X-ray transparent microfluidic platforms for membrane protein crystallization with microseeds

Jeremy M. Schieferstein, Ashtamurthy S. Pawate, Michael J. Varel, Sudipto Guha, Ieva Astrauskaite, Robert B. Gennis, Paul J.A. Kenis

Research output: Contribution to journalArticle

Abstract

Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating structure-function relationships. Microseeding, the process of transferring sub-microscopic crystal nuclei from initial screens into new crystallization experiments, is an effective, yet underutilized approach to grow crystals suitable for X-ray crystallography. Here, we report simplified methods for crystallization of membrane proteins that utilize microseeding in X-ray transparent microfluidic chips. First, a microfluidic method for introduction of microseed dilutions into metastable crystallization experiments is demonstrated for photoactive yellow protein and cytochrome bo3 oxidase. As microseed concentration decreased, the number of crystals decreased while the average size increased. Second, we demonstrate a microfluidic chip for microseed screening, where many crystallization conditions were formulated on-chip prior to mixing with microseeds. Crystallization composition, crystal size, and diffraction data were collected and mapped on phase diagrams, which revealed that crystals of similar diffraction quality and size typically grow in distinct regions of the phase diagram.

Original languageEnglish (US)
Pages (from-to)944-954
Number of pages11
JournalLab on a chip
Volume18
Issue number6
DOIs
StatePublished - Mar 21 2018

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ASJC Scopus subject areas

  • Bioengineering
  • Biochemistry
  • Chemistry(all)
  • Biomedical Engineering

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