Microseed matrix screening: more hits in less time

Moroz O.V.¹, Blagova E.B.¹, Friis E.P. ², Davies G.J.¹ and Wilson K.S.¹

¹Department of Chemistry, York Structural Biology Laboratory, University of York, York, UK
²Novozymes A/S, Bagsvaerd, Denmark

Microseed matrix screening (MMS) is an approach in macromolecular crystallisation where crushed crystals from one hit, often poor, are used as nucleation points in conditions different from the initial ones.  The technique was introduced by Ireton & Stoddard in 2004 (1) and subsequently automated by D'Arcy et al. (2). The rationale behind this approach is in separating nucleation from crystal growth, leading to new hits and better diffracting crystals. A number of groups have reported success in using the technique, with results not only from self-seeding (with crushed crystals of the same protein), but also cross seeding with a homologous protein, as well as seeding of a complex with crystals of one of the components (3,4,5). The approach was recently extended to crystallisation of membrane proteins, using lipid cubic phase seed stock (6).

We present here examples of successful application of MMS in crystallisation of a number of industrially important enzymes: proteases, lipases and amylases. In our experience, MMS often increases the number of hits, improves the quality of crystals and shortens the time required for growth of diffraction quality crystals.

1. Ireton G.C.& Stoddard B.L. Acta Cryst. D60, 601-5 (2004).

2. D'Arcy A, Villard F, & Marsh M. Acta Cryst. D63, 550-4 (2007).

3. Obmolova G, et al., Acta Cryst. D66, 927-33 (2010). 

4. Shaw Stewart P.D., et al., Cryst. Growth Des. 11, 3432–3441 (2011).  

5. D’Arcy A. et al., Acta Cryst. F70, 1117-1126 (2014)

6. Kolek S.A. et al., Acta Cryst. F72, 307-312 (2016)