CryoEM single particle reconstruction with a complex-valued particle stack

Abstract

Single particle reconstruction (SPR) in cryoEM is an image processing task with an elaborate hierarchy that starts with a large number of very noisy multi-frame images. Efficient representation of the intermediary image structures is critical for keeping the calculations manageable. One such intermediary structure is called a particle stack and contains cut-out images of particles in square boxes of predefined size. The micrograph that is the source of the boxed images is usually corrected for motion between frames prior to particle stack creation. However, the contrast transfer function (CTF) or its Fourier Transform point spread function (PSF) are not considered at this step. Historically, the particle stack was intended for large particles and for a tighter PSF, which is characteristic of lower resolution data. The field now performs analyses of smaller particles and to higher resolution, and these conditions result in a broader PSF that requires larger padding and slower calculations to integrate information for each particle. Consequently, the approach to handling structures such as the particle stack should be reexamined to optimize data processing. Here we propose to use as a source image for the particle stack an exit-wave-reconstruction-based image, in which CTF correction is implicitly applied as a real component of the image. The final CTF correction that we later refine and apply has a very narrow PSF, and so cutting out particles from micrographs that were approximately corrected for CTF does not require extended buffering, i.e. the boxes during the analysis only have to be large enough to encompass the particle. The Fourier Transform of an exit-wave reconstruction creates an image that has complex values. This is a complex value image considered in real space, opposed to standard SPR data processing where complex numbers appear only in Fourier space. This extension of the micrograph concept provides multiple advantages because the particle box size can be small while calculations crucial for high resolution reconstruction such as Ewald sphere correction, aberration refinement, and particlespecific defocus refinement can be performed on the small box data. Highlights{blacksquare} A complex-valued particle stack facilitates flexible and fast data processing {blacksquare}A real-valued, compact particle stack can be used at intermediate steps {blacksquare}Whole micrograph padding is used from the beginning of data analysis