Microfluidizer apparatus

Sth1 constructs and complexes were cloned and expressed as described^{24 (link)}. For Sth1_301-1097P1Swap, the Sth1 fragments 301–645 and 693-1097 were primer-extended to add S. solfataricus Rad54 residues 610–644. A silent EcoRI site was introduced during extension to ligate the two Sth1 fragments. For protein purification, cells were resuspended in lysis buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 5% glycerol, 25 mM imidazole, and 4 mM benzamidine), lysed using a Microfluidizer apparatus (Microfluidics), and clarified by centrifugation. Lysates were purified on a Ni-NTA affinity column (Qiagen) and washed extensively with lysis buffer. Proteins were eluted with 250 mM imidazole and bound to a HiTrap Heparin HP column (GE Healthcare) equilibrated in sample buffer (20 mM HEPES pH 7.5, 200 mM NaCl, 5% glycerol, 2 mM dithiothreitol (DTT), and 4 mM benzamidine). Proteins were eluted using a 200–800 mM NaCl gradient, and further purified on a SD-200 gel filtration column (GE Healthcare) equilibrated with sample buffer. To reconstitute the Rtt102-Arp7/9 ternary complex, Rtt102 and Arp7/9^{24 (link)} were mixed at a 2:1 molar ratio and purified through a SD-200 gel filtration column equilibrated in sample buffer (without benzamidine).

Wild-type and TMBS mutant Tmod3 in pTXB1 were expressed in E. coli BL21 (DE3) cells (Invitrogen) grown in Terrific Broth medium at 37C until the OD600 reached a value of 1–1.5. Expression was induced with 0.3 mM IPTG, and was carried out for 16 h at 19C. Cells were harvested by centrifugation, re-suspended in chitin buffer (HEPES-20mM-pH 7.5, NaCl-500mM, EDTA-1mM, DTT-1mM, PMSF-1mM), and lysed using a microfluidizer apparatus (Microfluidics). Proteins were first purified on a chitin affinity column, and eluted after selfcleavage of the intein induced by incubation with 40 mM DTT for 24 h. Proteins were then purified through a SD200HL 26/600 gel filtration column (GE Healthcare) in HEPES-20mM-pH 7.5, NaCl-200mM, EDTA-1mM, DTT-1mM. Fractions containing Tmod were dialyzed overnight to HEPES-20mM, NaCl-50mM, loaded on an ion exchange sourceQ column (Pharmacia), and eluted with a 50–500 mM NaCl gradient. CapZ protein was purified as described [18 (link)]. Briefly, both subunits (alpha and beta) were cloned into both cloning sites of pRSFDuet-1 vector (Novagen) which includes a 6x-His tag and a TEV cleavage site [18 (link)]. Purification was carried out as described previously [18 (link)]. Actin [58 (link)] and tropomyosin [59 (link)] were purified as described.