2018
104.
Jones, Christopher G.; Martynowycz, Michael W.; Hattne, Johan; Fulton, Tyler J.; Stoltz, Brian M.; Rodriguez, Jose A.; Nelson, Hosea; Gonen, Tamir
The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination
ChemRxiv, 2018.
@unpublished{G.Jones2018,
title = {The CryoEM Method MicroED as a Powerful Tool for Small Molecule Structure Determination},
author = {Christopher G. Jones and Michael W. Martynowycz and Johan Hattne and Tyler J. Fulton and Brian M. Stoltz and Jose A. Rodriguez and Hosea Nelson and Tamir Gonen},
url = {https://chemrxiv.org/engage/chemrxiv/article-details/60c73f18469df44234f429c7},
doi = {10.26434/chemrxiv.7215332.v1},
year = {2018},
date = {2018-10-17},
urldate = {2018-10-17},
publisher = {American Chemical Society (ACS)},
abstract = {In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule’s structure requires X-ray and/or neutron diffraction studies. In practice, however, x-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the CryoEM method MicroED to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high quality MicroED data from nanocrystals (~100x100x100 nm, ~10–15 g) resulting in atomic resolution (<1 Å) crystal structures in minutes.},
howpublished = {ChemRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
In the many scientific endeavors that are driven by organic chemistry, unambiguous identification of small molecules is of paramount importance. Over the past 50 years, NMR and other powerful spectroscopic techniques have been developed to address this challenge. While almost all of these techniques rely on inference of connectivity, the unambiguous determination of a small molecule’s structure requires X-ray and/or neutron diffraction studies. In practice, however, x-ray crystallography is rarely applied in routine organic chemistry due to intrinsic limitations of both the analytes and the technique. Here we report the use of the CryoEM method MicroED to provide routine and unambiguous structural determination of small organic molecules. From simple powders, with minimal sample preparation, we could collect high quality MicroED data from nanocrystals (~100x100x100 nm, ~10–15 g) resulting in atomic resolution (<1 Å) crystal structures in minutes.
103.
Martynowycz, Michael W.; Zhao, Wei; Hattne, Johan; Jensen, Grant J.; Gonen, Tamir
Collection of continuous rotation MicroED Data from Ion Beam Milled Crystals of Any Size
bioRxiv, 2018.
@unpublished{Martynowycz2018,
title = {Collection of continuous rotation MicroED Data from Ion Beam Milled Crystals of Any Size},
author = {Michael W. Martynowycz and Wei Zhao and Johan Hattne and Grant J. Jensen and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/425611},
doi = {10.1101/425611},
year = {2018},
date = {2018-09-24},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract 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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
102.
Jinming, Ma; Lei, Hsiang-Ting; Gonen, Tamir
A conformational change in the N terminus of SLC38A9 signals mTORC1 activation
bioRxiv, 2018.
@unpublished{Jinming2018,
title = {A conformational change in the N terminus of SLC38A9 signals mTORC1 activation},
author = {Ma Jinming and Hsiang-Ting Lei and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/339937},
doi = {10.1101/339937},
year = {2018},
date = {2018-06-06},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract 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. One Sentence Summary N-plug inserted state of SLC38A9 reveals mechanisms of mTORC1 activation and arginine-enhanced luminal amino acids efflux.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
101.
Lei, Hsiang-Ting; Ma, Jinming; Martinez, Silvia Sanchez; Gonen, Tamir
Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state
In: Nat. Struct. Mol. Biol., vol. 25, no. 6, pp. 522–527, 2018.
@article{pmid29872228,
title = {Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state},
author = {Hsiang-Ting Lei and Jinming Ma and Silvia Sanchez Martinez and Tamir Gonen},
doi = {10.1038/s41594-018-0072-2},
year = {2018},
date = {2018-06-05},
journal = {Nat. Struct. Mol. Biol.},
volume = {25},
number = {6},
pages = {522--527},
abstract = {Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. 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 transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Recent advances in understanding intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on solute carrier 38, family A member 9 (SLC38A9), a lysosomal transporter responsible for the binding and translocation of several essential amino acids. 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 transmembrane helix 1 (TM1) of drSLC38A9, which was anchored at the groove between TM5 and TM7. These anchoring interactions were mediated by the highly conserved WNTMM motif in TM1, and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing the mTORC1 signaling pathway to amino acids and for maintenance of lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporters.
100.
