title: Generating isomorphous heavy-atom derivatives by a quick-soak method. Part II: phasing of new structures.
abstract: A quick-soak method has been applied to generate de novo heavy-atom phasing to solve two new protein structures, a type II transforming growth factor beta receptor (TBRII) and a natural killer cell receptor-ligand complex, NKG2D-ULBP3. In the case of TBRII, a crystal derivatized for only 10 min in saturated HgCl(2) provided adequate phasing for structure determination. Comparison between HgCl(2) derivatives generated by 10 min soaking and by 12 h soaking revealed similar phasing statistics. The shorter soak, however, resulted in a derivative more isomorphous to the native than the longer soak as judged by changes in the unit-cell parameter a upon derivatization as well as by the quality of a combined SIRAS electron-density map. In the case of the NKG2D-ULBP3 structure, all overnight soaks in heavy-atom solutions resulted in crystal lattice disorder and only the quick soaks preserved diffraction. Despite fragile lattice packing, the quick-soaked K(2)PtCl(4) derivative was isomorphous with the native crystal and the electron-density map calculated from combined SIR and MAD phases is better than that calculated from MAD phases alone. Combined with mass-spectrometry-assisted solution heavy-atom derivative screening and the use of synchrotron radiation, the quick-soak derivatization has the potential to transform the time-consuming conventional heavy-atom search into a real-time 'on-the-fly' derivatization process that will benefit high-throughput structural genomics.
authors.affiliation: Structural Immunology Section, Laboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, 12441 Parklawn Drive, Rockville, MD 20852, USA. psun@nih.gov
authors.forename: Peter D
authors.initials: PD
authors.lastname: Sun
authors.forename: Sergei
authors.initials: S
authors.lastname: Radaev
mesh_terms: D002352:Carrier Proteins / Q000737:chemistry; D018360:Crystallography, X-Ray / Q000295:instrumentation / Q000379:methods*; D004583:Electrons; D005583:Fourier Analysis; D058851:GPI-Linked Proteins; D023281:Genomics; D015395:Histocompatibility Antigens Class I / Q000737:chemistry; D006801:Humans; D036341:Intercellular Signaling Peptides and Proteins; D007694:Killer Cells, Natural / Q000378:metabolism; D008024:Ligands; D013058:Mass Spectrometry / Q000379:methods; D008627:Mercuric Chloride / Q000494:pharmacology; D008958:Models, Molecular; D055655:NK Cell Lectin-Like Receptor Subfamily K; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II; D011971:Receptors, Immunologic / Q000737:chemistry; D055607:Receptors, Natural Killer Cell; D018125:Receptors, Transforming Growth Factor beta / Q000737:chemistry*; D013997:Time Factors
chemical_list: D002352:Carrier Proteins; D058851:GPI-Linked Proteins; D015395:Histocompatibility Antigens Class I; D036341:Intercellular Signaling Peptides and Proteins; C527085:KLRK1 protein, human; D008024:Ligands; D055655:NK Cell Lectin-Like Receptor Subfamily K; D011971:Receptors, Immunologic; D055607:Receptors, Natural Killer Cell; D018125:Receptors, Transforming Growth Factor beta; C423229:ULBP3 protein, human; D008627:Mercuric Chloride; D017346:Protein Serine-Threonine Kinases; D000077294:Receptor, Transforming Growth Factor-beta Type II