Capabilities of the Falcon III detector for single-particle structure determination.

Highlights • The Falcon III camera has integrating and counting mode acquisition. • Maps were obtained with resolutions of 97% of Nyquist frequency in the counting mode. • Reconstructions of medium sized complexes (0.5 MDa) gain from the counting mode. • The structure of TMV was determined at 2.3 Å resolution from data collected in integrating mode. Abstract Direct electron detectors are an essential asset for the resolution revolution in electron cryo microscopy of biological objects. The direct detectors provide two modes of data acquisition; the counting mode in which single electrons are counted, and the integrating mode in which the signal that arises from the incident electrons is integrated. While counting mode leads to far higher detective quantum efficiency at all spatial frequencies, the integrating mode enables faster data acquisition at higher exposure rates. For optimal throughput at best possible resolution it is important to understand when the better performance in counting mode becomes essential for solving a structure and when the lower detective quantum efficiency in integrating mode can be compensated by increasing the number of particles in the data set. Here, we provide a case study of the Falcon III camera, which has counting mode capability at exposure rates of <0.9 e−/Px² and integrating mode capability at exposure rates above 10 e−/Px². We found that counting mode gives better resolution for medium sized complexes such as the β-galactosidase (465 kDa) (2.2 Å, 97% of Nyquist vs. 2.4 Å, 89% of Nyquist) with data sets of similar size. However, for larger particles such as Hepatitis B virus capsid like particles (4.8 MDa) we did not find any resolution gain in counting mode. [ABSTRACT FROM AUTHOR]