John Rose

I have over 35 years’ experience in diffraction based structural biology. I began my career in 1980 as a post doc in B.C. Wang’s lab at the VA Medical Center in Pittsburgh. My project focused on the isolation and purification the protein neurophysin (neurophysin is involved in the storage of the hormones oxytocin and vasopressin) from porcine posterior pituitary glands for crystallization trials. As part of these studies I also synthesized the hormone analogues Phe-Tyr amide and Tyr-Phe amide in the laboratory of Klaus Hofmann at the University of Pittsburgh, since these peptides were needed for the crystallization trials. Once I had crystals I learned an important lesson - data quality depends both on the crystal and the X-ray source. Because of source problems I took a keen interest in the care and maintenance of the lab’s diffraction instrumentation.  Today, I have over 30 years’ experience in managing/maintaining X-ray facilities at the University of Pittsburgh, the Institute of Molecular Biology, Academia Sinica (IMB), Taiwan and the University of Georgia. I also serve as the Director of the UGA X-ray Diffraction Core facility and the Assistant Director of SER-CAT, one of the nation’s premiere synchrotron facilities for the collection of X-ray diffraction data.

Although my formal training was small molecule crystallography my post-doctoral work with gave me a solid foundation in all phases of the macromolecular crystal structure determination process from producing and purifying protein to validating the final refined protein structure. During this time B.C. Wang developed his solvent flattening phasing process that revolutionized the field. His ISAS method relying on single-wavelength anomalous scattering data (SAD) reduced the need for multiple ISOMORPHOUS heavy atom derivatives since all necessary data could, in most cases be collected on a single crystal containing an anomalous scatterer.  These anomalous scatterers could be metal ion natively present in the protein or anomalous scatterers introduced by crystal soaking or (later by seleomethionine labeling). Under B.C.'s watchful eye and using cocrystals of a neurophysin complexed with p-iodo Phe-Tyr amide Liqing Chen and I were able to phase the first ISAS structure in 1991. This was followed up by the crystal structure of the neurophysin-oxytocin complex in 1996 by molecular replacement.

The ultimate goal of SAD phasing was the ability to generate phases from crystals of the native protein using the weak anomalous signal from the sulfur atoms present in cysteine and methionine residues (now termed Native-SAD).  Over the next decade we focused on developing methods and procedures to increase the anomalous signal to noise in Native-SAD data and in making SAD a first-choice method for de novo X-ray structure determination. The idea was that if we could solve a Native SAD structure, then most all other elements became available for SAD phasing. During this time, we solved several crystal structures including Ferrochelatase by Fe-SAD, the transcription factor sc‐mtTFB by Xe-SAD culminating in the structure determination of photo protein obelin by Native-SAD. Today a majority of de novo protein structures are determined from single-wavelength anomalous scattering data. 

To be continued ...