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Analysis of high-resolution electron diffraction patterns from purple membrane labelled with heavy-atoms
1MRC Laboratory of Molecular Biology, Cambridge, England.
Journal of Molecular Biology
|June 5, 1990
Summary
Determining bacteriorhodopsin structure was difficult due to missing phase data. Heavy-atom labeling shows promise for phasing, but current accuracy is insufficient for reliable maps.
Area of Science:
- Structural biology
- Biophysics
- Crystallography
Background:
- Bacteriorhodopsin structure determination is crucial for understanding membrane protein function.
- Previous efforts were limited by the lack of 3D phase information at high resolution.
- Heavy-atom labeling is a potential method for phasing via multiple isomorphous replacement.
Purpose of the Study:
- To screen heavy-atom compounds for labeling bacteriorhodopsin.
- To evaluate the feasibility of using heavy-atom derivatives for phasing.
- To improve electron diffraction data collection and analysis methods.
Main Methods:
- Screening of approximately 30 heavy-atom compounds.
- Collection of electron diffraction data at -120°C to minimize radiation damage.
- Analysis of difference Fourier maps and intensity merging for phase calculation.
Main Results:
- Two heavy-atom compounds were selected for 3D data collection at 3 Å resolution.
- Native data were collected to 2.7 Å resolution.
- Phasing power was found to be too small for a reliably interpretable map due to data accuracy limitations.
Conclusions:
- Heavy-atom labeling shows potential for bacteriorhodopsin structure determination.
- Current data accuracy and derivative phasing power are insufficient for direct interpretation.
- Heavy-atom derivative data may aid in confirming phases from other methods like electron microscopy.