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79 Publications

Showing 1-10 of 79 results
09/07/17 | The role of disulfide bond replacements in analogues of the tarantula toxin ProTx-II and their effects on inhibition of the voltage-gated sodium ion channel Nav1.7.
Wright ZV, McCarthy S, Dickman R, Reyes FE, Sanchez-Martinez S, Cryar A, Kilford I, Hall A, Takle AK, Topf M, Gonen T, Thalassinos K, Tabor AB
Journal of the American Chemical Society. 2017 Sep 07:. doi: 10.1021/jacs.7b06506

Spider venom toxins, such as Protoxin-II (ProTx-II), have recently received much attention as selective Nav1.7 channel blockers, with potential to be developed as leads for the treatment of chronic nocioceptive pain. ProTx-II is a 30-amino acid peptide with three disulfide bonds that has been reported to adopt a well-defined inhibitory cystine knot (ICK) scaffold structure. Potential drawbacks with such peptides include poor pharmacodynamics and potential scrambling of the disulfide bonds in vivo. In order to address these issues, in the present study we report the solid-phase synthesis of lanthionine-bridged analogues of ProTx-II, in which one of the three disulfide bridges is replaced with a thioether linkage, and evaluate the biological properties of these analogues. We have also investigated the folding and disulfide bridging patterns arising from different methods of oxidation of the linear peptide precursor. Finally, we report the X-ray crystal structure of ProTx-II to atomic resolution; to our knowledge this is the first crystal structure of an ICK spider venom peptide not bound to a substrate.

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06/24/17 | MicroED structure of Au146(p-MBA)57 at subatomic resolution reveals a twinned FCC cluster.
Vergara S, Lukes DA, Martynowycz MW, Santiago U, Plascencia-Villa G, Weiss SC, de la Cruz MJ, Black DM, Alvarez MM, Lopez-Lozano X, Barnes CO, Lin G, Weissker H, Whetten RL, Gonen T, Calero G
Journal of Physical Chemistry Letters. 2017 Oct 31;8(5523-30):arXiv:1706.07902 [physics.atm-clus]. doi: 10.1021/acs.jpclett.7b02621

Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. We report the structure of Au146(p-MBA)57 at subatomic resolution (0.85 {\AA}) using electron diffraction (MicroED) and atomic resolution by X-ray diffraction. The 146 gold atoms may be decomposed into two constituent sets consisting of 119 core and 27 peripheral atoms. The core atoms are organized in a twinned FCC structure whereas the surface gold atoms follow a C2 rotational symmetry about an axis bisecting the twinning plane. The protective layer of 57 p-MBAs fully encloses the cluster and comprises bridging, monomeric, and dimeric staple motifs. Au146(p-MBA)57 is the largest cluster observed exhibiting a bulk-like FCC structure as well as the smallest gold particle exhibiting a stacking fault.

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06/22/17 | Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets.
Hughes MP, Sawaya MR, Goldschmidt L, Rodriguez JA, Cascio D, Gonen T, Eisenberg DS
bioRxiv. 2017 Jun 22:. doi: 10.1101/153817

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 Å 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.

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06/22/17 | Taking the measure of MicroED.
Rodriguez JA, Eisenberg DS, Gonen T
Current Opinion in Structural Biology. 2017 Jun 22;46:79-86. doi: 10.1016/

It is now possible to routinely determine atomic resolution structures by electron cryo-microscopy (cryoEM), facilitated in part by the method known as micro electron-diffraction (MicroED). Since its initial demonstration in 2013, MicroED has helped determine a variety of protein structures ranging in molecular weight from a few hundred Daltons to several hundred thousand Daltons. Some of these structures were novel while others were previously known. The resolutions of structures obtained thus far by MicroED range from 3.2Å to 1.0Å, with most better than 2.5Å. Crystals of various sizes and shapes, with different space group symmetries, and with a range of solvent content have all been studied by MicroED. The wide range of crystals explored to date presents the community with a landscape of opportunity for structure determination from nano crystals. Here we summarize the lessons we have learned during the first few years of MicroED, and from our attempts at the first ab initio structure determined by the method. We re-evaluate theoretical considerations in choosing the appropriate crystals for MicroED and for extracting the most meaning out of measured data. With more laboratories worldwide adopting the technique, we speculate what the first decade might hold for MicroED.

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06/21/17 | A method to minimize condenser lens-induced hysteresis effects in a JEOL JEM-3200FSC microscope to enable stable cryoEM low-dose operations.
de la Cruz MJ, Martynowycz M, Hattne J, Shi D, Gonen T
bioRxiv. 2017 Jun 21:. doi: 10.1101/153395

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.

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06/20/17 | MicroED structures from micrometer thick protein crystals.
Martynowycz M, Glynn C, Miao J, de la Cruz MJ, Hattne J, Shi D, Cascio D, Rodriguez J, Gonen T
bioRxiv. 2017 Jun 20:. doi: 10.1101/152504

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.

