A gradual desiccation method for improving the efficiency of protein crystallization screening

In vapor diffusion protein crystallization screening, it has been reported that replacing the reservoir solution with desiccant can increase the crystallization success rate. Therefore, the desiccation method is a potentially powerful method in practical protein crystallization screening. However, this method is difficult to apply broadly because the optimal amount of desiccant for a specific screening task is unknown. Utilizing an unsuitable amount of desiccant can result in even worse screening results than would be obtained from the traditional vapor diffusion method. Here, it is shown that by employing a modified strategy, named the gradual desiccation method, the problem can be solved without knowing the optimal amount of desiccant, and the crystallization success rate can be further increased compared with the one-time desiccation method.

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A simple and efficient innovation of the vapor-diffusion method for controlling nucleation and growth of large protein crystals

Journal of Applied Crystallography ◽

10.1107/s0021889801004666 ◽

2001 ◽

Vol 34 (3) ◽

pp. 388-391 ◽

Cited By ~ 7

Author(s):

Genpei Li ◽

Ye Xiang ◽

Ying Zhang ◽

Da-Cheng Wang

Keyword(s):

Crystallization Process ◽

Protein Crystallization ◽

Protein Crystals ◽

Diffusion Method ◽

Capillary Barrier ◽

Large Protein ◽

Vapor Diffusion ◽

Crystallization Experiments ◽

Water Vaporization ◽

Improved Technique

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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A double cell for controlling nucleation and growth of protein crystals

Journal of Applied Crystallography ◽

10.1107/s0021889888012270 ◽

1989 ◽

Vol 22 (2) ◽

pp. 115-118 ◽

Cited By ~ 13

Author(s):

M. Przybylska

Keyword(s):

Crystal Growth ◽

Protein Crystallization ◽

Heavy Atom ◽

Nucleation And Growth ◽

The Other ◽

Crystal Nucleation ◽

Diffusion Method ◽

Vapour Diffusion ◽

Reservoir Solution ◽

The Stability

A simple device for protein crystallization is described that consists of two connected cells, one for the hanging- or sitting-drop vapour diffusion method and the other for changing the concentration of the reservoir solution. It has been found useful for decoupling crystal nucleation from crystal growth, for improving the size and the stability of crystals, and in the preparation of heavy-atom derivatives.

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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 simple technique to convert sitting-drop vapor diffusion into hanging-drop vapor diffusion by solidifying the reservoir solution with agarose

Journal of Applied Crystallography ◽

10.1107/s0021889809028805 ◽

2009 ◽

Vol 42 (5) ◽

pp. 975-976 ◽

Cited By ~ 7

Author(s):

Tae Woong Whon ◽

Yong-Hwan Lee ◽

Dong-Shan An ◽

Hyun Kyu Song ◽

Song-Gun Kim

Keyword(s):

Protein Crystallization ◽

Glucose Isomerase ◽

Diffusion Experiment ◽

Hanging Drop ◽

Vapor Diffusion ◽

Egg White Lysozyme ◽

Simple Protocol ◽

Crystallization Experiments ◽

Reservoir Solution ◽

Hen Egg White

A simple protocol to convert sitting-drop vapor-diffusion plating into a hanging-drop vapor-diffusion experiment in protein crystallization is reported. After making a sitting-drop plate, agarose solution was added to solidify the reservoir solution, and the plates were incubated upside down. Crystallization experiments with hen egg white lysozyme, thaumatin and glucose isomerase showed that the `upside-down sitting-drop' method could produce single crystals with all the benefits of the hanging-drop crystallization method.

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Replacing a reservoir solution with desiccant in vapor diffusion protein crystallization screening

Journal of Applied Crystallography ◽

10.1107/s0021889810030803 ◽

2010 ◽

Vol 43 (5) ◽

pp. 1021-1026 ◽

Cited By ~ 6

Author(s):

Qin-Qin Lu ◽

Da-Chuan Yin ◽

Rui-Qing Chen ◽

Si-Xiao Xie ◽

Yong-Ming Liu ◽

...

Keyword(s):

Protein Crystallization ◽

Crystal Formation ◽

Protein Solution ◽

Vapor Diffusion ◽

Modified Method ◽

Reservoir Solution ◽

The Cost ◽

Drop Technique

This paper presents a modification to the conventional vapor diffusion (hanging- or sitting-drop) technique for protein crystallization screening. In this modified method, the reservoir solution is replaced with a desiccant to allow for a larger range of protein solution concentrations, thereby providing more opportunities for crystal formation. This method was tested in both reproducibility and screening studies, and the results showed that it significantly improves the efficiency and reduces the cost of protein crystallization screens.

