title: Initiating heavy-atom-based phasing by multi-dimensional molecular replacement.
abstract: To obtain an electron-density map from a macromolecular crystal the phase problem needs to be solved, which often involves the use of heavy-atom derivative crystals and concomitant heavy-atom substructure determination. This is typically performed by dual-space methods, direct methods or Patterson-based approaches, which however may fail when only poorly diffracting derivative crystals are available. This is often the case for, for example, membrane proteins. Here, an approach for heavy-atom site identification based on a molecular-replacement parameter matrix (MRPM) is presented. It involves an n-dimensional search to test a wide spectrum of molecular-replacement parameters, such as different data sets and search models with different conformations. Results are scored by the ability to identify heavy-atom positions from anomalous difference Fourier maps. The strategy was successfully applied in the determination of a membrane-protein structure, the copper-transporting P-type ATPase CopA, when other methods had failed to determine the heavy-atom substructure. MRPM is well suited to proteins undergoing large conformational changes where multiple search models should be considered, and it enables the identification of weak but correct molecular-replacement solutions with maximum contrast to prime experimental phasing efforts.
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark.
authors.forename: Bjørn Panyella
authors.initials: BP
authors.lastname: Pedersen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark.
authors.forename: Pontus
authors.initials: P
authors.lastname: Gourdon
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark.
authors.forename: Xiangyu
authors.initials: X
authors.lastname: Liu
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark.
authors.forename: Jesper Lykkegaard
authors.initials: JL
authors.lastname: Karlsen
authors.affiliation: Centre for Membrane Pumps in Cells and Disease, Danish National Research Foundation, Department of Molecular Biology and Genetics, Aarhus University, Gustav Wieds Vej 10C, DK-8000 Aarhus, Denmark.
authors.forename: Poul
authors.initials: P
authors.lastname: Nissen
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D003460:Crystallization; D018360:Crystallography, X-Ray / Q000379:methods; D004583:Electrons; D016952:Legionella pneumophila / Q000737:chemistry*; D011487:Protein Conformation; D012984:Software
chemical_list: D001426:Bacterial Proteins; C071263:CopA protein, Bacteria
keywords: experimental phasing; heavy-atom substructure; molecular replacement