Ab initio crystallographic structure determination of insulin from protein to electron density without crystal handling.
Insulin crystals suitable for cryogenic data collection and structure determination by single-wavelength anomalous scattering (SAS) were obtained by a self-optimization screening process in a single capillary tube without manipulation of the crystals at any time. Using the counter-diffusion crystallization technique, screening for optimal conditions for crystal growth, incorporation of a strong anomalous scattering halide and cryogenic solution took place simultaneously in a single capillary tube. The crystals in the capillaries can be placed directly in the cryostream for data collection using a conventional home-laboratory X-ray source. High-redundancy data were used to obtain a Patterson solution from the anomalous signal of iodine. As a result, the anomalous scattering-atom position was determined and the phase calculated, giving rise to an initial electron-density map at 2.4 A resolution. This entire procedure from crystal growth to the determination of an initial structure was performed within four weeks.
Gavira
José A
JA
Laboratory for Structural Biology and the Department of Biological Sciences, University of Alabama in Huntsville, 35899, USA.
Toh
Diana
D
Lopéz-Jaramillo
Javier
J
García-Ruíz
Juan M
JM
Ng
Joseph D
JD
D000818:Animals; D001665:Binding Sites; D002417:Cattle; D018360:Crystallography, X-Ray / Q000379:methods*; D004583:Electrons; D007328:Insulin / Q000737:chemistry*; D007455:Iodine / Q000737:chemistry; D008958:Models, Molecular; D010179:Pancreas / Q000378:metabolism; D013455:Sulfur / Q000737:chemistry; D013997:Time Factors
D007328:Insulin; D013455:Sulfur; D007455:Iodine