Multi-particle cryo-EM refinement withMvisualizes ribosome-antibiotic complex at 3.7 Å inside cells

Abstract

Cryo-electron microscopy (cryo-EM) enables macromolecular structure determination in vitro and in situ. In addition to aligning individual particles, accurate registration of sample motion and 3D deformation during exposures is crucial for achieving high resolution. Here we describe M, a software tool that establishes a reference-based, multi-particle refinement framework for cryo-EM data and improves the results of structure determination. M provides a unified optimization framework for both in vitro frame series and in situ tomographic tilt series data. We show that tilt series data can provide the same resolution as frame series, indicating that the alignment step no longer limits the resolution obtainable from tomographic data. In combination with Warp and RELION, M improves upon previous methods, and resolves a 70S ribosome bound to an antibiotic inside bacterial cells at a nominal resolution of 3.7 Å. Thus, computational tools are now available to resolve structures from tomographic in situ cryo-EM data at residue level.Correspondence should be addressed to D.T. (dteguno@mpibpc.mpg.de), P.C. (patrick.cramer@mpibpc.mpg.de), and J.M. (julia.mahamid@embl.de). Figure 1| Th e Warp-RELION-M pipeline for frame and tilt series cryo-EM data refi nement Electron microscopy data are pre-processed on-the-fl y in Warp, which then exports particles as images or sub-tomograms. Particles are imported in RELION, where they can be subjected to a multitude of processing strategies, resulting in 3D reference maps, global particle pose alignments, and class assignments. Th e particle population encompassing all classes is then imported in M, where reference-based frame or tilt image alignments are performed simultaneously with further refi nement of particle poses and CTF parameters to improve resolution. Finally, M produces high-resolution reconstructions that can be used for model building. Alternatively, the improved alignments can be used in Warp to re-export particles for further, more accurate classifi cation in RELION.

Routine sub-2.5 Å cryo-EM structure determination of B-family G protein-coupled receptors

“…During the experiment setup, there is a natural urge to use higher defocus because it makes the molecules of the sample easier to see in the images. However, during processing, strongly delocalized high-resolution signals require more care and are more difficult to recover (Tegunov et al, 2020). Our experience showed that using lower defocus values, and the associated much lower image contrast, did not lessen the ability to locate and accurately align the particles during reconstruction.…”

“…Current particle handling strategies in the most popular image processing packages impose a restriction on the minimum particle box size as a function of the maximum defocus in the dataset that will preserve delocalized high-resolution components within the box. The recently introduced M package overcomes this limitation by performing a CTF sign correction on a larger area before extracting the particle box (Tegunov et al, 2020). This was shown to solve the resolution limitation of higher defocus values and could allow their use in the future without detrimental effects.…”

Challenges in solving structures from radiation-damaged tomograms of protein nanocrystals assessed by simulation

“…Despite these technical challenges, recent successes in cryo-ET of noncrystalline specimens support applying this technique to solve structures from nanocrystals to high resolution. In particular, subtomogram averages of purified immature HIV-1 Gag particles to 3.1Å and in situ ribosomes to 3.7Å demonstrate that high-resolution signal is available in tilt-series data [20,21]. As with SPR, only particles with high molecular weight can be aligned with sufficient accuracy for this high-resolution information to be retained during subtomogram averag-ing.…”

“…While the use of nanocrystals facilitates determining relative particle orientations, the low molecular weight of the constituent proteins makes finding a common phase origin -the other step of particle alignment -more challenging. High-resolution subtomogram averaging has exclusively targeted high molecular weight proteins, and with two exceptions, isolated particles in solvent [19][20][21]70]. These characteristics facilitate finding a common phase origin from each particle's center of mass.…”

“…In cryo-ET, a tilt-series of projection images is collected from a sample embedded in vitreous ice and reconstructed into a tomogram, a volume that contains 3D structural information about the specimen. This method has traditionally been used to study cellular ultrastructure, and the reconstruction approach of subtomogram averaging has recently determined nearatomic resolution (<5Å) structures of select purified proteins and ribosomes in situ [19][20][21]. Like microED, cryo-ET is suitable for protein nanocrystals, with an expected optimal sample thickness of 50-300 nm [22,23].…”