Liu, Shian; Gonen, Tamir
MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter
In: Commun Biol, vol. 1, pp. 38, 2018.
@article{pmid30167468,
title = {MicroED structure of the NaK ion channel reveals a Na⁺ partition process into the selectivity filter},
author = {Shian Liu and Tamir Gonen},
doi = {10.1038/s42003-018-0040-8},
year = {2018},
date = {2018-05-03},
journal = {Commun Biol},
volume = {1},
pages = {38},
abstract = {Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Sodium (Na+) is a ubiquitous and important inorganic salt mediating many critical biological processes such as neuronal excitation, signaling, and facilitation of various transporters. The hydration states of Na+ are proposed to play critical roles in determining the conductance and the selectivity of Na+ channels, yet they are rarely captured by conventional structural biology means. Here we use the emerging cryo-electron microscopy (cryoEM) method micro-electron diffraction (MicroED) to study the structure of a prototypical tetrameric Na+-conducting channel, NaK, to 2.5 Å resolution from nano-crystals. Two new conformations at the external site of NaK are identified, allowing us to visualize a partially hydrated Na+ ion at the entrance of the channel pore. A process of dilation coupled with Na+ movement is identified leading to valuable insights into the mechanism of ion conduction and gating. This study lays the ground work for future studies using MicroED in membrane protein biophysics.
99.
Hattne, Johan; Shi, Dan; Glynn, Calina; Zee, Chih-Te; Gallagher-Jones, Marcus; Martynowycz, Michael W; Rodriguez, Jose A; Gonen, Tamir
Analysis of Global and Site-Specific Radiation Damage in Cryo-EM
In: Structure, vol. 26, no. 5, pp. 759–766, 2018.
@article{pmid29706530,
title = {Analysis of Global and Site-Specific Radiation Damage in Cryo-EM},
author = {Johan Hattne and Dan Shi and Calina Glynn and Chih-Te Zee and Marcus Gallagher-Jones and Michael W Martynowycz and Jose A Rodriguez and Tamir Gonen},
doi = {10.1016/j.str.2018.03.021},
year = {2018},
date = {2018-05-01},
journal = {Structure},
volume = {26},
number = {5},
pages = {759--766},
abstract = {Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Micro-crystal electron diffraction (MicroED) combines the efficiency of electron scattering with diffraction to allow structure determination from nano-sized crystalline samples in cryoelectron microscopy (cryo-EM). 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.
98.
Ma, Jinming; Lei, Hsiang-Ting; Reyes, Francis E.; Sanchez-Martinez, Silvia; Sarhan, Maen; Gonen, Tamir
Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS
bioRxiv, 2018.
@unpublished{Ma2018,
title = {Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS},
author = {Jinming Ma and Hsiang-Ting Lei and Francis E. Reyes and Silvia Sanchez-Martinez and Maen Sarhan and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/293811},
doi = {10.1101/293811},
year = {2018},
date = {2018-04-03},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract 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 fromMethanococcus 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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
97.
Lei, Hsiang-Ting; Ma, Jinming; Martinez, Silvia Sanchez; Gonen, Tamir
Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state
bioRxiv, 2018.
@unpublished{Lei2018,
title = {Crystal structure of arginine-bound lysosomal transporter SLC38A9 in the cytosol-open state},
author = {Hsiang-Ting Lei and Jinming Ma and Silvia Sanchez Martinez and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/288233},
doi = {10.1101/288233},
year = {2018},
date = {2018-03-24},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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 . },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
96.
Guenther, Elizabeth L; Ge, Peng; Trinh, Hamilton; Sawaya, Michael R; Cascio, Duilio; Boyer, David R; Gonen, Tamir; Zhou, Hong Z; Eisenberg, David S
Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2
In: Nat. Struct. Mol. Biol., vol. 25, no. 4, pp. 311–319, 2018.
