The FUSION protein crystallization screen

Abstract

The success and speed of atomic structure determination of biological macromolecules by X-ray crystallography depends critically on the availability of diffraction-quality crystals. However, the process of screening crystallization conditions often consumes large amounts of sample and time. An innovative protein crystallization screen formulation called FUSION has been developed to help with the production of useful crystals. The concept behind the formulation of FUSION was to combine the most efficient components from the three MORPHEUS screens into a single screen using a systematic approach. The resulting formulation integrates 96 unique combinations of crystallization additives. Most of these additives are small molecules and ions frequently found in crystal structures of the Protein Data Bank (PDB), where they bind proteins and complexes. The efficiency of FUSION is demonstrated by obtaining high yields of diffraction-quality crystals for seven different test proteins. In the process, two crystal forms not currently in the PDB for the proteins α-amylase and avidin were discovered.

Keywords: X-ray crystallography; crystallization screens; macromolecular crystallization; protein crystals.

Figure 1

Systematic sampling of the two sets of additive mixes (sets 1 and 2) across the standard 96-well plate layout, forming 96 unique combinations of additives. For example, the combination {1,a} in well A1 corresponds to the two mixes ‘divalent metal cations 1’ (labelled ‘1’ in Table 1 ▸) and ‘alcohols’ (labelled ‘a’ in Table 2 ▸).

Figure 2

The ‘backbone’ of the FUSION screen, formed with the four precipitant mixes (P1–P4) and three titrated buffer systems (pH1–pH3) from the original MORPHEUS screen. For example, the combination {P1,pH1} in well A1 corresponds to ‘precipitant mix 1’ and ‘buffer system 1’, titrated to pH 6.5 (Table 3 ▸). The combination ‘{P1, pH1}’ is shown in bold to indicate the shift in positions across the 12 columns, in contrast to the original MORPHEUS 3D grid screen, where a single combination of precipitant and buffer populates each column (Gorrec, 2009 ▸). As a result, there are four of each mix of precipitants and three of each buffer system across each row, while there are two of each mix of precipitants and two to three of each buffer system in each column.