Growth Of High-quality Protein Crystals Research Articles

Existence of twisting in dislocation-free protein single crystals

The growth of high-quality protein crystals is a prerequisite for the structure analysis of proteins by X-ray diffraction. However, dislocation-free perfect crystals such as silicon and diamond have been so far limited to only two kinds of protein crystals, such as glucose isomerase and ferritin crystals. It is expected that many other high-quality or dislocation-free protein crystals still exhibit some imperfection. The clarification of the cause of imperfection is essential for the improvement of crystallinity. Here, we explore twisting as a cause of the imperfection in high-quality protein crystals of hen egg-white lysozyme crystals with polymorphisms (different crystal forms) by digital X-ray topography with synchrotron radiation. The magnitude of the observed twisting is 10−6 to 10−5°/μm which is more than two orders smaller than 10−3 to 104°/μm in other twisted crystals owing to technique limitations with optical and electron microscopy. Twisting is clearly observed in small crystals or in the initial stage of crystal growth. It is uniformly relaxed with crystal growth and becomes smaller in larger crystals. Twisting is one of main residual defects in high-quality crystals and determines the crystal perfection. Furthermore, it is presumed that the handedness of twisting can be ascribed to the anisotropic interaction of chiral protein molecules associated with asymmetric units in the crystal forms. This mechanism of twisting may correspond to the geometric frustration proposed as a primary mechanism of twisting in molecular crystals. Our finding provides insights for the understanding of growth mechanism and the growth control of high-quality crystals.

「きぼう」を利用した高品質タンパク質結晶生成実験の現状と 合理的な宇宙実験へのアプローチ

「きぼう」を利用した高品質タンパク質結晶生成実験の現状と 合理的な宇宙実験へのアプローチ

Technology of high quality protein crystals’ obtaining aboard the ISS at execution of joint Japanese–Russian experiments

A series of space experiments aboard the International Space Station (ISS) associated with high-quality Protein Crystal Growth (PCG) in microgravity conditions can be considered as a unique and one of the best examples of fruitful collaboration between Japanese and Russian scientists and engineers in space, which includes also other ISS International Partners. X-ray diffraction is still the most powerful tool to determine the protein three dimensional structure necessary for Structure based drug design (SBDD). The major purpose of the experiment is to grow high quality protein crystals in microgravity for X-ray diffraction on Earth. Within one and a half decade, Japan and Russia have established an efficient process over PCG in space to support latest developments over drug design and structural biology. One of the keys for success of the experiment lies in how precisely pre-launch preparations are made. Japanese party provides flight equipment for crystallization and ensures the required environment to support the experiment aboard of the ISS’s Kibo module, and also mainly takes part of the experiment ground support such as protein sample characterization, purification, crystallization screening, and solution optimization for microgravity experiment. Russian party is responsible for integration of the flight items equipped with proteins and precipitants on board Russian transportation space vehicles (Soyuz or Progress), for delivery them at the ISS, transfer to Kibo module, and returning the experiments’ results back on Earth aboard Soyuz manned capsule. Due to close cooperation of the parties and solid organizational structure, samples can be launched at the ISS every half a year if the ground preparation goes smoothly. The samples are crystallized using counter diffusion method at 20 degree C for 1–2.5 months. After samples return, the crystals are carefully taken out from the capillary, and frozen for X-ray diffraction at SPring8 facility in Japan. Extensive support of researchers from both countries is also a part of this process. The paper analyses details of the PCG experiment scheme, unique and reliable technology of its execution, and contains examples of the application. Key words: International Space Station, Protein crystals, Microgravity, International collaboration.

Crystal growth of high-quality protein crystals under the application of an external electric field

Crystal growth of high-quality protein crystals under the application of an external electric field

Recent Advance in High Quality Protein Crystal Growth Experiment on the International Space Station by JAXA

Recent Advance in High Quality Protein Crystal Growth Experiment on the International Space Station by JAXA

X-ray transparent microfluidic platforms for membrane protein crystallization with microseeds.

Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating structure-function relationships. Microseeding, the process of transferring sub-microscopic crystal nuclei from initial screens into new crystallization experiments, is an effective, yet underutilized approach to grow crystals suitable for X-ray crystallography. Here, we report simplified methods for crystallization of membrane proteins that utilize microseeding in X-ray transparent microfluidic chips. First, a microfluidic method for introduction of microseed dilutions into metastable crystallization experiments is demonstrated for photoactive yellow protein and cytochrome bo3 oxidase. As microseed concentration decreased, the number of crystals decreased while the average size increased. Second, we demonstrate a microfluidic chip for microseed screening, where many crystallization conditions were formulated on-chip prior to mixing with microseeds. Crystallization composition, crystal size, and diffraction data were collected and mapped on phase diagrams, which revealed that crystals of similar diffraction quality and size typically grow in distinct regions of the phase diagram.

