Goniometer Head Research Articles

Femtosecond Laser Processing of Agarose Gel Surrounding Protein Crystals for Development of an Automated Crystal Capturing System

Protein crystals must be captured to be mounted onto the goniometer head of X-ray diffraction equipment for structural analysis. However, this capturing operation has to be performed manually under microscopic observation. Crystallographers often face problems with this operation because protein crystals are very soft and fragile. Here, we crystallized elastase, thaumatin, glucose isomerase, and lysozyme in 2.0% (w/v) agarose gels and applied a femtosecond laser to process the agarose gel surrounding the protein crystals. A software-based operation system was established to enable automated laser processing. This new approach allows high-speed, high-precision, and reproducible processing of the gel without unsealing the crystallization trays. The processed gel containing crystals could be captured using a nylon loop without difficulty, followed by mounting the crystal onto the goniometer head of the X-ray diffraction equipment. X-ray diffraction analysis of such crystals suggested that the processed agarose gel with a thickness of approximately <0.2 mm has little effect on the background X-ray scattering. Furthermore, the effect of laser irradiation was investigated by X-ray diffraction and subsequent structural analyses, which demonstrated that the quality of the diffraction data and obtained electron density was essentially the same as that obtained before laser irradiation. On the other hand, the manually processed gel-grown crystals gave higher values on the background X-ray scattering. These comparative experimental results show clear advantages of our laser processing system. This approach leads to the possibility that protein crystals can be captured reproducibly without affecting any later crystallographic analysis, thereby providing an automated system for crystal capture.

Establishment of a computer-controlled retroreflection measurement facility to characterize photometric properties of retroreflectors

Determination of photometric properties of retroreflectors used especially on cars, motorways and clothes for safety of traffic is very important. To characterize the retroreflection properties of such materials, a retroreflection measurement system was constructed at TÜBİTAK-UME. The measurement system is composed of a lighting projector, a two-dimensional goniometer, reference and observation photometer heads with transimpedance amplifiers and an analog digital converter card with a developed software. The system is capable of measuring both luminous intensity and retroreflection coefficients of retroreflective materials at observation angles varying from 0.1° to 5°.

A versatile sample-environment cell for non-ambient X-ray scattering experiments

A compact reaction cell is described forin-situexperiments requiring control of both the temperature of the sample and the atmosphere over the sample. The cell incorporates an optional furnace capable of temperatures of up to ∼1273 K. The compact design and ability of the cell to mount directly on a standard goniometer head allows portability to a large number of diffraction instruments at synchrotron sources.

High-throughput operation of sample-exchange robots with double tongs at the Photon Factory beamlines

Sample-exchange robots that can exchange cryo-pins bearing protein crystals out of experimental hutches according to user instructions have been developed. The robots were designed based on the SAM (Stanford Synchrotron Research Laboratory automated mounting) system. In order to reduce the time required for the sample exchange, the single tongs of the SAM system were modified and a double-tongs system that can hold two cryo-pins at the same time was developed. Robots with double tongs can move to the goniometer head holding the next cryo-pin with one set of tongs, dismount the experimented cryo-pin with the other set, and then mount the next pin onto the goniometer head without leaving the diffractometer area. Two different types of tongs have been installed: single tongs at beamlines BL-5A and AR-NW12A, and a double-tongs system at beamline BL-17A of the Photon Factory. The same graphical user interface software for operation of the sample-exchange robots is used at all beamlines, however, so that users do not need to consider differences between the systems. In a trial, the robot with double tongs could exchange samples within 10 s.

A plate holder for non-destructive testing of mesophase crystallization assays

Here, we describe a goniometer holder to mount standard 96-well crystallization plates directly onto the goniometer head of an oscillation camera. This attachment was designed to check crystallization conditions straight from the crystallization plates under X-rays, and was proven to be useful for checking small crystals and solutions that destabilize monoolein-based lipidic cubic phase (LCP) crystallization experiments. A quick procedure for setting up LCP assays employing commercially available instruments is also reported.

