title: Application of DEN refinement and automated model building to a difficult case of molecular-replacement phasing: the structure of a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum.
abstract: Phasing by molecular replacement remains difficult for targets that are far from the search model or in situations where the crystal diffracts only weakly or to low resolution. Here, the process of determining and refining the structure of Cgl1109, a putative succinyl-diaminopimelate desuccinylase from Corynebacterium glutamicum, at ∼3 Å resolution is described using a combination of homology modeling with MODELLER, molecular-replacement phasing with Phaser, deformable elastic network (DEN) refinement and automated model building using AutoBuild in a semi-automated fashion, followed by final refinement cycles with phenix.refine and Coot. This difficult molecular-replacement case illustrates the power of including DEN restraints derived from a starting model to guide the movements of the model during refinement. The resulting improved model phases provide better starting points for automated model building and produce more significant difference peaks in anomalous difference Fourier maps to locate anomalous scatterers than does standard refinement. This example also illustrates a current limitation of automated procedures that require manual adjustment of local sequence misalignments between the homology model and the target sequence.
authors.affiliation: Department of Molecular and Cellular Physiology, Stanford University, USA. brunger@stanford.edu
authors.forename: Axel T
authors.initials: AT
authors.lastname: Brunger
authors.forename: Debanu
authors.initials: D
authors.lastname: Das
authors.forename: Ashley M
authors.initials: AM
authors.lastname: Deacon
authors.forename: Joanna
authors.initials: J
authors.lastname: Grant
authors.forename: Thomas C
authors.initials: TC
authors.lastname: Terwilliger
authors.forename: Randy J
authors.initials: RJ
authors.lastname: Read
authors.forename: Paul D
authors.initials: PD
authors.lastname: Adams
authors.forename: Michael
authors.initials: M
authors.lastname: Levitt
authors.forename: Gunnar F
authors.initials: GF
authors.lastname: Schröder
mesh_terms: D000581:Amidohydrolases / Q000032:analysis* / Q000737:chemistry; D000595:Amino Acid Sequence; D057205:Automation, Laboratory / Q000379:methods*; D048230:Corynebacterium glutamicum / Q000201:enzymology*; D018360:Crystallography, X-Ray / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016415:Sequence Alignment; D012984:Software; D040681:Structural Homology, Protein
chemical_list: D000581:Amidohydrolases; C054685:N-succinyl-l,l-diaminopimelic acid desuccinylase