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Three-dimensional reconstruction of Negatively Stained Tarantula Thick Filament.

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Presentation on theme: "Three-dimensional reconstruction of Negatively Stained Tarantula Thick Filament."— Presentation transcript:

1

2 Three-dimensional reconstruction of Negatively Stained Tarantula Thick Filament

3 43.5 nm Negatively Stained Tarantula Thick Filament

4 =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

5 FFTFFT

6 Three-dimensional Reconstruction (MRC) Electron Micrographs 3D Map Correction of Filaments Alignment of Filaments Combining Filaments Indexing in Fourier space

7 Three-dimensional Reconstruction (Part I) Making the FFT Indexing Correction of Filaments

8 Making the FFT FFTFFT

9 Near Side Layer Line 0: J 0 ; J 12 ; J -12 Layer Line 1: J 4 ; J 16 ; J -8 Layer Line 2: J 8 ; J -4; J -16 Layer Line 3: J 0 ; J 12 ; J -12 Far Side... Near Far Indexing Look for the reflections from the helical structure 1/43.5nm -1 1/14.5nm -1

10 Integrated Intensity Y Spacing (Transform steps) Indexing 1) Plot the sum of the intensity along a column in the FFT

11 Amplitudes (0-9) e.g.: Convert amplitudes from -114 to 250 to a 0 to 9 range. -114. 250 0....90....9 Phases (A-Z) e.g.: Convert phases from 0 to 360 Deg. To a 36 letters and numbers Indexing 2) Plot the FFT 99999999...55544467789999887766543322233456677889999877644555... f f f ff f f f...bbbaaacddef f f f eeddccba9988899abccddeef f f feddcaabbb...

12 Output the FFT All Layer Lines from the Near Side in a File All Layer Lines from the Far Side in a File

13 Correction of: - Tilt - Shift - Shear Correction of Filaments Y Z X

14 Produce a reference Fitting Average or Bessel Separation Output the 3D-Map Three-dimensional Reconstruction (Part II)

15 Produce a Reference Filament Select the main portion from each Layer Line of one filament to be used as a reference. 4 th

16 Alignment by: - Rotation - Z Shift - Radial Scale Filament Alignment Y Z X Reference

17 Combining Filaments Average Average of Amplitude and Phases of each Layer Line of all filaments Bessel Saparation Average after separating the contributions of the near and far side to the Bessel Functions of each Layer Lines

18 Finnal Bessels on the 3Dmap Layer Line 0: J 0 ; J 12 Layer Line 1: J 4 ; J -8 Layer Line 2: J -4 ; J 8 Layer Line 3: J 0 ; J -12 Layer Line 4: J 4 ; J -8 Layer Line 5: J -4 ; J 8 Layer Line 6: J 0 ; J -12 Layer Line 7: J 4 ; J -8 Layer Line 8: J -4 Layer Line 9: J 0

19 Regla de selección Las “layerlines” apareceran a una distancia Z a lo largo del meridiano: z = n/P + m/p donde m y n son enteros. La verdadera repetición R de la éelice ocurrira luego de t vueltas de la hélice si Pt/p=u es entero u es el número de monómeros en t vueltas de la hélice dando una repetición R. Por tanto: R = Pt = pu Definimos entonces las “layerlines” como: z = l/R = m/p + n/P de donde: l = zR = mR/p + nR/P = m(pu)/p + n(Pt)/P = mu + nt


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