8SDK

The MicroED structure of proteinase K crystallized by suspended drop crystallization

Summary for 8SDK

• Entry DOI: 10.2210/pdb8sdk/pdb
• EMDB information: 40351
• Descriptor: Proteinase K, SULFATE ION, CALCIUM ION, ... (4 entities in total)
• Functional Keywords: microed, suspended drop crystallization, enzyme, hydrolase
• Biological source: Parengyodontium album
• Total number of polymer chains: 1
• Total formula weight: 29066.92

Authors

Gillman, C.,Nicolas, W.J.,Martynowycz, M.W.,Gonen, T. (deposition date: 2023-04-06, release date: 2023-05-31, Last modification date: 2024-10-23)

Primary citation

Gillman, C.,Nicolas, W.J.,Martynowycz, M.W.,Gonen, T.
Design and implementation of suspended drop crystallization.
Iucrj, 10:430-436, 2023

PubMed 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.

PubMed: 37223996
DOI: 10.1107/S2052252523004141

Experimental method

ELECTRON CRYSTALLOGRAPHY (2.1 Å)