title: Protein purification and crystallization artifacts: The tale usually not told.
abstract: The misidentification of a protein sample, or contamination of a sample with the wrong protein, may be a potential reason for the non-reproducibility of experiments. This problem may occur in the process of heterologous overexpression and purification of recombinant proteins, as well as purification of proteins from natural sources. If the contaminated or misidentified sample is used for crystallization, in many cases the problem may not be detected until structures are determined. In the case of functional studies, the problem may not be detected for years. Here several procedures that can be successfully used for the identification of crystallized protein contaminants, including: (i) a lattice parameter search against known structures, (ii) sequence or fold identification from partially built models, and (iii) molecular replacement with common contaminants as search templates have been presented. A list of common contaminant structures to be used as alternative search models was provided. These methods were used to identify four cases of purification and crystallization artifacts. This report provides troubleshooting pointers for researchers facing difficulties in phasing or model building.
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Jerzy Haber Institute of Catalysis and Surface Chemistry, Polish Academy of Sciences, Niezapominajek 8, Krakow, 30-239, Poland.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.
authors.forename: Ewa
authors.initials: E
authors.lastname: Niedzialkowska
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.
authors.forename: Olga
authors.initials: O
authors.lastname: Gasiorowska
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.
authors.forename: Katarzyna B
authors.initials: KB
authors.lastname: Handing
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.|Center for Structural Genomics of Infectious Diseases (CSGID), Chicago, Illinois, 60611.
authors.forename: Karolina A
authors.initials: KA
authors.lastname: Majorek
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.
authors.forename: Przemyslaw J
authors.initials: PJ
authors.lastname: Porebski
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.|Center for Structural Genomics of Infectious Diseases (CSGID), Chicago, Illinois, 60611.|New York Structural Genomics Research Consortium (NYSGRC), Bronx, New York, 10461.
authors.forename: Ivan G
authors.initials: IG
authors.lastname: Shabalin
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 6044, Charlottesville, Virginia, 22908.
authors.forename: Ewelina
authors.initials: E
authors.lastname: Zasadzinska
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.
authors.forename: Marcin
authors.initials: M
authors.lastname: Cymborowski
authors.affiliation: Department of Molecular Physiology and Biological Physics, University of Virginia School of Medicine, 1340 Jefferson Park Avenue, Jordan Hall, Room 4223, Charlottesville, Virginia, 22908.|Midwest Center for Structural Genomics (MCSG), Argonne, Illinois, 60439.|Center for Structural Genomics of Infectious Diseases (CSGID), Chicago, Illinois, 60611.|New York Structural Genomics Research Consortium (NYSGRC), Bronx, New York, 10461.
authors.forename: Wladek
authors.initials: W
authors.lastname: Minor
mesh_terms: D000123:Acetyltransferases / Q000737:chemistry / Q000302:isolation & purification; D000818:Animals; D016477:Artifacts; D001426:Bacterial Proteins / Q000737:chemistry / Q000302:isolation & purification; D003460:Crystallization / Q000379:methods*; D012321:DNA-Directed RNA Polymerases / Q000737:chemistry / Q000302:isolation & purification; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry* / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D015203:Reproducibility of Results; D012808:Sigma Factor / Q000737:chemistry / Q000302:isolation & purification; D013211:Staphylococcus aureus / Q000737:chemistry; D000077022:Survivin; D014981:Xenopus / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011506:Proteins; D011994:Recombinant Proteins; D012808:Sigma Factor; D000077022:Survivin; D029867:Xenopus Proteins; C470844:ZinT protein, E coli; C472712:birc5.2 protein, Xenopus; D000123:Acetyltransferases; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases
keywords: YadF (carbonic anhydrase); YodA (metal-binding lipocalin); crystallization artifacts; protein purification artifacts; reproducibility