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Author Notes:

Correspondence e-mail: eric.ortlund@emory.edu

We thank N. T. Seyfried in the Center for Neurodegenerative Diseases’ Proteomics Core at Emory University for his help in acquiring the MS data for protein identification.

Data were collected on the Southeast Regional Collaborative Access Team (SER-CAT) 22-ID beamline at the Advanced Photon Source, Argonne National Laboratory. Supporting institutions may be found at http://www.ser-cat.org/members.html.

Subject:

Research Funding:

The research reported in this publication was supported by the National Institute of Diabetes and Digestive and Kidney Diseases of the National Institutes of Health under award No. RO1DK095750 to EAO and by AHA predoctoral grant 12PRE12060583 and an Emory–National Institute of Environmental Health Sciences Graduate and Postdoctoral Training in Toxicology grant (T32ES012870) to PMM.

The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

Use of the Advanced Photon Source was supported by the US Department of Energy, Office of Science, Office of Basic Energy Sciences under Contract No. W-31-109-Eng-38.

Keywords:

  • glycerol dehydrogenase
  • Serratia

Journal Title:

Acta Crystallographica Section F: Structural Biology Communications

Volume:

Volume 70, Number 2

Publisher:

, Pages 166-172

Type of Work:

Article | Final Publisher PDF

Abstract:

The 1.90 Å resolution X-ray crystal structure of glycerol dehydrogenase derived from contaminating bacteria present during routine Escherichia coli protein expression is presented. This off-target enzyme showed intrinsic affinity for Ni2+-Sepharose, migrated at the expected molecular mass for the target protein during gel filtration and was crystallized before it was realised that contamination had occurred. In this study, it is shown that liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) can efficiently identify the protein composition of crystals in a crystallization experiment as part of a structure-determination pipeline for an unknown protein. The high-resolution X-ray data enabled sequencing directly from the electron-density maps, allowing the source of contamination to be placed within the Serratia genus. Incorporating additional protein-identity checks, such as tandem LC-MS/MS, earlier in the protein expression, purification and crystallization workflow may have prevented the unintentional structure determination of this metabolic enzyme, which represents the first enterobacterial glycerol dehydrogenase reported to date.

Copyright information:

© International Union of Crystallography 2014

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