Crystal Mounting Research Articles

Automated harvesting and processing of protein crystals through laser photoablation.

Currently, macromolecular crystallography projects often require the use of highly automated facilities for crystallization and X-ray data collection. However, crystal harvesting and processing largely depend on manual operations. Here, a series of new methods are presented based on the use of a low X-ray-background film as a crystallization support and a photoablation laser that enable the automation of major operations required for the preparation of crystals for X-ray diffraction experiments. In this approach, the controlled removal of the mother liquor before crystal mounting simplifies the cryocooling process, in many cases eliminating the use of cryoprotectant agents, while crystal-soaking experiments are performed through diffusion, precluding the need for repeated sample-recovery and transfer operations. Moreover, the high-precision laser enables new mounting strategies that are not accessible through other methods. This approach bridges an important gap in automation and can contribute to expanding the capabilities of modern macromolecular crystallography facilities.

Two way street - complementary methods.

Two way street - complementary methods.

Acoustic transfer of protein crystals from agarose pedestals to micromeshes for high-throughput screening.

Acoustic droplet ejection (ADE) is an emerging technology with broad applications in serial crystallography such as growing, improving and manipulating protein crystals. One application of this technology is to gently transfer crystals onto MiTeGen micromeshes with minimal solvent. Once mounted on a micromesh, each crystal can be combined with different chemicals such as crystal-improving additives or a fragment library. Acoustic crystal mounting is fast (2.33 transfers s(-1)) and all transfers occur in a sealed environment that is in vapor equilibrium with the mother liquor. Here, a system is presented to retain crystals near the ejection point and away from the inaccessible dead volume at the bottom of the well by placing the crystals on a concave agarose pedestal (CAP) with the same chemical composition as the crystal mother liquor. The bowl-shaped CAP is impenetrable to crystals. Consequently, gravity will gently move the crystals into the optimal location for acoustic ejection. It is demonstrated that an agarose pedestal of this type is compatible with most commercially available crystallization conditions and that protein crystals are readily transferred from the agarose pedestal onto micromeshes with no loss in diffraction quality. It is also shown that crystals can be grown directly on CAPs, which avoids the need to transfer the crystals from the hanging drop to a CAP. This technology has been used to combine thermolysin and lysozyme crystals with an assortment of anomalously scattering heavy atoms. The results point towards a fast nanolitre method for crystal mounting and high-throughput screening.

Датчик гидростатического давления на основе микрокристаллов антимонида галлия

Currently, silicon and germanium, the most common materials in the production of discrete semiconductor devices and integrated circuits, do not always meet all the requirements to the sensing elements of mechanical quantities sensors. Therefore, it is logical to research the properties of other semiconductor materials that could be used as sensing elements in such sensors. A3B5 semiconductor compounds seem promising for such purpose. Effect of hydrostatic pressure up to 5000 bar on the resistance of n-type antimonide gallium whiskers doped by Se or Te was studied. Coefficient of hydrostatic pressure for this crystals was determined, it equals Kh = (16,5—20,0)•10–5 bar–1 at 20°N. Temperature dependence of resistance and coefficient Kh for this crystals in the temperature range ±60°N was studied. Design of the developed hydrostatic pressure sensor based on GaSb whiskers and its characteristics are presented. The possibility to decrease the temperature dependence of sensitive element resistance by mounting GaSb whiskers on the substrates fabricated from materials with different temperature coefficient of expansion was examined. It was shown that mounting of GaSb crystals on Cu substrate gives the optimal result, in this case the temperature coefficient decrease to 0,05%•°N–1, that leads to decrease of output temperature dependence. The main advantages of developed pressure sensor are: the simplified design in comparison with pressure sensors with strain gauges mounted on spring elements; the high sensitivity to pressure that is constant in the wide pressure range; the improvement of sensors metrological characteristics owing to hysteresis absence. The possible application fields of developed sensors are measuring of high and extremely high pressure, chemical and oil industries, measuring of pressure in oil bore-holes, investigation of explosive processes.

X-ray Transparent Microfluidic Chip for Mesophase-Based Crystallization of Membrane Proteins and On-Chip Structure Determination.

