title: Electron crystallography of bacteriorhodopsin with millisecond time resolution.
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-A 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.
authors.affiliation: Laboratory of Biochemistry, National Cancer Institute, Bethesda, Maryland 20892, USA. sriram@mrc-imb.ac.uk
authors.forename: S
authors.initials: S
authors.lastname: Subramaniam
authors.forename: R
authors.initials: R
authors.lastname: Henderson
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry*; D020285:Cryoelectron Microscopy; D003461:Crystallography / Q000379:methods*; D004583:Electrons; D005583:Fourier Analysis; D019617:Halobacterium salinarum; D008958:Models, Molecular; D011487:Protein Conformation; D017434:Protein Structure, Tertiary
chemical_list: D001436:Bacteriorhodopsins