Selenourea: a convenient phasing vehicle for macromolecular X-ray crystal structures

Zhipu Luo

doi:10.1038/srep37123

Abstract

Majority of novel X-ray crystal structures of proteins are currently solved using the anomalous diffraction signal provided by selenium after incorporation of selenomethionine instead of natural methionine by genetic engineering methods. However, selenium can be inserted into protein crystals in the form of selenourea (SeC(NH2)2), by adding the crystalline powder of selenourea into mother liquor or cryo-solution with native crystals, in analogy to the classic procedure of heavy-atom derivatization. Selenourea is able to bind to reactive groups at the surface of macromolecules primarily through hydrogen bonds, where the selenium atom may serve as acceptor and amide groups as donors. Selenourea has different chemical properties than heavy-atom reagents and halide ions and provides a convenient way of phasing crystal structures of macromolecules.

Highlights

Because of the availability of large number of structures of macromolecules stored in the Protein Data Bank[1] (PDB), the majority of X-ray crystal structures of proteins and nucleic acids are nowadays solved by the Molecular Replacement technique
The “classic” approach, used since the early days of protein crystallography, involves derivatization of native crystals by prolonged soaking in solutions or co-crystallization with various reagents containing heavy metals[3], such as Hg, Pt, Au etc. Variations of this approach involve, for example, the use of the heavy-metal clusters[4], especially suitable for structures of very large macromolecular complexes, the gaseous xenon or krypton pressurized into native crystals[5], or the short soaking in salts of halides[6] (Br or I)
It is possible to obtain useful anomalous phasing signal from sulfur of Cys and Met naturally occurring in proteins[7,8,9] or from phosphorus in nucleic acids[10]

Summary

Zhipu Luo

Majority of novel X-ray crystal structures of proteins are currently solved using the anomalous diffraction signal provided by selenium after incorporation of selenomethionine instead of natural methionine by genetic engineering methods. The tests have shown that it is possible to effectively introduce selenium into native crystals by soaking them in the presence of selenourea (SeU, SeC(NH2)2), Fig. 1 This simple compound, similar to well known urea (Supplementary Fig. 1), penetrates through the crystal solvent channels and binds to certain functions at the surface of biomolecules, in analogy to the heavy-atom or halide derivatization. The SeU molecule is small, of the size smaller than most of the heavy-metal complexes used for classic derivatization of proteins, and in analogy to small halide ions, rapidly diffuse through the solvent channels of macromolecular crystals It can be used in a wide pH range, at least from 4 to 9. The examples shown in the Methods section ascertain that SeU can be successfully and conveniently used as a practical and applicable vehicle for phasing novel crystal structures of macromolecules by the SAD (or MAD) approach through the anomalous signal of selenium

Methods

Findings

Additional Information