Hui-Meng Lu

A containerless levitation setup for liquid processing in a superconducting magnet

Containerless processing of materials is considered beneficial for obtaining high quality products due to the elimination of the detrimental effects coming from the contact with container walls. Many containerless processing methods are realized by levitation techniques. This paper describes a containerless levitation setup that utilized the magnetization force generated in a gradient magnetic field. It comprises a levitation unit, a temperature control unit, and a real-time observation unit. Known volume of liquid diamagnetic samples can be levitated in the levitation chamber, the temperature of which is controlled using the temperature control unit. The evolution of the levitated sample is observed in real time using the observation unit. With this setup, containerless processing of liquid such as crystal growth from solution can be realized in a well-controlled manner. Since the levitation is achieved using a superconducting magnet, experiments requiring long duration time such as protein crystallization and simulation of space environment for living system can be easily succeeded.

A strategy for selecting the pH of protein solutions to enhance crystallization

The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.

Interferometry measurement of protein concentration evolution during crystallization and dissolution with improved reliability and versatility

Interferometry is a widely used technique for real-time monitoring of concentration variation during processes in a solution like crystal growth and dissolution. This paper reports a protein concentration measurement system using a conventional Mach–Zehnder interferometer that allows monitoring the time course evolution of the protein concentration distribution during the processes of crystallization or dissolution. The demonstrated method showed improved reliability and versatility in that it (1) enables measurements under versatile temperature conditions and (2) eliminates the unpredictable errors caused by the environmental disturbances. The measurement system described in this paper can also be applied in measurements in other solution systems.

Phylogenetic relationship of 16 Oedipodidae species (Insecta: Orthoptera) based on the 16S rRNA gene sequences

Abstract The sequences of the mitochondrial 16S rRNA gene of 16 Oedipodidae species were amplified and sequenced. All sequences were aligned and analyzed and the phylogenetic relationships were inferred. The properties of 16S gene in Oedipodidae showed typical patterns of many insects such as a high A+T content and variable distance‐dependent transition/transversion ratios. The 0.2 weight for sites of loops may be advisable for phylogeny reconstruction using the maximum parsimony method. The phylogenetic analysis results do not support the current subfamily classification systems of Oedipodidae. Bryodemellinae and Bryodeminae are closely related and should be merged as one subfamily. The status of Oedipodinae and Locustinae is also problematic.

A protein crystallisation screening kit designed using polyethylene glycol as major precipitant

Crystallisation of proteins is usually achieved with the help of chemical agents. Because there are few general guidelines in determining what agents will help to crystallise a specific protein, suitable crystallisation agents are often found via exhaustive trial-and-error tests by mixing many chemical agents (the collection of which is called a crystallisation screening kit) one-by-one with the protein. Currently, many commercially available crystallisation screening kits have been developed and utilised in practical crystallisation screen experiments. However, information regarding the design of new screening kits has yet to be expanded using a large amount of experimental data. Here, we show the step-by-step design processes of a polyethylene glycol-based screening kit. It was found that the screening performance could be improved by modifying the crystallisation screening kits according to the accumulated data (such as those in the Biological Macromolecule Crystallisation Database (BMCD)), the screening test results and existing knowledge. The screening kit designed in this paper can be used for practical protein crystallisation screen experiments and the method can be used in the design of other crystallisation screening kits.

Correlation between Protein Sequence Similarity and Crystallization Reagents in the Biological Macromolecule Crystallization Database

The protein structural entries grew far slower than the sequence entries. This is partly due to the bottleneck in obtaining diffraction quality protein crystals for structural determination using X-ray crystallography. The first step to achieve protein crystallization is to find out suitable chemical reagents. However, it is not an easy task. Exhausting trial and error tests of numerous combinations of different reagents mixed with the protein solution are usually necessary to screen out the pursuing crystallization conditions. Therefore, any attempts to help find suitable reagents for protein crystallization are helpful. In this paper, an analysis of the relationship between the protein sequence similarity and the crystallization reagents according to the information from the existing databases is presented. We extracted information of reagents and sequences from the Biological Macromolecule Crystallization Database (BMCD) and the Protein Data Bank (PDB) database, classified the proteins into different clusters according to the sequence similarity, and statistically analyzed the relationship between the sequence similarity and the crystallization reagents. The results showed that there is a pronounced positive correlation between them. Therefore, according to the correlation, prediction of feasible chemical reagents that are suitable to be used in crystallization screens for a specific protein is possible.

Design of shared instruments to utilize simulated gravities generated by a large-gradient, high-field superconducting magnet.

A high-field superconducting magnet can provide both high-magnetic fields and large-field gradients, which can be used as a special environment for research or practical applications in materials processing, life science studies, physical and chemical reactions, etc. To make full use of a superconducting magnet, shared instruments (the operating platform, sample holders, temperature controller, and observation system) must be prepared as prerequisites. This paper introduces the design of a set of sample holders and a temperature controller in detail with an emphasis on validating the performance of the force and temperature sensors in the high-magnetic field.

A quality comparison of protein crystals grown under containerless conditions generated by diamagnetic levitation, silicone oil and agarose gel. Erratum

High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the R-merge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

Correlation Between Protein Sequence Similarity and X-Ray Diffraction Quality in the Protein Data Bank

As the most widely utilized technique to determine the 3-dimensional structure of protein molecules, X-ray crystallography can provide structure of the highest resolution among the developed techniques. The resolution obtained via X-ray crystallography is known to be influenced by many factors, such as the crystal quality, diffraction techniques, and X-ray sources, etc. In this paper, the authors found that the protein sequence could also be one of the factors. We extracted information of the resolution and the sequence of proteins from the Protein Data Bank (PDB), classified the proteins into different clusters according to the sequence similarity, and statistically analyzed the relationship between the sequence similarity and the best resolution obtained. The results showed that there was a pronounced correlation between the sequence similarity and the obtained resolution. These results indicate that protein structure itself is one variable that may affect resolution when X-ray crystallography is used.

Measurement of contact angles in a simulated microgravity environment generated by a large gradient magnetic field

The contact angle is an important parameter that is essential for studying interfacial phenomena. The contact angle can be measured using commercially available instruments. However, these well-developed instruments may not function or may be unsuitable for use in some special environments. A simulated microgravity generated by a large gradient magnetic field is such an environment in which the current measurement instruments cannot be installed. To measure the contact angle in this environment, new tools must be designed and manufactured to be compatible with the size and physical environment. In this study, we report the development and construction of a new setup that was specifically designed for use in a strong magnetic field to measure the contact angle between a levitated droplet and a solid surface. The application of the setup in a large gradient magnetic field was tested, and the contact angles were readily measured.