@article{pmid29531287,
title = {Atomic-level evidence for packing and positional amyloid polymorphism by segment from TDP-43 RRM2},
author = {Elizabeth L Guenther and Peng Ge and Hamilton Trinh and Michael R Sawaya and Duilio Cascio and David R Boyer and Tamir Gonen and Hong Z Zhou and David S Eisenberg},
doi = {10.1038/s41594-018-0045-5},
year = {2018},
date = {2018-03-12},
journal = {Nat. Struct. Mol. Biol.},
volume = {25},
number = {4},
pages = {311--319},
abstract = {Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Proteins in the fibrous amyloid state are a major hallmark of neurodegenerative disease. Understanding the multiple conformations, or polymorphs, of amyloid proteins at the molecular level is a challenge of amyloid research. Here, we detail the wide range of polymorphs formed by a segment of human TAR DNA-binding protein 43 (TDP-43) as a model for the polymorphic capabilities of pathological amyloid aggregation. Using X-ray diffraction, microelectron diffraction (MicroED) and single-particle cryo-EM, we show that the 247DLIIKGISVHI257 segment from the second RNA-recognition motif (RRM2) forms an array of amyloid polymorphs. These associations include seven distinct interfaces displaying five different symmetry classes of steric zippers. Additionally, we find that this segment can adopt three different backbone conformations that contribute to its polymorphic capabilities. The polymorphic nature of this segment illustrates at the molecular level how amyloid proteins can form diverse fibril structures.
95.
Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Yeates, Todd O
Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system
In: Proc. Natl. Acad. Sci. U.S.A., vol. 115, no. 13, pp. 3362–3367, 2018.
@article{pmid29507202,
title = {Near-atomic cryo-EM imaging of a small protein displayed on a designed scaffolding system},
author = {Yuxi Liu and Shane Gonen and Tamir Gonen and Todd O Yeates},
doi = {10.1073/pnas.1718825115},
year = {2018},
date = {2018-03-05},
urldate = {2018-03-27},
journal = {Proc. Natl. Acad. Sci. U.S.A.},
volume = {115},
number = {13},
pages = {3362--3367},
abstract = {Current single-particle cryo-electron 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 suitable for 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-Å 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 cryo-EM to proteins of typical size in the cell.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Current single-particle cryo-electron 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 suitable for 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-Å 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 cryo-EM to proteins of typical size in the cell.
94.
Martynowycz, Michael W; Gonen, Tamir
From electron crystallography of 2D crystals to MicroED of 3D crystals
In: Curr Opin Colloid Interface Sci, vol. 34, pp. 9–16, 2018.
@article{pmid30166936,
title = {From electron crystallography of 2D crystals to MicroED of 3D crystals},
author = {Michael W Martynowycz and Tamir Gonen},
doi = {10.1016/j.cocis.2018.01.010},
year = {2018},
date = {2018-03-01},
journal = {Curr Opin Colloid Interface Sci},
volume = {34},
pages = {9--16},
abstract = {Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Electron crystallography is widespread in material science applications, but for biological samples its use has been restricted to a handful of examples where two-dimensional (2D) crystals or helical samples were studied either by electron diffraction and/or imaging. Electron crystallography in cryoEM, was developed in the mid-1970s and used to solve the structure of several membrane proteins and some soluble proteins. In 2013, a new method for cryoEM was unveiled and named Micro-crystal Electron Diffraction, or MicroED, which is essentially three-dimensional (3D) electron crystallography of microscopic crystals. This method uses truly 3D crystals, that are about a billion times smaller than those typically used for X-ray crystallography, for electron diffraction studies. There are several important differences and some similarities between electron crystallography of 2D crystals and MicroED. In this review, we describe the development of these techniques, their similarities and differences, and offer our opinion of future directions in both fields.
93.
Krotee, Pascal; Griner, Sarah L; Sawaya, Michael R; Cascio, Duilio; Rodriguez, Jose A; Shi, Dan; Philipp, Stephan; Murray, Kevin; Saelices, Lorena; Lee, Ji; Seidler, Paul; Glabe, Charles G; Jiang, Lin; Gonen, Tamir; Eisenberg, David S
In: J. Biol. Chem., vol. 293, no. 8, pp. 2888–2902, 2018.
