An electron density map of concanavalin A at 2.4-A resolution has been produced by X-ray crystallographic methods with five heavy atom derivatives. The molecule is a tetramer with all subunits identical and each containing a single polypeptide chain of 231 amino acid residues. The course of the entire backbone has been traced and three different regions of /3 structure involve about 57% of the residues. One of these /3-structure regions contains six strands of polypeptide chain and is related by a crystallographic twofold rotation axis to an additional six strands of the second c \*_>oncanavalin A (Con A1) exhibits some unusual biological properties as a result of its ability to bind various carbohydrates. It agglutinates erythrocytes from certain animal species, starch granules, and some bacteria and yeasts (Sumner and Howell, 1936a), and has been shown to precipitate various glycogens, dextrans, mannans, glycoproteins, and blood group substances (Sumner and Howell, 1936b; So and Goldstein, 1968; Leon and Young, 1970; and Lloyd et al., 1969). Carbohydrates with the minimum specificity for Con A binding contain hexose residues with the D-ara6//zo-pyranoside configuration2 at C-3, C-4, and C-5 (Goldstein et al., 1965).Con A induces transformation of lymphocytes by reversibly binding to specific sites on the cell surface (Powell and Leon, 1970; Novogrodsky and Katchalski, 1971) and inhibits phagocytosis by polymorphonuclear leucocytes (Berlin, 1972). It also agglutinates embryonic tissue cells (Moscona, 1971) and various neoplastic cells in tissue cultures (Inbar and Sachs, 1969), whereas binding sites on the corresponding adult cells and normal cells appear to be masked. Studies by Inbar et al. (1971) indicate that the site for Con A on the cell surface membrane of hamster cells has two components, one which actually binds the Con A and the other which is responsible for the agglutination.The purpose of our investigation is to determine the complete three-dimensional structure of Con A by X-ray crystallographic techniques and we report here features of the molecule, mainly the course of the polypeptide chain, subunit interactions, regions of /3 structure, the carbohydrate binding site, and the Mn2J~and Ca2^sites as interpreted from our electron density map at 2.4-A resolution. Experimental ProcedureCon A, purified and crystallized as previously described (Hardman et a!., 1971a), was used to collect the lower resolut From the

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The carbohydrate binding site of concanavalin A has been identified in crystals of the concanavalin A-methyl alpha-D-mannopyranoside complex and is 35 A from the iodophenol binding site (K. D. Hardman and C. F. Ainsworth (1973), Biochemistry 12,4442), which has been postulated to be adjacent to the carbohydrate-specific binding site (Edelman et al. (1972), Proc. Natl. Acad. Sci. U.S.A. 69, 2580). The crystals are orthorhombic in space group C222(1) and crystal denisty measurements indicate a protein mass of four monomers (molecular weight of 104 000) per asymmetric unit. However, the electron density map contains eight monomers/asymmetric unit, revealing lattice disorder. The electron density map with a nominal resolution of 6 A has been solved using three heavy-atom derivatives and the position and orientation of each monomer established. Atomic coordinates of the native protein which has previously been determined (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103) were transposed into this new space group and the gross conformations of the monomers, dimers, and tetramers were found to be very similar to the previous structure. However, some minor differences were apparent even at this resolution. After crystal growth, the methyl alpha-D-mannopyranoside was replaced by o-iodophenyl beta-D-glucopyranoside or methyl 2-iodoacetimido-2-deoxy-alpha-D-glucopyranoside in separate experiments, and difference electron density maps were calculated. The highest peaks for both iodinated sugar derivatives associated with each monomer agreed within a few angstroms of each other and were found near side chains Tyr-12 and -100 and Asp-16 and -208. This region is 10-14 A from the manganese, in good agreement with nuclear magnetic resonance (NMR) studies in solution (C. F. Brewer et al. (1973), Biochemistry 12, 4448) and with the site predicted from crosslinked 1222 crystal studies (K. D. Hardman (1973), Adv. Exp. Med. Biol. 40, 103).

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The structure of a multisubunit protein (immunoglobulin light chain) was solved in three crystal forms, differing only in the solvent of crystallization. The three structures were obtained at high ionic strength and low pH, high ionic strength and high pH, and low ionic strength and neutral pH. The three resulting "snapshots" of possible structures show that their variable-domain interactions differ, reflecting their stabilities under specific solvent conditions. In the three crystal forms, the variable domains had different rotational and translational relationships, whereas no alteration of the constant domains was found. The critical residues involved in the observed effect of the solvent are tryptophans and histidines located between the two variable domains in the dimeric structure. Tryptophan residues are commonly found in interfaces between proteins and their subunits, and histidines have been implicated in pHdependent conformation changes. The quaternary structure observed for a multisubunit protein or protein complex in a crystal may be influenced by the interactions of the constituents within the molecule or complex and/or by crystal packing interactions. The comparison of buried surface areas and hydrogen bonds between the domains forming the molecule and between the molecules forming the crystals suggest that, for this system, the interactions within the molecule are most likely the determining factors.The immunoglobulin light chain is a multidomain protein; it consists of two domains, the N-terminal variable (V) domain and the C-terminal constant (C) domain. The two domains are connected by a flexible "switch" peptide. Many light chains form dimers by homologous association of the V and C domains of the two chains. The angle between the local twofold axes of the V domain dimer and the C domain dimer defines the "elbow" bend. In antigen binding fragments light chains are associated with heavy chains. The same surface of the light chain domain that is used for formation of antigen binding fragment with heavy chain forms the light chain dimer interface.We have previously determined the structure of the AI type human light chain dimer Loc in two crystal forms (1, 2) crystallized from ammonium sulfate (LocAS) and from distilled water (LocW). We found that the V-domain interactions as well as the elbow bends differed in the two structures. We have now refined these structures using high-resolution data. We have also crystallized the molecule in a third form from NaKSO4 and determined and refined its structure (LocNaKS).This third structure differs from the previous two in the relative positions of the V domains; it appears to be a transition between the two. The three structures allow interpretation of how the solvent of crystallization, its ionic strength, and pH influence the domain interactions in this molecule. The observed modulation of quaternary structures by solvent composition may be relevant to crystallographic analysis of other multidomain proteins as well as protein-ligand and proteinp...