Purpose: We proposed the de novo determination of the atomic structure of protein assemblies in living cells. To achieve this, we intended to obtain diffraction from heavy atom labeled protein assemblies (nanocrystals) inside living cells. We devised several complementary methods for heavy atom incorporation into protein assemblies inside whole, living cells, which we propose will allow us to uniquely determine the atomic structure of these assemblies within the cells; a technique complementary to a variety of live cell imaging approaches. Our new method selectively incorporated heavy atoms, primarily Iodine, onto protein complexes in living cells by genetic means, in the form of non-natural amino acids (I-Phe, I-Tyr), or through a combined use of electroporation and an incorporation catalyst; chloramine-T or tetrachloro-diphenylglycoluril. By these means, we attempted to recover single or multiple isomorphous differences between labeled and native data sets, as well as any detectable anomalous differences in these data.
The following samples were tested at SACLA: 1) Bacillus Sphaericus BinAB protein complex nanocrystals (native) 2) Bacillus Sphaericus BinAB protein complex nanocrystals (3Iodo-tyrosine) 3) Bacillus Sphaericus BinAB protein complex nanocrystals (3Iodo-Phenylalanine) 4) Bacillus Thuringiensis protein complex nanocrystals (native) 5) Bacillus Thuringiensis protein complex nanocrystals (3Iodo-tyrosine)
Summary of experiment/measurement results
Sample 1) Collected and Processed a full data set at 2.8A resolution Sample 2) Collected and process a data set 98% complete at 3.4 A resolution Sample 3) Collected and process a data set 85% complete at 3.0 A resolution Sample 4) Collected and process a data set 95% complete at 3.0 A resolution Sample 5) Collected and process a data set 73% complete at 3.4 A resolution Currently we are in the process of extracting phasing information from the Anomalous signal of the Iodo atoms. |