A high-throughput strategy to screen 2D crystallization trials of membrane proteins

Martin Vink, KD Derr, James Love, David L. Stokes, Iban Ubarretxena-Belandia

Research output: Contribution to journalArticle

35 Scopus citations

Abstract

Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography.

Original languageEnglish (US)
Pages (from-to)295-304
Number of pages10
JournalJournal of Structural Biology
Volume160
Issue number3
DOIs
Publication statusPublished - Dec 1 2007

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Keywords

  • 96-Well format
  • Crystallization block
  • Dialysis block
  • Electron crystallography
  • High-throughput screening
  • Membrane protein reconstitution
  • Membrane proteins
  • Negative staining
  • Two-dimensional (2D) crystals

ASJC Scopus subject areas

  • Structural Biology

Cite this

Vink, M., Derr, KD., Love, J., Stokes, D. L., & Ubarretxena-Belandia, I. (2007). A high-throughput strategy to screen 2D crystallization trials of membrane proteins. Journal of Structural Biology, 160(3), 295-304. https://doi.org/10.1016/j.jsb.2007.09.003