Crystal Growth of High-Quality Protein Crystals under the Presence of an Alternant Electric Field in Pulse-Wave Mode, and a Strong Magnetic Field with Radio Frequency Pulses Characterized by X-ray Diffraction

The first part of this research was devoted to investigating the effect of alternate current (AC) using four different types of wave modes (pulse-wave) at 2 Hz on the crystal growth of lysozyme in solution. The best results, in terms of size and crystal quality, were obtained when protein crystals were grown under the influence of electric fields in a very specific wave mode (“breathing” wave), giving the highest resolution up to 1.34 Å in X-ray diffraction analysis compared with controls and with those crystals grown in gel. In the second part, we evaluated the effect of a strong magnetic field of 16.5 Tesla combined with radiofrequency pulses of 0.43 μs on the crystal growth in gels of tetragonal hen egg white (HEW) lysozyme. The lysozyme crystals grown, both in solution applying breathing-wave and in gel under the influence of this strong magnetic field with pulses of radio frequencies, produced the larger-in-size crystals and the highest resolution structures. Data processing and refinement statistics are very good in terms of the resolution, mosaicity and Wilson B factor obtained for each crystal. Besides, electron density maps show well-defined and distinctly separated atoms at several selected tryptophan residues for the crystal grown using the “breathing wave pulses”.

High quality protein crystal growth under microgravity in JAXA PCG project

High quality protein crystal growth under microgravity in JAXA PCG project

High-quality Protein Crystal Growth under Space Environment

High-quality Protein Crystal Growth under Space Environment

New developments in crystal growth from solutions: Oxides, proteins, and nitrides

Abstract Controlling the nucleation and growth processes is important for growing large, high-quality crystals of oxides, proteins, and nitrides from a solution. We must understand how the solution conditions should be controlled, and determine a universal principle that is applicable to the growth of various high-quality crystals. In this report, we will present the effects of solution stirring and nucleation control on the solution growth of CsLiB 6 O 10 (CLBO), proteins, and GaN crystals with respect to the following issues: (1) effect of solution stirring on the growth of high-quality CLBO crystals, (2) high-quality protein crystal growth by means of forced nucleation induced by laser irradiation and the solution-stirring method, and (3) high-quality 2-in GaN crystal by Na flux liquid-phase epitaxy method.

Pre-assembled clusters distort crystal nucleation kinetics in supersaturated lysozyme solutions

Efficient determination of three-dimensional protein structures is critical for unraveling structure–function relationships and for supporting targeted drug design. A major impediment to these efforts is our lack of control over the nucleation and growth of high-quality protein crystals for X-ray structure determinations. While basic research on protein crystal growth mechanisms has provided valuable new insights, studies of crystal nucleation have been plagued by inconsistent and outright contradictory results. Using dynamic light scattering and SDS gel electrophoresis, we have investigated possible causes of these inconsistencies. We find that commercial sources of lyophilized hen-egg white lysozyme (HEWL) used in nucleation studies contain significant populations of large (∼ 100 nm), pre-assembled lysozyme clusters that can readily evade standard assays of sample purity. In supersaturated solutions, these clusters act as heterogeneous nucleation centers that enhance the rate of crystal nucleation and significantly deteriorate the quality of macroscopic crystals.

329 Effects of hypothalamically elicited restlessness behavior on the distribution and surface expression of the adhesion molecules of leukocytes in the cat

Controlling the nucleation and growth processes is important for growing large, high-quality crystals of oxides, proteins, and nitrides from a solution. We must understand how the solution conditions should be controlled, and determine a universal principle that is applicable to the growth of various high-quality crystals.In this report, we will present the effects of solution stirring and nucleation control on the solution growth of CsLiB6O10 (CLBO), proteins, and GaN crystals with respect to the following issues: (1) effect of solution stirring on the growth of high-quality CLBO crystals, (2) high-quality protein crystal growth by means of forced nucleation induced by laser irradiation and the solution-stirring method, and (3) high-quality 2-in GaN crystal by Na flux liquid-phase epitaxy method.