Machine learning approaches to cryoEM density modification differentially affect biomacromolecule and ligand density quality.

The application of machine learning to cryogenic electron microscopy (cryoEM) data analysis has added a valuable set of tools to the cryoEM data processing pipeline. As these tools become more accessible and widely available, the implications of their use should be assessed. We noticed that machine learning map modification tools can have differential effects on cryoEM densities. In this perspective, we evaluate these effects to show that machine learning tools generally improve densities for biomacromolecules while generating unpredictable results for ligands. This unpredictable behavior manifests both in quantitative metrics of map quality and in qualitative investigations of modified maps. The results presented here highlight the power and potential of machine learning tools in cryoEM, while also illustrating some of the risks of their unexamined use.

Chemical tools for study and modulation of biomolecular phase transitions.

Biomolecular phase transitions play an important role in organizing cellular processes in space and time. Methods and tools for studying these transitions, and the intrinsically disordered proteins (IDPs) that often drive them, are typically less developed than tools for studying their folded protein counterparts. In this perspective, we assess the current landscape of chemical tools for studying IDPs, with a specific focus on protein liquid-liquid phase separation (LLPS). We highlight methodologies that enable imaging and spectroscopic studies of these systems, including site-specific labeling with small molecules and the diverse range of capabilities offered by inteins and protein semisynthesis. We discuss strategies for introducing post-translational modifications that are central to IDP and LLPS function and regulation. We also investigate the nascent field of noncovalent small-molecule modulators of LLPS. We hope that this review of the state-of-the-art in chemical tools for interrogating IDPs and LLPS, along with an associated perspective on areas of unmet need, can serve as a valuable and timely resource for these rapidly expanding fields of study.

Real-time observation of structure and dynamics during the liquid-to-solid transition of FUS LC.

A subset of the proteins found in pathological protein fibrils also exhibit tendencies for liquid-liquid phase separation (LLPS) both in vitro and in cells. The mechanisms underlying the connection between these phase transitions have been challenging to study due to the heterogeneous and dynamic nature of the states formed during the maturation of LLPS protein droplets into gels and solid aggregates. Here, we interrogate the liquid-to-solid transition of the low-complexity domain of the RNA-binding protein FUS (FUS LC), which has been shown to adopt LLPS, gel-like, and amyloid states. We employ magic-angle-spinning NMR spectroscopy, which has allowed us to follow these transitions in real time and with residue-specific resolution. We observe the development of ß-sheet structure through the maturation process and show that the final state of FUS LC fibrils produced after LLPS is distinct from that grown from fibrillar seeds. We also apply our methodology to FUS LC G156E, a clinically relevant FUS mutant that exhibits accelerated fibrillization rates. We observe significant changes in dynamics during the transformation of the FUS LC G156E construct and begin to unravel the sequence specific contributions to this phenomenon with computational studies of the phase-separated state of FUS LC and FUS LC G156E.

Fused Split Inteins: Tools for Introducing Multiple Protein Modifications.

The split inteins from the DnaE cyanobacterial family are efficient and versatile tools for protein engineering and chemical biology applications. Their ultrafast splicing kinetics allow for the efficient production of native proteins from two separate polypeptides both in vitro and in cells. They can also be used to generate proteins with C-terminal thioesters for downstream applications. In this chapter, we describe a method based on a genetically fused version of the DnaE intein Npu for the preparation of doubly modified proteins through recombinant expression. In particular, we provide protocols for the recombinant production of modified ubiquitin through amber suppression where fused Npu is used (1) as a traceless purification tag or (2) as a protein engineering tool to introduce C-terminal modifications for subsequent attachment to other proteins of interest. Our purification protocol allows for quick and facile separation of truncated products and eliminates the need for engineering protease cleavage sites. Our approach can be easily adapted to different proteins and applications where the simultaneous presence of internal and C-terminal modifications is desirable.