A mini-goniometer for X-ray diffraction studies down to 4 K on four-circle diffractometers equipped with two-dimensional detectors

A `universal' low-temperature device for laboratory X-ray diffractometers equipped with two-dimensional detectors has been developed. Single-crystal data collections can be performed down to 4 K. Owing to its original design, the completeness of the data set is not affected by the limited number of accessible orientations of the sample. Classical structure analysis can therefore be performed as well as high-resolution (high-angle) studies for electron-density analysis. Derived from an idea of Argoud &amp; Muller [J. Appl. Cryst.(1989),22, 584–591], the sample is mounted on a holder magnetically coupled to the diffractometer φ axis. The coupling is achieved by mounting a master magnet in place of the usual goniometer head. This magnet drives a slave magnet fixed on the crystal holder: a two-axis mini-goniometer. This low-temperature arrangement is adaptable to any kappa-geometry single-crystal diffractometer equipped with a two-dimensional detector, and can be placed into various types of cryostat. This paper reports the home-made mechanical design and the performance of this device.

フォトンファクトリー構造生物ビームラインにおける自動化に向けた取り組み

The Photon Factory currently operates four synchrotron beamlines for protein crystallography and two more beamlines are scheduled to be constructed in the next years. Over the last years these beamlines have been upgraded and equipped with a fully automated beamline control system based on a robotic sample changer. The current system allows for remote operation, controlled from the user's area, of sample mounting, centering and data collection of pre-frozen crystals mounted in Hampton-type cryo-loops on goniometer head. New intuitive graphical user interfaces have been developed so as to control the complete beamline operation. Furthermore, algorithms for automatic sample centering based on pattern matching and X-ray beam scanning are being developed and combined with newly developed diffraction evaluation programs in order to complete entire automation of the data collection.

The Crystal Structure of Mitochondrial (Type 1A) Peptide Deformylase Provides Clear Guidelines for the Design of Inhibitors Specific for the Bacterial Forms

Peptide deformylase (PDF) inhibitors have a strong potential to be used as a new class of antibiotics. However, recent studies have shown that the mitochondria of most eukaryotes, including humans, contain an essential PDF, PDF1A. The crystal structure of the Arabidopsis thaliana PDF1A (AtPDF1A), considered representative of PDF1As in general, has been determined. This structure displays several similarities to that of known bacterial PDFs. AtPDF1A behaves as a dimer, with the C-terminal residues responsible for linking the two subunits. This arrangement is similar to that of Leptospira interrogans PDF, the only other dimeric PDF identified to date. AtPDF1A is the first PDF for which zinc has been identified as the catalytic ion. However, the zinc binding pocket does not differ from the binding pockets of PDFs with iron rather than zinc. The crystal structure of AtPDF1A in complex with a substrate analog revealed that the substrate binding pocket of PDF1A displays strong modifications. The S1' binding pocket is significantly narrower, due to the creation of a floor from residues present in all PDF1As but not in bacterial PDFs. A true S3' pocket is created by the residues of a helical CD-loop, which is very long in PDF1As. Finally, these modified substrate binding pockets modify the position of the substrate in the active site. These differences provide guidelines for the design of bacterial PDF inhibitors that will not target mitochondrial PDFs.

An Unusual, His-dependent Family I Pyrophosphatase from Mycobacterium tuberculosis

Soluble inorganic pyrophosphatases (PPases) comprise two evolutionarily unrelated families (I and II). These two families have different specificities for metal cofactors, which is thought to be because of the fact that family II PPases have three active site histidines, whereas family I PPases have none. Here, we report the structural and functional characterization of a unique family I PPase from Mycobacterium tuberculosis (mtPPase) that has two His residues (His21 and His86) in the active site. The 1.3-A three-dimensional structure of mtPPase shows that His86 directly interacts with bound sulfate, which mimics the product phosphate. Otherwise, mtPPase is structurally very similar to the well studied family I hexameric PPase from Escherichia coli, although mtPPase lacks the intersubunit metal binding site found in E. coli PPase. The cofactor specificity of mtPPase resembles that of E. coli PPase in that it has high activity in the presence of Mg2+, but it differs from the E. coli enzyme and family II PPases because it has much lower activity in the presence of Mn2+ or Zn2+. Replacements of His21 and His86 in mtPPase with the residues found in the corresponding positions of E. coli PPase had either no effect on the Mg2+- and Mn2+-supported reactions (H86K) or reduced Mg2+-supported activity (H21K). However, both replacements markedly increased the Zn2+-supported activity of mtPPase (up to 11-fold). In the double mutant, Zn2+ was a 2.5-fold better cofactor than Mg2+. These results show that the His residues in mtPPase are not essential for catalysis, although they determine cofactor specificity.

