Watershed provides a controlled humidity environment for a wide range of common sample preparation tasks, including

• separating crystals from each other, from precipitate, and from protein skins that may form over crystallization drops;
• soaking crystals in solutions containing cryoprotectants, heavy atoms or ligands;
• harvesting crystals from drops onto X-ray sample mounts;
• blotting or wicking away excess liquid from crystals to reduce background X-ray scatter and increase sample cooling rates during plunge cooling;
• placing crystals in MiTeGen’s MicroRT capillaries or glass capillaries for room temperature data collection; and
• adjusting the hydration of crystals to a desired relative humidity, which may be different from their as-grown relative humidity, to modify protein structure or optimize diffraction properties.

分水岭由一个水箱、一个控制单元和两个工作站组成，可提供 50% 到 >90% RH 之间的可控相对湿度。

加湿室工作站接受 18 或 22 毫米盖玻片或标准显微镜载玻片上的液滴。可以使用标准晶体环、测角仪底座和磁棒在工作站内采集和操作晶体，同时使用长距离体视显微镜从上方观察。安装后，可以将样品转移到含有潮湿空气的冷冻管中，然后可以将样品 + 冷冻管取出并运输到例如冷冻冷却装置。

定向喷嘴工作站可与多孔结晶板中的晶体一起使用，以提供指向被检查孔的加湿气流。该工作站还可以与实验室 X 射线源一起使用，以在衍射数据收集过程中可控地改变晶体的水合作用。

阅读 MiTeGen Watershed 用户手册以了解更多信息

维持和控制样品水合作用是生物分子晶体学以及细胞生物学、辅助生殖和相关领域的关键问题。 Watershed 为这个问题提供了一种方便、可靠、低成本的解决方案。

当操作盖玻片、载玻片或多孔微孔板上的液滴中包含的晶体时，蒸发和蛋白质降解会导致形成沉淀物和蛋白质表皮，导致晶体聚集和粘附，以及晶体开裂和其他损坏。

蒸发限制了给定液滴在晶体操作和收获过程中暴露在空气中的时间，然后液滴和其中的剩余晶体变得无法使用。

晶体浸泡在含有冷冻保护剂、重原子化合物或配体的溶液中的蒸发会导致不确定性，并导致晶体内这些溶质浓度的晶体与晶体之间的变化。

在这些步骤中以及在晶体学环或支架上晶体收获过程中的蒸发会导致晶体衍射特性的退化以及晶胞尺寸和蛋白质结构的晶体与晶体之间的变化。

生长后处理过程中的晶体脱水是室温 X 射线数据收集中晶体非同构的压倒性原因，也是低温非同构的重要原因。当仔细控制晶体水合时，1% 或更多的典型室温晶胞体积变化会降低到小于 0.2%。

另一方面，对于大约 5-10% 的蛋白质晶体，适度控制脱水可以改善整体晶体顺序和衍射特性，并在晶体衍射不足时提供有用的补救方法。

Watershed 提供了一种方便、低成本的解决方案，用于在晶体收获、操作、安装和 X 射线数据收集期间控制水合环境。

By using Watershed,

• Drop and sample working times can be increased by a factor of 10 or more relative to typical laboratory environments having relative humidities below 50%.
• More crystals can be harvested from each drop before it becomes unusable.
• Crystal isomorphism is improved, improving data quality and sometimes reducing the number of crystals required for structure determination.
• Perturbations of protein structures away from their biologically relevant forms are reduced.
• Quality and reproducibility of data are improved.

Watershed mixes high-humidity, room-temperature air (generated by bubbling dry air through room temperature water) with dry air to obtain an air stream with a desired relative humidity.

Relative Humidity Range:  72% to >90% RH
Water content of the compressed air supply affects the lowest achievable humidity. 72% RH tested with 23°C water and 6% RH air source.

Water tank capacity:  2.5 gallons / 10 liters

Requirements:

Power source:  110-125 VAC, 50-60 Hz, preferably with GFCI protection

Source of dry air or nitrogen at a pressure of 25 psi

Distilled or deionized water

Control unit:

Dimensions:  5.3 (H) x 14.0 (W) x 10.0 (D) inches / 135 x 357 x 255 mm

Weight:  11.5 lbs / 5.2 kg

Water Tank:

Dimensions:  10.2 (H) x 14.0 (W) x 9.3 (D) inches / 260 x 357 x 235 mm

Weight:  5.0 lbs / 2.3 kg empty; 26 lbs / 11.8 kg full

Humidified Enclosure Workstation:

Dimensions:  2.6 (H) x 4.5 (W) x 2.2 (D) inches / 66 x 115 x 55 mm

Accepts two 18 mm or 22 mm round cover slips or one 25 x 75 mm microscope slide

Required microscope working distance:  1.8 inches / 45 mm minimum

Positionable Nozzle Workstation:

Magnetic base diameter:  2 inches / 54 mm

Positionable tubing length:  19 inches / 500 mm

“Improved reproducibility of unit-cell parameters in macromolecular cryocrystallography by limiting dehydration during crystal mounting,”  Christopher Farley, Geoffry Burks, Thomas Siegert and Douglas H. Juers, Acta Cryst. D70, 2111-2124 (2014).

“A generic protocol for protein crystal dehydration using the HC1b humidity controller,” Carina M. C. Lobley, James Sandy, Juan Sanchez-Weatherby, Marco Mazzorana, Tobias Krojer, Radosław P. Nowak, and Thomas L. Sorensen, Acta Cryst. D72, 629-640 (2016).

“Automation and Experience of Controlled Crystal Dehydration: Results from the European Synchrotron HC1 Collaboration,” Matthew W. Bowler, Uwe Mueller, Manfred S. Weiss, Juan Sanchez-Weatherby, Thomas L-M. Sorensen, Marjolein M. G. M. Thunnissen,, Thomas Ursby, Alexandre Gobbo,,Silvia Russi, Michael G. Bowler, Sandor Brockhauser, Olof Svensson, and Florent Cipriani, Crystal Growth and Design 15, 1043-1054 (2015).

“Humidity control and hydrophilic glue coating applied to mounted protein crystals improves X-ray diffraction experiments,” Seiki Baba, Takeshi Hoshino, Len Ito and Takashi Kumasaka, Acta Cryst. D69, 1839-1849 (2013).

“Measurement of the equilibrium relative humidity for common precipitant concentrations: facilitating controlled dehydration experiments,” by M. J. Wheeler, S. Russi, M. G. Bowler, and M. W. Bowler, Acta Cryst. F 68, 111-114, (2011).

“Improving diffraction by humidity control: a novel device compatible with X-ray beamlines.”  Juan Sanchez-Weatherby, Matthew W. Bowler, Julien Huet, Alexandre Gobbo, Franck Felisaz, Bernard Lavault, Raphael Moya, Jan Kadlec, Raimond B. G. Ravelli, and Florent Cipriani, Acta Cryst. D65, 1237-1246 (2009).

“A novel free-mounting system for protein crystals: transformation and improvement of diffraction power by accurately controlled humidity changes,”  Reiner Kiefersauer, Manuel E. Than, Holger Dobbek, Lothar Gremer, Marcos Melero, Stefan Strobl, Joao M. Dieas, Twefik Soulimane, and Robert Huber, J. Appl. Cryst. 33, 1223-1230 (2000).

“A design of crystal mounting cell that allows the controlled variation of humidity at the protein crystal during X-ray diffraction,” M. G. Pickford, E. F. Garman, E. Y. Jones, and D. I. Stuart, J.Appl. Cryst. 26, 465-466 (1993).