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X-ray Transparent Microfluidic Platforms for Membrane Protein Crystallization with Microseeds

Abstract

Crystallization of membrane proteins is a critical step for uncovering atomic resolution 3-D structures and elucidating the structure-function relationship. 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 demonstarte 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 revelaed that crystals of similar diffraction quality and size typically grow in distinct regions of the phase diagram.

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Publication details

The article was received on 21 Oct 2017, accepted on 08 Feb 2018 and first published on 08 Feb 2018


Article type: Paper
DOI: 10.1039/C7LC01141E
Citation: Lab Chip, 2018, Accepted Manuscript
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    X-ray Transparent Microfluidic Platforms for Membrane Protein Crystallization with Microseeds

    J. Schieferstein, A. Pawate, M. Varel, S. Guha, L. Astrauskaite, R. B. Gennis and P. Kenis, Lab Chip, 2018, Accepted Manuscript , DOI: 10.1039/C7LC01141E

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