Fine-rotation attachment to standard goniometers

A new type of fine-rotation stage has been constructed and tested. It can be attached to standard goniometers used in X-ray and neutron crystallography. The device consists of a shaft and a bar that is fitted tightly to a hole traversing the shaft. The diameter of the shaft is 5 to 10 times larger than the diameter of the bar and the length of the bar is about 5 times larger than the height of the shaft. The bottom of the shaft is attached to the top plate of the goniometer and a goniometer head can be fitted to the other end of the shaft. The free end of the bar is pushed tangentially by a linear actuator to produce a torsion moment at the shaft. The dimensions and materials of the prototype were chosen such that a 1 mm bend of the bar corresponded to a torsion angle of the shaft of about 20 µrad. The rotation angle was measured using a double-crystal diffractometer in the non-dispersive setting, with MoKα1radiation from a fine-focus X-ray tube. Accurately known angular deviations were produced by refraction in a prism and the shifts in the rocking-curve position were measured. The measured torsion angle agreed within 4% with the value calculated from the elastic constants and dimensions of the device. The repeatability of the angle was ±20 nrad (0.004 arcsec).

Cocrystallization with Acetylene. The 1 : 1 Complex with Benzene: Crystal Growth, X‐Ray Diffraction and Molecular Simulations

AbstractThe crystal structure of an unusual 1 : 1 molecular complex between benzene and acetylene, two very small and apolar molecules, has been determined by X‐ray‐analysis of crystals grown by first mixing the two liquids under conditions of low temperature and high pressure in a capillary, followed by repeated zone melting to form crystals directly on the goniometer head of a diffractometer. Each acetylene molecule is clamped between two parallel benzene rings, with its molecular axis apparently perpendicular to the benzene planes. Closer inspection of thermal‐motion data from the diffraction experiment suggests that the acetylene molecule undergoes a wobbling, or precession, motion between the two rings so that it is perpendicular to them only in a time‐averaged sense. The results of quantum‐chemical calculations on isolated molecular dimers and trimers support this conclusion. In addition, the calculation of separate coulombic, dispersion, polarization and repulsion contributions to intermolecular bonding reveals that the CH⋅⋅⋅π‐bond interaction between acetylene and benzene in a T‐shaped dimer consists of a mixture of coulombic and polarization interactions. In the benzeneacetylene cocrystal, its magnitude is quantitatively comparable with that of other dispersive interactions. 5.4 ns Molecular‐dynamics simulations of the liquid mixture reveal that the two components are persistently miscible, a possible key to the formation of the cocrystal. No structure is, however, observed in the solution during the relatively short simulation time.

SYSTEMATICS IN THE STRUCTURE AND XANES SPECTRA OF PYROXENES, AMPHIBOLES, AND MICAS AS DERIVED FROM ORIENTED SINGLE CRYSTALS

