title: Structure determination of the minimal complex between Tfb5 and Tfb2, two subunits of the yeast transcription/DNA-repair factor TFIIH: a retrospective study.
abstract: Tfb5 interacts with the Tfb2 subunit of the general transcription factor TFIIH to ensure efficient nucleotide-excision repair in eukaryotes. The crystal structure of the complex between Tfb5 and the C-terminal region of Tfb2 (Tfb2C) from Saccharomyces cerevisiae has recently been reported. Here, the structure-determination process is described as a case study. Although crystals were obtained readily, it was not possible to determine experimental phases from a first crystal form (Tfb2(412-513)-Tfb5(2-72)) that diffracted to 2.6 A resolution. Shortening of the Tfb2C from its N-terminus was decisive and modified the crystal packing, leading to a second crystal form (Tfb2(435-513)-Tfb5(2-72)). These crystals diffracted to 1.7 A resolution with excellent mosaicity and allowed structure determination by conventional approaches using heavy atoms. The refined structure from the second crystal form was used to solve the structure of the first crystal form by molecular replacement. Comparison of the two structures revealed that the N-terminal region of Tfb2C and (to a lesser extent) the C-terminal region of Tfb5 contributed to the crystal packing. A detailed analysis illustrates how variation in domain boundaries influences crystal packing and quality.
authors.affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/UDS, BP 163, Illkirch, France.
authors.forename: Denis E
authors.initials: DE
authors.lastname: Kainov
authors.forename: Vincent
authors.initials: V
authors.lastname: Cura
authors.forename: Marc
authors.initials: M
authors.lastname: Vitorino
authors.forename: Helène
authors.initials: H
authors.lastname: Nierengarten
authors.forename: Pierre
authors.initials: P
authors.lastname: Poussin
authors.forename: Bruno
authors.initials: B
authors.lastname: Kieffer
authors.forename: Jean
authors.initials: J
authors.lastname: Cavarelli
authors.forename: Arnaud
authors.initials: A
authors.lastname: Poterszman
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018360:Crystallography, X-Ray; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D021122:Protein Subunits / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000737:chemistry* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000378:metabolism; D016415:Sequence Alignment; D051758:Transcription Factor TFIIH / Q000737:chemistry* / Q000378:metabolism
chemical_list: D021122:Protein Subunits; D029701:Saccharomyces cerevisiae Proteins; C517875:Tfb2 protein, S cerevisiae; C517874:Tfb5 protein, S cerevisiae; D051758:Transcription Factor TFIIH