The highly ordered crystal structure of crambin has been solved at 1.5 Å resolution directly from the diffraction data of a native crystal at a wavelength remote from the sulphur absorption edge. The molecule has three disulphide bridges among its 46 amino acid residues, of which 46% are in helices and 17% are in a β-sheet. Crambin is shown to be an amphipathic protein, inasmuch as its six charged groups are segregated from hydrophobic surface elements. Phasing methods used here will also apply elsewhere.Bijvoet's observation 1 that anomalous scattering might aid in solving the phase problem has had many implications 2 , including suggestions that departures from Friedel's law of diffraction symmetry might suffice to determine directly the atomic structures of crystals containing heavy atoms [3][4][5] . Anomalous-scattering methods have also been widely used in protein crystallography [6][7][8][9][10][11] , particularly as an adjunct to isomorphous-replacement phasing. We have now combined elements from these two traditions to solve the structure of crambin by exploiting the anomalous scattering of sulphur atoms at a single wavelength (1.54 Å of CuKα) far removed from the absorption edge of sulphur (5.02 Å).Crambin is a small protein found in the embryonic tissue (cotyledons and hypocotyledons) of seeds from Crambe abyssinica, a relative of mustard and rape commonly known as Abyssinian cabbage. It is hydrophobic in that organic solvents are required to solubilize it, Van Etten et al. 12 characterized crambin after noticing that crystals formed during evaporation of an aqueous acetone extract of defatted seed meal. The remarkable crystalline order observed by Teeter for this protein (strong diffraction from spacings <0.88 Å) 13 correlates well with the unprecedented structural stability seen in solution by Llinás et al. 14 using NMR spectroscopy. The function of crambin is not known. However, the recently completed chemical sequence 15 reveals an unmistakable homology with the plant toxins purothionin 16 and viscotoxin 17 . Crambin itself is not toxic when fed to rats 18 .We set out to solve the crystal structure of crambin in view of its exceptional potential for providing detailed structural information and in the hope of shedding light on the function of this hydrophobic protein. When our efforts to prepare heavy-atom derivatives failed, we explored alternatives to isomorphous replacement for phase determination. Our experience HHS Public Access Author Manuscript Author ManuscriptAuthor ManuscriptAuthor Manuscript in using anomalous scattering to locate the iron atoms in myohaemerythrin 19 led us to contemplate an attempt based on the six sulphur atoms in crambin. An estimate of the magnitude of Bijvoet differences to be expected from crambin gave a value about half that found with myohaemerythrin and showed that sulphur scattering would account for 98% of the expected anomalous signal. The expected contribution of the sulphur partial structure to the protein diffraction pattern proved to be 29% (〈|F ...

Refinement of the diffraction data at 2.5Aresolution from orthorhombic crystals of yeast tRNAPhe has poced to t oint where spermine and magnesium ions can elocated in the d rence electron density map. Two spermine molecules are found: one is located in the major groove at one end of the anticodon stem; the other is near the variable loop and curls around phosphate 10 in a region where the polynucleotide chain takes a sharp turn. Four distinct magnesium ions have been identified: one in the anticodon loop, two in the D loop, and one coordinated with phosphates 8, 9, 11, and 12, where the polynucleotide chain is coiled. The conformation of the anticodon stem and loop is stabilized by the cations at the end of the molecule. The positions of these ions may be related to aspects of the biological activity of tRNA. The spermine and magnesium ions appear to be important in maintaining the overall folding of the tRNA molecule. The polyamines are widely distributed in biological systems. Because of their polycationic nature, they bind to the highly negative nucleic acids (1, 2). They have profound effects on protein synthesis in general and on tRNA folding in particular (3,4). The crystal structure analysis of yeast tRNAPhe has been made possible by the observation that the addition of spermine to yeast tRNAPhe produces crystals that yield diffraction patterns of nearly 2-A resolution (5, 6). In contrast, virtually all other tRNA crystals have somewhat disordered crystal lattices (7). The ordered yeast tRNAPhe lattice has made possible the elucidation of the structure of this molecule in both orthorhombic (8) and monoclinic crystal forms (9, 10). We have continued to study the structural details of yeast tRNAPhe in the orthorhombic lattice by the use of refinement procedures. The present refinement of the structure has produced a very good agreement between the observed and calculated data. Consequently, from the differences between the calculated and observed diffraction data, we can visualize the remaining components in the electron density map. This has allowed us to identify portions of electron density that we have interpreted as belonging to spermine and magnesium ions as well as some probable water sites. Two spermine molecules and four magnesium ions have been identified, and several additional tentative sites have been observed. The positions of both the spermine and magnesium in different parts of the tRNA molecule have lead to the conclusion that these cations both play a significant role in stabilizing the three-dimensional folding of the molecule. These observations suggest possible roles for these ions in modifying the biological activity of tRNA molecules during protein synthesis. METHODSRefinement of the yeast tRNAPhe structure had been previously accomplished by a real space refinement procedure in which rigid constraints are applied to the bond angles and bond lengths 64 of the molecular model being refined (11). This procedure produced a reasonable model with a residual error (R value) of 28% a...

