Rapid tilt-series acquisition for electron cryotomography

Using a new Titan Krios stage equipped with a single-axis holder, we developed two methods to accelerate the collection of tilt-series. We demonstrate a continuous-tilting method that can record a tilt-series in seconds, but with loss of details finer than ～4?nm. We also demonstrate a fast-incremental method that can record a tilt-series several-fold faster than current methods and with similar resolution. We characterize the utility of both methods in real biological electron cryotomography workflows. We identify opportunities for further improvements in hardware and software and speculate on the impact such advances could have on structural biology.     

Consideration of sample motion in cryo-tomography based on alignment residual interpolation

Recently, it has been shown that the resolution in cryo-tomography could be improved by considering the sample motion in tilt-series alignment and reconstruction, where a set of quadratic polynomials were used to model this motion. One requirement of this polynomial method is the optimization of a large number of parameters, which may limit its practical applicability. In this work, we propose an alternative method for modeling the sample motion. Starting from the standard fiducial-based tilt-series alignment, the method uses the alignment residual as local estimates of the sample motion at the 3D fiducial positions. Then, a scattered data interpolation technique characterized by its smoothness and a closed-form solution is applied to model the sample motion. The motion model is then integrated in the tomographic reconstruction. The new method improves the tomogram quality similar to the polynomial one, with the important advantage that the determination of the motion model is greatly simplified, thereby overcoming one of the major limitations of the polynomial model. Therefore, the new method is expected to make the beam-induced motion correction methodology more accessible to the cryoET community.     

TomoAlign: A novel approach to correcting sample motion and 3D CTF in CryoET

TomoAlign is a software package that integrates tools to mitigate two important resolution limiting factors in cryoET, namely the beam-induced sample motion and the contrast transfer function (CTF) of the microscope. The package is especially focused on cryoET of thick specimens where fiducial markers are required for accurate tilt-series alignment and sample motion estimation. TomoAlign models the beam-induced sample motion undergone during the tilt-series acquisition. The motion models are used to produce motion-corrected subtilt-series centered on the particles of interest. In addition, the defocus of each particle at each tilt image is determined and can be corrected, resulting in motion-corrected and CTF-corrected subtilt-series from which the subtomograms can be computed. Alternatively, the CTF information can be passed on so that CTF correction can be carried out entirely within external packages like Relion. TomoAlign serves as a versatile tool that can streamline the cryoET workflow from initial alignment of tilt-series to final subtomogram averaging during in situ structure determination.