Electron crystallography of bacteriorhodopsin with millisecond time resolution

Sriram Subramaniam, Richard Henderson

Research output: Contribution to journalArticlepeer-review

Abstract

The goal of time-resolved crystallographic experiments is to capture dynamic 'snapshots' of molecules at different stages of a reaction pathway. In recent work, we have developed approaches to determine determined light- induced conformational changes in the proton pump bacteriorhodopsin by electron crystallographic analysis of two-dimensional protein crystals. For this purpose, crystals of bacteriorhodopsin were deposited on an electron microscopic grid and were plunge-frozen in liquid ethane at a variety of times after illumination. Electron diffraction patterns were recorded either from unilluminated crystals or from crystals frozen as early as 1 ms after illumination and used to construct projection difference Fourier maps at 3.5- Å resolution to define light-driven changes in protein conformation. As demonstrated here, the data are of a sufficiently high quality that structure factors obtained from a single electron diffraction pattern of a plunge- frozen bacteriorhodopsin crystal are adequate to obtain an interpretable difference Fourier map. These difference maps report on the nature and extent of light-induced conformational changes in the photocycle and have provided incisive tools for understanding the molecular mechanism of proton transport by bacteriorhodopsin.

Original languageEnglish (US)
Pages (from-to)19-25
Number of pages7
JournalJournal of Structural Biology
Volume128
Issue number1
DOIs
StatePublished - Dec 1 1999
Externally publishedYes

Keywords

  • Electron diffraction
  • High-resolution electron microscopy
  • Intermediates
  • Membrane protein
  • Photocycle
  • Two-dimensional crystals

ASJC Scopus subject areas

  • Structural Biology

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