By reaching near-atomic resolution for a wide range of specimens, single-particle cryo-EM structure determination is transforming structural biology. However, the necessary calculations come at large computational costs, which has introduced a bottleneck that is currently limiting throughput and the development of new methods. Here, we present an implementation of the RELION image processing software that uses graphics processors (GPUs) to address the most computationally intensive steps of its cryo-EM structure determination workflow. Both image classification and high-resolution refinement have been accelerated more than an order-of-magnitude, and template-based particle selection has been accelerated well over two orders-of-magnitude on desktop hardware. Memory requirements on GPUs have been reduced to fit widely available hardware, and we show that the use of single precision arithmetic does not adversely affect results. This enables high-resolution cryo-EM structure determination in a matter of days on a single workstation.

Here, we describe the third major release of RELION. CPU-based vector acceleration has been added in addition to GPU support, which provides flexibility in use of resources and avoids memory limitations. Reference-free autopicking with Laplacian-of-Gaussian filtering and execution of jobs from python allows non-interactive processing during acquisition, including 2D-classification, de novo model generation and 3D-classification. Per-particle refinement of CTF parameters and correction of estimated beam tilt provides higher resolution reconstructions when particles are at different heights in the ice, and/or coma-free alignment has not been optimal. Ewald sphere curvature correction improves resolution for large particles. We illustrate these developments with publicly available data sets: together with a Bayesian approach to beam-induced motion correction it leads to resolution improvements of 0.2-0.7 angstrom compared to previous RELION versions.

Oxygenic photosynthesis produces oxygen and builds a variety of organic compounds, changing the chemistry of the air, the sea and fuelling the food chain on our planet. The photochemical reactions underpinning this process in plants take place in the chloroplast. Chloroplasts evolved ~1.2 billion years ago from an engulfed primordial diazotrophic cyanobacterium, and chlororibosomes are responsible for synthesis of the core proteins driving photochemical reactions. Chlororibosomal activity is spatiotemporally coupled to the synthesis and incorporation of functionally essential co-factors, implying the presence of chloroplast-specific regulatory mechanisms and structural adaptation of the chlororibosome^1,2. Despite recent structural information^3,4,5,6, some of these aspects remained elusive. To provide new insights into the structural specialities and evolution, we report a comprehensive analysis of the 2.9–3.1 Å resolution electron cryo-microscopy structure of the spinach chlororibosome in complex with its recycling factor and hibernation-promoting factor. The model reveals a prominent channel extending from the exit tunnel to the chlororibosome exterior, structural re-arrangements that lead to increased surface area for translocon binding, and experimental evidence for parallel and convergent evolution of chloro- and mitoribosomes.

The introduction of direct detectors and the automation of data collection in cryo-EM have led to a surge in data, creating new opportunities for advancing computational processing. In particular, on-the-fly workflows that connect data collection with three-dimensional reconstruction would be valuable for more efficient use of cryo-EM and its application as a sample-screening tool. Here, accelerated on-the-fly analysis is reported with optimized organization of the data-processing tools, image acquisition and particle alignment that make it possible to reconstruct the three-dimensional density of the 70S chlororibosome to 3.2 angstrom resolution within 24 h of tissue harvesting. It is also shown that it is possible to achieve even faster processing at comparable quality by imposing some limits to data use, as illustrated by a 3.7 angstrom resolution map that was obtained in only 80 min on a desktop computer. These on-the-fly methods can be employed as an assessment of data quality from small samples and extended to high-throughput approaches.

The pyruvate dehydrogenase complex (PDC) is a central component of all aerobic respiration, connecting glycolysis to mitochondrial oxidation of pyruvate. Despite its central metabolic role, its precise composition and means of regulation remain unknown. To explain the variation in stoichiometry reported for the E3-recruiting protein X (PX) in the fungal PDC, we established cryo-EM reconstructions of the native and recombinant PDC from the filamentous fungus and model organism Neurospora crassa. We find that the PX C-terminal domain localizes interior to the E2 core. Critically, we show that two distinct arrangements of a trimeric oligomer exists, which both result in strict tetrahedral symmetry of the PDC core interior. Both oligomerization and volume occlusion of the PDC interior by PX appears to limit its binding stoichiometry, which explains the variety of stoichiometries found previously for S. cerevisiae. This also suggests that the PX oligomer stability and size are potential mechanisms to dynamically adjust PDC compostion in response to external cues. Moreover, we find that the site where PX binds is conserved within fungi but not mammals, suggesting that it could be therapeutically targeted. To this end, we also show that a PX knockout results in loss of activity through dysfunctional E3 recruitment, leading to severely impaired N. crassa growth on sucrose. The fungal PDC is thus shown to be fundamentally similar to the mammalian PDC in function but subject to other conditions of possible regulation, conditioned by a steric restrictions imposed by the symmetry of the PDC and its components.