In order to assess the extent of X-ray anisotropy in biaxial minerals, Fe XANES spectra were acquired on single crystals of pyroxenes, amphiboles, and micas with the X-ray beam polarized along the X, Y, and Z optical orientations. Specifically, single crystals of enstatite (En), augite (Aug), aegirine (Ae), kaersutite (Krs; three different compositions), dioctahedral (Ms and Ill), and trioctahedral (Ann and Phl) micas were selected. Each crystal was oriented morphologically and with the aid of EXCALIBR using a spindle stage equipped polarized light microscope, and their XANES spectra were acquired at beamline X26a, NSLS, using a special geometry of the beam to accommodate the goniometer head and a 20 � 30 � m X-ray beam. As would be expected from visible and IR spectra of these minerals, the intensity of both pre-edge and main edge peaks is variable as a function of orientation. Structural similarities among these mineral groups result in similarities among their XANES spectra. Spectra acquired along the length of the chains (c for pyroxene, c for amphibole, and a for sheet silicates) are similar, with corresponding changes in the optical directions, such that Ydioct ≈ Ztrioct ≈ ZKrs ≈ ZEn ≈ XAe (clinopyroxene is intermediate). Spectra taken along the b crystallographic axes (along the layer of octahedra, across the I-beam) are similar for all groups (Zdioct ≈ Ytrioct ≈ YKrs ≈ XEn ≈ YAug), and more similar for the amphiboles and micas. Lastly, spectra acquired along the stacking direction, which is the a crystallographic direction for pyroxene and amphiboles and the c crystallographic direction in sheet silicates, are analogous. From these relationships, assignment of specific transitions to individual features in XANES spectra are facilitated. Furthermore, errors on use of pre-edge positions for determination of Fe 3+ /� Fe in minerals can be better constrained.

Use of the spindle stage for orientation of single crystals for microXAS: Isotropy and anisotropy in Fe-XANES spectra

A new method has been developed to orient 50–100 μm size single crystals to obtain XANES or EXAFS spectra along any crystallographic orientation relative to a polarized synchrotron X-ray beam. The method uses a modified spindle stage and an X-ray goniometer head to hold a crystal and the glass fiber on which it is mounted. The orientation of the crystallographic axes within the crystal can be determined by X-ray diffraction in all cases or with the use of the polarizing light microscope if the minerals are biaxial. Once an optical orientation direction has been located, it can be oriented parallel to the polarization direction of the synchrotron source for spectral acquisition. Mineral samples representing the three optical classes (i.e., isotropic, uniaxial, and biaxial) were oriented along their a , b , and c crystallographic axes, and Fe-XANES spectra were obtained. In all cases the interactions of polarized X-rays with the crystal structures are analogous to the interactions of visible light (e.g., as represented by refractive index). For the isotropic almandine sample the XANES spectra were similar at all orientations. For the uniaxial buergerite and scapolite samples the spectra were similar along the a and b axes and different along c . A detailed set of spectra acquired at multiple (known) optical orientations in the scapolite crystal demonstrates that spectral features vary in intensity as a function of orientation, with maxima and minima when the X-rays are polarized along optical orientations. For the biaxial fayalite sample, the XANES spectra differed along all three crystallographic axes, as expected by analogy to optical properties.

The Crystal Structure of the Endothelial Protein C Receptor and a Bound Phospholipid

The endothelial cell protein C receptor (EPCR) shares approximately 20% sequence identity with the major histocompatibility complex class 1/CD1 family of molecules, accelerates the thrombin-thrombomodulin-dependent generation of activated protein C, a natural anticoagulant, binds to activated neutrophils, and can undergo translocation from the plasma membrane to the nucleus. Blocking protein C/activated protein C binding to the receptor inhibits not only protein C activation but the ability of the host to respond appropriately to bacterial challenge, exacerbating both the coagulant and inflammatory responses. To understand how EPCR accomplishes these multiple tasks, we solved the crystal structure of EPCR alone and in complex with the phospholipid binding domain of protein C. The structures were strikingly similar to CD1d. A tightly bound phospholipid resides in the groove typically involved in antigen presentation. The protein C binding site is outside this conserved groove and is distal from the membrane-spanning domain. Extraction of the lipid resulted in loss of protein C binding, which could be restored by lipid reconstitution. CD1d augments the immune response by presenting glycolipid antigens. The EPCR structure is a model for how CD1d binds lipids and further suggests additional potential functions for EPCR in immune regulation, possibly including the anti-phospholipid syndrome.

