Crystal Mounting Research Articles

X-ray spectroscopy and X-ray diffraction at wavelengths near theK-absorption edge of phosphorus

Phosphorus is an abundant element in living organisms. It is traceable by its X-ray absorption spectrum which shows a strong white line at its K-edge, comparable with that observed for the L(III) edges of rare earth ions. With purple membrane, the variation of the imaginary part of the anomalous dispersion of phosphorus is found to be close to 20 anomalous electron units. Anomalous diffraction experiments at wavelengths near the K-absorption edge of phosphorus confirm this result. The spatial distribution of lipids derived from anomalous diffraction agrees with earlier results from neutron diffraction. Test experiments on single crystals of the carrier protein using 5.76 A photons gave a first low-resolution diffraction pattern. Various techniques of crystal mounting were attempted. In addition, fluorescence measurements on a solution of threonine synthase appear to hint at a change of the phosphate environment of the cofactor upon activator binding.

Using cryoloops for X-ray data collection from protein crystals at room temperature: A simple applicable method

Although cryoloops are now routinely used for X-ray data collection from protein crystals in cryocooling condition, it is still necessary to collect X-ray diffraction data from protein crystals at room temperature under such circumstances as to find resolution limit and/or to avoid damage of protein crystals at cryogenic temperature (e.g. 100 K). Here, we show that a cryoloop, which is accompanied by a glass capillary cap to maintain humid environment of crystal in the cryoloop, can be used not only to examine protein or non-protein crystals but also to collect X-ray diffraction data for structural analysis from protein crystals at room temperature. The size of cryoloop should be carefully chosen so that the crystal does not move in the cryoloop. This crystal mounting method can be time-saving compared to the traditional method to mount a crystal in a glass capillary tube.

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

A new sample mounting technique for room-temperature macromolecular crystallography

A new method for mounting protein crystals and other environmentally sensitive samples for room-temperature diffraction measurements is described. A crystal is retrieved using a microfabricated sample mount as recently reported, and the mount is inserted into a modified goniometer-compatible base. A transparent thin-wall polyester tube sealed at one end and filled with stabilizing liquid is then drawn over the crystal and sealed to the goniometer base. Compared with mounting using glass capillaries, this method can provide lower-background X-ray scattering, especially at higher resolutions; dramatically improved ease of crystal mounting with minimal chance of damage; accurate and reproducible crystal positioning relative to the goniometer base; improved crystal visibility and ease of alignment, especially for very small crystals; and compatibility with high-throughput approaches. Crystals can be rapidly screened and eliminated earlier in the data collection pipeline, and the cause of poor low-temperature diffraction can be diagnosed.

Ultrahigh vacuum apparatus for quartz crystal microbalance measurements in the temperature range 4–400 K

We describe an apparatus for friction experiments on molecularly thin films carried out with the quartz crystal microbalance technique (QCM) in ultrahigh vacuum (UHV) and at a temperature as low as 4 K. Through the implementation in the UHV chamber of standard cryogenic procedures, the temperature of the quartz can be controlled with a resolution better than 5 mK below 10 K. The main chamber is provided with a stainless steel jacket that allows a quick change of the various temperature inserts (cryocooler head, liquid nitrogen insert, Peltier-cell stage, etc.) that span the working range 4–400 K, without ever breaking the vacuum. The crystal mounting is compatible with UHV conditions and with the Omicron standard and guarantees a good thermal and electrical contact over the entire temperature range. Once inserted in the copper sample holder, the quartz frequency and amplitude stabilities are found to be as good as the best recorded values achieved with QCMs mounted in standard high-vacuum, liquid bath cryostats. This apparatus can also be used for general QCM investigations in surface science, like the determination of film growth in deposition and sputter processes, the stoichiometry of submonolayer oxide films, and the measurement of adsorbate sticking probability.

