Matrix Seeding

What is Matrix Seeding? This is a technique where seeding is used to find hits in an initial screen, rather than being used as an optimisation strategy. Crystal formation is nucleation limited, and matrix seeding helps overcome this by providing nucleation points. Any crystals (or almost crystals) from the protein of interest (or something similar) from an initial screen can be used as seed stock. This has proven to be a powerful way of generating new crystal hits and leads.

How do I do it? Generally, one sets up seeded drops with 10% of the final volume of the drop consisting of microseed stock; for example a matrix microseeded drop might contain 150 nL protein, 120 nL reservoir solution, and 30 nL seed stock. Interestingly, matrix microseeding can reveal new leads and crystals which don’t have the same symmetry (space group) as the seed crystals.

Too much of a good thing: The image above illustrates what happens if your initial matrix seeded condition has excess nucleation (Left). Here one simply lowers the seed concentration (dilutes the seed stock) and tries again. As you do this, you should see less nucleation and (hopefully) larger crystal forms (Middle & Right). This is a great example provided from D’Arcy et al.

Iterative Seeding: The image below illustrates what can be achieved by multiple rounds of matrix microseeding. The star-burst crystals in the image on the left are an awkward morphology, with many one-dimensional crystals growing from the same nucleation point. Turning these into seed stock and microseeding into a sparse screen returned crystals in a different condition (middle), the process repeated again returned the crystals in the right image. Images again from D’Arcy et al.

What are the caveats? As you are adding seeds to a sparse screen (which will have a lot of different chemical combinations), you might generate a lot of salt crystals. This will be more of a problem if your seed stock contains a divalent metal, like magnesium. The phosphate salts of divalent metals are notoriously insoluble, and you will likely get a lot of (false) hits in phosphate-containing conditions. Similarly, if your seed solution contains sulfate you will get false positives in calcium-containing conditions. Other combinations (HEPES and zinc, for example) will also give salt crystals. Fortunately, if your protein contains tryptophan you can use UV imaging to help distinguish salt crystals from protein crystals.

Reference: D’Arcy, A., Bergfors, T., Cowan-Jacob, S.W., and Marsh, M. Microseed matrix screening for optimization in protein crystallization: what have we learned? Acta Crystallographica Section F Structural Biology Communications 70, (2014) 1117–1126

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