Crystallization of RNA polymerase I subcomplex A14/A43 by iterative prediction, probing and removal of flexible regions.
The removal of flexible protein regions is generally used to promote crystallization, but advanced strategies to quickly remove multiple flexible regions from proteins or protein complexes are lacking. Here, it is shown how a protein heterodimer with multiple flexibilities, the RNA polymerase I subcomplex A14/A43, could be crystallized with the use of an iterative procedure of predicting flexible regions, experimentally testing and improving these predictions and combining deletions of flexible regions in a stepwise manner. This strategy should enable the crystallization of other proteins and subcomplexes with multiple flexibilities, as required for hybrid structure solution of large macromolecular assemblies.
Geiger
Sebastian R
SR
Gene Center Munich, Center for Integrated Protein Science (CIPSM) and Department of Chemistry and Biochemistry, Ludwig-Maximilians-Universität München, Feodor-Lynen-Strasse 25, 81377 Munich, Germany.
Kuhn
Claus D
CD
Leidig
Christoph
C
Renkawitz
Jörg
J
Cramer
Patrick
P
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D019295:Computational Biology; D003460:Crystallization / Q000379:methods*; D018360:Crystallography, X-Ray; D019281:Dimerization; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012318:RNA Polymerase I / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D012319:RNA Polymerase II / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D029701:Saccharomyces cerevisiae Proteins; D012319:RNA Polymerase II; C470553:RPB7 protein, S cerevisiae; D012318:RNA Polymerase I