Synchrotron microcrystal native-SAD phasing at a low energy

Abstract

De novo structural evaluation of native biomolecules from single-wavelength anomalous diffraction (SAD) is a challenge because of the weakness of the anomalous scattering. The anomalous scattering from relevant native elements – primarily sulfur in proteins and phosphorus in nucleic acids – increases as the X-ray energy decreases toward their K-edge transitions. Thus, measurements at a lowered X-ray energy are promising for making native SAD routine and robust. For microcrystals with sizes less than 10 µm, native-SAD phasing at synchrotron microdiffraction beamlines is even more challenging because of difficulties in sample manipulation, diffraction data collection and data analysis. Native-SAD analysis from microcrystals by using X-ray free-electron lasers has been demonstrated but has required use of thousands of thousands of microcrystals to achieve the necessary accuracy. Here it is shown that by exploitation of anomalous microdiffraction signals obtained at 5 keV, by the use of polyimide wellmounts, and by an iterative crystal and frame-rejection method, microcrystal native-SAD phasing is possible from as few as about 1 200 crystals. Our results show the utility of low-energy native-SAD phasing with microcrystals at synchrotron microdiffraction beamlines.

Structural Basis for Ca2+-Dependent Activation of a Plant Metacaspase

“…We hypothesized that microcrystals may allow for large conformational changes without compromising their diffraction. We therefore prepared wild-type AtMC4 microcrystals and solved Ca 2+ -treated structure by using the method we recently developed 23 . Ca 2+ -treated microcrystals changed the space group, shrunk the unit cell dimensions, and diffracted X-rays better than the non-treated large crystals (Extended Data Table 1).…”

“…We then moved microcrystals back to cold room. To manipulate microcrystals for microdiffraction data collection, we used a pipette to aspire microcrystal slurries, put them on the custom-made MiTiGen wellmounts 23 , and flash-frozen them into liquid nitrogen.…”

“…The C139A mutant structure was determined by singleisomorphous replacement with anomalous scattering (SIRAS). To enhance anomalous signals from Se sites at a low resolution of 4 Å, we used an iterative crystal and frame rejection technique that we developed for microcrystals 23 . The assembled data were used for substructure determination by program SHELXD 33 .…”

“…Structure determination from Ca 2+ -treated microcrystals. We solved the Ca 2+treated structure by combining partial data from 12 microcrystals using a modified data assembly method that we have developed 23 . Briefly, we collected 132 partial data sets, each from a Ca 2+ -treated microcrystals.…”

“…Among these merged data sets, group 11 displays the lowest refined R free (0.35) and contains data merged from 14 microcrystals. This data was further optimized by using the iterative crystal and frame reject method 23 . The optimized data used 338 frames from 12 microcrystals.…”