Ribosome structures to near-atomic resolution from thirty thousand cryo-EM particles

Abstract

Although electron cryo-microscopy (cryo-EM) single-particle analysis has become an important tool for structural biology of large and flexible macro-molecular assemblies, the technique has not yet reached its full potential. Besides fundamental limits imposed by radiation damage, poor detectors and beam-induced sample movement have been shown to degrade attainable resolutions. A new generation of direct electron detectors may ameliorate both effects. Apart from exhibiting improved signal-to-noise performance, these cameras are also fast enough to follow particle movements during electron irradiation. Here, we assess the potentials of this technology for cryo-EM structure determination. Using a newly developed statistical movie processing approach to compensate for beam-induced movement, we show that ribosome reconstructions with unprecedented resolutions may be calculated from almost two orders of magnitude fewer particles than used previously. Therefore, this methodology may expand the scope of high-resolution cryo-EM to a broad range of biological specimens.DOI: http://dx.doi.org/10.7554/eLife.00461.001

Cryo-electron microscopy for structural biology: current status and future perspectives

“…Direct electron detection enhances the imaging quality because it eliminates electron-photon-electron data transfer and, more importantly, it can perform continuous imaging at high speeds. The video-like image capture, in combination with computer image processing, is able to eliminate most image blurring caused by instabilities in the sample or stage [5,6]. Last year, this significant technical breakthrough contributed to a dozen solved protein structures (and their atomic models), with resolutions beyond 4 Å. Yifan Cheng et al benefitted tremendously from this breakthrough in the TRVP1 structure determination ( Figure 6).…”

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

“…When using integration mode, the typical exposure time is relatively short, around 1 s. These 'movies' contain mostly charge-induced motion [13]. For K2 Summit operating in counting mode, coincidence loss is a major concern, and the optimal total exposure time is significantly longer.…”

“…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]. Collection of image stacks enables correction of stage-or beam-induced motion and significantly improves image quality by recovering the high-resolution signal deteriorated by that motion [4,13,14]. Furthermore, it provides an effective way to deal with rapid radiation damage, allowing images to be recorded with a significantly higher total electron dose because high-resolution noise from the later frames may be removed or down-weighted at a later stage of image processing [4,15].…”

“…Various tools are now available to correct image motion recorded in 'movie' stacks, such as Motioncorr [4], movie processing in RELION [13,27], optical flow [28] and alignparts lmbfgs [29]. Furthermore, tools that are used to align tilt series from tomographic data can be repurposed to align movie frames of single-particle data recorded with DDDs [18].…”

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.…”