Data Collection with the DE-12 Camera Nikolaus Grigorieff - - PowerPoint PPT Presentation

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Data Collection with the DE-12 Camera Nikolaus Grigorieff - - PowerPoint PPT Presentation

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Data Collection with the DE-12 Camera Nikolaus Grigorieff Brandeis University & TSRI Exposure Series 1 2 3 8 electrons/ 2 8 electrons/ 2 8 electrons/ 2 Rotavirus DLP Mapping Rotations Exposure 1 2 Longest vectors

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SLIDE 1

Data Collection with the DE-12 Camera

Nikolaus Grigorieff Brandeis University & TSRI

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SLIDE 2

1 2

Exposure Series

3

Rotavirus DLP

8 electrons/Å2 8 electrons/Å2 8 electrons/Å2

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SLIDE 3

Mapping Rotations

Exposure 1 → 2 Longest vectors correspond to ~2 deg rotations.

50 100 150 0.5 1 1.5 2

# particles Rotation angle [°]

∆Dose = 8 e-/Å2 Max at 0.38°

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SLIDE 4

Movies

Recorded with direct electron detector DE-12 (Direct Electron)

Frame rate = 40 fps Dose/frame = 0.5 e-/Å2 Duration = 1.5 s

  • No. of frames = 60

Total dose = 30 e-/Å2 1 movie = 720 MB (1 byte/pixel) → Data Tsunami! Brilot et al. 2012

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SLIDE 5

Each averaged frame corresponds to 0.25 s. Dose/frame = 5 e-/Å2

10-Frame Averages

Brilot et al. 2012

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SLIDE 6

Rotations Over Time

2.5° deg Vectors

Brilot et al. 2012

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SLIDE 7

Translations Over Time

25 Å Vectors

Brilot et al. 2012

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SLIDE 8

Different Corner

Brilot et al. 2012

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SLIDE 9

60-frame average (no alignment)

Frame Alignment

60-frame average (translational alignment)

Rotation: ~1 deg in this area → signal at 10 Å attenuated by 70% (B-factor = 500 Å2)

Brilot et al. 2012

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SLIDE 10

Larger Data Set

  • Microscope:

FEI TF20 (200 kV)

  • Detector:

DE-12 (4k x 3k, direct)

  • Frame rate:

25 fps

  • Frames:

18 (2 dark + 16 light)

  • Dose/frame:

2 electrons/Å2

  • Total dose:

32 electrons/Å2

  • Grids:

C-flat 1.2/1.3 μm

  • Pixel size:

1.42 Å

  • Movies collected:

501

  • Particles:

1915 total, 807 used

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SLIDE 11

Rotavirus DLP Data Set

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SLIDE 12

Rotation Histograms

50 100 150 0.5 1 1.5 2

# particles Rotation angle [°]

∆Dose = 8 e-/Å2 Max at 0.38°

100 200 300 400 500 600 0.5 1 1.5 2

# particles Rotation angle [°]

∆Dose = 8 e-/Å2 Max at 0.17°

100 200 300 400 500 0.5 1 1.5 2

# particles Rotation angle [°]

∆Dose = 24 e-/Å2 Max at 0.27° Entire hole exposed One side of hole exposed

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SLIDE 13

50 100 150 200 250 1 2 3 4 5 6 7 8

# particles Shift [Å]

50 100 150 200 250 1 2 3 4 5 6 7 8

# particles Shift [Å]

Translation Histograms

∆Dose = 8 e-/Å2 Max at 20 Å/sec ∆Dose = 8 e-/Å2 Max at 15 Å/sec Beginning of exposure End of exposure Sample drift ~2 Å/sec (Leginon)

20 40 60 80 100 2 4 6 8 10 12 14 16

# particles Shift [Å]

∆Dose = 24 e-/Å2 Max at 17 Å/sec

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SLIDE 14

Rotations and Translations

2.5° deg Rotations 10 Å Translations

∆Dose = 24 e-/Å2

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SLIDE 15

Increased Resolution

100 Å 10 Å

DLP VP6

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SLIDE 16

Improved Density

No frame alignment With frame alignment (~800 particles) 2008 map (~8,000 particles)

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SLIDE 17

Beam Damage

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SLIDE 18

24 electrons/Å2

High-Dose Imaging

Flu hemagglutinin

(2FK0, Stevens et al. 2006)

Trimer = 180 kDa

Melody Campbell Peter Lee (unpublished)

104 electrons/Å2 Voltage = 200 kV Defocus = 1.5 μm

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SLIDE 19
  • Brandeis

Axel Brilot, James Chen

  • Scripps

Melody Campbell, Anchi Cheng, Arne Moeller, Dmitry Lyumkis, David Veesler, Peter Lee, Ian Wilson, Clint Potter, Bridget Carragher

  • Harvard Med. School

Junhua Pan, Steve Harrison

  • MRC-LMB

Richard Henderson

  • Financial Support:

HHMI, NIH

Acknowledgements