Abstract
Structure determination for amyloid fibrils presents many challenges due to the high variability exhibited by fibrils and heterogeneous morphologies present, even in single samples. Mass per unit length (MPL) estimates can be used to differentiate amyloid fibril morphologies and provide orthogonal evidence for helical symmetry parameters determined by other methods. In addition, MPL data can provide insight on the arrangement of subunits in a fibril, especially for more complex fibrils assembled with multiple parallel copies of the asymmetric unit or multiple twisted protofilaments. By detecting only scattered electrons, which serve as a relative measure of total scattering, and therefore protein mass, dark field imaging gives an approximation of the total mass of protein present in any given length of fibril. When compared with a standard of known MPL, such as Tobacco Mosaic Virus (TMV), MPL of the fibrils in question can be determined. The program suite MpUL-multi was written for rapid semi-automated processing of TB-TEM dark field data acquired using this method. A graphical user interface allows for simple designation of fibrils and standards. A second program averages intensities from multiple TMV molecules for accurate standard determination, makes multiple measurements along a given fibril, and calculates the MPL.
Highlights
Determination of amyloid fibril structures by electron microscopy is generally performed using helical reconstruction methods such as iterative real space helical reconstruction (IHRSR)[8] or using programs such as SPRING9, or FREALIX10
Mass per unit length (MPL) measurements by scanning transmission electron microscopy (STEM) have been used to differentiate fibril morphologies and validate biochemical models for amyloid fibrils composed of a variety of proteins including Calcitonin[17], Amyloid beta[18], and IAPP19,20
Whilst STEM using a calibrated detector remains the “gold standard” for determination of MPL, Chen et al.[21] presented an alternative method using tilted beam transmission electron microscopy (TB-TEM), which can be performed with a standard transmission EM
Summary
Where If and Itmv are the uncorrected intensity measurements for the fibril and TMV standard, Bf and Btmv are corresponding background intensity measurements for each intensity measurement, and K is the MPL of TMV, taken form the literature as 131 kDa nm−1 22. The individual 1 px wide measurements from all subregions of the fibrils are combined to calculate the mean MPL. In centering the line along which the measurements are made on the fibrils. This was tested by making a series of measurements of various widths on a test image containing bars with standardized widths and pixel values (Fig. 2)
Sign-in/Register to view highlights & summary Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
