scholarly journals Methods for Obtaining Better Diffractive Protein Crystals: From Sample Evaluation to Space Crystallization

Crystals ◽  
2020 ◽  
Vol 10 (2) ◽  
pp. 78
Author(s):  
Yoshinobu Hashizume ◽  
Koji Inaka ◽  
Naoki Furubayashi ◽  
Masayuki Kamo ◽  
Sachiko Takahashi ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Quality Evaluation ◽  
Protein Crystallization ◽  
Protein Crystals ◽  
Microgravity Environment ◽  
Theoretical Understanding ◽  
Protein Sample ◽  
Space Experiments ◽  
Flash Cooling ◽  
Crystallization Conditions

In this paper, we present a summary on how to obtain protein crystals from which better diffraction images can be produced. In particular, we describe, in detail, quality evaluation of the protein sample, the crystallization conditions and methods, flash-cooling protection of the crystal, and crystallization under a microgravity environment. Our approach to protein crystallization relies on a theoretical understanding of the mechanisms of crystal growth. They are useful not only for space experiments, but also for crystallization in the laboratory.

scholarly journals Methods to Grow Better Diffractive Protein Crystals Acquired through Space Experiments

2019 ◽  
Author(s):  
Yoshinobu Hashizume ◽  
Koji Inaka ◽  
Naoki Furubayashi ◽  
Masayuki Kamo ◽  
Sachiko Takahashi ◽  
...  
Keyword(s):  
Quality Evaluation ◽  
Protein Crystals ◽  
Microgravity Environment ◽  
Protein Sample ◽  
Space Experiments ◽  
Flash Cooling ◽  
Crystallization Conditions

We summarize how to obtain protein crystals from which better diffraction images can be obtained. In particular, we describe in detail the quality evaluation of the protein sample, the crystallization methods and crystallization conditions, the flash-cooling protection of the crystal, and the crystallization under a microgravity environment.

scholarly journals Methods to Grow better Diffractive Protein Crystals Acquired through Space Experiments

2019 ◽  
Author(s):  
Yoshinobu Hashizume ◽  
Koji Inaka ◽  
Naoki Furubayashi ◽  
Masayuki Kamo ◽  
Sachiko Takahashi ◽  
...  
Keyword(s):  
Quality Evaluation ◽  
Protein Crystals ◽  
Microgravity Environment ◽  
Protein Sample ◽  
Crystallization Method ◽  
Space Experiments ◽  
Crystallization Conditions

We summarize how to obtain protein crystals from which better diffraction images can be obtained. In particular, the quality evaluation of the protein sample, the crystallization method and crystallization conditions, the freezing protection of the crystal, and the crystallization in the microgravity environment are described in detail.

scholarly journals The Study of the Mechanism of Protein Crystallization in Space by Using Microchannel to Simulate Microgravity Environment

Crystals ◽  
2018 ◽  
Vol 8 (11) ◽  
pp. 400 ◽  
Author(s):  
Yong Yu ◽  
Kai Li ◽  
Hai Lin ◽  
Ji-Cheng Li
Keyword(s):  
Growth Rates ◽  
Simulated Microgravity ◽  
Crystal Surface ◽  
Protein Crystallization ◽  
Experimental Results ◽  
Protein Crystals ◽  
Microgravity Environment ◽  
Microgravity Conditions ◽  
Lysozyme Crystals ◽  
Lysozyme Crystal

Space is expected to be a convection-free, quiescent environment for the production of large-size and high-quality protein crystals. However, the mechanisms by which the diffusion environment in space improves the quality of the protein crystals are not fully understood. The interior of a microfluidic device can be used to simulate a microgravity environment to investigate the protein crystallization mechanism that occurs in space. In the present study, lysozyme crystals were grown in a prototype microchannel device with a height of 50 μm in a glass-polydimethylsiloxane (PDMS)-glass sandwich structure. Comparative experiments were also conducted in a sample pool with a height of 2 mm under the same growth conditions. We compared the crystal morphologies and growth rates of the grown crystals in the two sample pools. The experimental results showed that at very low initial supersaturation, the morphology and growth rates of lysozyme crystals under the simulated microgravity conditions is similar to that on Earth. With increasing initial supersaturation, a convection-free, quiescent environment is better for lysozyme crystal growth. When the initial supersaturation exceeded a threshold, the growth of the lysozyme crystal surface under the simulated microgravity conditions never completely transform from isotropic to anisotropic. The experimental results showed that the convection may have a dual effect on the crystal morphology. Convection can increase the roughness of the crystal surface and promote the transformation of the crystal form from circular to tetragonal during the crystallization process.

Overcoming merohedral twinning in crystals of bacteriorhodopsin grown in lipidic mesophase

2009 ◽  
Vol 66 (1) ◽  
pp. 26-32 ◽  
Author(s):  
Valentin Borshchevskiy ◽  
Rouslan Efremov ◽  
Ekaterina Moiseeva ◽  
Georg Büldt ◽  
Valentin Gordeliy
Keyword(s):  
Crystal Growth ◽  
Membrane Protein ◽  
Slow Growth ◽  
Protein Crystals ◽  
Domain Formation ◽  
Ratio Distribution ◽  
Model Calculations ◽  
Growth Defects ◽  
Twin Domain ◽  
Crystallization Conditions