On the High-Temperature Phase Transition of Gd5Si2Ge2

The first-order monoclinic-to-orthorhombic (beta-->gamma) phase transition of the giant magnetocaloric material Gd(5)Si(2)Ge(2) was studied using in situ high-temperature single-crystal X-ray diffraction. A special crystal mounting procedure was developed to avoid crystal contamination by oxygen or nitrogen at high temperatures. The elastic beta-->gamma transformation occurs at 300-320 degrees C during heating, and it is reversible during fast and slow heating and slow cooling but irreversible during rapid cooling. Contrary to theoretical predictions, the macroscopic distribution of the Si and Ge atoms remains the same in both the orthorhombic gamma-polymorph and the monoclinic beta-phase. It appears that interstitial impurities may affect stability of both the monoclinic and orthorhombic phases. In the presence of small amounts of air, the beta-->gamma transformation is complete only at 600 degrees C. The interslab voids, which can accommodate impurity atoms, have been located in the structure, and an effect of partially filling these voids with oxygen or nitrogen atoms on the beta-gamma transition is discussed.

Calcineurin A-α But Not A-β Is Required for Normal Kidney Development and Function

Calcineurin is an important signaling molecule in the kidney and may be involved in a variety of processes. The phosphatase subunit of calcineurin (CnA) has three isoforms, alpha, beta, and gamma. In this study, we investigated the effect of loss of calcineurin A-alpha (CnA-alpha) or calcineurin A-beta (CnA-beta) on the development and function of the kidney. Total calcineurin expression and activity was significantly reduced in whole kidney homogenates from both CnA-alpha -/- and CnA-beta -/- mice. Kidneys of CnA-beta -/- mice appear normal and the mice develop with no phenotypic abnormalities. In contrast, kidneys of CnA-alpha -/- animals fail to fully develop. In particular, postnatal maturation of the nephrogenic zone (NZ) is defective. Within the NZ, glomeruli also fail to mature and lack mesangial cells. In addition to alterations in development, there is an absence of proliferation and an increase of cell death in the NZ with loss of CnA-alpha. Finally, increased collagen deposition is observed and serum creatinine levels are significantly increased in CnA-alpha -/- animals compared to wild-type littermates, indicating that kidney function is impaired. In summary, absence of CnA-alpha but not CnA-beta leads to a defect in normal maturation of the NZ and glomeruli, alterations in the cell cycle, and impaired kidney function.

Automated Sample Mounting and Alignment System for Biological Crystallography at a Synchrotron Source

High-throughput data collection for macromolecular crystallography requires an automated sample mounting and alignment system for cryo-protected crystals that functions reliably when integrated into protein-crystallography beamlines at synchrotrons. Rapid mounting and dismounting of the samples increases the efficiency of the crystal screening and data collection processes, where many crystals can be tested for the quality of diffraction. The sample-mounting subsystem has random access to 112 samples, stored under liquid nitrogen. Results of extensive tests regarding the performance and reliability of the system are presented. To further increase throughput, we have also developed a sample transport/storage system based on “puck-shaped” cassettes, which can hold sixteen samples each. Seven cassettes fit into a standard dry shipping Dewar. The capabilities of a robotic crystal mounting and alignment system with instrumentation control software and a relational database allows for automated screening and data collection to be developed.

A quick and gentle method for mounting crystals in capillaries

The capillary mounting of protein crystals is still necessary under some circumstances, despite its being supplanted by loop mounting/flash freezing as the favored method for crystal mounting prior to data collection. Traditional capillary mounting methods require a degree of dexterity and finesse that causes apprehension among many crystallographers. Here a simple method for capillary mounting that uses a loop for depositing the crystal in the capillary is described.

A simple and rapid method for mounting protein crystals at room temperature

Cryocooling of protein crystals for X-ray data collection has now become a routine method in the majority of biostructural laboratories. The improvement of facilities at synchrotron sources and their increased use has made it essential to have properly frozen crystals for optimal data collection. Although in general crystals can be cooled without significant damage, there are often cases in which crystals with slight disorder or twinning problems suffer considerably during the freezing process. In other cases, poor or mosaic diffraction may be blamed on the cryoprotectant or cooling protocol. Many crystals may be wasted in searching for the best freezing conditions when the intrinsic quality of the crystals is poor. In principle, the collection of room-temperature diffraction data would provide a reference that would allow the detection of crystal damage caused by addition of cryoprotectant or by cryocooling. In practice, however, many investigators are reluctant to do this, one reason being that capillary mounting of crystals is a tedious method, especially for those who are new to crystallography. Here a simplified method for mounting crystals at room temperature is reported, which requires little expertise and no expensive equipment.

