One model of the three - dimensional fold of the fibril consisting of five b - sheets per monomer has been proposed [ 13b ] based on ss - NMR and cryo - electron microscopic data . However , long - range restraints at the molecular level in support of this model are still missing . Recently , we have reported pulsed EPR distance measurements to determine the intra - molecular distance between the extremal b - strands in aS fibrils . [ 15 ] A pair - labeling strategy was introduced to infer the orientation of the standard methanethiosulfonate ( MTSL ) label with respect to the plane of the b - strand , as the detected spin - bearing nitroxide of MTSL is located about 0.5 nm from the Ca of the amino acid the spin label is attached to . The resulting distance of ( 4.5 AE 0.5 ) nm between the extremal strands was consistent with the size of the subfilament measured in AFM and cryo - electron microscopic studies . [ 12 , 13b ] Nevertheless the dipolar oscillations observed in the pulsed EPR data , used to extract distances , were very weak and experiments at two different EPR frequencies were required to exclude artifacts . Similar observations were reported in other recent pulsed EPR distance measurements on amyloid fibrils of human islet polypeptide and tau protein . [ 16a , b ] The challenge of these experiments is caused by the required labeling procedure combined with the structural instability of these proteins with respect to point mutations . [ 17 ] To date , despite the wealth of NMR and other spectroscopic data , no EPR distance measurements on aS fibrils have been reported by other groups . In the present study we illustrate how an optimized sample preparation has provided large signals in EPR distance measurements for several selected mutation sites . The result permitted to exploit a labeling strategy to measure distances between largely conserved b - strand regions and to obtain vectorial information on the spatial arrangements of the labels within the strands . The distances and two - dimensional coordinates reported here provide new long - range restraints for the structure of aS fibrils .