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

FFTFFTFFTFFT

Three-dimensional Reconstruction

(MRC)

Electron Micrographs

3D Map

Correction of Filaments

Alignment of Filaments

Combining Filaments

Indexing in Fourier space

Three-dimensional Reconstruction

(Part I)

•Making the FFT•Indexing•Correction of Filaments

Making the FFT

FFTFFTFFTFFT

•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

• Correction of:- Tilt- Shift- Shear

Correction of Filaments

Y

Z

X

•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

• Alignment by:- Rotation- Z Shift- Radial Scale

Filament Alignment

Y

Z

X

Reference

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

•Finnal Bessels on the 3Dmap

Layer Line 0: J0; J12

Layer Line 1: J4; J-8Layer Line 2: J-4; J8Layer Line 3: J0; J-12 Layer Line 4: J4; J-8Layer Line 5: J-4; J8Layer Line 6: J0; J-12

Layer Line 7: J4; J-8Layer Line 8: J-4Layer Line 9: J0