We have developed a complete framework for alignment, classification, and averaging of volumes derived by electron tomography that is computationally efficient and effectively accounts for the missing wedge that is inherent to limited angle electron tomography. Modeling the missing data as a multiplying mask in reciprocal space we have shown that the effect of the missing wedge can be accounted for seamlessly in all alignment and classification operations. The development of these tools has proved to be critical for our successful effort in determining the structure of trimeric HIV-1 envelope glycoproteins. The speed of data processing has increased by about 10-fold over the course of the last year, without which we could not have undertaken the systematic effort of analyzing structural variations across a wide variety of HIV and SIV strains. Progress in image processing and segmentation has also continued with a view to complete automation in the data processing pipeline. The effective use of denoising, when used with care is an enormously powerful tool for the automated interpretation of complex 3D data sets at high throughput.
Chemical definition of complex protein assemblies is integral to interpreting 3D structure. The methods we have developed for the determination of absolute amounts and the relative stoichiometry of proteins in a mixture using fluorescence and mass spectrometry is now used in many other laboratories in a wide variety of applications. Fusing a unique genetically coded spectroscopic signal element with concatenated proteotypic peptides thus provides a powerful method to accurately quantify and determine the relative stoichiometry of multiple proteins present in complexes or mixtures that cannot be readily assessed using classical gravimetric, enzymatic, or antibody-based technologies.
In a new direction for the lab, we have begun to explore the potential of imaging mass spectrometry to reveal structural signatures of cells at submicron resolution with the aim of identifying chemical differences between normal and diseased or malignant cells. Our goal is to image chemical moieties within intact mammalian cells using secondary ion mass spectrometry, with simultaneous mapping of elemental and molecular species along with intrinsic nuclear and membrane-specific cellular markers. Results from imaging of both the cell surface and cell interior exposed by site-specific focused ion beam milling demonstrate that in-plane resolutions of ~ 400 nm and can be achieved. The results from mapping cell surface phosphocholine, the nuclear marker DAPI, and several other molecular ions present in the cells establish that spatially-resolved chemical signatures of individual cells can be derived from multivariate analysis and classification of the molecular images obtained at different m/z ratios. The methods we have developed for specimen preparation and chemical imaging of cell interiors lay the foundation for obtaining 3D molecular signatures of unstained mammalian cells, with particular relevance for probing the subcellular distribution of drugs and metabolites.
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