Development of novel liquid phase qcrs sensor technology

This paper discusses the development of novel liquid phase quartz crystal resonant sensor technologies for label free, real-time detection of pharmaceutical applications. New crystal oscillator technologies are introduced alongside crystal mounting and flow cell designs circumventing more traditional approaches to yield greater robustness, reliability and sensitivity. Data for standardised long-term experiments is presented and analysed with respect to reproducibility and stability and comparisons made with surface plasmon resonance.

Automated mounting, centering and screening of crystals for high-throughput protein crystallography.

A fully automated system for screening protein crystals for X-ray diffraction analysis has been designed and is being installed on the beamline BW6 at DORIS in Hamburg, Germany. The system includes robotic mounting of flash-frozen crystals from a storage dewar, centering and alignment of the sample both by optical and X-ray (scattering and fluorescence) techniques, assessment of the diffraction quality of the sample, and SAD/MAD or non-conventional diffraction data acquisition with high-throughput data rates. The system covers all experimental steps required for protein x-ray structure analysis and provides a powerful means for structural genomics projects.

The TB structural genomics consortium crystallization facility: towards automation from protein to electron density.

The crystallization facility of the TB (Tuberculosis) structural genomics consortium, one of nine NIH sponsored p50 structural genomic centres, provides TB consortium members with automated crystallization, data collection and basic molecular replacement (MR) structure solution up to bias minimized electron density maps. Crystallization setup of up to ten proteins per day follows the CRYSTOOL combinatorial screen protocol using a modular and affordable robotic design with an open architecture. Components include screen preparation, plate setup, automated image acquisition and analysis, and optimisation design. A new 96 well crystallization plate has been designed for optimal robotic handling while maintaining ease of manual crystal harvesting. Robotic crystal mounting, screening, and data collection are conducted in-house and at the Advanced Light Source (ALS) in Berkeley. A simple automated protocol based on MR and homology based structure prediction automatically solves modestly difficult problems. Multiple search models are evaluated in parallel MR and the best multi-segment rigid body refined MR solution is subjected to simulated annealing torsion angle molecular dynamics using CNS, bringing even marginal MR solutions within the convergence radius of the subsequent highly effective bias removal and map reconstruction protocol, Shake&wARP, used to generate electron density for initial rebuilding. Real space correlation plots allow rapid assessment of local structure quality. Modular design of robotics and automated scripts using publicly available programs for structure solution allow for efficient high throughput crystallography - at a reasonable cost.

Automated mounting of cryo-cooled crystals on Stanford synchrotron radiation laboratory beamline 11-1

Automated mounting of cryo-cooled crystals on Stanford synchrotron radiation laboratory beamline 11-1

Automated crystal mounting and alignment system at the advanced light source

Automated crystal mounting and alignment system at the advanced light source

Design and performance of a stable first crystal mount for a cryogenically cooled Si monochromator at the Advanced Photon Source

We present a new design for mounting a cryogenically cooled Si crystal which gives greatly improved beam stability. The design has been successfully implemented at the University of Michigan, Howard University, Bell Laboratories-Lucent Technologies Collaborative Access Team (MHATT-CAT) 7ID Beamline of the Advanced Photon Source. Before the installation of the new crystal mount, our Si (lll) cryogenically cooled monochromator was sensitive to the pressure fluctuations of the liquid nitrogen coolant, such that the angle of incidence on the first crystal varied linearly with the applied pressure in the cooling lines, causing beam motion of about 250 μm, 60 m from the source. The key element of the design is a symmetrically positioned cooling manifold which balances the forces caused by pressure fluctuations. With this new mount, the typical beam stability is now about 10 μm, comparable to the source stability.

