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The hidden treasure in your data: phasing with unexpected weak anomalous scatterers from routine data sets.
Raghurama P Hegde1, Alexander A Fedorov2, J Michael Sauder3
1Division of Biological Sciences, Poornaprajna Institute of Scientific Research, #4, 16th Cross, Sadashivnagar, Bangalore 560 080, India.
Summary
Fortuitously found calcium atoms in crystallization solutions enabled de novo protein structure determination using X-ray diffraction. This method works even without optimized data collection, offering a new approach for solving complex protein structures.
Area of Science:
- Structural Biology
- X-ray Crystallography
- Biophysics
Background:
- Single-wavelength anomalous dispersion (SAD) typically requires specific experimental conditions and highly redundant data for structure determination.
- Native sulfur atoms or other light elements (Z ≤ 20) are often used, necessitating long-wavelength X-rays for sufficient anomalous signal.
- Standard SAD protocols often overlook the potential anomalous signal from common crystallization additives.
Purpose of the Study:
- To investigate the utility of serendipitously acquired anomalous signals from calcium atoms for de novo protein structure determination.
- To demonstrate that standard X-ray diffraction data, not optimized for anomalous scattering, can drive structure solution.
- To explore the feasibility of using intrinsic sulfur atoms for structure determination under specific data collection conditions.
Main Methods:
- Utilized anomalous signal from surface-bound calcium atoms present in crystallization solutions for two protein structure determinations.
- Collected X-ray diffraction data at wavelengths of 0.98 Å and 1.74 Å, with the latter optimized for sulfur but exploiting calcium's signal.
- Performed computational substructure solution using SHELXD on data with varying completeness and anomalous multiplicity.
Main Results:
- Successful de novo structure determination was achieved for two proteins by exploiting anomalous signals from fortuitously bound calcium atoms.
- Data collected at ~1.0 Å, with a sulfur f'' value of 0.28 electrons, proved sufficient for structure determination from intrinsic sulfur atoms in a strongly diffracting crystal.
- SHELXD successfully generated substructure solutions from high-exposure data with 70% completeness to 3.5 Å resolution and low anomalous multiplicity.
Conclusions:
- Fortuitous anomalous scatterers, such as calcium, in crystallization solutions can be effectively utilized for de novo structure determination.
- This approach bypasses the need for specifically optimized data collection strategies for anomalous signal enhancement.
- Evaluating existing X-ray diffraction datasets for the presence of fortuitous anomalous signals could streamline structure solution and atom assignment.