Crystallization from lipidic mesophase matrices is a promising route to diffraction-quality crystals and structures of membrane proteins. The microfluidic approach reported here eliminates two bottlenecks of the standard mesophase-based crystallization protocols: (i) manual preparation of viscous mesophases and (ii) manual harvesting of often small and fragile protein crystals. In the approach reported here, protein-loaded mesophases are formulated in an X-ray transparent microfluidic chip using only 60 nL of the protein solution per crystallization trial. The X-ray transparency of the chip enables diffraction data collection from multiple crystals residing in microfluidic wells, eliminating the normally required manual harvesting and mounting of individual crystals. We validated our approach by on-chip crystallization of photosynthetic reaction center, a membrane protein from Rhodobacter sphaeroides, followed by solving its structure to a resolution of 2.5 Å using X-ray diffraction data collected on-chip under ambient conditions. A moderate conformational change in hydrophilic chains of the protein was observed when comparing the on-chip, room temperature structure with known structures for which data were acquired under cryogenic conditions.

A new protein crystal mounting method using humid air and glue coating

Cryopreservation of protein crystals are a useful to reduce radiation damage for synchrotron experiments, the cryoloop mounting method using cryoprotectant agents is the widely used. However, Protein crystals are fragile, and they have often trouble to find a condition suitable for cryo-cooling. X-ray diffraction experiments at room temperature can evaluate the quality of the protein crystal and perform structural analysis without being affected shrink of the crystal by cryo-cooled and addition cryoprotectant agents. And, conventional humidity controlled method is possible to improve resolution of limited protein crystals [1]. However, protein crystals of these diffraction experiments cannot apply same mounting method. We developed a new crystal mounting method, the humid air and glue-coating (HAG) mounting method, which involves a combination of controlled adjustable humid air and water-soluble polymer glue for crystal coating [2]. By coating with the water-soluble polymer glue, most crystals exposed to the controlled humid air were stable at room temperature and could be cryocooled under optimized humidity without additional cryoprotectant agents. For example, the crystals of the bacterial hydrolase RsbQ was mechanically very fragile and sensitive to environmental changes. Thus, RsbQ crystal cannot apply cryoloop mount with cryoprotectant agents. By using the HAG method, we were able to obtain 1.4 Å data and solve its structure. For another example, membrane protein crystal was improved resolution to optimize humidity. The crystals by using HAG method reproducibly showed crystal lattice transformation in response to a change in humidity, thus using this method a series of isomorphic crystals can be prepared. We introduce HAG method, and demonstrate its success with various protein crystals.

Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting.

In macromolecular cryocrystallography unit-cell parameters can have low reproducibility, limiting the effectiveness of combining data sets from multiple crystals and inhibiting the development of defined repeatable cooling protocols. Here, potential sources of unit-cell variation are investigated and crystal dehydration during loop-mounting is found to be an important factor. The amount of water lost by the unit cell depends on the crystal size, the loop size, the ambient relative humidity and the transfer distance to the cooling medium. To limit water loss during crystal mounting, a threefold strategy has been implemented. Firstly, crystal manipulations are performed in a humid environment similar to the humidity of the crystal-growth or soaking solution. Secondly, the looped crystal is transferred to a vial containing a small amount of the crystal soaking solution. Upon loop transfer, the vial is sealed, which allows transport of the crystal at its equilibrated humidity. Thirdly, the crystal loop is directly mounted from the vial into the cold gas stream. This strategy minimizes the exposure of the crystal to relatively low humidity ambient air, improves the reproducibility of low-temperature unit-cell parameters and offers some new approaches to crystal handling and cryoprotection.

Cryogenic neutron protein crystallography: routine methods and potential benefits

The use of cryocooling in neutron diffraction has been hampered by several technical challenges, such as the need for specialized equipment and techniques. This article reports the recent development and deployment of equipment and strategies that allow routine neutron data collection on cryocooled crystals using off-the-shelf components. This system has several advantages compared to a closed displex cooling system, such as fast cooling coupled with easier crystal mounting and centering. The ability to routinely collect cryogenic neutron data for analysis will significantly broaden the range of scientific questions that can be examined by neutron protein crystallography. Cryogenic neutron data collection for macromolecules has recently become available at the new Biological Diffractometer BIODIFF at the FRM II and the Macromolecular Diffractometer (MaNDi) at the Spallation Neutron Source, Oak Ridge National Laboratory. To evaluate the benefits of a cryocooled neutron structure, a full neutron data set was collected on the BIODIFF instrument on a Toho-1 β-lactamase structure at 100 K.

