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72 Publications

Showing 1-10 of 72 results
02/13/17 | Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED.
de la Cruz MJason, Hattne J, Shi D, Seidler P, Rodriguez J, Reyes FE, Sawaya MR, Cascio D, Weiss SC, Kim SKyung, Hinck CS, Hinck AP, Calero G, Eisenberg D, Gonen T
Nature Methods. 2017 Feb 13:. 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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09/19/16 | 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 Sep 19:. 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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07/25/16 | Atomic resolution structure determination by the cryo-EM method MicroED.
Liu S, Hattne J, Reyes FE, Sanchez-Martinez S, de la Cruz MJason, Shi D, Gonen T
Protein Science : a Publication of the Protein Society. 2016 Jul 25:. doi: 10.1002/pro.2989

The electron cryo-microscopy (cryoEM) method MicroED has been rapidly developing. In this review we highlight some of the key steps in MicroED from crystal analysis to structure determination. We compare and contrast MicroED and the latest X-ray based diffraction method the X-ray free electron laser (XFEL). Strengths and shortcomings of both MicroED and XFEL are discussed. Finally, all current MicroED structures are tabulated with a view to the future. This article is protected by copyright. All rights reserved.

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07/22/16 | Accurate design of megadalton-scale two-component icosahedral protein complexes.
Bale JB, Gonen S, Liu Y, Sheffler W, Ellis D, Thomas C, Cascio D, Yeates TO, Gonen T, King NP, Baker D
Science (New York, N.Y.). 2016 Jul 22;353(6297):389-94. doi: 10.1126/science.aaf8818

Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

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06/16/16 | High-resolution macromolecular structure determination by MicroED, a cryo-EM method.
Rodriguez JA, Gonen T
Methods in Enzymology. 2016 Jun 16:. doi: 10.1016/bs.mie.2016.04.017

Microelectron diffraction (MicroED) is a new cryo-electron microscopy (cryo-EM) method capable of determining macromolecular structures at atomic resolution from vanishingly small 3D crystals. MicroED promises to solve atomic resolution structures from even the tiniest of crystals, less than a few hundred nanometers thick. MicroED complements frontier advances in crystallography and represents part of the rebirth of cryo-EM that is making macromolecular structure determination more accessible for all. Here we review the concept and practice of MicroED, for both the electron microscopist and crystallographer. Where other reviews have addressed specific details of the technique (Hattne et al., 2015Shi et al., 2016 and Shi et al., 2013), we aim to provide context and highlight important features that should be considered when performing a MicroED experiment.

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06/15/16 | Design of a hyperstable 60-subunit protein icosahedron.
Hsia Y, Bale JB, Gonen S, Shi D, Sheffler W, Fong KK, Nattermann U, Xu C, Huang P, Ravichandran R, Yi S, Davis TN, Gonen T, King NP, Baker D
Nature. 2016 Jun 15:. doi: 10.1038/nature18010

The icosahedron is the largest of the Platonic solids, and icosahedral protein structures are widely used in biological systems for packaging and transport. There has been considerable interest in repurposing such structures for applications ranging from targeted delivery to multivalent immunogen presentation. The ability to design proteins that self-assemble into precisely specified, highly ordered icosahedral structures would open the door to a new generation of protein containers with properties custom-tailored to specific applications. Here we describe the computational design of a 25-nanometre icosahedral nanocage that self-assembles from trimeric protein building blocks. The designed protein was produced in Escherichia coli, and found by electron microscopy to assemble into a homogenous population of icosahedral particles nearly identical to the design model. The particles are stable in 6.7 molar guanidine hydrochloride at up to 80 degrees Celsius, and undergo extremely abrupt, but reversible, disassembly between 2 molar and 2.25 molar guanidinium thiocyanate. The icosahedron is robust to genetic fusions: one or two copies of green fluorescent protein (GFP) can be fused to each of the 60 subunits to create highly fluorescent 'standard candles' for use in light microscopy, and a designed protein pentamer can be placed in the centre of each of the 20 pentameric faces to modulate the size of the entrance/exit channels of the cage. Such robust and customizable nanocages should have considerable utility in targeted drug delivery, vaccine design and synthetic biology.

