Measurement of amyloid fibril mass-per-length by tilted-beam transmission electron microscopy.
We demonstrate that accurate values of mass-per-length (MPL), which serve as strong constraints on molecular structure, can be determined for amyloid fibrils by quantification of intensities in dark-field electron microscope images obtained in the tilted-beam mode of a transmission electron microscope. MPL values for fibrils formed by residues 218-289 of the HET-s fungal prion protein, for 2-fold- and 3-fold-symmetric fibrils formed by the 40-residue beta-amyloid peptide, and for fibrils formed by the yeast prion protein Sup35NM are in good agreement with previous results from scanning transmission electron microscopy. Results for fibrils formed by the yeast prion protein Rnq1, for which the MPL value has not been previously reported, support an in-register parallel beta-sheet structure, with one Rnq1 molecule per 0.47-nm beta-sheet repeat spacing. Since tilted-beam dark-field images can be obtained on many transmission electron microscopes, this work should facilitate MPL determination by a large number of research groups engaged in studies of amyloid fibrils and similar supramolecular assemblies.
Chen
Bo
B
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD 20892, USA.
Thurber
Kent R
KR
Shewmaker
Frank
F
Wickner
Reed B
RB
Tycko
Robert
R
D016229:Amyloid beta-Peptides / Q000737:chemistry / Q000648:ultrastructure*; D005656:Fungal Proteins / Q000737:chemistry / Q000648:ultrastructure; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D006801:Humans; D046529:Microscopy, Electron, Transmission / Q000379:methods*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000737:chemistry / Q000648:ultrastructure*; D010454:Peptide Termination Factors / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D011328:Prions / Q000737:chemistry / Q000648:ultrastructure; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics / Q000648:ultrastructure
D016229:Amyloid beta-Peptides; D005656:Fungal Proteins; C083891:HET-S protein, Podospora anserina; D010446:Peptide Fragments; D010454:Peptide Termination Factors; D011328:Prions; C404807:RNQ1 protein, S cerevisiae; D011993:Recombinant Fusion Proteins; C068068:SUP35 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; C078592:amyloid beta-protein (1-40); D049452:Green Fluorescent Proteins
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