Monique S. Straub

Understanding and ultimately predicting the function of proteins is one of the frontiers in structural biology. This will only be possible if it becomes feasible to routinely observe proteins on the fast timescales on which they perform their tasks. Recently, laser flash melting and revitrification experiments have improved the time resolution of cryo-electron microscopy (cryo-EM) to microseconds, rendering it fast enough to observe the domain motions of proteins that are frequently linked to function. However, observations have been limited to a time window of just a few hundred microseconds. Here, we introduce time-resolved cryo-EM experiments based on jet vitrification that combine microsecond resolution with an observation window of up to seconds. We use a short laser pulse to initiate protein dynamics, and as they unfold, vitrify the sample with a jet of a liquid cryogen to arrest the dynamics at that point in time. We demonstrate that our approach affords near-atomic spatial resolution and a time resolution of 21 µs. This allows us to observe the photoinduced dynamics of the light-driven sodium pump Er NaR on the microsecond to millisecond timescale. Our experiments significantly expand the ability of cryo-EM to observe protein dynamics across multiple timescales.

Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air–water interface of the sample in a limited number of orientations. This makes it challenging to obtain high-resolution reconstructions, or may even cause projects to fail altogether. We have previously observed that laser flash melting and revitrification of cryo-EM samples reduces preferred orientation for large, symmetric particles. Here we demonstrate that our method can in fact be used to scramble the orientation of proteins of a range of sizes and symmetries. The effect can be enhanced for some proteins by increasing the heating rate during flash melting or by depositing amorphous ice onto the sample prior to revitrification. This also allows us to shed light onto the underlying mechanism. Our experiments establish a set of tools for overcoming preferred orientation that can be easily integrated into existing workflows.

Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting in loss of function of the enzyme methylmalonyl-CoA mutase (MMUT). Despite acute and persistent neurological symptoms, the pathogenesis of MMA in the central nervous system is poorly understood, which has contributed to a dearth of effective brain specific treatments. Here we utilised patient-derived induced pluripotent stem cells and in vitro differentiation to generate a human neuronal model of MMA. We reveal strong evidence of mitochondrial dysfunction caused by deficiency of MMUT in patient neurons. By employing patch-clamp electrophysiology, targeted metabolomics, and bulk transcriptomics, we expose an altered state of excitability, which is exacerbated by application of dimethyl-2-oxoglutarate, and we suggest may be connected to metabolic rewiring. Our work provides first evidence of mitochondrial driven neuronal dysfunction in MMA, which through our comprehensive characterisation of this paradigmatic model, enables first steps to identifying effective therapies.

Sample preparation remains a bottleneck for protein structure determination by cryo-electron microscopy. A frequently encountered issue is that proteins adsorb to the air-water interface of the sample in a limited number of orientations. This makes it challenging to obtain high-resolution reconstructions or may even cause projects to fail altogether. We have previously observed that laser flash melting and revitrification of cryo samples reduces preferred orientation for large, symmetric particles. Here, we demonstrate that our method can in fact be used to scramble the orientation of proteins of a range of sizes and symmetries. The effect can be enhanced for some proteins by increasing the heating rate during flash melting or by depositing amorphous ice onto the sample prior to revitrification. This also allows us to shed light onto the underlying mechanism. Our experiments establish a set of tools for overcoming preferred orientation that can be easily integrated into existing workflows.

Methylmalonic aciduria (MMA) is an inborn error of metabolism resulting in loss of function of the enzyme methylmalonyl-CoA mutase (MMUT). Despite acute and persistent neurological symptoms, the pathogenesis of MMA in the central nervous system is poorly understood, which has contributed to a dearth of effective brain specific treatments. Here we utilised patient-derived induced pluripotent stem cells and in vitro differentiation to generate a human neuronal model of MMA. We reveal strong evidence of mitochondrial dysfunction caused by deficiency of MMUT in patient neurons. By employing patch-clamp electrophysiology, targeted metabolomics, and bulk transcriptomics, we expose an altered state of excitability, which is exacerbated by application of 2-dimethyloxoglutarate, and we suggest may be connected to metabolic rewiring. Our work provides first evidence of mitochondrial driven neuronal dysfunction in MMA, which through our comprehensive characterisation of this paradigmatic model, enables first steps to identifying effective therapies.

The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe²⁺ and Mn²⁺ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg²⁺ uptake in prokaryotes and another the transport of Al³⁺ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg²⁺ selectivity (Ramanadane et al., 2022). Here, we have addressed the structural and functional properties of a putative Al³⁺ transporter from Setaria italica. We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al³⁺ and Ga³⁺, which are both presumable substrates. Its cryo-electron microscopy (cryo-EM) structure displays an occluded conformation that is closer to an inward- than an outward-facing state, with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.

The transport of transition metal ions by members of the SLC11/NRAMP family constitutes a ubiquitous mechanism for the uptake of Fe ²⁺ and Mn ²⁺ across all kingdoms of life. Despite the strong conservation of the family, two of its branches have evolved a distinct substrate preference with one mediating Mg ²⁺ uptake in prokaryotes and another the transport of Al ³⁺ into plant cells. Our previous work on the SLC11 transporter from Eggerthella lenta revealed the basis for its Mg ²⁺ selectivity (Ramanadane et al., 2022). Here we have addressed the structural and functional properties of a putative Al ³⁺ transporter from Setaria italica. We show that the protein transports diverse divalent metal ions and binds the trivalent ions Al ³⁺ and Ga ³⁺ , which are both presumable substrates. Its cryo-EM structure displays an occluded conformation that is closer to an inward-than an outward-facing state with a binding site that is remodeled to accommodate the increased charge density of its transported substrate.

TMEM16F, a member of the conserved TMEM16 family, plays a central role in the initiation of blood coagulation and the fusion of trophoblasts. The protein mediates passive ion and lipid transport in response to an increase in intracellular Ca²⁺. However, the mechanism of how the protein facilitates both processes has remained elusive. Here we investigate the basis for TMEM16F activation. In a screen of residues lining the proposed site of conduction, we identify mutants with strongly activating phenotype. Structures of these mutants determined herein by cryo-electron microscopy show major rearrangements leading to the exposure of hydrophilic patches to the membrane, whose distortion facilitates lipid diffusion. The concomitant opening of a pore promotes ion conduction in the same protein conformation. Our work has revealed a mechanism that is distinct for this branch of the family and that will aid the development of a specific pharmacology for a promising drug target.

Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca²⁺ and Mg²⁺, which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H⁺ serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg²⁺. The protein transports Mg²⁺ and Mn²⁺ but not Ca²⁺ by a mechanism that is not coupled to H⁺. Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.

Members of the ubiquitous SLC11/NRAMP family catalyze the uptake of divalent transition metal ions into cells. They have evolved to efficiently select these trace elements from a large pool of Ca ²⁺ and Mg ²⁺ , which are both orders of magnitude more abundant, and to concentrate them in the cytoplasm aided by the cotransport of H ⁺ serving as energy source. In the present study, we have characterized a member of a distant clade of the family found in prokaryotes, termed NRMTs, that were proposed to function as transporters of Mg ²⁺ . The protein transports Mg ²⁺ and Mn ²⁺ but not Ca ²⁺ by a mechanism that is not coupled to H ⁺ . Structures determined by cryo-EM and X-ray crystallography revealed a generally similar protein architecture compared to classical NRAMPs, with a restructured ion binding site whose increased volume provides suitable interactions with ions that likely have retained much of their hydration shell.