A new view on crystal harvesting.

X-ray crystallography typically requires the mounting of crystals, which can make the sample difficult to manipulate when it is small and the microscope objective is close to the crystallization plate. By simply moving the objective to the bottom of a clear crystallization plate (inverting the normal view), crystals were able to be manipulated and harvested from wells having a 0.9 mm diameter and 5.0 mm depth. The mounting system enabled the structural solution of the 187 amino acid N-terminal domain of Saccharomyces cerevisiae glutaminyl-tRNA synthetase from crystals that appeared during high-throughput screening but proved recalcitrant to scale-up and optimization. While not a general mounting solution, the simple expedient of removing the objective lens from the area where manipulation and harvesting occur greatly facilitates the manual, or even automated, process.

湿度調整と水溶性ポリマーのコーティングを用いたタンパク質結晶マウント法

A novel protein crystal mounting method was developed using controlled humid air and hydrophobic polymer glue for crystal coating. The glue-coated crystals under the optimized humid air were quite stable at room temperature and could be cryocooled. This method is particularly useful when applied to fragile protein crystals known to be sensitive to environmental change. And, the crystals by using HAG method reproducibly showed crystal lattice transformation in response to a change in humidity, thus using this method a series of isomorphic crystals can be prepared.

Graphene as a protein crystal mounting material to reduce background scatter.

The overall signal-to-noise ratio per unit dose for X-ray diffraction data from protein crystals can be improved by reducing the mass and density of all material surrounding the crystals. This article demonstrates a path towards the practical ultimate in background reduction by use of atomically thin graphene sheets as a crystal mounting platform for protein crystals. The results show the potential for graphene in protein crystallography and other cases where X-ray scatter from the mounting material must be reduced and specimen dehydration prevented, such as in coherent X-ray diffraction imaging of microscopic objects.

Data collection and processing

Data collection and processing

Design, Build & Test of a Double Crystal Monochromator for Beamlines I09 & I23 at the Diamond Light Source

A high stability Double Crystal Monochromator has been developed at The Diamond Light Source for beamlines I09 and I23. The design specification was a cryogenic, fixed exit, energy scanning monochromator, operating over an energy range of 2.1 – 25 keV using a Si(111) crystal set. The novel design concepts are the direct drive, air bearing Bragg axis, low strain crystal mounts and the cooling scheme. The instrument exhibited superb stability and repeatability on the B16 Test Beamline. A 20 keV Si(555), 1.4 μrad rocking curve was demonstrated. The DCM showed good stability without any evidence of vibration or Bragg angle nonlinearity.

Diamond crystal optics for self-seeding of hard X-rays in X-ray free-electron lasers

We report design, fabrication, and results of X-ray topography characterization of thin synthetic type IIa diamond crystal plates and crystal-holder assemblies developed for the Hard X-ray Self-Seeding project at the Linac Coherent Light Source. The goal of the project was to achieve generation of fully coherent hard X-rays using the self-seeding concept and the single-crystal diamond wake monochromator [Geloni et al., J. Mod. Opt. 58, 1391 (2011)]. High crystal quality, crystal thickness of ≈0.1–0.2mm and strain-free crystal mount were the main requirements. Nearly defect-free diamond plates of (001) orientation, with thicknesses of 0.1mm and 0.15mm, and of a trapezoidal shape were fabricated and preliminarily evaluated. The plates were further characterized using X-ray topography. These tests helped to minimize strain in crystals induced by mounting in crystal holders and to determine defect-free crystal regions. Self-seeding experiments were conducted at the Linac Coherent Light Source using the diamond plates and crystal-holder assemblies selected by our studies. Fully coherent 8.33-keV X-rays with 5×10−5 relative bandwidth were produced [Amann et al., Nat. Photonics 6, 693 (2012)].

