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Three-dimensional Three-dimensional reconstructionreconstruction of of Negatively Stained Negatively Stained Tarantula Thick Tarantula Thick FilamentFilament
Three-dimensional Three-dimensional reconstructionreconstruction of of Negatively Stained Negatively Stained Tarantula Thick Tarantula Thick FilamentFilament
43.5 nm43.5 nm43.5 nm43.5 nmNegatively Stained Negatively Stained Tarantula Thick Tarantula Thick FilamentFilament
Negatively Stained Negatively Stained Tarantula Thick Tarantula Thick FilamentFilament
=14.5 nm =14.5 nm Subunit separation
Helical repeat 43.5 nm =
Subunit separation 1/43.5nm-1=0.023nm-1=
=1/14.5nm-1
43.5nm/14.5nm=3
1/p Subunit separation
1/P Helical repeat
Three-dimensional Reconstruction
(MRC)
Electron Micrographs
3D Map
Correction of Filaments
Alignment of Filaments
Combining Filaments
Indexing in Fourier space
•Near SideLayer Line 0: J0; J12; J-12 Layer Line 1: J4; J16; J-8Layer Line 2: J8; J-4; J-16
Layer Line 3: J0; J12; J-12 •Far Side...
Near
Far
Indexing
• Look for the reflections from the helical structure
1/43.5nm-1
1/14.5nm-
1
Inte
gra
ted
Inte
nsi
ty
Y Spacing (Transform steps)
IndexingIndexing 1) Plot the sum of 1) Plot the sum of the intensity along a the intensity along a column in the FFTcolumn in the FFT
•Amplitudes (0-9) e.g.: Convert amplitudes from -114 to 250 to a 0 to 9 range.
-114....250
0....9
•Phases (A-Z) e.g.: Convert phases from 0 to 360 Deg. To a 36 letters and numbers
IndexingIndexing 2) Plot the 2) Plot the
FFTFFT
...5554446778999999998877665433222334566778899999999877644555......5554446778999999998877665433222334566778899999999877644555... ...bbbaaacddef f f ff f f f eeddccba9988899abccddeef f f ff f f feddcaabbb......bbbaaacddef f f ff f f f eeddccba9988899abccddeef f f ff f f feddcaabbb...
Output the FFT
•All Layer Lines from the Near Side in a File•All Layer Lines from the Far Side in a File
•Produce a reference•Fitting•Average or Bessel Separation•Output the 3D-Map
Three-dimensional
Reconstruction(Part II)
Produce a Reference Filament
•Select the main portion from each Layer Line of one filament to be used as a reference.
4th
Combining Filaments
AverageAverage•Average of Amplitude and Phases of each Layer Line of all filaments
Bessel SaparationBessel Saparation•Average after separating the contributions of the near and far side to the Bessel Functions of each Layer Lines