title: Solving the RNA polymerase I structural puzzle.
abstract: Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: María
authors.initials: M
authors.lastname: Moreno-Morcillo
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Nicholas M I
authors.initials: NM
authors.lastname: Taylor
authors.affiliation: Department of Structural Chemistry, Georg-August-University, Tammannstrasse 4, 37077 Göttingen, Germany.
authors.forename: Tim
authors.initials: T
authors.lastname: Gruene
authors.affiliation: SOLEIL Synchrotron, L'Orme de Merisiers, Saint Aubin, Gif-sur-Yvette, France.
authors.forename: Pierre
authors.initials: P
authors.lastname: Legrand
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Umar J
authors.initials: UJ
authors.lastname: Rashid
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Federico M
authors.initials: FM
authors.lastname: Ruiz
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Steuerwald
authors.affiliation: Structural and Computational Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.
authors.forename: Christoph W
authors.initials: CW
authors.lastname: Müller
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, 28040 Madrid, Spain.
authors.forename: Carlos
authors.initials: C
authors.lastname: Fernández-Tornero
mesh_terms: D020134:Catalytic Domain; D003460:Crystallization; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism; D008958:Models, Molecular; D011487:Protein Conformation; D055503:Protein Multimerization; D012318:RNA Polymerase I / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
chemical_list: D004247:DNA; D012318:RNA Polymerase I
keywords: RNA polymerase I; low-resolution structure determination; multi-subunit complexes; transcription