A fibre-based crystal mounting technique for protein cryocrystallography

The CryoFibre, a crystal mounting tool, has been developed for protein cryocrystallography. The technique attaches single crystals to the tips of polyester fibres, allowing removal of excess liquid around each crystal. Single-wavelength anomalous dispersion phasing using a Cu Kα X-ray source (Cu SAD) was applied to crystals from five proteins without any derivatization, demonstrating a clear improvement in the success rate of Cu SAD compared with the conventional loop technique. In addition, a xylanase crystal on the surface of a synthetic zeolite as a hetero-epitaxic nucleant was directly mounted on the CryoFibre without separation treatment of the crystal from the zeolite. The crystal had a lower mosaicity than that observed using the conventional technique, indicating that the fibre technique is suitable for high-quality data collection from zeolite-mediated crystals.

Direct cryocooling of naked crystals: are cryoprotection agents always necessary?

Over the last 20 years cryocrystallography has revolutionized the field of macromolecular crystallography, greatly reducing radiation damage and allowing the collection of complete data sets at synchrotron sources. However, in order to cool crystals to 100 K cryoprotective agents must usually be added to prevent the formation of crystalline ice, which disrupts the macromolecular crystal lattice and often results in a degradation of diffraction quality. This process can involve the extensive testing of solution compositions and soaking protocols tofind suitable conditions that maintain diffraction quality. In this study, it is demonstrated that when some crystals of macromolecules are mounted in the complete absence of surrounding liquid no crystalline ice is formed and the diffraction resolution, merging R factors and mosaic spread values are comparable to those of crystals cryocooled in the presence of a cryoprotectant. This potentially removes one of the most onerous manual steps in the structure-solution pipeline and could alleviate some of the foreseen difficulties in the automation of crystal mounting.

Novel microfocus X-ray sources for high-pressure crystallography

Beam Characteristics of the IμS The Ag-IμS delivers a focused and monochromatic X-ray beam with a FWHM of 0.09 mm in the image focus. In contrast to the typical top-hat shaped beam profile that is produced by sources coupled to a flat graphite monochromator, the beam profile from the IμS has a symmetrical Gaussian-shaped intensity distribution. As a consequence, the flux density increases with decreasing sample size (Fig. 1), thus leading to strong diffracted intensities especially for small samples. Furthermore, the convergent beam from the IμS reduces the background of the data by minimizing the scattering from peripherals, such as conventional crystal mounts or gaskets. The integrated intensity of the original Ag-IμS, which is available since 2009, is at least about three times higher than the intensity of a 1.5 kW Ag sealed tube system. The new Ag-IμSHigh Brilliance has an improved X-ray optical design that results in a further intensity gain of 50% when compared to the original Ag-IμS.

Polymeric strontium ranelate nonahydrate

The title compound, poly[[μ-aqua-tetra­aqua{μ-5-[bis­(carboxyl­atometh­yl)amino]-3-carboxyl­atomethyl-4-cyano­thio­phene-2-carboxyl­ato}distrontium(II)] tetra­hydrate], [Sr2(C12H6N2O8S)(H2O)5]·3.79H2O, crystallizes with nine- and eight-coordinated Sr2+ cations. They are bound to seven of the eight ranelate O atoms and five of the water mol­ecules. The SrO8 and SrO9 polyhedra are inter­connected by edge-sharing, forming hollow layers parallel to (011). The layers are, in turn, inter­connected by ranelate anions, forming a metal–organic framework (MOF) structure with channels along the a axis. The four water mol­ecules not coordinated to strontium are located in these channels and hydrogen bonded to each other and to the ranelates. Part of the water H atoms are disordered. The compound dehydrates very easily and 0.210 (4) water mol­ecules out of nine were lost during crystal mounting causing additional disorder in the water structure.

Diffraction study of protein crystals grown in cryoloops and micromounts.

Protein crystals are usually grown in hanging or sitting drops and generally get transferred to a loop or micromount for cryocooling and data collection. This paper describes a method for growing crystals on cryoloops for easier manipulation of the crystals for data collection. This study also investigates the steps for the automation of this process and describes the design of a new tray for the method. The diffraction patterns and the structures of three proteins grown by both the new method and the conventional hanging-drop method are compared. The new setup is optimized for the automation of the crystal mounting process. Researchers could prepare nanolitre drops under ordinary laboratory conditions by growing the crystals directly in loops or micromounts. As has been pointed out before, higher levels of supersaturation can be obtained in very small volumes, and the new method may help in the exploration of additional crystallization conditions.

Crystal Direct: a new system for automatic crystal mounting and diffraction analysis

Crystal Direct: a new system for automatic crystal mounting and diffraction analysis