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Structure Determination from Lipidic Cubic Phase Embedded Microcrystals by MicroED.

Structure (London, England : 1993) (2020-08-01)
Lan Zhu, Guanhong Bu, Liang Jing, Dan Shi, Ming-Yue Lee, Tamir Gonen, Wei Liu, Brent L Nannenga
ABSTRACT

The lipidic cubic phase (LCP) technique has proved to facilitate the growth of high-quality crystals that are otherwise difficult to grow by other methods. However, the crystal size optimization process could be time and resource consuming, if it ever happens. Therefore, improved techniques for structure determination using these small crystals is an important strategy in diffraction technology development. Microcrystal electron diffraction (MicroED) is a technique that uses a cryo-transmission electron microscopy to collect electron diffraction data and determine high-resolution structures from very thin micro- and nanocrystals. In this work, we have used modified LCP and MicroED protocols to analyze crystals embedded in LCP converted by 2-methyl-2,4-pentanediol or lipase, including Proteinase K crystals grown in solution, cholesterol crystals, and human adenosine A2A receptor crystals grown in LCP. These results set the stage for the use of MicroED to analyze microcrystalline samples grown in LCP, especially for those highly challenging membrane protein targets.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Iodoacetamide, BioUltra
Sigma-Aldrich
Proteinase K from Tritirachium album, lyophilized powder, BioUltra, ≥30 units/mg protein, for molecular biology
Sigma-Aldrich
Cholesterol, Sigma Grade, ≥99%
Sigma-Aldrich
Lipase from Candida rugosa, Type VII, ≥700 unit/mg solid
Sigma-Aldrich
1-Oleoyl-rac-glycerol, ≥99%
Sigma-Aldrich
Bovine Serum Albumin, heat shock fraction, protease free, fatty acid free, essentially globulin free, pH 7, ≥98%
Sigma-Aldrich
Monoclonal ANTI-FLAG® M2-FITC antibody produced in mouse, clone M2, purified immunoglobulin, buffered aqueous solution