An approach to rapid protein crystallization using nanodroplets

Abstract

An approach that enables up to a two order of magnitude reduction in the amount of protein required and a tenfold reduction in the amount of time required for vapor-diffusion protein crystallization is reported. A prototype high-throughput automated system was used for the production of diffractionquality crystals for a variety of proteins from a screen of 480 conditions using drop volumes as small as 20 nL. This approach results in a signi®cant reduction in the time and cost of protein structure determination, and allows for larger and more ef®cient screens of crystallization parameter space. The ability to produce diffraction-quality crystals rapidly with minimal quantities of protein enables high-throughput efforts in structural genomics and structure-based drug discovery.

A novel Plasmodium-specific prodomain fold regulates the malaria drug target SUB1 subtilase

“…Initial identification of crystallization conditions was carried out using the vapour diffusion method in a Cartesian technology workstation. Sitting drops were set using 200 nl of 1:1 mixture of PvSUB1 and a crystallization solution (672 different conditions, commercially available), equilibrating against 150 ml reservoir in a Greiner plate 51 . Optimization of initial hits was pursued manually in Linbro plates with a hanging drop setup.…”

Polarization-resolved second-harmonic generation microscopy as a method to visualize protein-crystal domains

“…Crystalline samples with multiple domains are not always easily identifiable by bright-field imaging, especially for the specific case of twinning. Consequently, rapid and nondestructive identification of crystalline domains could significantly improve the productive throughput of synchrotron facilities (Chayen & Saridakis, 2008;Santarsiero et al, 2002;Walter et al, 2003;Chayen, 2003;Bergfors, 2003;Stojanoff et al, 2011;Kisselman et al, 2011).…”

Crystal structure of an Apo mRNA decapping enzyme (DcpS) from Mouse at 1.83 Å resolution

“…The appropriate fractions were pooled, further purified using a Superdex 200 size exclusion column (SEC; Amersham Biosciences) with elution in Crystallization Buffer [20 mM Tris, pH 7.9, 150 mM NaCl, 0.25 mM TCEP], and concentrated for crystallization assays to 16 mg/mL by centrifugal ultrafiltration (Millipore). The protein was crystallized using the nanodroplet vapor diffusion method 13 with standard JCSG crystallization protocols. 5 The crystallization reagent contained 20% polyethylene glycol (PEG)-3350 and 0.2 M potassium fluoride at pH 7.2.…”