The water structure has been analyzed for a model of the protein crambin refined against 0.945-x-ray diffraction data. Crystals contain 32% solvent by volume, and 77% of the solvent molecules have been located-i.e., 2 ethanol molecules and 64 water molecules with 10-14 alternate positions. Many water oxygen atoms found form chains between polar groups on the surface of the protein. However, a cluster of pentagonal arrays made up of 16 water molecules sits at a hydrophobic, intermolecular cleft and forms a cap around the methyl group of leucine-18. Several waters in the cluster are hydrogen-bonded directly to the protein. Additional closed circular arrays, which include both protein atoms and other water oxygen atoms, form next to the central cluster. This water array stretches in the b lattice direction between groups of three ionic side chains.Water is the most abundant molecule in living systems and plays an important role in their structure and function. From crystal structures of proteins (1-3), the following picture of tightly bound internal and surface water molecules has emerged (4, 5). First, internal water is found singly (6), in clusters (7), or bound to metal ions (8, 9). It either may stabilize the protein structure by connecting charged or polar groups, or both, or may serve a catalytic function (10). Second, surface water links polar groups at the intra-and intermolecular protein surface; 2-3 times more water hydrogen bonds are made to main-chain -CO groups than to -NH groups (4, 5) in both crystal structures and simulations, reflecting in part the greater capacity of -CO to form hydrogen bonds. Many surface waters bind at turn regions (11). Here, fewer secondary-structure, backbone hydrogen bonds are formed, and the side chains are often hydrophilic (12). Little if any ordered surtace water has been reported at nonpolar side chains, perhaps because such water may be mobile as inferred from computer simulations (13). Finally, less ordered water (about 30% of that in the first shell) is seen in the second shell (14).Water-protein interactions may be important for protein folding (4). Klotz proposed that hydrophobic residues would organize surface water into five-membered ring arrays analogous to the water clathrate (cage) hydrate structures (15). However, protein crystal structures reveal that many hydrophobic groups fold instead into the protein interior away from water, as suggested by Kauzmann (16). Nevertheless, 40-50%o are found on the protein surface in contact with solvent. The folded structure may be a balance between removal of hydrophobic groups from water to form van der Waals attractive contacts and the penalty for burial of polar groups which might not be able to form hydrogen bonds. The arrangement of water at surface hydrophobic sites is not known. If five-membered ring structures were formed (15) and were not hydrogen-bonded to the protein, they might become disordered or vibrate strongly. Such waters would contribute little scattering to an x-ray experiment.Crystals of the h...

The charge density distribution of a protein has been refined experimentally. Diffraction data for a crambin crystal were measured to ultra-high resolution (0.54 Å) at low temperature by using shortwavelength synchrotron radiation. The crystal structure was refined with a model for charged, nonspherical, multipolar atoms to accurately describe the molecular electron density distribution. The refined parameters agree within 25% with our transferable electron density library derived from accurate single crystal diffraction analyses of several amino acids and small peptides. The resulting electron density maps of redistributed valence electrons (deformation maps) compare quantitatively well with a high-level quantum mechanical calculation performed on a monopeptide. This study provides validation for experimentally derived parameters and a window into charge density analysis of biological macromolecules.T he electronic charge density distribution of a molecule carries information (1) that determines its intermolecular interactions. For example, the charge distribution of an enzyme complements that of the substrate it recognizes and binds. The electrostatic potential and electric moments derivable from the charge density (1-3) provide maps that can guide the design of molecules for specified interactions. Furthermore, powerful insights into the nature and strength of hydrogen bonding and ionic interactions result from analysis of the electron density gradient and Laplacian (4-6). Extension of such analyses to proteins would permit a unique understanding of the driving forces between biomolecules as well as the subtleties of enzymatic reactions (7).Experimental electron density distributions are obtained by analysis of single-crystal x-ray diffraction data measured to ultra-high resolution, typically to a diffraction resolution limit d min Ϸ 0.5 Å (1,8,9). The crystallographic studies usually map and analyze the deformation density, which is the difference between the actual electron density of the molecule and the density calculated for the promolecule, a molecular superposition of spherical, neutral, i.e., free, atoms. The deformation density thus reveals the redistribution of valence electron density caused by chemical bonding and intermolecular interactions and also is used to calibrate theoretical electron density calculations (10). However, a difficulty in crystallography is the separation of the anisotropic atomic mean-square displacements from the static molecular electron distribution (11). Proper experimental deconvolution requires very accurate diffraction data to ultrahigh resolution. Thus, charge density studies have so far been limited to small-unit-cell crystals, and proteins still await study.We have shown (12, 13) that effective thermal displacement deconvolution and meaningful deformation density distributions can be achieved for larger structures at lower resolution by transferring average experimental electron density parameters. We have built a database of such parameters derived from ultra-high ...