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The effect of diluting crystallization droplets on protein crystallization in vapor diffusion method

Crystal Research and Technology ◽

10.1002/crat.201100178 ◽

2011 ◽

pp. n/a-n/a

Author(s):

Qin-Qin Lu ◽

Da-Chuan Yin ◽

Si-Xiao Xie ◽

Yong-Ming Liu ◽

Rui-Qing Chen

Keyword(s):

Protein Crystallization ◽

Diffusion Method ◽

Vapor Diffusion

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Crystal structure analyses of oxygenase component of resorcinol hydroxylase

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314095515 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C448-C448

Author(s):

Tomomi Fujii ◽

Kazutaka Kobayashi ◽

Takae Yamauchi ◽

Masahiro Yoshida ◽

Tadao Oikawa ◽

...

Keyword(s):

Crystal Structure ◽

Catalytic Mechanism ◽

Structural Basis ◽

Diffusion Method ◽

Flavin Reductase ◽

Vapor Diffusion ◽

Space Group ◽

Terminal Domain ◽

Loop Region ◽

Reservoir Solution

The resorcinol hydroxylase is involved in the first step of the resorcinol catabolic pathway and catalyzes hydroxylation of resorcinol to hydroxyquinol. The enzyme belongs to the two-component flavin-diffusible monooxygenase family and acts in the coexistence of two components: an oxygenase and a flavin reductase. The oxygenase component hydroxylates the substrate using molecular oxygen and reduced flavin produced by the reductase. To understand the structural basis for the catalytic mechanism, we analyzed the crystal structure of the oxygenase component (GraA) from Rhizobium sp. strain MTP-10005. The GraA subunit has 409 amino acid residues. Apo-form crystals were obtained in the tetragonal space group I4122 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and K2HPO4. Holo-form crystals were obtained in the trigonal space group P3221 by a sitting-drop vapor-diffusion method with a reservoir solution of PEG3350 and KNO3. Both structures were determined by molecular replacement and refined at 2.3 Å and 3.2 Å resolutions, respectively. GraA is a homotetramer with three molecular two-fold axes identical to crystallographic two-fold axes in the apo-form crystal. In the holo-form crystal, four tetramers exist in the asymmetric unit and each subunit binds one FAD. The subunit consists of three domains. The N-terminal domain has an α-structure mainly of antiparallel α-helices; the central domain has a β-structure of two β-sheets stacked together; the C-terminal domain has a four-helix-bundle structure of long antiparallel α-helices involved in tetramer formation. In the holo-form, the FAD is located in the space that is encompassed by these three domains. The loop region of 13 residues, which is disordered in the apo-form, is ordered and covers FAD of another subunit. The turn portion of the loop occludes the entrance of the putative active site.

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A simple vapor-diffusion method enables protein crystallization inside the HARE serial crystallography chip

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798321003855 ◽

2021 ◽

Vol 77 (6) ◽

Author(s):

Brenna Norton-Baker ◽

Pedram Mehrabi ◽

Juliane Boger ◽

Robert Schönherr ◽

David von Stetten ◽

...

Keyword(s):

Structure Determination ◽

Protein Crystallization ◽

Diffusion Method ◽

High Quality ◽

Protein Variant ◽

Simple Method ◽

Batch Crystallization ◽

Vapor Diffusion ◽

Require Time ◽

Serial Crystallography

Fixed-target serial crystallography has become an important method for the study of protein structure and dynamics at synchrotrons and X-ray free-electron lasers. However, sample homogeneity, consumption and the physical stress on samples remain major challenges for these high-throughput experiments, which depend on high-quality protein microcrystals. The batch crystallization procedures that are typically applied require time- and sample-intensive screening and optimization. Here, a simple protein crystallization method inside the features of the HARE serial crystallography chips is reported that circumvents batch crystallization and allows the direct transfer of canonical vapor-diffusion conditions to in-chip crystallization. Based on conventional hanging-drop vapor-diffusion experiments, the crystallization solution is distributed into the wells of the HARE chip and equilibrated against a reservoir with mother liquor. Using this simple method, high-quality microcrystals were generated with sufficient density for the structure determination of four different proteins. A new protein variant was crystallized using the protein concentrations encountered during canonical crystallization experiments, enabling structure determination from ∼55 µg of protein. Additionally, structure determination from intracellular crystals grown in insect cells cultured directly in the features of the HARE chips is demonstrated. In cellulo crystallization represents a comparatively unexplored space in crystallization, especially for proteins that are resistant to crystallization using conventional techniques, and eliminates any need for laborious protein purification. This in-chip technique avoids harvesting the sensitive crystals or any further physical handling of the crystal-containing cells. These proof-of-principle experiments indicate the potential of this method to become a simple alternative to batch crystallization approaches and also as a convenient extension to canonical crystallization screens.

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