With over 60,000 protein structures available in the Protein Data Bank, it is frequently possible use one of them to obtain starting phase information and to solve new crystal structures. Molecular replacement1–4 procedures, which search for placements of a starting model within the crystallographic unit cell that best account for the measured diffraction amplitudes, followed by automatic chain tracing methods5–8, have allowed the rapid solution of large numbers of protein structures. Despite extensive work9–14, molecular replacement or the subsequent rebuilding usually fail with more divergent starting models based on remote homologues with less than 30% sequence identity. Here we show that this limitation can be substantially reduced by combining algorithms for protein structure modeling with those developed for crystallographic structure determination. An approach integrating Rosetta structure modeling with Autobuild chain tracing yielded high-resolution structures for 8 of 13 X-ray diffraction datasets that could not be solved in the laboratories of expert crystallographers and that remained unsolved after application of an extensive array of alternative approaches. We estimate the new method should allow rapid structure determination without experimental phase information for over half the cases where current methods fail, given diffraction datasets of better than 3.2Å resolution, four or fewer copies in the asymmetric unit, and the availability of structures of homologous proteins with >20% sequence identity.

Gs (Geobacter sulfurreducens) can transfer electrons to the exterior of its cells, a property that makes it a preferential candidate for the development of biotechnological applications. Its genome encodes over 100 cytochromes and, despite their abundance and key functional roles, to date there is no structural information for these proteins in solution. The trihaem cytochrome PpcA might have a crucial role in the conversion of electronic energy into protonmotive force, a fundamental step for ATP synthesis in the presence of extracellular electron acceptors. In the present study, 15N-labelled PpcA was produced and NMR spectroscopy was used to determine its solution structure in the fully reduced state, its backbone dynamics and the pH-dependent conformational changes. The structure obtained is well defined, with an average pairwise rmsd (root mean square deviation) of 0.25 Å (1 Å=0.1 nm) for the backbone atoms and 0.99 Å for all heavy atoms, and constitutes the first solution structure of a Gs cytochrome. The redox-Bohr centre responsible for controlling the electron/proton transfer was identified, as well as the putative interacting regions between PpcA and its redox partners. The solution structure of PpcA will constitute the foundation for studies aimed at mapping out in detail these interacting regions.

The structure of a cytochrome c(7) (PpcA) from Geobacter sulfurreducens was determined by X-ray diffraction at 1.45 A resolution; the R factor is 18.2%. The protein contains a three-heme core that is surrounded by 71 amino acid residues. An unusual feature of this cytochrome is that it has 17 lysine residues, but only nine hydrophobic residues that are larger than alanine. The details of the structure are described and compared with those of cytochrome c(7) from Desulfuromonas acetoxidans and with cytochromes c(3). The two cytochrome c(7) molecules have sequences that are 46% identical, but the arrangements of the hemes in the two structures differ; the rms deviation of all alpha-carbons is 2.5 A. These cytochromes can reduce various metal ions. The reduction site of the chromate ion in D. acetoxidans is occupied by a sulfate ion in the crystal structure of PpcA. We identified four additional homologues of cytochrome c(7) in the G. sulfurreducens genome and three polymers of c(7)-type domains. Of the polymers, two have four repeats and one has nine repeats. On the basis of sequence alignments, one of the hemes in each of the cytochrome c(7)-type domains does not have the bis-histidine coordination. The packing of the molecules in the crystal structure of PpcA suggests that the polymers have an elongated conformation and might form a "nanowire".

The most common form of systemic amyloidosis originates from antibody light chains. The large number of amino acid variations that distinguish amyloidogenic from nonamyloidogenic light chain proteins has impeded our understanding of the structural basis of light-chain fibril formation. Moreover, even among the subset of human light chains that are amyloidogenic, many primary structure differences are found. We compared the thermodynamic stabilities of two recombinant k4 light-chain variable domains~V L s! derived from amyloidogenic light chains with a V L from a benign light chain. The amyloidogenic V L s were significantly less stable than the benign V L . Furthermore, only the amyloidogenic V L s formed fibrils under native conditions in an in vitro fibril formation assay. We used site-directed mutagenesis to examine the consequences of individual amino acid substitutions found in the amyloidogenic V L s on stability and fibril formation capability. Both stabilizing and destabilizing mutations were found; however, only destabilizing mutations induced fibril formation in vitro. We found that fibril formation by the benign V L could be induced by low concentrations of a denaturant. This indicates that there are no structural or sequence-specific features of the benign V L that are incompatible with fibril formation, other than its greater stability. These studies demonstrate that the V L b-domain structure is vulnerable to destabilizing mutations at a number of sites, including complementarity determining regions~CDRs!, and that loss of variable domain stability is a major driving force in fibril formation.

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