Sample purity is central to in vitro studies of protein function and regulation, and to the efficiency and success of structural studies using techniques such as x-ray crystallography and cryo-electron microscopy (cryo-EM). Here, we show that mass photometry (MP) can accurately characterize the heterogeneity of a sample using minimal material with high resolution within a matter of minutes. To benchmark our approach, we use negative stain electron microscopy (nsEM), a popular method for EM sample screening. We include typical workflows developed for structure determination that involve multi-step purification of a multi-subunit ubiquitin ligase and chemical cross-linking steps. When assessing the integrity and stability of large molecular complexes such as the proteasome, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP over current methods for rapid sample characterization, prioritization and workflow optimization.

The viscoelastic response of complex fluids is length-and time-scale dependent, encoding information on intrinsic dynamic correlations and mesoscopic structure. We study the length scale above which bulk viscoelasticity sets in, and the material response that precedes it at shorter distances. We show that the crossover between these two regimes may appear at a surprisingly large distance. We generalize the framework of microrheology to include both regimes and apply it to F-actin networks, thereby extracting their dynamic correlation length from their bulk and local viscoelastic properties. PACS numbers: 47.57.Qk, 87.16.dm, 87.16.dj, 87.16.Ln Most fluids in nature and industry are complex, or structured [1], in the sense that they include mesoscopic elements in between the molecular and macroscopic scales. For example, in suspensions, micron-scale solid particles are dispersed in a molecular fluid, and in polymer gels the polymer chains form a network embedded within a molecular solvent. Consequently, the response of complex fluids to stress is characterized by intermediate length and time scales.The bulk viscoelastic response of such materials is commonly measured using macrorheology [2]. Similar information, for a wider frequency range and smaller material quantity, can be extracted from microrheology by following the motions of embedded tracer particles [3][4][5][6][7][8]. In one-point (1P) microrheology [3-5] the thermal fluctuations of a single particle are used to infer the viscoelastic properties of the medium via a generalized Stokes-Einstein relation (GSER). It has been found that this measurement is affected by the local environment of the tracer particle [9,10], and thus may fail to reproduce the material's bulk response. Two-point (2P) microrheology [6] overcomes this obstacle by tracking the correlated motions of particle pairs as a function of their separation. 2P measurements have focused on asymptotically large separations, where the pair correlation has a universal form due to momentum conservation.The current work addresses two questions: i) Beyond what length scale does the bulk viscoelastic behavior emerge? ii) What is the material response at smaller length scales? We find that the leading correction to the asymptotic behavior at large distances, referred to hereafter as the subdominant response, may be unexpectedly large, causing the bulk response to set in at surprisingly large distances. The physical origin of the subdominant response, which is unique to complex fluids, is different from that of the asymptotic one. It is related as well to a conservation law (of fluid mass rather than momentum), resulting in a generic system-independent form. The study of this distinctive regime leads to a more com-plete description of the complex-fluid response.We first derive the generic form of the subdominant response and, subsequently, confirm the general predictions in a specific theoretical example, the two-fluid model of polymer gels [8,11,12]. Extending the framework of microrheology to include...

Eukaryotic cells rely on their cytoskeleton to carry out coordinated motion, to transport materials within them, and to interact mechanically with their environment. To adapt to the changing requirements, the cell's cytoskeleton constantly remodels through the action of myosin II motor clusters that interact with numerous actin filaments simultaneously. Here we study the various roles of myosin II clusters in the formation and evolution of in vitro actomyosin networks as a model system for the cell's cytoskeleton.In our experiments the motor clusters can vary in size between 14 and 144 myosin II molecules and apply forces ranging from several to tens of piconewtons. During the initial process of network formation the motor clusters become embedded within the network structure, where they act as internal active cross-linkers. Myosin II clusters enhance the nucleation of network filaments/bundles in a concentration dependent manner, in the presence of the passive bundling protein fascin, thus functioning as a 'network co-nucleator'. As network formation is achieved, myosin II turns into a 'network reorganizer', where it takes part in remodeling and coarsening of the overall network structure. As a result of the strong confinement (the motor clusters within the network bundles exhibit high processivity with a fraction of attached motors p att $ 0.15), their effect on the nucleation and reorganization of the actin network is enhanced, rendering even small clusters of 14 myosin II molecules efficient. The stresses building-up in the networks lead to complex dynamics and can drive their contraction and rupture, depending on the motor concentration and cluster size. Above a certain concentration, the severing and disassembly properties of the motors dominate, and they function as 'network disassembly agents'. Myosin II motors are shown to be unique motors that function as complex machines that can perform a diversity of tasks, thereby regulating the nature of the assembled network and facilitating its formation.

The mechanical properties of polymer gels based on cytoskeleton proteins (e.g. actin) have been studied extensively due to their significant role in biological cell motility and in maintaining the cell's structural integrity. Microrheology is the natural method of choice for such studies due to its economy in sample volume, its wide frequency range, and its spatial sensitivity. In microrheology, the thermal motion of tracer particles embedded in a complex fluid is used to extract the fluid's viscoelastic properties. Comparing the motion of a single particle to the correlated motion of particle pairs, it is possible to extract viscoelastic properties at different length scales. In a recent study, a crossover between intermediate and bulk response of complex fluids was discovered in microrheology measurements of reconstituted actin networks. This crossover length was related to structural and mechanical properties of the networks, such as their mesh size and dynamic correlation length. Here we capitalize on this result giving a detailed description of our analysis scheme, and demonstrating how this relation can be used to extract the dynamic correlation length of a polymer network. We further study the relation between the dynamic correlation length and the structure of the network, by introducing a new length scale, the average filament length, without altering the network's mesh size. Contrary to the prevailing assumption, that the dynamic correlation length is equivalent to the mesh size of the network, we find that the dynamic correlation length increases once the filament length is reduced below the crossover distance.

The linear ubiquitin chain assembly complex (LUBAC) is the only known ubiquitin ligase for linear/Met1-linked ubiquitin chain formation. One of the LUBAC components, HOIL-1L, was recently shown to catalyse oxyester bond formation between ubiquitin and some substrates. However, oxyester bond formation in the context of LUBAC has not been directly observed. Here, we present the first 3D reconstruction of human LUBAC obtained by electron microscopy and report its generation of heterotypic ubiquitin chains containing linear linkages with oxyester-linked branches. We found that this event depends on HOIL-1L catalytic activity. By cross-linking mass spectrometry showing proximity between the catalytic RBR domains, a coordinated ubiquitin relay mechanism between the HOIP and HOIL-1L ligases is suggested. In mouse embryonic fibroblasts, these heterotypic chains were induced by TNF, which is reduced in cells expressing an HOIL-1L catalytic inactive mutant. In conclusion, we demonstrate that LUBAC assembles heterotypic ubiquitin chains by the concerted action of HOIP and HOIL-1L.