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04/25/17 | An Amidase_3 domain-containing N-acetylmuramyl-L-alanine amidase is required for mycobacterial cell division.
Senzani S, Li D, Bhaskar A, Ealand C, Chang J, Rimal B, Liu C, Joon Kim S, Dhar N, Kana B
Scientific Reports. 2017 Apr 25;7(1):1140. doi: 10.1038/s41598-017-01184-7

Mycobacteria possess a multi-layered cell wall that requires extensive remodelling during cell division. We investigated the role of an amidase_3 domain-containing N-acetylmuramyl-L-alanine amidase, a peptidoglycan remodelling enzyme implicated in cell division. We demonstrated that deletion of MSMEG_6281 (Ami1) in Mycobacterium smegmatis resulted in the formation of cellular chains, illustrative of cells that were unable to complete division. Suprisingly, viability in the Δami1 mutant was maintained through atypical lateral branching, the products of which proceeded to form viable daughter cells. We showed that these lateral buds resulted from mislocalization of DivIVA, a major determinant in facilitating polar elongation in mycobacterial cells. Failure of Δami1 mutant cells to separate also led to dysregulation of FtsZ ring bundling. Loss of Ami1 resulted in defects in septal peptidoglycan turnover with release of excess cell wall material from the septum or newly born cell poles. We noted signficant accumulation of 3-3 crosslinked muropeptides in the Δami1 mutant. We further demonstrated that deletion of ami1 leads to increased cell wall permeability and enhanced susceptiblity to cell wall targeting antibiotics. Collectively, these data provide novel insight on cell division in actinobacteria and highlights a new class of potential drug targets for mycobacterial diseases.

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02/13/17 | Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.
de la Cruz MJ, Hattne J, Shi D, Seidler P, Rodriguez J, Reyes FE, Sawaya MR, Cascio D, Weiss SC, Kim SK, Hinck CS, Hinck AP, Calero G, Eisenberg D, Gonen T
Nature Methods. 2017 Feb 13;14(4):399-402. doi: 10.1038/nmeth.4178

Traditionally, crystallographic analysis of macromolecules has depended on large, well-ordered crystals, which often require significant effort to obtain. Even sizable crystals sometimes suffer from pathologies that render them inappropriate for high-resolution structure determination. Here we show that fragmentation of large, imperfect crystals into microcrystals or nanocrystals can provide a simple path for high-resolution structure determination by the cryoEM method MicroED and potentially by serial femtosecond crystallography.

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01/03/17 | Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity.
Krotee P, Rodriguez JA, Sawaya MR, Cascio D, Reyes FE, Shi D, Hattne J, Nannenga BL, Oskarsson ME, Philipp S, Griner S, Jiang L, Glabe CG, Westermark GT, Gonen T, Eisenberg DS
eLife. 2017 Jan 03;6:. doi: 10.7554/eLife.19273

hIAPP fibrils are associated with Type-II Diabetes, but the link of hIAPP structure to islet cell death remains elusive. Here we observe that hIAPP fibrils are cytotoxic to cultured pancreatic β-cells, leading us to determine the structure and cytotoxicity of protein segments composing the amyloid spine of hIAPP. Using the cryoEM method MicroED, we discover that one segment, 19-29 S20G, forms pairs of β-sheets mated by a dry interface that share structural features with and are similarly cytotoxic to full-length hIAPP fibrils. In contrast, a second segment, 15-25 WT, forms non-toxic labile β-sheets. These segments possess different structures and cytotoxic effects, however, both can seed full-length hIAPP, and cause hIAPP to take on the cytotoxic and structural features of that segment. These results suggest that protein segment structures represent polymorphs of their parent protein and that segment 19-29 S20G may serve as a model for the toxic spine of hIAPP.

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10/04/16 | Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED.
Sawaya MR, Rodriguez J, Cascio D, Collazo MJ, Shi D, Reyes FE, Hattne J, Gonen T, Eisenberg DS
Proceedings of the National Academy of Sciences of the United States of America. 2016 Oct 04;113(40):11232-6. doi: 10.1073/pnas.1606287113

Electrons, because of their strong interaction with matter, produce high-resolution diffraction patterns from tiny 3D crystals only a few hundred nanometers thick in a frozen-hydrated state. This discovery offers the prospect of facile structure determination of complex biological macromolecules, which cannot be coaxed to form crystals large enough for conventional crystallography or cannot easily be produced in sufficient quantities. Two potential obstacles stand in the way. The first is a phenomenon known as dynamical scattering, in which multiple scattering events scramble the recorded electron diffraction intensities so that they are no longer informative of the crystallized molecule. The second obstacle is the lack of a proven means of de novo phase determination, as is required if the molecule crystallized is insufficiently similar to one that has been previously determined. We show with four structures of the amyloid core of the Sup35 prion protein that, if the diffraction resolution is high enough, sufficiently accurate phases can be obtained by direct methods with the cryo-EM method microelectron diffraction (MicroED), just as in X-ray diffraction. The success of these four experiments dispels the concern that dynamical scattering is an obstacle to ab initio phasing by MicroED and suggests that structures of novel macromolecules can also be determined by direct methods.

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