Biology subjects
Publications and source records attributed to Gonen, T..
We developed a procedure for the cryoEM method MicroED using SerialEM. With this approach, SerialEM coordinates stage rotation, microscope operation, and camera functions for automated continuous-rotation MicroED data collection. More than 300 datasets can be collected overnight in this way, facilitating high-throughput MicroED data collection for large-scale data analyses.
Microcrystal electron diffraction (MicroED) allows for macromolecular structure solution from nanocrystals. To create crystals of suitable size for MicroED data collection, sample preparation typically involves sonication or pipetting a slurry of crystals from a crystallization drop. The resultant crystal fragments are fragile and the quality of the data that can be obtained from them is sensitive to subsequent sample preparation for cryoEM as interactions in the water-air interface can damage crystals during blotting. Here, we demonstrate the use of a focused ion beam to generate lamellae of macromolecular protein crystals for continuous rotation MicroED that are of ideal thickness, easy to locate, and require no blotting optimization. In this manner, crystals of nearly any size may be scooped and milled to ideal dimensions prior to data collection, thus streamlining the methodology for sample preparation for MicroED.
mTORC1 is a central signal hub that integrates multiple environmental cues, such as cellular stresses, energy levels, nutrients and certain amino acids, to modulate metabolic status and cellular responses. Recently, SLC38A9, a lysosomal amino acid transporter, has emerged as a sensor for luminal arginine levels and as an activator of mTOCRC1. The activation of mTORC1 occurs through the N-terminal domain of SLC38A9. Here, we determined the crystal structure of SLC38A9 and surprisingly found its N-terminal fragment inserted deep into the transporter, bound in the substrate binding pocket where normally arginine would bind. Compared with our recent arginine bound structure of SLC38A9, a significant conformational change of the N-terminal domain was observed. A ball-and-chain model is proposed for mTORC1 activation where in the starved state the N-terminal domain of SLC38A9 is buried deep in the transporter but in the fed state the N-terminal domain could be released becoming free to bind the Rag GTPase complex and to activate mTORC1. This work provides important new insights into how SLC38A9 senses the fed state and activates the mTORC1 pathways in response to dietary amino acids.\n\nOne Sentence SummaryN-plug inserted state of SLC38A9 reveals mechanisms of mTORC1 activation and arginine-enhanced luminal amino acids efflux.
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.
Amino acid-dependent activation of mechanistic target of rapamycin complex 1 (mTORC1) is essential to reflect nutrient availabilities in cells for cell growth and metabolism1. Solute carrier 38 family A member 9 (SLC38A9) is the lysosomal transporter responsible for amino acid sensing in the mTORC1 signaling pathway2-4. Here we present the first crystal structure of SLC38A9 from Danio rerio in complex with arginine. As captured in the cytosol-open state, the bound arginine was locked in a transitional state stabilized by the transmembrane helix 1 (TM1) of SLC38A9 which was anchored at the grove between transmembrane helix 5 and 7 inside the transporter. The key motif WNTMM on TM1, contributing to the anchoring interactions, is highly conserved in various species. Mutations in WNTMM motif abolished arginine transport by SLC38A9. The underlying mechanism of substrate binding is critical for both sensitizing mTORC1 signaling pathway to amino acids and for maintaining amino acid homeostasis across lysosomal membranes2.
Micro-crystal electron diffraction (MicroED) is an emerging method in cryo-EM for structure determination using nanocrystals. It has been used to solve structures of a diverse set of biomolecules and materials, in some cases to sub-atomic resolution. However, little is known about the damaging effects of the electron beam on samples during such measurements. We assess global and site-specific damage from electron radiation on nanocrystals of proteinase K and of a prion hepta-peptide and find that the dynamics of electron-induced damage follow well-established trends observed in X-ray crystallography. Metal ions are perturbed, disulfide bonds are broken, and acidic side chains are decarboxylated while the diffracted intensities decay exponentially with increasing exposure. A better understanding of radiation damage in MicroED improves our assessment and processing of all types of cryo-EM data.
HIV-1 protease (PR) cleavage of the Gag polyprotein triggers the assembly of mature, infectious particles. Final cleavage of Gag occurs at the junction helix between the capsid protein CA and the SP1 spacer peptide. Here we used MicroED to delineate the binding interactions of the maturation inhibitor bevirimat (BVM) using very thin frozen-hydrated, three-dimensional microcrystals of a CTD-SP1 Gag construct with and without bound BVM. The 2.9-[A] MicroED structure revealed that a single BVM molecule stabilizes the 6-helix bundle via both electrostatic interactions with the dimethysuccinyl moiety and hydrophobic interactions with the pentacyclic triterpenoid ring. These results provide insight into the mechanism of action of BVM and related maturation inhibitors that will inform further drug discovery efforts. This study also demonstrates the capabilities of MicroED for structure-based drug design.\n\nOne-sentence summaryThe MicroED structure of HIV-1 Gag CTD-SP1 at 2.9 [A] resolution reveals the molecular basis for binding of the maturation inhibitor bevirimat that will inform further drug discovery efforts.
