage-induced phasing (RIP) technique. Inspection of the crystal structures and electron-density maps demonstrated that covalent bonds between selenium and carbon at all sites located in the core of the proteins or in a hydrophobic environment were much more susceptible to UV radiation-induced cleavage than other bonds typically present in Mse proteins. The rapid UV radiation-induced bond cleavage opens a reliable new paradigm for phasing when no tunable X-ray source is available. The behaviour of Mse derivatives of various proteins provides novel insights and an initial basis for understanding the mechanism of selenium-specific UV radiation damage.

Solving X-ray protein structures without a crystal: using X-ray Free Electron Laser, the fourth generation synchrotron light sources

A synchrotron light source is a source of electromagnetic radiation artificially produced by specialized electron accelerators. Compared to the commonly used in-house X-ray sources, it is wavelength adjustable, much stronger and more focused. In the last two decades, synchrotron usage has become the mainstream for X-ray protein structure determination. Taking the advantage of micro-focus light beams of the third generation synchrotron, the size of a usable protein crystal for data collection decreases to micron level, which increases the rate of macromolecular structure determination to about 10 new protein data bank entries per day. The fourth generation synchrotron light sources not only inherit the above advantages of traditional synchrotron but also develop them to a higher level: you do not even need a protein crystal for atomic resolution X-ray structure determination by using the nano-focus beam line at the fourth generation synchrotron! This is obviously exciting news for those structural biologists working on membrane proteins and large macromolecular complexes which are notorious for crystallization. X-ray Free Electron Laser (XFEL), provided by the fourth generation synchrotron light sources, has femtosecond pulse length and both peak and average brightness largely exceeding the beam of 3rd generation sources. XFEL is a laser that shares the same optical properties as conventional lasers such as emitting a beam consisting of coherent electromagnetic radiation which can reach high power, but it uses a relativistic electron beam as the lasing medium, which moves freely through a magnetic structure. In other words, it is coherent in phase and has very high brightness. The world's first XFEL, called Linac Coherent Light Source (LCLS), was completed at Stanford's Linear Accelerator Center in 2009 (Hand, 2009). Theoretically, a beam emitted by this machine will have “a brightness that is 10 billion times greater than that of any existing X-ray source on earth,” reported by the press office at the Argonne National Laboratory. At an XFEL, a nano-focus beam line is available. Together with the incredible intensity, a crystal with 1 μm might be large enough to yield the necessary scattering data. Of course the radiation damage will cause serious problems here. So people need to change their strategy for collecting a complete data set from rotating one crystal during exposure while collecting and merging the images from maybe hundreds of different crystals. It is unlikely that a nano-crystal could survive from one shot of XFEL. The very short pulse of XFEL also provides a way out of the damage problem. Compared to the picosecond pulse of a conventional synchrotron, XFEL could provide femtosecond pulse length. A computer simulation showed that one can rapidly image a sample with such a short pulse before the atoms in the molecule may be destroyed (Neutze et al., 2004). The most revolutionary impact of an XFEL on structural biology is the collection of X-ray scattering data without crystals. The basic idea of processing X-ray scattering data of single molecule is developed from electron microscopy (EM) studies (Huldt et al., 2003). In EM research, the images are sorted and averaged by the cross correlation between them. Then the mutual orientation of the averaged images can be determined based upon the idea that any two projections through a three-dimensional object have at least one same line. It is very similar in X-ray studies that any two scattering images share at least one curve in common where the Ewald spheres intersect in reciprocal space. By sorting and averaging the X-ray scattering images collected from

High‐Resolution Crystal Structure of a Lasso Peptide

Lasso peptides are a group of a growing class of bioactive peptides of microbial origin. The first crystal structure of a member of this family, the glucagon receptor antagonist BI-32169, shows that the fold is built predominantly by regular secondary structural elements and a tight network of hydrogen bonds that are partially shielded from solvent by hydrophobic amino acid side chains. This results in an extraordinary stable structure that is resistant to thermal unfolding or proteolytic digestion, which facilitates its biological function.

Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of the collagen-binding region of RspB fromErysipelothrix rhusiopathiae

RspB is a surface adhesin of Erysipelothrix rhusiopathiae. A recombinant form of the collagen-binding region of this protein, RspB((31-348)), has been overexpressed in Escherichia coli in native and selenomethionine-derivative forms and purified using affinity and gel-permeation chromatography. Thin plate-like crystals were obtained by the hanging-drop vapour-diffusion method using the same condition for both forms. The native crystals diffracted to a resolution of 2.5 A using an in-house X-ray source, while the selenomethionine-derivative crystals diffracted to a resolution of 2.2 A using synchrotron radiation. The crystals belonged to the monoclinic space group P2(1), with unit-cell parameters a = 46.19, b = 66.65, c = 101.72 A, beta = 94.11 degrees .