Acta Cryst. (2003). D59, 1081±1083 Uppsten et al.  Ribonucleotide reductase nrdE crystals 1081 crystallization papers Acta Crystallographica Section D Biological Crystallography ISSN 0907-4449 Expression and preliminary crystallographic studies of R1E, the large subunit of ribonucleotide reductase from Salmonella typhimurium Malin Uppsten, a Mathias FaÈrnega Êrdh, a ² Albert Jordan, b S. Ramaswamy a ³ and Ulla Uhlin a * a Swedish University of Agricultural Sciences, Department of Molecular Biology, Box 590, Uppsala Biomedical Center, S-751 24 Uppsala, Sweden, and b Department of Genetics and Microbiology, Autonomous University of Barcelona, E-08193 Barcelona, Spain ² Formerly Mathias Eriksson (until 24 July 1999). Present address: Karo Bio AB, Novum, S-141 57 Huddinge, Sweden. ³ Present address: Department of Biochemistry, The University of Iowa, Iowa City, IA 52242, USA. Correspondence e-mail: ulla@xray.bmc.uu.se # 2003 International Union of Crystallography Printed in Denmark ± all rights reserved The nrdE gene product R1E, the large subunit of the class 1b Salmonella typhimurium ribonucleotide reductase, has been over- expressed, puri®ed and crystallized. Initially, the protein crystallized in two orthorhombic space groups, C222 1 and P2 1 2 1 2, using tartrate and PEG 6000 as precipitants, respectively. Better diffracting crystals belonging to the tetrahedral space group P4 3 2 1 2 were obtained using sodium malonate as precipitant. The P4 3 2 1 2 crystals could only be obtained after seeding from a drop containing C222 1 crystals grown in sodium tartrate. Thus, streak-seeding resulted in crystals of a supergroup to C222 1 . Data to 2.8 A Ê resolution have been collected on the P4 3 2 1 2 crystals which contained one R1E subunit in the asymmetric unit. Received 6 January 2003 Accepted 26 March 2003 1. Introduction Ribonucleotide reductase (RNR) catalyses the de novo synthesis of all four deoxyribo- nucleotide building blocks for DNA synthesis by reducing the corresponding ribonucleotides. This synthesis is carefully transcriptionally and allosterically regulated in most organisms. The substrate-reduction mechanism involves the use of a radical (Ehrenberg & Reichard, 1972; Reichard & Ehrenberg, 1983). Depending on the availability of cofactors and oxygen, three different types of probably unrelated radical- generation mechanisms have evolved and de®ne three classes of RNRs (Reichard, 1993, 1997). The most well studied enzyme is that from Escherichia coli, which is the prototype for class I reductases, which are present in eukaryotes, several viruses and bacteria. It consists of two homodimeric proteins, R1 and R2. Protein R2 contains the radical-generating machinery. It contains a diferric iron centre and a buried tyrosyl radical in its active form (Larsson & Sjo È berg, 1986). R1 contains the active site, redox-active cysteinyl residues and two allosteric binding sites. The binding of ATP or dATP to one of the allosteric effector sites, called the overall activity site, determines whether the enzyme is active or inhibited, respectively (Thelander & Reichard, 1979). The second effector-binding site changes the substrate speci®city by binding different dNTPs or ATP. This site is tuned to produce balanced levels of dNTPs appropriate for the particular organism. The structures of R1 (Uhlin & Eklund, 1994) and R2 (Nordlund et al., 1990) from E. coli have been determined. In this paper, we describe the expression, puri®cation and crystallization of the large subunit of a differently regulated class I ribo- nucleotide reductase. This enzyme has now been discovered in many organisms, particu- larly pathogenic bacteria; the enzymes from different sources share limited sequence similarities and have a similar molecular composition. The most interesting difference compared with the normal class I enzymes is the lack of negative regulation by high concentrations of dATP (Eliasson et al., 1996). This suggests that the enzymes lack the allo- steric overall activity site. From the structure of E. coli R1 in complex with the ATP analogue AMPPNP, the overall activity site was shown to be located in the ®rst 100 residues of the N-terminal domain (Eriksson et al., 1997). The R1 chains of the new class I are considerably shorter in the N-terminus. To distinguish the new type from the E. coli class I prototype, the old enzymes with two allosteric binding sites were named class Ia and the new enzymes with only one effector- binding site were named class Ib. Both types of reductases are present in E. coli and S. typhi- murium. The class Ib enzyme is fully functional in these organisms but seems not to be essen- tial, while the Ib form is the active RNR in Mycobacterium tuberculosis (Yang et al., 1994), Lactococcus lactis (Jordan et al., 1996) and Bacillus subtilus (Scotti et al., 1996); class Ib genes have also been found in Mycoplasma genitalium (Fraser et al., 1995) and M. pneu- monia (Himmelreich et al., 1997). The S. typhimurium genes nrdE and nrdF were the ®rst to be sequenced and cloned and the S. typhimurium enzyme is the prototype of