Acta Cryst. (1995). D51, 177-189 Crystal Structure of Narbonin at 1.8,& Resolution BY MICHAEL HENNIG,* SABINE PFEFFER-HENNIG,'[" ZBIGNIEW DAUTER AND KEITH S. WILSON European Molecular Biology Laboratory (EMBL) c/o DESY, Notkestrasse 85, 22603 Hamburg, Germany AND BERNHARD SCHLESIER AND VAN HAl NONG Institute of Plant Genetics and Crop Plant Research, Correnstrasse 3, 06466 Gatersleben, Germany (Received 14 February 1994; accepted 23 August 1994) 177 Abstract The three-dimensional structure of narbonin, a seed protein from Vicia narbonensis L, has been determined at 1.SA resolution. Phase information was obtained by multiple isomorphous replacement and optimized anomalous dispersion. The narbonin structure was ini- tially traced with only 17% amino-acid sequence infor- mation and preliminarily refined to a crystallographic R-factor of 16.5%. It is now refined to 15.9% using full sequence information derived from cDNA and after the addition of more solvent molecules. The monomeric molecule of narbonin is an eight-stranded parallel l;~- barrel surrounded by a-helices in a ~3/a-topology similar to that first observed in triose phosphate isomerase. Dif- ferences exist in the N-terminal part of the polypeptide chain, where the first helix is replaced by a loop and the second ~-strand is followed by an additional antiparallel a-sheet placed parallel on top of a-helices a3 and a4. Two short additional secondary structures are present. The first, an a-helix, is situated between the seventh 3- strand and the following helix, and the second, which is a 310 helix, between the eighth strand and the C- terminal helix. The most striking observation is the lack of a known enzymatic function for narbonin, because all TIM-like structures known so far are enzymes. I. Abbreviations TIM, triose phosphate isomerase; PRAI, N- (5'-phosphoribosyl)anthranilate isomerase; IGPS, indole-3-glycerol-phosphate synthase; TS, tryptophan synthase (a subunit); ARP, automated refinement procedure; MIR, multiple isomorphous replacement. 2. Introduction Storage globulins are a family of proteins of different size and structural organization, found in all legume seeds. They share several properties, such as similar * Correspondence address: Michael Hennig, Biozentrum, Depart- ment of Structural Biology, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland. Jr Present address: Sandoz AG, CH-4002 Basel, Switzerland. © 1995 International Union of Crystallography Printed in Great Britain - all rights reserved amino-acid composition, salting-in solubility, time of synthesis, and degradation and lack of enzymatic or biological activity. Subsequently, the classification of globulins from legume seeds was performed using their sedimentation coefficient (Derbyshire, Wright & Boulter, 1976). Narbonin is a member of the smaller 2S globulins, a monomer of 290 amino-acid residues and consists of a single polypeptide chain. The three-dimensional structures of two 7S globulins, phaseolin and canavalin, have been determined to 3.0 and 2.6/~, resolution, respectively (Lawrence, Suzuki, Varghese, Davis, Van Donkelaar, Tulloch & Colman, 1990; Ko, Ng & McPherson, 1993). Comparison of the two structures shows high similarity. They are composed of two domains, which are almost exactly related by twofold symmetry, with each domain being subdivided into two structural elements: an eight-stranded r-barrel with Swiss roll topology and an extended loop with a helix-turn-helix motif. The homology of the proteins is confirmed by 60% sequence identity. Until now, no three-dimensional structure for the 11S globulins has been published, although some llS globulins have been crystallized (Hennig & Schlesier, 1994). The crystal structure of narbonin from Vicia narbo- nensis L was determined at 1.8 A resolution by mul- tiple isomorphous replacement. Because of lack of se- quence information, a tentative amino-acid sequence was derived by examining the shapes of the side chains in the electron-density map. The structure was preliminarily refined to the crystallographic R-factor 16.5% and the tentative amino-acid sequence published (Hennig, Schle- sier, Dauter, Pfeffer, Betzel, Hrhne & Wilson, 1992; Hennig & Schlesier, 1993). Narbonin turned out to be folded as a /3/a-protein with r-strands and a-helices alternating to form an eight-stranded parallel /3-barrel surrounded by a ring of seven a-helices. This topology is similar to that first observed in triose phosphate iso- merase (Banner, Bloomer, Petsko, Phillips, Pogson, Wil- son, Corran, Furth, Milman, Offord, Priddle & Waley, 1975) and later found in many different enzymes. Here we present the refinement and analysis of the narbonin structure at 1.8 /~ resolution, considering the Acta Crystallographica Section D ISSN 0907-4449 ©1995