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 language | English (US) |
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Pages (from-to) | 19-25 |
Number of pages | 7 |
Journal | Journal of Structural Biology |
Volume | 128 |
Issue number | 1 |
DOIs | |
State | Published - Dec 1 1999 |
Externally published | Yes |
Keywords
- Electron diffraction
- High-resolution electron microscopy
- Intermediates
- Membrane protein
- Photocycle
- Two-dimensional crystals
ASJC Scopus subject areas
- Structural Biology