Structure determination of the nucleosome core particle by selenium SAD phasing

Area of Science:

• Structural Biology
• Molecular Biology
• Biochemistry

Background:

• The eukaryotic genome is organized into chromatin, with the nucleosome as its fundamental unit.
• Nucleosome structure is crucial for genome regulation but challenging to determine experimentally.
• Molecular replacement is the standard method for nucleosome structure determination via X-ray crystallography, but it has limitations.

Purpose of the Study:

• To develop a simple and widely applicable experimental phasing method for nucleosome structure determination.
• To overcome limitations of molecular replacement for noncanonical nucleosome structures.

Main Methods:

• Incorporation of selenomethionine into histones H3, H2A, and H2B.
• Reconstitution of selenomethionine-labeled histones into nucleosomes.
• Crystallization of reconstituted nucleosomes for X-ray crystallography.
• Structure determination using single-wavelength anomalous diffraction (SAD) phasing.

Main Results:

• Successfully reconstituted selenomethionine-incorporated nucleosomes.
• Determined nucleosome core particle structures containing 12 and 16 selenium atoms.
• Achieved structure resolution of 2.5 Å and 2.4 Å using SAD phasing.
• Demonstrated that nucleosomes can be phased with a relatively small number of selenium atoms.

Conclusions:

• A straightforward experimental phasing method for nucleosome structure determination has been established.
• This method, utilizing selenomethionine incorporation and SAD phasing, is particularly valuable for challenging, noncanonical nucleosome structures.
• The findings offer a new approach for advancing our understanding of chromatin organization and function.