The use of two novel methods to grow protein crystals by microdialysis and vapor diffusion in an agarose gel

The rate of water vaporization in the vapor-diffusion method is critical for the protein crystallization process. Present methods, however, allow little or no control of the equilibration rates. This paper presents a relatively simple innovation of the conventional vapor-diffusion method by introducing a capillary barrier (for hanging drop) or a punched film barrier (for both hanging and sitting drop) between drop and reservoir, which can be beneficial in controlling the water vaporization rate, thereby promoting growth of large protein crystals. The crystallization experiments for lysozyme, trichosanthin and a novel neurotoxin BmK Mu9 show that this modified vapor-controlling-diffusion method is very effective for producing large protein crystals. The improved technique can be routinely used as a method for the preparation of other macromolecular and small-molecule crystals whose crystallization involves vaporization of water.

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Fast preparation of a desiccant array for the gradual desiccation method in protein crystallization screening

Journal of Applied Crystallography ◽

10.1107/s0021889813009734 ◽

2013 ◽

Vol 46 (3) ◽

pp. 817-822 ◽

Cited By ~ 2

Author(s):

Qin-Qin Lu ◽

Xu-Zhuo Xie ◽

Yong-Ming Liu ◽

Hui-Meng Lu ◽

Da Chen ◽

...

Keyword(s):

Aqueous Solution ◽

High Throughput ◽

Protein Crystallization ◽

Protein Crystals ◽

Diffusion Method ◽

Screening Process ◽

Vapor Diffusion ◽

High Throughput Method ◽

Droplet Array

The gradual desiccation method (GDM) is a modification of the vapor diffusion method for protein crystallization screening. This method can dramatically increase the chances of obtaining protein crystals and is therefore potentially useful for practical protein crystallization screening. However, it is troublesome to prepare the desiccant for the GDM because each of the 96 desiccants must be of the same mass. Repeated manual weighing of the desiccant (at least 96 times for one plate) to obtain the same amount is required, and manual distribution of the weighed desiccants to the respective reservoir wells is also necessary. These procedures require a considerable amount of labor and thus lower the efficiency of the screening process. Additionally, they reduce the applicability of this method in routine protein crystallization screening. To solve this problem, a high-throughput method is proposed, which involves dispensing an aqueous solution of salts (a combination of CoCl2and AlCl3) into a droplet array (8 × 12, corresponding to the arrangement in a standard crystallization plate) on a piece of tape, then drying this array to obtain the final desiccant array. Simply covering and sealing this desiccant array over the crystallization droplets in the crystallization plate can give a perfect vapor diffusion screen. With this method, the labor and automation requirements of the GDM will be comparable to those of the conventional vapor diffusion method; furthermore, the amount of the desiccant can be easily and accurately controlled, allowing the GDM to be applied in daily protein crystallization screening.

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A crystallization technique for obtaining large protein crystals with increased mechanical stability using agarose gel combined with a stirring technique

Journal of Crystal Growth ◽

10.1016/j.jcrysgro.2015.11.008 ◽

2016 ◽

Vol 452 ◽

pp. 172-178 ◽

Cited By ~ 5

Author(s):

Mihoko Maruyama ◽

Yuki Hayashi ◽

Hiroshi Y. Yoshikawa ◽

Shino Okada ◽

Haruhiko Koizumi ◽

...

Keyword(s):

Mechanical Stability ◽

Protein Crystals ◽

Agarose Gel ◽

Large Protein

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

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s0907444913016296 ◽

2013 ◽

Vol 69 (10) ◽

pp. 1901-1910 ◽

Cited By ~ 11

Author(s):

Hui-Ling Cao ◽

Li-Hua Sun ◽

Jian Li ◽

Lin Tang ◽

Hui-Meng Lu ◽

...

Keyword(s):

Silicone Oil ◽

Crystal Quality ◽

Protein Crystals ◽

Agarose Gel ◽

Resolution Limit ◽

X Ray Diffraction ◽

High Quality ◽

X Ray ◽

Diamagnetic Levitation ◽

High Quality Protein

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 theRmerge. 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.

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Femtosecond Laser Processing of Agarose Gel Surrounding Protein Crystals for Development of an Automated Crystal Capturing System

Japanese Journal of Applied Physics ◽

10.1143/jjap.48.105502 ◽

2009 ◽

Vol 48 (10) ◽

pp. 105502 ◽

Cited By ~ 8

Author(s):

Shigeru Sugiyama ◽

Hitoshi Hasenaka ◽

Mika Hirose ◽

Noriko Shimizu ◽

Tomoya Kitatani ◽

...

Keyword(s):

Femtosecond Laser ◽

Laser Processing ◽

Protein Crystals ◽

Agarose Gel ◽

Femtosecond Laser Processing

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ATP-dependent RNA helicase domain of the ZC3H41 protein from Trypanosoma brucei: expression, purification and crystallization

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x20015204 ◽

2020 ◽

Vol 76 (12) ◽

pp. 604-608

Author(s):

Lee S. Izhaki-Tavor ◽

Moshe Dessau

Keyword(s):

Escherichia Coli ◽

Trypanosoma Brucei ◽

Structural Information ◽

Rna Helicase ◽

Protein Crystals ◽

Diffusion Method ◽

Helicase Domain ◽

Vapor Diffusion ◽

Bacterial System

A fragment of the Trypanosoma brucei ZC3H41 protein encompassing the ATP-dependent RNA helicase domain was successfully subcloned for expression in a bacterial system (Escherichia coli). Following expression, the protein was purified and crystallized using the vapor-diffusion method. The protein crystals were optimized at a 1:1 protein:reservoir solution ratio using PPGBA 2000. The optimized crystals diffracted to a d min of 3.15 Å. The collected data revealed preliminary structural information regarding this newly discovered protein.

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