Toward structure determination using membrane-protein nanocrystals and microcrystals

Mark S. Hunter, Petra Fromme

Research output: Contribution to journalArticle

44 Citations (Scopus)

Abstract

Membrane proteins are very important for all living cells, being involved in respiration, photosynthesis, cellular uptake and signal transduction, amongst other vital functions. However, less than 300 unique membrane protein structures have been determined to date, often due to difficulties associated with the growth of sufficiently large and well-ordered crystals. This work has been focused on showing the first proof of concept for using membrane protein nanocrystals and microcrystals for high-resolution structure determination. Upon determining that crystals of the membrane protein Photosystem I, which is the largest and most complex membrane protein crystallized to date, exist with only 100. unit cells with sizes of less than 200. nm on an edge, work was done to develop a technique that could exploit the growth of the Photosystem I nanocrystals and microcrystals. Femtosecond X-ray protein nanocrystallography was developed for use at the first high-energy X-ray free electron laser, the LCLS at SLAC National Accelerator Laboratory, in which a liquid jet brought fully-hydrated Photosystem I nanocrystals into the interaction region of the pulsed X-ray source. Diffraction patterns were recorded from millions of individual PSI nanocrystals and data from thousands of different, randomly oriented crystallites were integrated using Monte Carlo integration of the peak intensities. The short pulses (~70. fs) provided by the LCLS allowed the possibility to collect the diffraction data before the onset of radiation damage, exploiting the diffract-before-destroy principle. During the initial experiments at the AMO beamline using 6.9-Å wavelength, Bragg peaks were recorded to 8.5-Å resolution, and an electron-density map was determined that did not show any effects of X-ray-induced radiation damage [94]. Many additional techniques still need to be developed to explore the femtosecond nanocrystallography technique for experimental phasing and time-resolved X-ray crystallography experiments. The first proof-of-principle results for the femtosecond nanocrystallography technique indicate the incredible potential of the technique to offer a new route to the structure determination of membrane proteins.

Original languageEnglish (US)
Pages (from-to)387-404
Number of pages18
JournalMethods
Volume55
Issue number4
DOIs
StatePublished - Dec 2011

Fingerprint

Microcrystals
Nanoparticles
Nanocrystals
Membrane Proteins
Photosystem I Protein Complex
X-Rays
Radiation damage
X rays
Electrons
Radiation
Cell Respiration
X ray lasers
Signal transduction
Crystals
Photosynthesis
X ray crystallography
Free electron lasers
X Ray Crystallography
Beam plasma interactions
Growth

Keywords

  • Femtosecond nanocrystallography
  • Membrane proteins
  • Protein nanocrystals
  • Structure determination
  • X-ray crystallography
  • XFEL

ASJC Scopus subject areas

  • Molecular Biology
  • Biochemistry, Genetics and Molecular Biology(all)

Cite this

Toward structure determination using membrane-protein nanocrystals and microcrystals. / Hunter, Mark S.; Fromme, Petra.

In: Methods, Vol. 55, No. 4, 12.2011, p. 387-404.

Research output: Contribution to journalArticle

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