A novel microseeding method for the crystallization of membrane proteins in lipidic cubic phase

Abstract

Random microseed matrix screening (rMMS), in which seed crystals are added to random crystallization screens, is an important breakthrough in soluble protein crystallization that increases the number of crystallization hits that are available for optimization. This greatly increases the number of soluble protein structures generated every year by typical structural biology laboratories. Inspired by this success, rMMS has been adapted to the crystallization of membrane proteins, making LCP seed stock by scaling up LCP crystallization conditions without changing the physical and chemical parameters that are critical for crystallization. Seed crystals are grown directly in LCP and, as with conventional rMMS, a seeding experiment is combined with an additive experiment. The new method was used with the bacterial integral membrane protein OmpF, and it was found that it increased the number of crystallization hits by almost an order of magnitude: without microseeding one new hit was found, whereas with LCP-rMMS eight new hits were found. It is anticipated that this new method will lead to better diffracting crystals of membrane proteins. A method of generating seed gradients, which allows the LCP seed stock to be diluted and the number of crystals in each LCP bolus to be reduced, if required for optimization, is also demonstrated.

Lipidic cubic phase serial femtosecond crystallography structure of a photosynthetic reaction centre

“…Seeding is a common technique in vapour-diffusion crystallization that is widely used to improve crystal growth. Seeding has been less common in LCP crystallization due to difficulties in crushing LCP crystals to form a smooth distribution of crystal seeds, although innovative approaches have been applied (Kolek et al, 2016). We instead developed a new protocol for seeding LCP setups to produce microcrystals for SFX based upon our earlier vapour-diffusion microcrystals (Dods et al, 2017).…”

Seeding Techniques and Optimization of Solution Crystallization Processes

“…They concluded that a series of diluted seed stock mixtures could effectively control the concentration or the number of submicrometer-sized seeds added. Microseed matrix seeding, which greatly increases the number of crystallization hits and improves the crystal quality for structure determination, has been intensively studied. − Macroseeding can also be used in single-crystal cultivation, which can be handled one-by-one in solution, while for the crystallization of pharmaceuticals and fine chemicals, macroseeding is widely used on both the laboratory and industrial scales. Although submicrometer-sized seeds have been used in specific fields, such as crystallization of biological macromolecules, the small-sized seeds can easily introduce crystal agglomeration.…”

Towards an Optimal Sample Delivery Method for Serial Crystallography at XFEL

“…This also facilitates easier tracking of crystallization progress with microscopic imaging. Seeding has achieved great success for soluble protein and is also feasible for crystals grown in LCP [78]. However, adaptation of a seeding protocol for serial crystallography sample preparation has not yet been reported.…”