Monochromator crystal mounting stage

An insertion device beamline at the Advanced Photon Source had received a second insertion device “undulator A” and consequently needed an upgrade of the optics. The decision to use diamond optics required a mechanical modification of the double crystal monochromator and its crystal alignment stages. Specifications for the tilt alignment accuracy are developed. A crystal alignment stage with independent tilt and translation is described. A new crystal holder was developed, which reduces the temperature of the diamond crystal by over 100 °C and allows for strain-free mounting.

Instrumentation in X-ray crystallography: Past, present and future

This paper deals with the very great changes in X–ray crystallographic techniques and apparatus over a period of approximately the last 60 years. This is not a general history; it is a personal account of the developments with which I have been directly involved; it is, therefore, biased towards apparatus developments in the field of macromolecular crystallography in which I have worked during most of this period. The bias needs little excuse: many of the new techniques of X–ray crystallography were devised initially for large–molecule structure determinations which had most need of such advances in order to be feasible at all. Among them are the uses of computers in calculating electron density maps, the construction of automatic diffractometers and microdensitometers, the introduction of rotating-anode X–ray generators and of microfocus X–ray tubes, the development of electronic X–ray area detectors, the pioneering work on the use of synchrotron radiation for diffraction studies, the building of three–dimensional atomic models by computer and the complete automation of the mounting, selection and alignment of crystals on the diffractometer.

Insulin-like Growth Factor-I Induces Renal Cell Hypertrophy via a Calcineurin-dependent Mechanism

Insulin-like growth factor-I (IGF-I) may play an important role in the development of renal hypertrophy. In this study we determined the effect of IGF-I on cultured mesangial cells (MCs) and examined activation of key signaling pathways. IGF-I induced hypertrophy as determined by an increase in cell size and an increase in protein to DNA ratio and increased accumulation of extracellular matrix (ECM) proteins. IGF-I also activated both Erk1/Erk2 MAPK and phosphatidylinositol 3-kinase (PI3K) in MCs. Inhibition of either MAPK or PI3K, however, had no effect on IGF-I-induced hypertrophy or ECM production. Next, we examined the effect of IGF-I on activation of the calcium-dependent phosphatase calcineurin. IGF-I treatment stimulated calcineurin activity and increased the protein levels of calcineurin and the calcineurin binding protein, calmodulin. Cyclosporin A, an inhibitor of calcineurin, blocked both IGF-I-mediated hypertrophy and up-regulation of ECM. In addition, calcineurin resulted in sustained Akt activation, indicating possible cross-talk with other signaling pathways. Finally, IGF-I treatment resulted in the calcineurindependent nuclear localization of NFATc1. Therefore, IGF-I induces hypertrophy and increases ECM accumulation in MCs. IGF-I-mediated hypertrophy is associated with activation of Erk1/Erk2 MAPK and PI3K but does not require either of these pathways. Instead, IGF-I mediates hypertrophy via a calcineurin-dependent pathway.

A new radiative microfurnace for X-ray single-crystal diffractometry

Microfurnaces available today for X-ray single-crystal investigation (radiative, gas-flame, gas-flow) are generally temperature-monitored by a thermocouple placed in a different site with respect to the crystal. The new F1 microfurnace aims at the following goals: fast crystal mounting; access to a large portion of reciprocal lattice; low absorption of direct beam; efficient in situ temperature measurement; fast power supply regulation; high temperature stability; controlled atmosphere. In the F1, the crystal is directly glued to a thermocouple with refractory cement. The heating body, fixed on the χ and radially shiftable along the ϕ axis circle, hosts the Pt winding and connections of both gas flow and power supply, and is enclosed in a thin pyrolitic boron nitride (PBN) shield. Temperature stability is within ± 1°C. Calibration of the F1 was carried out by collecting X-ray data from a single-crystal synthetic periclase at tem- peratures of 28, 150, 300, 450, 600, 700, 900 and 1000°C, and yielded the equation: a = 0.0000625 (7) (T(K) - 273) + 4.2083 (4), based on the ratio between cell edge and temperature. Structural refinements gave structural parame- ters and thermal expansion values at the investigated temperature. The mean coefficient of linear expansion up to 1000°C was 14.3 × 10-6 °C.