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

Data Collection with the DE-12 Camera Nikolaus Grigorieff Brandeis University & TSRI

Exposure Series 1 2 3 8 electrons/Å 2 8 electrons/Å 2 8 electrons/Å 2 Rotavirus DLP

Mapping Rotations Exposure 1 → 2 Longest vectors correspond to ~2 deg rotations. 150 ∆ Dose = 8 e - /Å 2 Max at 0.38° 100 # particles 50 0 0 0.5 1 1.5 2 Rotation angle [°]

Movies Recorded with direct electron detector DE-12 (Direct Electron) Frame rate = 40 fps Dose/frame = 0.5 e - /Å 2 Duration = 1.5 s No. of frames = 60 Total dose = 30 e - /Å 2 1 movie = 720 MB (1 byte/pixel) → Data Tsunami! Brilot et al. 2012

10-Frame Averages Each averaged frame corresponds to 0.25 s. Dose/frame = 5 e - /Å 2 Brilot et al. 2012

Rotations Over Time Vectors 2.5° deg Brilot et al. 2012

Translations Over Time Vectors 25 Å Brilot et al. 2012

Different Corner Brilot et al. 2012

Frame Alignment 60-frame average 60-frame average (no alignment) (translational alignment) Rotation: ~1 deg in this area → signal at 10 Å attenuated by 70% (B-factor = 500 Å 2 ) Brilot et al. 2012

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/Å 2 • Total dose: 32 electrons/Å 2 • Grids: C-flat 1.2/1.3 μm • Pixel size: 1.42 Å • Movies collected: 501 • Particles: 1915 total, 807 used

Rotavirus DLP Data Set

Rotation Histograms 600 Entire hole exposed ∆ Dose = 8 e - /Å 2 500 150 Max at 0.17° 400 # particles 300 100 # particles ∆ Dose = 8 e - /Å 2 200 Max at 0.38° 100 50 0 0 0.5 1 1.5 2 ∆ Dose = 24 e - /Å 2 Rotation angle [°] 500 0 Max at 0.27° 0 0.5 1 1.5 2 400 # particles Rotation angle [°] 300 200 100 One side of hole exposed 0 0 0.5 1 1.5 2 Rotation angle [°]

Translation Histograms 250 250 Beginning of exposure End of exposure 200 200 # particles # particles ∆ Dose = 8 e - /Å 2 ∆ Dose = 8 e - /Å 2 150 150 Max at 20 Å/sec Max at 15 Å/sec 100 100 50 50 0 0 0 1 2 3 4 5 6 7 8 0 1 2 3 4 5 6 7 8 Shift [Å] Shift [Å] 100 ∆ Dose = 24 e - /Å 2 80 Max at 17 Å/sec # particles 60 40 20 Sample drift ~2 Å/sec (Leginon) 0 0 2 4 6 8 10 12 14 16 Shift [Å]

Rotations and Translations ∆ Dose = 24 e - /Å 2 Rotations 2.5° deg Translations 10 Å

Increased Resolution DLP 100 Å VP6 10 Å

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

Beam Damage

High-Dose Imaging 104 electrons/Å 2 24 electrons/Å 2 Voltage = 200 kV Defocus = 1.5 μm Flu hemagglutinin (2FK0, Stevens et al. 2006) Trimer = 180 kDa Melody Campbell Peter Lee (unpublished)

Acknowledgements • 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