Ab initio structure solution of a proteolytic fragment using ARCIMBOLDO.
Crystal structure determination requires solving the phase problem. This can be accomplished using ab initio direct methods for small molecules and macromolecules at resolutions higher than 1.2 Å, whereas macromolecular structure determination at lower resolution requires either molecular replacement using a homologous structure or experimental phases using a derivative such as covalent labeling (for example selenomethionine or mercury derivatization) or heavy-atom soaking (for example iodide ions). Here, a case is presented in which crystals were obtained from a 30.8 kDa protein sample and yielded a 1.6 Å resolution data set with a unit cell that could accommodate approximately 8 kDa of protein. Thus, it was unclear what had been crystallized. Molecular replacement with pieces of homologous proteins and attempts at iodide ion soaking failed to yield a solution. The crystals could not be reproduced. Sequence-independent molecular replacement using the structures available in the Protein Data Bank also failed to yield a solution. Ultimately, ab initio structure solution proved successful using the program ARCIMBOLDO, which identified two α-helical elements and yielded interpretable maps. The structure was the C-terminal dimerization domain of the intended target from Mycobacterium smegmatis. This structure is presented as a user-friendly test case in which an unknown protein fragment could be determined using ARCIMBOLDO.
['Abendroth|Jan|J|0000-0002-8229-3243', 'Sankaran|Banumathi|B|', 'Myler|Peter J|PJ|0000-0002-0056-0513', 'Lorimer|Donald D|DD|', 'Edwards|Thomas E|TE|0000-0002-0474-8003']
D000595:Amino Acid Sequence D001426:Bacterial Proteins D001665:Binding Sites D003001:Cloning, Molecular D018360:Crystallography, X-Ray D030562:Databases, Protein D004926:Escherichia coli D020558:GTP Phosphohydrolases D015870:Gene Expression D005822:Genetic Vectors D006153:Guanosine Diphosphate D008958:Models, Molecular D020102:Mycobacterium smegmatis D010446:Peptide Fragments D011485:Protein Binding D000072756:Protein Conformation, alpha-Helical D000072757:Protein Conformation, beta-Strand D054730:Protein Interaction Domains and Motifs D055503:Protein Multimerization D059748:Proteolysis D011994:Recombinant Proteins D012984:Software
proteolytic fragments ARCIMBOLDO Mycobacterium smegmatis ab initio structure determination infectious diseases structural genomics