CHARMM (Chemistry at HARvard Macromolecular Mechanics) is a highly flexible computer program which uses empirical energy functions to model macromolecular systems. The program can read or model build structures, energy minimize them by first-or second-derivative techniques, perform a normal mode or molecular dynamics simulation, and analyze the structural, equilibrium, and dynamic properties determined in these calculations. The operations that CHARMM can perform are described, and some implementation details are given. A set of parameters for the empirical energy function and a sample run are included.

Microbial secondary metabolism constitutes a rich source of antibiotics, chemotherapeutics, insecticides and other high-value chemicals. Genome mining of gene clusters that encode the biosynthetic pathways for these metabolites has become a key methodology for novel compound discovery. In 2011, we introduced antiSMASH, a web server and stand-alone tool for the automatic genomic identification and analysis of biosynthetic gene clusters, available at http://antismash.secondarymetabolites.org. Here, we present version 3.0 of antiSMASH, which has undergone major improvements. A full integration of the recently published ClusterFinder algorithm now allows using this probabilistic algorithm to detect putative gene clusters of unknown types. Also, a new dereplication variant of the ClusterBlast module now identifies similarities of identified clusters to any of 1172 clusters with known end products. At the enzyme level, active sites of key biosynthetic enzymes are now pinpointed through a curated pattern-matching procedure and Enzyme Commission numbers are assigned to functionally classify all enzyme-coding genes. Additionally, chemical structure prediction has been improved by incorporating polyketide reduction states. Finally, in order for users to be able to organize and analyze multiple antiSMASH outputs in a private setting, a new XML output module allows offline editing of antiSMASH annotations within the Geneious software.

A biosynthetic antibody binding site, which incorporated the variable domains of anti-digoxin monoclonal antibody 26-10 in a single polypeptide chain (Mr = 26,354), was produced in Escherichia cofi by protein engineering. This variable region fragment (Fv) analogue comprised the 26-10 heavy-and light-chain variable regions (VH and VL) connected by a 15-amino acid linker to form a single-chain Fv (sFv). The sFv was designed as a prolyl-VH-(linker)-VL sequence of 248 amino acids. A 744-base-pair DNA sequence corresponding to this sFv protein was derived by using an E. colt codon preference, and the sFv gene was assembled starting from synthetic oligonucleotides. The sFv polypeptide was expressed as a fusion protein in E. colt, using a leader derived from the trp LE sequence. The sFv protein was obtained by acid cleavage of the unique Asp-Pro peptide bond engineered at the junction of leader and sFv in the fusion protein [(leader)-Asp-Pro-VH-(linker)-VL]. After isolation and renaturation, folded sFv displayed specificity for digoxin and related cardiac glycosides similar to that of natural 26-10 Fab fragments. Binding between afirmity-purified sFv and digoxin exhibited an association constant [Ka = (3.2 ± 0.9) x 107 M -1] that was about a factor of 6 smaller than that found for 26-10 Fab fragments [K. = (1.9 @ 0.2) x 108 M 'I under the same buffer conditions, consisting of 0.01 M sodium acetate, pH 5.5/0.25 M urea.It is known that antigen binding fragments of antibodies (1,2) can be refolded from denatured states with recovery of their specific binding activity (3)(4)(5)(6). The smallest such fragment that contains a complete binding site is termed Fv, consisting of an Mr 25,000 heterodimer of the VH and VL domains (2, 5-11). Givol and coworkers were the first to prepare an Fv by peptic digestion of murine IgA myeloma MOPC 315 (2). However, subsequent development of general cleavage procedures for Fv isolation has met with limited success (7-11). As a result, the Mr 50,000 Fab (1) has remained the only monovalent binding fragment used routinely in biomedical applications.An Fv analogue was constructed in which both heavy-and light-chain variable domains (VH and VL) were part of a single polypeptide chain. Synthetic genes for the 26-10 anti-digoxin VH and VL regions were designed to permit their connection through a linker segment, as well as other manipulations (12,13 MATERIALS AND METHODSModel Antibody. The digoxin binding site of the IgG2a,K monoclonal antibody 26-10 has been analyzed by MudgettHunter and colleagues (14-16). The 26-10 V region sequences were determined from both protein sequencing (17) (14) and has a well-defined specificity profile (15) (Fig. 1).Gene Synthesis. Design of the 744-base sequence for the synthetic sFv gene was derived from the sFv protein sequence by choosing codons preferred by E. coli (25). Synthetic genes encoding the trp promoter-operator, the modified trp LE leader peptide (MLE), and VH were prepared largely as described (26). The gene encoding VH was assembled from 46...

Using X-ray coordinates of antigen-antibody complexes McPC 603, D1.3, and HyHEL-5, we made semiquantitative estimates of Gibbs free energy changes (delta G) accompanying noncovalent complex formation of the McPC 603 Fv fragment with phosphocholine and the D1.3 or HyHEL-5 Fv fragments with hen egg white lysozyme. Our empirical delta G function, which implicitly incorporates solvent effects, has the following components: hydrophobic force, solvent-modified electrostatics, changes in side-chain conformational entropy, translational/overall rotational entropy changes, and the dilutional (cratic) entropy term. The calculated delta G ranges matched the experimentally determined delta G of McPC 603 and D1.3 complexes and overestimated it (i.e., gave a more negative value) in the case of HyHEL-5. Relative delta G contributions of selected antibody residues, calculated for HyHEL-5 complexes, agreed with those determined independently in site-directed mutagenesis experiments. Analysis of delta G attribution in all three complexes indicated that only a small number of amino acids probably contribute actively to binding energetics. These form a subset of the total antigen-antibody contact surface. In the antibodies, the bottom part of the antigen binding cavity dominated the energetics of binding whereas in lysozyme, the energetically most important residues defined small (2.5-3 nm2) "energetic" epitopes. Thus, a concept of protein antigenicity emerges that involves the active, attractive contributions mediated by the energetic antigenic epitopes and the passive surface complementarity contributed by the surrounding contact area. The D1.3 energetic epitope of lysozyme involved Gly 22, Gly 117, and Gln 121; the HyHEL-5 epitope consisted of Arg 45 and Arg 68. These are also the essential antigenic residues determined experimentally. The above positions belong to the most protruding parts of the lysozyme surface, and their backbones are not exceptionally flexible. Least-squares analysis of six different antibody binding regions indicated that the geometry of the VH-VL interface beta-barrel is well conserved, giving no indication of significant changes in domain-domain contacts upon complex formation.