TY - JOUR

T1 - Solving the RNA polymerase I structural puzzle

AU - Moreno-Morcillo, María

AU - Taylor, Nicholas M I

AU - Gruene, Tim

AU - Legrand, Pierre

AU - Rashid, Umar J

AU - Ruiz, Federico M

AU - Steuerwald, Ulrich

AU - Müller, Christoph W

AU - Fernández-Tornero, Carlos

PY - 2014

Y1 - 2014

N2 - 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.

AB - 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.

KW - Catalytic Domain

KW - Crystallization

KW - Crystallography, X-Ray

KW - DNA/metabolism

KW - Models, Molecular

KW - Protein Conformation

KW - Protein Multimerization

KW - RNA Polymerase I/chemistry

U2 - 10.1107/S1399004714015788

DO - 10.1107/S1399004714015788

M3 - Journal article

C2 - 25286842

VL - 70

SP - 2570

EP - 2582

JO - Acta Crystallographica Section D: Structural Biology

JF - Acta Crystallographica Section D: Structural Biology

SN - 2059-7983

IS - Pt 10

ER -