“What we have achieved is a huge jump in resolution of this complex,?said Frank. “Even so, this resolution would not allow us to study the complex in atomic detail, or even see individual helices.?He said the results from the cryo-EM analysis were informed by detailed x-ray crystallographic data on the PCC structure done by other researchers. In x-ray crystallography, an x-ray beam is directed through crystals of a target protein. As the x-rays pass through the crystal, they are diffracted. Researchers can then analyze the diffraction pattern to determine the atomic structure of the protein.
The analysis by Frank and his colleagues revealed that each channel consists of two PCC subunits joined in a clamshell arrangement. The cryo-EM data also revealed two different arrangements of the PCC -- one that was apparently in the functional, or “translocating?state, and one in a non-translocating state.
X-ray crystallography data from the lab of HHMI investigator Tom A. Rappaport suggested that the halves of the PCC clamshell were joined in a back-to-back arrangement. However, said Frank, x-ray crystallographic structures often do not represent the arrangements of proteins in their native functional state.
Thus, he and his colleagues applied a computational analytical method called “normal mode-based flexible fitting?(NMFF) to model how well the two possible channel structures could explain the structural data from cryo-EM. The NMFF method was developed and applied by co-authors Florence Tama and Charles Brooks of the Scripps Research Institute. The technique provides dynami