Electron counting and beam-induced motion correction enable near-atomic-resolution single-particle cryo-EM

Abstract

In recent work with large high symmetry viruses, single particle electron cryomicroscopy (cryoEM) has reached the milestone of determining near atomic resolution structures by allowing direct fitting of atomic models into experimental density maps. However, achieving this goal with smaller particles of lower symmetry remains extraordinarily challenging. Using a newly developed single electron counting detector, we confirm that electron beam induced motion significantly degrades resolution and, importantly, show how the combination of rapid readout and nearly noiseless electron counting allow image blurring to be corrected to subpixel accuracy. Thus, intrinsic image information can be restored to high resolution (Thon rings visible to ~3 Å). Using this approach we determined a 3.3 Å resolution structure of a ~700 kDa protein with D7 symmetry showing clear side chain density. Our method greatly enhances image quality and data acquisition efficiency - key bottlenecks in applying near atomic resolution cryoEM to a broad range of protein samples.

Single particle electron cryomicroscopy: trends, issues and future perspective

“…Electron counting can be done on any of the current CMOS detectors but is too slow for normal use unless the frame rate is high. A reasonably high frame rate is already available commercially with the Gatan K2/Summit detector (Li et al 2013a). Any implementation of electron counting will benefit from minimising missed or overlapping events, which requires a high signal-to-noise ratio and a high frame rate.…”

Single-particle cryo-EM data acquisition by using direct electron detection camera

“…In general, photographic film provides a better detective quantum efficiency (DQE, a measure of camera performance as a function of spatial frequency) at high frequency than scintillator-based cameras [4,5]. It also has a larger size than all digital cameras.…”

“…These cameras use a thin layer of phosphor scintillator to convert signals from incident electrons to photons that are then recorded by the cameras. They typically have a poorer high-frequency DQE but a better low-frequency DQE than film [4,5]. Thus, images recorded using cameras with scintillator often have better contrast than photographic film recorded under the same condition.…”

“…All DDD cameras have significantly higher DQEs than photographic film and scintillator-based cameras [4,5,11]. The high frame rate of DDD cameras enables the recording of cryo-EM data as dose-fractionated image stacks instead of as a single micrograph [12].…”

The Diamond Light Source and the challenges ahead for structural biology: some informal remarks

“…Whereas XFEL requires large dedicated facilities similar in scale of investment to synchrotrons, cryo-electron microscopy (cryoEM) with the use of highly sensitive direct electron detectors is already capable of producing near-atomic resolution structures with no crystals at all [30,31]. In almost the inverse of crystallography, it is easier to determine structures by cryoEM for larger particles rather than smaller, because of the greater signal and the resulting ability to align individual particles.…”