Different defocus images with their spatial frequency resolution. Figure 1 -a shows a typical image taken at 0.8 μm defocus with JEOL 400 kV electron cryo-microscope with a LaB6 gun. This image has low contrast but contains a detectable signal above noise at a relatively high spatial frequency out to 1/7 Å-1 (b). The image contrast can be enhanced by setting the defocus value to a larger value (i.e. 2.6 μm as shown in c). However, such an image contains much lower resolution data as shown in (d), the detectable signal is present out to spatial frequency (1/12Å-1). In order to obtain a high-resolution reconstruction, we would use images taken at condition similar to that of (a).