A NEW METHOD TO DETERMINE THE OPTICAL ORIENTATION OF BIAXIAL MINERALS: A MATHEMATICAL APPROACH

A mathematical approach has been developed to determine the optical orientation of biaxial minerals by combining spindle stage and X-ray-diffractometer measurements. First, a goniometer head, with crystal affixed, is screwed onto a spindle stage, and its orientation matrix for the biaxial indicatrix is determined. Next, the same goniometer head is transferred to a single-crystal X-ray diffractometer, where the unit cell and, in turn, an orientation matrix for the reciprocal crystallographic axes are determined. The necessary transformations and calculations are then developed to relate these two orientation matrices mathematically. Examples for two orthorhombic minerals of known optical orientation, one monoclinic mineral of unknown optical orientation, and one triclinic mineral of known optical orientation are given. Several measurements were repeated to test the accuracy, reproducibility, and systematic errors of the method; these measurements yielded an accuracy and reproducibility of the method to within 1.0°. Once a suitable crystal has been selected and mounted on a goniometer head, all the required measurements and calculations can be performed in approximately one hour.

A laboratory use image plate camera for surface and interface structure analysis

We designed a new X-ray camera with an image plate (IP) detector so as to study surface and interface structures. It is made up of two goniometers with coaxial setup and a curved IP cassette that traverses up and down. The axis of rotation is vertical and perpendicular to the incident beam. The sample is mounted on one of the goniometer. The other goniometer carries an arm. This arm is intended to hold a hemicylindrical metal sleeve with a narrow slot or with crossed slits. Owing to the slit screen, of which rotation can be synchronized with the sample rotation and the IP translation, the background was reduced by half. Consequently, we could detect X-ray surface scattering of the order of 10−6. On the other hand, if the slit screen is held stationary, an easy measurement of diffuse X-ray reflectivity was found to be possible by combining the IP translation and the sample rotation. Since the goniometer and goniometer head are intended for experimental setup of heavy and large devices, vacuum chambers or cryostats can easily be installed. Therefore, the IP camera enables to measure the diffraction from surfaces under various extreme conditions.

An improved goniometer head for high-temperature single-crystal X-ray diffraction

A reliable and inexpensive goniometer head has been designed. Its stability, resulting from its compact construction, makes it very suitable for accurate measurements. Moreover, its space-saving design facilitates its application in X-ray data collection using charge-coupled device (CCD) detectors. This head has been improved for high-temperature measurements and has been tested by comparison of accurate K0.88Rb0.12TiOPO4data collected both at room temperature and at 973 K on the same crystal. The excellent structure results obtained at 973 K during the 360 h of measurements prove the stability of the goniometer head.

A hinge goniometer head

A stable diffractometer goniometer head for mounting crystals enclosed in high-pressure cells, cryostats furnaces, or other heavy crystal-environment attachments, has been designed. Its construction is based on a hinge, thus it is called a hinge goniometer head, and allows adjustments in three directions. Under a load of 9.8 N perpendicular to the φ axis, the goniometer head yields less than 5 µm.

Digitally controlled high-precision thermostat for X-ray investigations on lyotropic and thermotropic liquid crystals

The construction of a special hot air generation system for X-ray diffraction investigations of lyotropic and thermotropic mesophases at different temperatures is described. The sample is positioned at the center of the X-ray diffractometer contained in a long cylindrical capillary, and resides at a small goniometer head used to adjust the precise position and inclination of the sample. It was decided to use air as the medium and initially the medium was intended to flow axially. A stream of air is blown from a small and high rectangular opening sideways toward the sample. The gas streaming out of the vessel has to pass through a vortex cell. The air leaving the vortex cell forms a core of whirling gas with constant temperature. A temperature sensor is mounted longitudinally above the sample and is positioned inside the same airflow core. The sensor provides the input signal for a microprocessor-based controller which regulates the power of the heating or cooling system in the inlet tube providing constant temperature in the gas core.

“Millikelvin-stabilized cell” for x-ray diffraction measurements with a function of sensing thermal anomalies

Based on our technique of fine temperature control, we developed a new “millikelvin (mK)-stabilized cell” having a function of sensing thermal anomalies for the detailed x-ray diffraction study of solid phase transitions. It is easily mounted on a commercially available goniometer head. The performance was tested using CsPbCl3 single crystal, in which a phase transition is known to take place at 320 K (47°C) from tetragonal to cubic. An abrupt variation in the distance between some two reciprocal points in the precession pattern was clearly detected in a 0.1 K temperature range centered at 47°C along with two thermal anomalies observed. The “mK-stabilized cell” will be very useful to investigate a mechanism of phase transitions occurring in a narrow temperature range.