We have used GroEL as a test bed to show that it is possible to achieve maps with a resolution on the order of 5-6Å, sufficient to discern the handedness of some α-helices, using relatively standard instrumentation (Tecnai F20, 4K×4K CCD, side entry cold stage) and a completely automated approach.
Our data illustrate one of the major issues in cryoEM and SPR: how to accurately assess resolution. We show that we get different values for resolution from the FSC0.5 and rmeasure methods. The resolution reported by rmeasure, however, tended to be more consistent with the observable details of the reconstructions than the FSC0.5. This was particularly evident in the comparison of reconstructions from data collected at different accelerating voltages (,). Reconstructions that had the same resolution as reported by the FSC0.5 method had dramatically different levels of detail as observed by visual inspection. The resolution reported by rmeasure, on the other hand, was more consistent with the details observed in the reconstructions. It is unclear why rmeasure was a better reporter of resolution, but the most likely interpretation is that it is less sensitive to noise bias.
Our data also demonstrate the importance of collecting large numbers of images. For all of our datasets, the resolution increases dramatically from 100 particles to about 2000 particles (equivalent to about 28,000 asymmetric subunits of GroEL), after which it requires exponentially more particles for relatively modest increases in resolution (Supplemental Fig. S4
). Nevertheless, while these later improvements in resolution are small, they are worth fighting for as they increase the confidence of interpretations based on secondary structure and other model building. We thus argue that it is always worth acquiring on the order of several tens of thousands of particles (corresponding to several hundreds of thousands of asymmetric units) for every dataset that will be subjected to single particle reconstruction. Achieving data throughput on this order has been the goal of the development of automated data acquisition methods in our lab (Suloway et al., 2005
Our highest resolution reconstruction of GroEL resulted from data collected at 120keV. This is likely the result of improved performance of the digital CCD cameras at the lower keV ranges. However, the influence of contrast on the alignment of particles and the effect of dose are complicating factors and it is clear that it will require additional experiments undertaken under more careful and exacting conditions before we can draw any conclusions on this issue.
At an accelerating voltage of 120keV, a dose of 19e-/Å2 appears to degrade the high-resolution features of the reconstruction; indicted by the fact that helices are not as clearly defined. We anticipated that when the number of particles were limiting (less than 10,000) the higher-dose data would have higher resolution because the images have higher signal-to-noise ratios, however in this case, the opposite is true. It is possible that the individual particles are starting to be degraded and they are losing features by which to classify them. In contrast, at an accelerating voltage of 200keV, a dose of 19e-/Å2 does not appear to make much difference. The resolution is very close to the lower-dose datasets, and similar features can be observed in the reconstructions, although it is possible that more features can be observed in the higher-dose dataset. The more likely conclusion is that we need to control the data acquisition parameters far more carefully to fully understand this issue.
We also showed that keeping track of particle orientation, as measured by the jump in Euler angle between iterations, was useful in eliminating bad particles from the reconstruction and improving the resolution. This method might be most useful in reducing the numbers of particles in the dataset at an early stage of the iterations and thus save considerable amounts of CPU time, particularly for very large data sets. The mean Euler jump measured across all particles also appears to be a good indicator of the quality of the data that is independent of number of particles and thus may be used to quickly identify problems in incoming data while the grid is still in the microscope. We intend to explore these issues further.
What will it take to achieve even higher resolution? One of the strongest resolution-limiting factors for our data is the high-frequency falloff characteristic of the CCD camera. We were able to considerably improve the resolutions of our reconstructions by increasing the magnification at which the data were collected. The results agree well with predictions from a detective quantum efficiency (DQE) (Mooney, 2007
) plot measured for our CCD camera (Supplemental Fig. S1
). The DQE plots also predict that we could further improve resolution by collecting data at higher magnifications such as 143,000X or 200,000X, though the improved DQE at higher magnification would need to be balanced against the fewer particles one could feasibly collect in any given session.
We have made a modest start at beginning to quantify some of the factors that affect the resolution of single particle reconstructions. We are well aware however that there is much more work to be done. Even for a relatively simple investigation like the dependence of resolution on image dose, factors like beam coherence and exposure time must be carefully measured and controlled (Chen et al., 2008
), and the performance of the particle alignment algorithms as a function of image contrast must be factored in and considered in the context of the magnification and defocus range of the images. Finding an optimal solution in this multi-parameter space requires careful experimental design built on a solid mathematical and statistical foundation. This will be the focus of future work in our lab.
Maps of the unfiltered volume and amplitude corrected volume of the highest-resolution 120keV, 100kX dataset have been deposited in the EM databank under the accession numbers 1457 and 1458, respectively.