Structures from Anomalous Diffraction of Native Biological Macromolecules

Abstract

Crystal structure analyses for biological macromolecules without known structural relatives entail solving the crystallographic phase problem. Typical de novo phase evaluations depend on incorporating heavier atoms than those found natively; most commonly, multi- or single-wavelength anomalous diffraction (MAD or SAD) experiments exploit selenomethionyl proteins. Here we realize routine structure determination using intrinsic anomalous scattering from native macromolecules. We devised robust procedures for enhancing signal-to-noise in the slight anomalous scattering from generic native structures by combining data measured from multiple crystals at lower-than-usual x-ray energy. Using this multi-crystal SAD method (5–13 equivalent crystals), we determined structures at modest resolution (2.8Å-2.3Å) for native proteins varying in size (127–1148 unique residues) and number of sulfur sites (3–28). With no requirement for heavy-atom incorporation, such experiments provide an attractive alternative to selenomethionyl SAD experiments.

An overview of heavy-atom derivatization of protein crystals

“…For weaker reflections the small differences between the Friedel pairs may be masked by noise. Thus, for selenomethionine-labelled crystals that diffract to medium or low resolution the identification of Se sites may not be possible without additional information and the phasing power may be too low to provide useful phase information (Liu et al, 2012). However, in these cases an initial HA derivative may allow the identification of selenium sites in selenomethionine-derivatized protein crystals, which can be useful for improving phasing and/or for tracing the chain.…”

“…In an ideal case, all of the data sets collected for native and derivatives would have identical unit-cell parameters and all of the molecules positioned in the same orientation with no site-specific radiation damage. In practice, the contents and dimensions of unit cells often vary substantially and it may be necessary to collect a range of native and HA data sets and then to cluster data sets with similar unit cells and calculate phases and maps with different combinations to obtain the best phases and interpretable maps (Liu et al, 2012;Giordano et al, 2012).…”

Crystallographic structure solution

“…Indeed, structures as large as 1148 independent amino acids have been phased at 2.8 Å from the inherent sulfur in the protein (Liu et al, 2012). This constitutes a very attractive development, which could become standard given its experimental straightforwardness.…”

Facilitating best practices in collecting anomalous scattering data forde novostructure solution at the ESRF Structural Biology Beamlines

“…The two data sets sets are processed separately and then scaled together to provide the final data set. Although, to our knowledge a systematic study on the beneficial use of inverse-beam geometry is not available, many examples of successful phasing experiments using this technique have been reported in the literature (Liu et al, 2012(Liu et al, , 2013Akey et al, 2014).…”