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05/11/16 | Modeling truncated pixel values of faint reflections in MicroED images.
Hattne J, Shi D, de la Cruz MJason, Reyes FE, Gonen T
Journal of Applied Crystallography. 2016 May 11;49(3):. doi: 10.1107/S1600576716007196

The weak pixel counts surrounding the Bragg spots in a diffraction image are important for establishing a model of the background underneath the peak and estimating the reliability of the integrated intensities. Under certain circumstances, particularly with equipment not optimized for low-intensity measurements, these pixel values may be corrupted by corrections applied to the raw image. This can lead to truncation of low pixel counts, resulting in anomalies in the integrated Bragg intensities, such as systematically higher signal-to-noise ratios. A correction for this effect can be approximated by a three-parameter lognormal distribution fitted to the weakly positive-valued pixels at similar scattering angles. The procedure is validated by the improved refinement of an atomic model against structure factor amplitudes derived from corrected micro-electron diffraction (MicroED) images.

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05/01/16 | The collection of MicroED data for macromolecular crystallography.
Shi D, Nannenga BL, de la Cruz MJason, Liu J, Sawtelle S, Calero G, Reyes FE, Hattne J, Gonen T
Nature Protocols. 2016 May;11(5):895-904. doi: 10.1038/nprot.2016.046

The formation of large, well-ordered crystals for crystallographic experiments remains a crucial bottleneck to the structural understanding of many important biological systems. To help alleviate this problem in crystallography, we have developed the MicroED method for the collection of electron diffraction data from 3D microcrystals and nanocrystals of radiation-sensitive biological material. In this approach, liquid solutions containing protein microcrystals are deposited on carbon-coated electron microscopy grids and are vitrified by plunging them into liquid ethane. MicroED data are collected for each selected crystal using cryo-electron microscopy, in which the crystal is diffracted using very few electrons as the stage is continuously rotated. This protocol gives advice on how to identify microcrystals by light microscopy or by negative-stain electron microscopy in samples obtained from standard protein crystallization experiments. The protocol also includes information about custom-designed equipment for controlling crystal rotation and software for recording experimental parameters in diffraction image metadata. Identifying microcrystals, preparing samples and setting up the microscope for diffraction data collection take approximately half an hour for each step. Screening microcrystals for quality diffraction takes roughly an hour, and the collection of a single data set is ∼10 min in duration. Complete data sets and resulting high-resolution structures can be obtained from a single crystal or by merging data from multiple crystals.

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03/07/16 | Data publication with the structural biology data grid supports live analysis.
Meyer PA, Socias S, Key J, Ransey E, Tjon EC, Buschiazzo A, Lei M, Botka C, Withrow J, Neau D, Rajashankar K, Anderson KS, Baxter RH, Blacklow SC, Boggon TJ, Bonvin AM, Borek D, Brett TJ, Caflisch A, Chang C, Chazin WJ, Corbett KD, Cosgrove MS, Crosson S, Dhe-Paganon S, Di Cera E, Drennan CL, Eck MJ, Eichman BF, Fan QR, Ferré-D'Amaré AR, Christopher Fromme J, Garcia KChristopher, Gaudet R, Gong P, Harrison SC, Heldwein EE, Jia Z, Keenan RJ, Kruse AC, Kvansakul M, McLellan JS, Modis Y, Nam Y, Otwinowski Z, Pai EF, Pereira PJosé, Petosa C, Raman CS, Rapoport TA, Roll-Mecak A, Rosen MK, Rudenko G, Schlessinger J, Schwartz TU, Shamoo Y, Sondermann H, Tao YJ, Tolia NH, Tsodikov OV, Westover KD, Wu H, Foster I, Fraser JS, Maia FR, Gonen T, Kirchhausen T, Diederichs K, Crosas M, Sliz P
Nature Communications. 2016 Mar 07;7:10882. doi: 10.1038/ncomms10882

Access to experimental X-ray diffraction image data is fundamental for validation and reproduction of macromolecular models and indispensable for development of structural biology processing methods. Here, we established a diffraction data publication and dissemination system, Structural Biology Data Grid (SBDG; data.sbgrid.org), to preserve primary experimental data sets that support scientific publications. Data sets are accessible to researchers through a community driven data grid, which facilitates global data access. Our analysis of a pilot collection of crystallographic data sets demonstrates that the information archived by SBDG is sufficient to reprocess data to statistics that meet or exceed the quality of the original published structures. SBDG has extended its services to the entire community and is used to develop support for other types of biomedical data sets. It is anticipated that access to the experimental data sets will enhance the paradigm shift in the community towards a much more dynamic body of continuously improving data analysis.

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