Design and implementation of suspended drop crystallization
Abstract
In this work, a novel crystal growth method termed suspended drop crystallization has been developed. Unlike traditional methods, this technique involves mixing protein and precipitant directly on an electron microscopy grid without any additional support layers. The grid is then suspended within a crystallization chamber designed in-house, allowing for vapor diffusion to occur from both sides of the drop. A UV-transparent window above and below the grid enables the monitoring of crystal growth via light, UV or fluorescence microscopy. Once crystals have formed, the grid can be removed and utilized for X-ray crystallography or microcrystal electron diffraction (MicroED) directly without having to manipulate the crystals. To demonstrate the efficacy of this method, crystals of the enzyme proteinase K were grown and its structure was determined by MicroED following focused ion beam/scanning electron microscopy milling to render the sample thin enough for cryoEM. Suspended drop crystallization overcomes many of the challenges associated with sample preparation, providing an alternative workflow for crystals embedded in viscous media, sensitive to mechanical stress and/or subject to preferred orientation on electron microscopy grids.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- May 25, 2023
- Source ID
- 10.1107/s2052252523004141
Entities
People
- Cody Gillman
- Michael W. Martynowycz
- Tamir Gonen
- William J. Nicolas
Organizations
- Defense Threat Reduction Agency
- Howard Hughes Medical Institute
- National Institute of Arthritis and Musculoskeletal and Skin Diseases
- National Institute of General Medical Sciences
- University of California, Los Angeles