STRUCTURE NOTE Crystal Structure of the YciO Protein From Escherichia coli Jia Jia, 1 Vladimir V. Lunin, 1 Ve ´ ronique Sauve ´, 1 Li-Wei Huang, 2 Allan Matte, 1 and Miroslaw Cygler 1 * 1 Biotechnology Research Institute, National Research Council of Canada, and Montreal Joint Centre for Structural Biology, Montreal, Quebec, Canada 2 Biophysics Group, Physics Division, Los Alamos National Laboratory, Los Alamos, New Mexico Introduction. The YciO protein of Escherichia coli is a member of a family of proteins that also includes YrdC, HypF in E. coli, YwlC in Bacillus subtilis, and Sua5 in Saccharomyces cerevisiae (PF01300 family in PFAM data- base 1 ). Sequences similar to YciO are found either as (a) independent proteins, (b) with C-terminal extensions, or (c) as domains in larger proteins (PFAM). The proteins YwlC from B. subtilis and Sua5 from S. cerevisiae are examples of the second category. Sua5 has been identified as having an essential role for normal growth on lactate or glycerol medium, although the precise function of the Sua5 protein remains unclear. 2 HypF represents the third cat- egory where this domain is located in the middle of the linear sequence. The hypF and hydN genes form the hydA locus in E. coli, which encodes functions necessary for the formation of hydrogenase activity. HypF, the hydrogenase maturation protein, most likely participates in the matura- tion of all three E. coli hydrogenase isozymes 1, 2, and 3 from their inactive precursor forms. 3 Both crystallographic and nucleic acid binding studies with E. coli YrdC revealed that this protein might exert its function through binding to double-stranded RNA. 4 This would imply a role for YciO/YrdC and their homologous domains in larger proteins as functioning in the regulation of either transcription or translation. To gain further insight into this family of proteins, we have determined the crystal structure of E. coli YciO at 2.1 Å resolution. Materials and Methods. The gene encoding E. coli yciO was cloned, and the protein was expressed and purified as an N-terminal His 6 -tag fusion with a thrombin cleavage site as described previously for E. coli MoeA. 5 Crystals of the fusion protein were obtained by the hang- ing drop vapor diffusion method by equilibrating drops containing 2 L of protein (15 mg/mL) in buffer (20 mM Tris, pH 7.5, 40 mM NH 4 SO 4 , 60 mM NH 4 Ac, 5 mM 2-mercaptoethanol) and 2 L of reservoir solution (10% [w/v] PEG3350, 0.1 M MES buffer, pH 6.5, 0.1 M MgAc 2 , 5% [v/v] ethylene glycol) suspended over 0.5 mL of reser- voir solution. The crystals belong to the orthorhombic system, space group P2 1 2 1 2 1 with unit cell dimensions a = 48.40, b = 68.77, c = 94.77 Å and one molecule in the asymmetric unit. Before data collection, the crystal was soaked for 15 s in a cryoprotecting solution of 23% (w/v) PEG3350, 0.1 M MES, pH 6.5, 0.1 M MgAc 2 , 5% (v/v) ethylene glycol, and 19% (w/v) glycerol, and was flash cooled in a cold stream of N 2 gas to 100 K. The structure of YciO was determined by MAD phasing from a SeMet-labeled protein crystal (Table I). Data were processed and scaled with HKL2000. 6 All expected Se sites were found (SOLVE 7 ), and the phases were calculated to 2.1 Å (figure of merit of 0.52). The electron density map after applying density modification (RESOLVE 8 ) allowed automatic tracing of 80% of the main-chain and built the side-chains with the program ARP/WARP. 9 The model was then adjusted manually by using the program O 10 and refined (CNS version 1.0 11 ) against data collected at the hard remote wavelength. During refinement, 4.8% of the reflections were set aside for the calculation of R free . Water molecules were initially added automatically with CNS and verified by visual inspection of the difference map. The final model contains all residues, 1–206, 127 water mol- ecules, and one sulfate ion with an R-factor of 0.211 and R free of 0.229. The model has good geometry with no residues in the disallowed regions of the Ramanchandran plot (Procheck 12 ). The coordinates of YciO are deposited in the Protein Data Bank with the accession code 1KK9. Results and Discussion. The YciO molecule has an overall / architecture and consists of a single domain containing a central 10-stranded -sheet flanked by six -helices (A–F) (Fig. 1). The -sheet can be subdivided into two sheets (1-8-2-3-7-4, 4-6-5-9-10) with a common middle strand 4. There are two helices on each side of the N-terminal part of the -sheet (A, E and B, F), whereas the remaining two helices (C, D) are on the same side of the C-terminal part of this sheet. Helices are positioned asymmetrically about the -sheet, with four of them (A, E, C, and D) packing against one face and two (B and F) against the other (Fig. 1). Analysis of sequences of proteins homologous to YciO identified residues that are highly conserved within this family. 4 We analyzed the residue conservation by using a *Correspondence to: Dr. Mirek Cygler, Biotechnology Research Institute, NRCC, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada. E-mail: mirek@bri.nrc.ca Received 19 March 2002; Accepted 21 March 2002 Published online 00 Month 2001 in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/prot.10178 PROTEINS: Structure, Function, and Genetics 49:139 –141 (2002) © 2002 WILEY-LISS, INC.