@article{pmid29282295,
title = {Common fibrillar spines of amyloid-β and human islet amyloid polypeptide revealed by microelectron diffraction and structure-based inhibitors},
author = {Pascal Krotee and Sarah L Griner and Michael R Sawaya and Duilio Cascio and Jose A Rodriguez and Dan Shi and Stephan Philipp and Kevin Murray and Lorena Saelices and Ji Lee and Paul Seidler and Charles G Glabe and Lin Jiang and Tamir Gonen and David S Eisenberg},
doi = {10.1074/jbc.M117.806109},
year = {2018},
date = {2018-02-23},
urldate = {2017-02-23},
journal = {J. Biol. Chem.},
volume = {293},
number = {8},
pages = {2888--2902},
abstract = {Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aβ and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aβ and hIAPP.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Amyloid-β (Aβ) and human islet amyloid polypeptide (hIAPP) aggregate to form amyloid fibrils that deposit in tissues and are associated with Alzheimer's disease (AD) and type II diabetes (T2D), respectively. Individuals with T2D have an increased risk of developing AD, and conversely, AD patients have an increased risk of developing T2D. Evidence suggests that this link between AD and T2D might originate from a structural similarity between aggregates of Aβ and hIAPP. Using the cryoEM method microelectron diffraction, we determined the atomic structures of 11-residue segments from both Aβ and hIAPP, termed Aβ(24-34) WT and hIAPP(19-29) S20G, with 64% sequence similarity. We observed a high degree of structural similarity between their backbone atoms (0.96-Å root mean square deviation). Moreover, fibrils of these segments induced amyloid formation through self- and cross-seeding. Furthermore, inhibitors designed for one segment showed cross-efficacy for full-length Aβ and hIAPP and reduced cytotoxicity of both proteins, although by apparently blocking different cytotoxic mechanisms. The similarity of the atomic structures of Aβ(24-34) WT and hIAPP(19-29) S20G offers a molecular model for cross-seeding between Aβ and hIAPP.
92.
Hughes, Michael P; Sawaya, Michael R; Boyer, David R; Goldschmidt, Lukasz; Rodriguez, Jose A; Cascio, Duilio; Chong, Lisa; Gonen, Tamir; Eisenberg, David S
Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks
In: Science, vol. 359, no. 6376, pp. 698–701, 2018.
@article{pmid29439243,
title = {Atomic structures of low-complexity protein segments reveal kinked β sheets that assemble networks},
author = {Michael P Hughes and Michael R Sawaya and David R Boyer and Lukasz Goldschmidt and Jose A Rodriguez and Duilio Cascio and Lisa Chong and Tamir Gonen and David S Eisenberg},
doi = {10.1126/science.aan6398},
year = {2018},
date = {2018-02-09},
journal = {Science},
volume = {359},
number = {6376},
pages = {698--701},
abstract = {Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Subcellular membraneless assemblies are a reinvigorated area of study in biology, with spirited scientific discussions on the forces between the low-complexity protein domains within these assemblies. To illuminate these forces, we determined the atomic structures of five segments from protein low-complexity domains associated with membraneless assemblies. Their common structural feature is the stacking of segments into kinked β sheets that pair into protofilaments. Unlike steric zippers of amyloid fibrils, the kinked sheets interact weakly through polar atoms and aromatic side chains. By computationally threading the human proteome on our kinked structures, we identified hundreds of low-complexity segments potentially capable of forming such interactions. These segments are found in proteins as diverse as RNA binders, nuclear pore proteins, and keratins, which are known to form networks and localize to membraneless assemblies.
91.
Nannenga, Brent L; Gonen, Tamir
MicroED: a versatile cryoEM method for structure determination
In: Emerg Top Life Sci, vol. 2, no. 1, pp. 1–8, 2018.
@article{pmid30167465,
title = {MicroED: a versatile cryoEM method for structure determination},
author = {Brent L Nannenga and Tamir Gonen},
doi = {10.1042/ETLS20170082},
year = {2018},
date = {2018-02-06},
journal = {Emerg Top Life Sci},
volume = {2},
number = {1},
pages = {1--8},
abstract = {Micro-electron diffraction, or MicroED, is a structure determination method that uses a cryo-transmission electron microscope to collect electron diffraction data from nanocrystals. This technique has been successfully used to determine the high-resolution structures of many targets from crystals orders of magnitude smaller than what is needed for X-ray diffraction experiments. In this review, we will describe the MicroED method and recent structures that have been determined. Additionally, applications of electron diffraction to the fields of small molecule crystallography and materials science will be discussed.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Micro-electron diffraction, or MicroED, is a structure determination method that uses a cryo-transmission electron microscope to collect electron diffraction data from nanocrystals. This technique has been successfully used to determine the high-resolution structures of many targets from crystals orders of magnitude smaller than what is needed for X-ray diffraction experiments. In this review, we will describe the MicroED method and recent structures that have been determined. Additionally, applications of electron diffraction to the fields of small molecule crystallography and materials science will be discussed.
90.
