A signalling protein resists forming large single crystals; the best crystals…
PHYSICS · Multi-part · Fermi University Physics · Biophysics & Medical Physics · Macromolecules, membranes and cell architecture · Macromolecules, membranes and cell architecture
Forces, Machines and Signals, Ch Seeing molecules: diffraction, cryo-electron microscopy and fluorescence
Problem
A 42kDa signalling protein resists forming large single crystals; the best crystals obtained are thin plates measuring 8μm×8μm×2μm. A folded protein occupies about 2.5A˚3 per dalton of mass. For comparison, a 0.1mm cube of a well-ordered 30kDa protein crystal holds about 1.3×1013 copies, each within the standard X-ray dose limit of 20 to 30MGy (at 100K), and such a crystal is known to give good diffraction data to atomic resolution. Cryo-electron microscopy instead images one frozen copy at a time, each surviving only a few tens of electrons per square angstrom of its own dose budget, and reaches useful resolution by computationally averaging many thousands of such single-particle images.
((a)) Estimate the number of protein copies contained in the plate crystal.
((b)) Assuming the same fixed X-ray dose per molecule applies to any crystal of this protein, and that the total diffracted signal obtainable before that dose limit is reached scales with the number of illuminated copies, estimate how many times weaker the plate crystal's total signal would be than that of the benchmark 0.1mm crystal above.
((c)) Part (b) compares copy numbers only. Explain why that comparison alone cannot decide whether X-ray crystallography with these plate crystals or single-particle cryo-electron microscopy is the more promising route to a structure for this protein, and name one further piece of information about the plates and one about the single particles that would decide it.