Nikolaus Grigorieff

Visualizing the structures of small proteins and complexes has been a longstanding challenge in single-particle cryo-electron microscopy (cryo-EM). Some of these targets have been successfully resolved by binding to antibody fragments (Fabs) or fusing with external scaffolds to increase their size. Recent advances in conventional single-particle techniques have enabled the determination of an increasing number of structures smaller than 100 kDa, achieving resolutions relevant to drug research. Compared to X-ray crystallography, cryo-EM preserves the near-native states of biomolecules, can resolve structural heterogeneity, and has the potential to apply to a wide range of targets. In this work, we demonstrate that the alignment and reconstruction of small macromolecular complexes can be improved using high-resolution structures as priors, combined with 2D template matching. Using this method, we reconstructed a previously intractable ∼43 kDa protein kinase and improved the density of its ligand-binding site. Our theoretical analysis predicts that this method can further extend single-particle cryo-EM to important drug-binding complexes well below 50 kDa.

Start codon selection extensively regulates the cellular proteome and is implicated in cancer and other diseases. The eIF5-mimic proteins (5MPs: 5MP1/BZW2 and 5MP2/BZW1) restrict non-AUG start codon usage and thereby reprogram proteoform expression from mRNAs with alternative start sites, such as the oncogenic c-Myc. Here, using in extracto cryo–electron microscopy (cryo-EM) and biochemical assays, we report how 5MP represses translation dependent on the sequence context at start codons. Cryo-EM structures of 48 S preinitiation complexes (PICs) reveal 5MP1 bound at the A site of the small ribosomal subunit. 5MPs act as initiation factors that stabilize an open PIC conformation, promoting mRNA scanning and inhibiting initiation at suboptimal start codons. We propose to designate 5MP as eIF1S (eukaryotic initiation factor 1 scanning). These findings reveal the structural basis for proteome modulation by 5MPs and may help correct dysregulated translation in disease.

Cryogenic electron microscopy (cryo-EM) made impressive progress in resolving cellular macromolecules and their detailed interactions. Single-particle cryo-EM traditionally relies on purified macromolecules and lacks the complexity of cellular environments, whereas in situ cryo-EM and cryogenic electron tomography (cryo-ET) require extensive sample preparation and data acquisition, presenting challenges in achieving high resolution. We describe cryo-EM of cellular lysates— in extracto cryo-EM—allowing the flexibility and high-resolution of cryo-EM in the context of cellular components. High-resolution 2D template matching (2DTM) yields ~2.2 Å maps of the mammalian translational apparatus. Elongating ribosome abundances in primate cell lines (MCF-7 and BSC-1) and rabbit reticulocyte lysates range from ~70% to ~10%, reflecting translational stress responses. Non-translating (hibernating) ribosomes carrying no mRNA feature numerous proteins shielding ribosomal functional centers. Elongation factor 2 (eEF2) is the most abundant hibernation factor bound to >95% of 80S ribosomes and, unexpectedly, to 60S subunits. eEF2•GDP is stabilized by interactions with the sarcin-ricin loop and protein uL14. Hibernating ribosomes also feature La-related protein 1 (LARP1) involved in initiation and mTOR signaling, eIF5A implicated in elongation and termination, and other factors, exposing the variety of hibernation scenarios. Our work underscores the efficiency and potential of in extracto cryo-EM to discover native cellular complexes and mechanisms at near-atomic resolution.

Visualizing the structures of small proteins and complexes has been a longstanding challenge in single-particle cryo-EM. Some of these targets have been successfully resolved by binding to antibody fragments (Fabs) or fusing with external scaffolds to increase their size. Recent advances in conventional single-particle techniques have enabled the determination of an increasing number of structures smaller than 100 kDa, achieving resolutions relevant to drug research. Compared to X-ray crystallography, cryo-EM preserves the near-native states of biomolecules, can resolve structural heterogeneity, and has the potential to apply to a wide range of targets. In this work, we demonstrate that the alignment and reconstruction of small macromolecular complexes can be improved using high-resolution structures as priors combined with 2D template matching. Using this method, we reconstructed a previously intractable ∼ 43 kDa protein kinase and improved the density of its ligand-binding site. Our theoretical analysis predicts that this method can further extend single-particle cryo-EM to important drug-binding complexes well below 50 kDa. Cryo-EM | 3D reconstruction | signal detection | template matching 61

The exposure of frozen biological samples to the high-energy electron beam in a cryo-electron microscope commonly leads to beam-induced sample motion and distortions. Previously, we described Unblur , software to correct for beam-induced motion based on the alignment of full frames in a movie collected during the beam exposure (Grant and Grigorieff, 2015). Here, we present Unbend , extending Unblur by accommodating more localized sample bending and distortions using a 3D cubic B-spline model. Unbend is integrated into our cis TEM software with a new local motion visualization panel. We processed movie frames from various in situ sample types, including whole cells, lamellae, and cell lysates, to analyze motion behavior across different specimen types. To quantify the improvement in high-resolution signal, we utilized the 2D template matching method to search large ribosomal subunits from the motion-corrected micrographs. Overall, the signal-to-noise ratio of detected particles improved by 3–8% across different samples compared with full-frame aligned micrographs, while the number of detected target particles increased by up to ~300%. Furthermore, we processed micrograph montages to study motion patterns across an entire sample, revealing considerable variance in distortion scale within the same sample, suggesting a complex underlying mechanism.