Current single particle electron cryo-microscopy (cryo-EM) techniques can produce images of large protein assemblies and macromolecular complexes at atomic level detail without the need for crystal growth. However, proteins of smaller size, typical of those found throughout the cell, are not presently amenable to detailed structural elucidation by cryo-EM. Here we use protein design to create a modular, symmetrical scaffolding system to make protein molecules of typical size amenable to cryo-EM. Using a rigid continuous alpha-helical linker, we connect a small 17 kDa protein (DARPin) to a protein subunit that was designed to self-assemble into a cage with cubic symmetry. We show that the resulting construct is amenable to structural analysis by single particle cryo-EM, allowing us to identify and solve the structure of the attached small protein at near-atomic detail, ranging from 3.5 to 5 [A] resolution. The result demonstrates that proteins considerably smaller than the theoretical limit of 50 kDa for cryo-EM can be visualized clearly when arrayed in a rigid fashion on a symmetric designed protein scaffold. Furthermore, because the amino acid sequence of a DARPin can be chosen to confer tight binding to various other protein or nucleic acid molecules, the system provides a future route for imaging diverse macromolecules, potentially broadening the application of cryoEM to proteins of typical size in the cell.\n\nSignificance statementNew electron microscopy methods are making it possible to view the structures of large proteins and nucleic acid complexes at atomic detail, but the methods are difficult to apply to molecules smaller than about 50 kDa, which is larger than the size of the average protein in the cell. The present work demonstrates that a protein much smaller than that limit can be successfully visualized when it is attached to a large protein scaffold designed to hold 12 copies of the attached protein in symmetric and rigidly defined orientations. The small protein chosen for attachment and visualization can be modified to bind to other diverse proteins, opening up a new avenue for imaging cellular proteins by cryo-EM.
Control of metabolism by compartmentation is a widespread feature of higher cells. Recent studies have focused on dynamic intracellular bodies such as stress granules, P-bodies, nucleoli, and metabolic puncta. These bodies appear as separate phases, some containing reversible, amyloid-like fibrils formed by interactions of low-complexity protein domains. Here we report five atomic structures of segments of low-complexity domains from granule-forming proteins, one determined to 1.1 [A] resolution by micro-electron diffraction. Four of these interacting protein segments show common characteristics, all in contrast to pathogenic amyloid: kinked peptide backbones, small surface areas of interaction, and predominate attractions between aromatic side-chains. By computationally threading the human proteome on three of our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such reversible interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, keratins, and cornified envelope proteins, consistent with the capacity of cells to form a wide variety of dynamic intracellular bodies.\n\nOne Sentence SummaryAtomic structures show transient membraneless organelles of cells formed by a new type of protein interaction akin to pathogenic amyloid fibrils.
Low dose imaging procedures are key for a successful cryoEM experiment (whether by electron cryotomography, single particle analysis, electron crystallography, or MicroED). We present a method to minimize magnetic hysteresis of the condenser lens system in the JEOL JEM-3200FSC transmission electron microscope (TEM) in order to maintain a stable optical axis for the beam path of low-dose imaging. The simple procedure involves independent voltage ramping of the CL1 and CL2 lenses immediately before switching to the focusing and exposure beam settings for data collection.\n\nHighlights O_LIEnables consistent horizontal beam position during low-dose mode switching\nC_LIO_LIFacilitates EM automated data collection for the JEOL JEM-3200FSC\nC_LIO_LIImproves beam stability in both TEM imaging and diffraction modes\nC_LI
Theoretical calculations suggest that crystals exceeding 100 nm thickness are excluded by dynamical scattering from successful structure determination using microcrystal electron diffraction (MicroED). These calculations are at odds with experimental results where MicroED structures have been determined from significantly thicker crystals. Here we systematically evaluate the influence of thickness on the accuracy of MicroED intensities and the ability to determine structures from protein crystals one micrometer thick. To do so, we compare ab initio structures of a human prion protein segment determined from thin crystals to those determined from crystals up to one micrometer thick. We also compare molecular replacement solutions from crystals of varying thickness for a larger globular protein, proteinase K. Our results indicate that structures can be reliably determined from crystals at least an order of magnitude thicker than previously suggested by simulation, opening the possibility for an even broader range of MicroED experiments.\n\nSummaryAtomic resolution protein structures can be determined by MicroED from crystals that surpass the theoretical maximum thickness limit by an order of magnitude.