Low-dose thickness measurement of glucose-embedded protein crystals by electron energy loss spectroscopy and STEM dark-field imaging

Abstract

Electron energy loss spectroscopy and dark-field imaging in a scanning transmission electrton microscope were used to determine the thickness of glucose-embedded crotoxin complex crystals. The results demonstrate the feasibility of identifying protein crystals with a thickness of half a unit cell (12.8 nm) under low-dose and low-temperature conditions. The accuracy of this method is limited by the amount of surface coating of the crystal's embedding glucose used for preserving the high-resolution structure of the protein. The histogram of the crystal thickness distribution and the spread of the anticipated crystal thickness allow us to make an estimate of the uncertainty in the glucose layer thickness. This approach can be incorporated as part of the experimental procedure in the three dimensional data collection for structure determination of protein crystals with variable thicknesses. The measurement can be done on areas approximately 200 nm in diameter so that crystals of suitable thickness can be pre-selected before the high-resolution data is recorded. Accurate determination of the crystal thickness will optimize the data collection efficiency by avoiding the collection and subsequent analysis of unmatchable data for the three-dimensional reconstruction.