Recent advancements in single particle electron cryomicroscopy (cryoEM) have demonstrated its capability for determining near atomic resolution three-dimensional (3D) structures of large protein assemblies with high symmetry, such as non-enveloped viruses with1–5
or, in one case, without6
icosahedral symmetry. This resolution is sufficient to allow the path of the polypeptide chain to be directly traced in the experimental density maps obviating the need for prior structural information. While in principle such resolutions should be obtainable for smaller macromolecular complexes or those without high symmetry7
, in practice, obtainable resolutions have been in the 5 ~ 10 Å regime, sufficient for docking crystal structures but not for de novo structure determination.
Owing to the sensitivity of the sample to radiation damage, single particle cryoEM requires averaging a large number of low dose images of the same sample. Combining this data into a 3D reconstruction requires that microscope aberration parameters be determined for each micrograph as well as precise 3D orientation and translation for each individual particle image. While the quality of microscopes have been steadily improved, two key limiting factors have been the quality of the image-recording medium and image blurring caused by either instability of the sample stage or motion induced by the illuminating electron beam. Given the very low signal-to-noise ratio (SNR), image quality can significantly limit how accurately these parameters can be determined, hence the concern about the quality of the image detector. Of paramount importance is the efficiency of recording high-resolution information and reducing noise introduced by the detection process. The dominant electron image recording media are either photographic film or scintillator based charge-coupled device (CCD) cameras. However, neither satisfies the need for both high resolution (film being better) and high throughput image acquisition (only possible with electronic detectors).
The recent development of a new generation of complementary metal-oxide-semiconductor (CMOS) cameras can overcome both limiting factors. These new cameras directly detect incoming electrons in the silicon without the need for a scintillator, affording the advantages of a traditional CCD camera with greatly improved detective quantum efficiency (DQE) at high frequency, comparable to photographic film8,9,10
. A number of recent efforts have characterized the performance of the DE-12 camera from Direct Electron, LP (San Diego, CA)11,12
, and showed that the 3D reconstruction calculated from data recorded by this camera reached a level that is close to its Nyquist limit11
Image blurring resulting from the beam striking the sample is a major limiting factor to resolution when imaging frozen hydrated biological samples13
. In practice, beam induced motion seems impossible to prevent14,15
, and deteriorates most images from “perfect” to marginal for high-resolution cryoEM16
. The effect can be approximated as Gaussian blurring and quantified by analogy with the crystallographic temperature factor17–19
, predicting a greater than 5-fold decrease in signal at 3 Å13
. Thus beam-induced motion is a major limiting factor to routine high-resolution single particle cryoEM.
The high frame rate of new CMOS based direct cameras (10~40 frames/sec) provides a means to correct for such image blurring by recording a “movie” throughout the exposure. That is, a single exposure is fractionated into a number of subframes with sufficiently short duration that the motion is frozen to an acceptable level13
. The drawback is that while the signal decreases with fractionation, camera readout noise on each subframe remains constant15
. Even so, in the case of large objects such as viruses20,21
particles, it has been possible to use the subframes as if they were independent images during the refinement resulting in an increase in resolution. However, for smaller objects the much lower SNR of each particle within a single subframe compromises the ability to accurately determine particle orientation. For this reason, we focused on a detector that minimizes both detection and readout noise as well as choosing to correct beam induced motion at the level of either the whole frame or large fractions of a frame.
While detection efficiency is significantly improved with direct detection, a statistically varying amount of energy is deposited for each electron event (Landau noise), adding noise to the otherwise high quality detection process. To surmount this problem, the K2 Summit direct electron detection camera from Gatan, Inc. was designed to allow practical detection of individual electron events, thereby eliminating the Landau noise problem23,24
. This requires very rapid frame rate and fast electron detection algorithm to minimize counting two electrons striking the same pixel in quick succession as a single event (coincidence loss). The K2 camera has a fixed internal frame rate of 400 frames/sec and the ability to centroid the electron peak (super-resolution mode) to determine its location to sub-pixel accuracy. Once detected, individual electron events are then digitally accumulated over time. By counting the primary electrons rather than simply integrating charge, as is done in traditional cameras, both the Landau noise and the readout noise of the detector can be effectively rejected, thereby dramatically improving the DQE.
Here, we demonstrate that the K2 operated in electron counting mode is superior to both photographic film and traditional CCD cameras for imaging frozen hydrated samples. Most importantly, the near noiseless readout allows for accurate registration of dose-fractionated subframes collected throughout the exposure to correct for beam-induced motion. To aid in the correction, we developed an algorithm that maximizes the self-consistency of the derived image shifts. Combining electron counting imaging and motion correction, we determined a ~3.3 Å resolution 3D reconstruction of the archaeal 20S proteasome (~700 kDa and D7 symmetry).