mode. The texture of the carbon film should be visualized in
the image.
7. Cycle the Search, Focus, and Exposure modes in sequence for a
few rounds until the beam is stable.
8. In Search mode, locate an area with intact carbon and an
appropriate density of crystals. Find a target crystal for imaging.
9. Use the wobbler tool to set the eucentric z-height of the stage.
Tilt the stage to the desired tilting angle.
10. Switch to Focus mode. To minimize the CTF modulation on
the image amplitudes and phases, less defocus will be applied.
Switch to Focus mode and use the focus knob to set a nominal
defocus to a value between À400 and À1200 nm.
11. While in focus mode, correct any astigmatism of the objective
lens and centered the beam using the beam-shift trackball. Wait
a few seconds for stabilization if the stage is moving.
12. Switch to Exposure mode, insert the digital recording media
and record the image. Set an exposure time that gives an
accumulating dose of about 10 e
À
/A ˚ 2 .
13. Switch back to Search mode, identify another crystal for imaging and repeat steps.
3.3 Initial Data
Quality Assessment
The quality of the recorded intensities can be evaluated from the
diffraction pattern or the power spectrum of the crystal image. The
diffraction peaks should be sharp and symmetric without splitting
or skewed. Also, the distribution of the diffraction peaks should be
isotropic, particularly the diffraction patterns or the images of tilted
crystal specimens. The highest resolution of the diffraction peaks
can be roughly estimated by the known unit cell parameters and
their Miller indices. For the study of lipid–protein interaction, one
will need high-resolution information (higher than 2.5 A ˚ resolution) to accurately model the atomic coordinates of the lipids.
4 Notes
1. Glucose is non-volatile and often used to embed crystal by the
back-injection method [34]. However, it was reported that
glucose embedding may not maintain the protein in its native
condition if the preparation is not carefully taken [48]. Trehalose was reported to preserve high-resolution information of
the 2D crystal samples [34, 49]. The concentration of trehalose
used for embedding is usually 1–7% (w/v) [11, 14, 27, 29],
which can be varied in different cases. Other sugars that are
commonly used are sucrose and tannic acid [35].
Electron Crystallography of Membrane Proteins
261
the image.
7. Cycle the Search, Focus, and Exposure modes in sequence for a
few rounds until the beam is stable.
8. In Search mode, locate an area with intact carbon and an
appropriate density of crystals. Find a target crystal for imaging.
9. Use the wobbler tool to set the eucentric z-height of the stage.
Tilt the stage to the desired tilting angle.
10. Switch to Focus mode. To minimize the CTF modulation on
the image amplitudes and phases, less defocus will be applied.
Switch to Focus mode and use the focus knob to set a nominal
defocus to a value between À400 and À1200 nm.
11. While in focus mode, correct any astigmatism of the objective
lens and centered the beam using the beam-shift trackball. Wait
a few seconds for stabilization if the stage is moving.
12. Switch to Exposure mode, insert the digital recording media
and record the image. Set an exposure time that gives an
accumulating dose of about 10 e
À
/A ˚ 2 .
13. Switch back to Search mode, identify another crystal for imaging and repeat steps.
3.3 Initial Data
Quality Assessment
The quality of the recorded intensities can be evaluated from the
diffraction pattern or the power spectrum of the crystal image. The
diffraction peaks should be sharp and symmetric without splitting
or skewed. Also, the distribution of the diffraction peaks should be
isotropic, particularly the diffraction patterns or the images of tilted
crystal specimens. The highest resolution of the diffraction peaks
can be roughly estimated by the known unit cell parameters and
their Miller indices. For the study of lipid–protein interaction, one
will need high-resolution information (higher than 2.5 A ˚ resolution) to accurately model the atomic coordinates of the lipids.
4 Notes
1. Glucose is non-volatile and often used to embed crystal by the
back-injection method [34]. However, it was reported that
glucose embedding may not maintain the protein in its native
condition if the preparation is not carefully taken [48]. Trehalose was reported to preserve high-resolution information of
the 2D crystal samples [34, 49]. The concentration of trehalose
used for embedding is usually 1–7% (w/v) [11, 14, 27, 29],
which can be varied in different cases. Other sugars that are
commonly used are sucrose and tannic acid [35].
Electron Crystallography of Membrane Proteins
261