Twinning is one of the most common crystal-growth defects in protein crystallography. There are neither efficient rational approaches for the growth of nontwinned protein crystals nor are there examples of systematic studies of the dependence of the twinning-ratio distribution on crystallization conditions. The description of the twinning phenomenon has been covered even less for membrane-protein crystals and is non-existent for crystals grown using lipidic phases (in meso). In the present work, possibilities for overcoming merohedral twinning are investigated for crystals of the membrane protein bacteriorhodopsin (bR) grownin meso. It is shown that traditional crystallization additives are not effective in the case of thein mesocrystallization of bR. The twinning ratio was determined for 310 crystals grown under different crystallization conditions. A correlation of the twinning ratio with the growth rate of the crystals was observed. Slow growth indicated that crystals had a noticeable chance of avoiding twinning. Model calculations were performed in order to rationalize this observation. The calculations confirmed the experimental observation that most crystals consist of two twin domains and showed that under this condition small changes in the probability of twin-domain formation lead to dramatic changes in the number of nontwinned crystals, which explains why slow crystal growth results in a considerable number of nontwinned crystals.

scholarly journals Novel Device and Strategy for Growing Large, High-Quality Protein Crystals by Controlling Crystallization Conditions

Crystals ◽  
2021 ◽  
Vol 11 (11) ◽  
pp. 1311
Author(s):  
Naoki Tanigawa ◽  
Sachiko Takahashi ◽  
Bin Yan ◽  
Masayuki Kamo ◽  
Naoki Furubayashi ◽  
...  
Keyword(s):  
Crystal Growth ◽  
Sodium Chloride ◽  
Protein Crystals ◽  
Hydrogen Atoms ◽  
High Quality ◽  
X Ray ◽  
Dry Air ◽  
Novel Device ◽  
High Quality Protein ◽  
Crystallization Conditions

Neutron diffraction experiments are informative for determining the locations of hydrogen atoms in protein molecules; however, much larger crystals are needed than those required for X-ray diffraction. Thus, additional techniques are required to grow larger crystals. Here, a unique crystallization device and strategy for growing large protein crystals are introduced. The device uses two micropumps to control crystal growth by altering the precipitant concentration and regulating the pinpoint injection of dry air flow to the crystallization cell. Furthermore, the crystal growth can be observed in real time. Preliminary microbatch crystallization experiments at various concentration ranges of polyethylene glycol (PEG) 4000 and sodium chloride were first performed to elucidate optimized crystallization conditions. Based on these results, a device to precisely control the sodium chloride and PEG concentrations and the supply of dry air to the crystallization cell was used, and 1.8 mm lysozyme and 1.5 mm alpha-amylase crystals with good reproducibility were obtained. X-ray data sets of both crystals were collected at room temperature at BL2S1 of the Aichi Synchrotron Radiation Center and confirmed that these crystals were of high quality. Therefore, this crystallization device and strategy were effective for growing large, high-quality protein crystals.

Combination of oils and gels for enhancing the growth of protein crystals

2002 ◽  
Vol 35 (1) ◽  
pp. 140-142 ◽  
Author(s):  
Abel Moreno ◽  
Emmanuel Saridakis ◽  
Naomi E. Chayen
Keyword(s):  
Crystal Growth ◽  
Protein Crystallization ◽  
Protein Crystals ◽  
Large Crystal

This note focuses on two different ways of enhancing the use of gels in protein crystallization by applying oils to the trials. Using a five-channel motorized syringe setup, crystals were grown in gelled microbatch drops under oil and compared with those grown under similar conditions in standard microbatch drops. The advantage of this technique over existing gel techniques is that numerous trials can be dispensed automatically, while consuming very small quantities of protein. The application of oil to improve the gel acupuncture technique was also investigated; crystal growth in the presence of an oil barrier was slower than in its absence, giving rise in each case to a single large crystal with no precipitation nor smaller crystals in the capillary.

scholarly journals Control of the rate of evaporation in protein crystallization by the `microbatch under oil' method

2008 ◽  
Vol 41 (5) ◽  
pp. 969-971 ◽  
Author(s):  
Boris Brumshtein ◽  
Harry M. Greenblatt ◽  
Anthony H. Futerman ◽  
Israel Silman ◽  
Joel L. Sussman
Keyword(s):  
Protein Crystallization ◽  
Fine Tuning ◽  
Protein Crystals ◽  
Water Evaporation ◽  
Protein Solutions ◽  
Concomitant Increase ◽  
Crystallization Conditions ◽  
Rate Of Evaporation ◽  
Nucleation Point

Microbatch crystallization under oil is a powerful procedure for obtaining protein crystals. Using this method, aqueous protein solutions are dispensed under liquid oil, and water evaporates through the layer of oil, with a concomitant increase in the concentrations of both protein and precipitant until the nucleation point is reached. A technique is presented for regulating the rate of water evaporation, which permits fine tuning of the crystallization conditions as well as preventing complete desiccation of the drops in the microbatch crystallization trays.

A method to stabilize reduced and/or gas-treated protein crystals by flash-cooling under a controlled atmosphere

1999 ◽  
Vol 32 (3) ◽  
pp. 505-509 ◽  
Author(s):  
Xavier Vernède ◽  
Juan Carlos Fontecilla-Camps
Keyword(s):  
Crystalline State ◽  
Protein Complexes ◽  
Protein Crystallization ◽  
Protein Crystals ◽  
Controlled Atmosphere ◽  
Cryogenic Temperatures ◽  
Redox States ◽  
Cryogenic Technique ◽  
Flash Cooling ◽  
Oxygen Sensitive

A customized glove box for protein crystallization under a controlled atmosphere is described along with a cryogenic technique adapted to freeze protein crystals inside the glove box and a very simple device for studying gas–protein complexes in the crystalline state at cryogenic temperatures. Using these techniques different redox states of oxygen-sensitive crystalline proteins have been stabilized and the interaction of hydrogenase with Xe, a model for the much lighter substrate molecular hydrogen, has been studied.

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