Vivaspin 500 mwco10 000

Preparation
of the HSA solution for crystallization was performed as described
previously.^{33 (link)} A stock solution of 50 mM
ARP for crystallization was prepared by dissolving in dimethyl sulfoxide
(DMSO). The ARP–HSA complex was formed by mixing the HSA solution
and the ARP stock solution at a 1:5 HSA–ARP molar ratio in
50 mM potassium phosphate pH 7.0 and 10% (v/v) DMSO, after which it
was incubated at 20 °C overnight. After incubation, the excess
unbound and insoluble ARP that was deposited was removed by centrifugation
(20 400g for 1 h at 20 °C), and the ARP–HSA
complex was washed with 50 mM potassium phosphate pH 7.0 by performing
four cycles of dilution and concentration using a Vivaspin 500 (MWCO
10 000, Sartorius) centrifugal concentrator. The ARP–HSA complex
solution was finally concentrated to an HSA concentration of 1.4 mM.
Co-crystallization of the ARP–HSA complex was performed using
the hanging–drop vapor diffusion method, and the ARP–HSA
crystals for the X-ray analysis were obtained by multiple rounds of
streak-seeding with droplets prepared by mixing 2 μL of the
ARP–HSA complex solution and 2 μL of the reservoir solution
containing 32% (w/v) polyethylene glycol 3350 and 50 mM potassium
phosphate pH 7.0 at 4 °C and pre-equilibrated for 1–3
days.