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The Shake-and-Bake method of structure determination is a new direct methods phasing algorithm based on a minimum-variance, phase invariant residual, which is referred to as the minimal principle. Previously, the algorithm had been applied only to known structures. This algorithm has now been applied to two previously unknown structures that contain 105 and 110 non-hydrogen atoms, respectively. This report focuses on (i) algorithmic and parametric optimizations of Shake-and-Bake and (ii) the determination of two previously unknown structures. Traditional tangent formula phasing techniques were unable to unravel these two new structures.

The crystal structure of the uncomplexed orthorhombic form of gramicidin A has been determined at 120 K and at 0.86 angstrom resolution. The pentadecapeptide crystallizes as a left-handed antiparallel double-stranded helical dimer with 5.6 amino acid residues per turn. The helix has an overall length of 31 angstroms and an average inner channel diameter of 4.80 angstroms. The channel of this crystalline form is void of ions or solvent molecules. The channel diameter varies form a minimum of 3.85 angstroms to a maximum of 5.47 angstroms and contains three pockets where the cross-channel contacts are 5.25 angstroms or greater. The range of variation seen for the φ and ψ torsion angles of the backbone of the helix suggests that these potential ion binding sites can be induced to travel the length of the channel in a peristaltic manner by cooperatively varying these angles. The indole rings of the eight tryptophan residues of the dimer are overlapped in three separate regions on the outer surface of the helix when viewed down the barrel of the channel. This arrangement would permit long-chained lipid molecules to nest parallel to the outer channel surface between these protruding tryptophan regions and act like molecular splines to constrain helical twist deformations of the channel.

Arginine vasopressin consists of a 20-membered, disulfide-linked macrocyclic ring system called pressinoic acid to which is attached a COOH-terminal tripeptide. The molecular conformation of pressinoic acid has been determined from single crystal x-ray diffraction data. The 20-membered macrocyclic ring, stabilized by two intramolecular hydrogen bonds, has a type I β-bend centered on Gln$^{4}$ and Asn$^{5}$ and a highly distorted type II′ bend centered on Phe$^{3}$ and Gln$^{4}$. In vasopressin the Asn$^{5}$ side chain extends away from the macrocyclic ring system and hydrogen bonds to the terminal tripeptide, but in pressinoic acid the Asn$^{5}$ side chain lies over the molecule and forms a strong hydrogen bond to the nitrogen of Tyr$^{2}$. The absence of pressor activity in pressinoic acid may be a result of both the loss of the COOH-terminal tripeptide and the incorrect orientation of the Asn$^{5}$ side chain. Whether this class of hormones has pressor or oxytocic activity is determined by the orientation of the Tyr$^{2}$ side chain, that is, whether it is extended away from or over the ring system, respectively. In pressinoic acid, the Tyr$^{2}$ side chain is in the expected ``pressor conformation,'' that is, extended away from the ring system, and is stabilized through a hydrophobic interaction with the Phe$^{3}$ side chain. Thus, the conformation of the pressinoic acid molecule partly explains the activity of vasopressin-like hormones.

The presence of multiple alpha,alpha-dialkyl amino acids such as alpha-methylalanine (alpha-aminoisobutyric acid, Aib) leads to predominantly helical structures, either with alpha-helical or 3(10)-helical hydrogen bonding patterns. The crystal structure of emerimicin-(1-9) benzyl ester (Ac-Phe-Aib-Aib-Aib-Val-Gly-Leu-Aib-Aib-OBzl) reported here shows essentially pure alpha-helical character, whereas other similar compounds show predominantly 3(10)-helical structures. The factors that govern helical preference include the inherent relative stability of the alpha-helix compared with the 3(10)-helix, the extra hydrogen bond seen with 3(10)-helices, and the enhanced electrostatic dipolar interaction of the 3(10)-helix when packed in a crystalline lattice. The balance of these forces, when combined with the steric requirements of the amino acid side chains, determines the relative stability of the two helical conformations under a given set of experimental conditions.

The crystal structure of the uncomplexed orthorhombic form of gramicidin A has been determined at 120 K and at 0.86 angstrom resolution. The pentadecapeptide crystallizes as a left-handed antiparallel double-stranded helical dimer with 5.6 amino acid residues per turn. The channel diameter varies from a minimum of 3.85 angstroms to a maximum of 5.47 angstroms and contains three pockets where the cross-channel contacts are 5.25 angstroms or greater.

A comparison of the monoclinic and orthorhombic crystal structures of the uncomplexed double-stranded, antiparallel, left-handed β-helix (5.6 amino acid residues per turn) ($\\uparrow\\downarrow\\beta^{5.6}$) conformers of gramicidin A reveals marked differences in the tryptophan side-chain orientations and the degree of helical uniformity of the dimer and in the manner in which these helical dimers associate with one another in the crystal. The helix of the orthorhombic dimer exhibits a regular pattern of bulges and constrictions that appears to be induced by crystal packing forces affecting tryptophan side chains that are aligned parallel to the helix axis. The monoclinic dimer is more uniform than the orthorhombic dimer as a consequence of π stacking interactions between dimers in which orientation of tryptophan side chains is normal to the helix axis to relieve the lateral crystal packing forces that may locally twist and deform the helix. It may be inferred from these observations that lipid interactions may be expected to destabilize the$\\uparrow\\downarrow\\beta^{5.6}$helix when it is inserted into a membrane bilayer.

The presence of multiple α,α-dialkyl amino acids such as α-methylalanine (α-aminoisobutyric acid, Aib) leads to predominantly helical structures, either with α-helical or 310-helical hydrogen bonding patterns. The crystal structure of emerimicin-(1-9) benzyl ester (Ac-Phe-Aib-Aib-Aib-Val-Gly-Leu-Aib-Aib-OBzl) reported here shows essentially pure α-helical character, whereas other similar compounds shows predominantly 310- helical structures. The factors that govern helical preference include the inherent relative stability of the α-helix compared with the 310-helix, the extra hydrogen bond seen with s10-helix when packed in a crystalline lattice. The balance of these forces, when combined with the steric requirements of the amino acid side chains, determines the relative stability of the two helical conformations under a given set of experimental conditions.