Hattne, Johan; Shi, Dan; Glynn, Calina; Zee, Chih-Te; Gallagher-Jones, Marcus; Martynowycz, Michael W.; Rodriguez, Jose A.; Gonen, Tamir
Analysis of global and site-specific radiation damage in cryo-EM
bioRxiv, 2018.
@unpublished{Hattne2018,
title = {Analysis of global and site-specific radiation damage in cryo-EM},
author = {Johan Hattne and Dan Shi and Calina Glynn and Chih-Te Zee and Marcus Gallagher-Jones and Michael W. Martynowycz and Jose A. Rodriguez and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/258178},
doi = {10.1101/258178},
year = {2018},
date = {2018-02-01},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Summary 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. },
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
89.
Gallagher-Jones, Marcus; Glynn, Calina; Boyer, David R; Martynowycz, Michael W; Hernandez, Evelyn; Miao, Jennifer; Zee, Chih-Te; Novikova, Irina V; Goldschmidt, Lukasz; McFarlane, Heather T; Helguera, Gustavo F; Evans, James E; Sawaya, Michael R; Cascio, Duilio; Eisenberg, David S; Gonen, Tamir; Rodriguez, Jose A
Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp
In: Nat. Struct. Mol. Biol., vol. 25, no. 2, pp. 131–134, 2018.
@article{pmid29335561,
title = {Sub-ångström cryo-EM structure of a prion protofibril reveals a polar clasp},
author = {Marcus Gallagher-Jones and Calina Glynn and David R Boyer and Michael W Martynowycz and Evelyn Hernandez and Jennifer Miao and Chih-Te Zee and Irina V Novikova and Lukasz Goldschmidt and Heather T McFarlane and Gustavo F Helguera and James E Evans and Michael R Sawaya and Duilio Cascio and David S Eisenberg and Tamir Gonen and Jose A Rodriguez},
doi = {10.1038/s41594-017-0018-0},
year = {2018},
date = {2018-01-15},
journal = {Nat. Struct. Mol. Biol.},
volume = {25},
number = {2},
pages = {131--134},
abstract = {The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the β2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The atomic structure of the infectious, protease-resistant, β-sheet-rich and fibrillar mammalian prion remains unknown. Through the cryo-EM method MicroED, we reveal the sub-ångström-resolution structure of a protofibril formed by a wild-type segment from the β2-α2 loop of the bank vole prion protein. The structure of this protofibril reveals a stabilizing network of hydrogen bonds that link polar zippers within a sheet, producing motifs we have named 'polar clasps'.
2017
88.
Purdy, Michael D.; Shi, Dan; Chrustowicz, Jakub; Hattne, Johan; Gonen, Tamir; Yeager, Mark
bioRxiv, 2017.
@unpublished{Purdy2017,
title = {MicroED Structures of HIV-1 Gag CTD-SP1 Reveal Binding Interactions with the Maturation Inhibitor Bevirimat},
author = {Michael D. Purdy and Dan Shi and Jakub Chrustowicz and Johan Hattne and Tamir Gonen and Mark Yeager},
url = {http://biorxiv.org/lookup/doi/10.1101/241182},
doi = {10.1101/241182},
year = {2017},
date = {2017-12-30},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract 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-Å 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. One-sentence summary The MicroED structure of HIV-1 Gag CTD-SP1 at 2.9 Å resolution reveals the molecular basis for binding of the maturation inhibitor bevirimat that will inform further drug discovery efforts.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
87.
Seidler, P M; Boyer, D R; Rodriguez, J A; Sawaya, M R; Cascio, D; Murray, K; Gonen, T; Eisenberg, D S
Structure-based inhibitors of tau aggregation
In: Nat Chem, vol. 10, no. 2, pp. 170–176, 2017.
@article{pmid29359764,
title = {Structure-based inhibitors of tau aggregation},
author = {P M Seidler and D R Boyer and J A Rodriguez and M R Sawaya and D Cascio and K Murray and T Gonen and D S Eisenberg},
doi = {10.1038/nchem.2889},
year = {2017},
date = {2017-11-20},
journal = {Nat Chem},
volume = {10},
number = {2},
pages = {170--176},
abstract = {Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aggregated tau protein is associated with over 20 neurological disorders, which include Alzheimer's disease. Previous work has shown that tau's sequence segments VQIINK and VQIVYK drive its aggregation, but inhibitors based on the structure of the VQIVYK segment only partially inhibit full-length tau aggregation and are ineffective at inhibiting seeding by full-length fibrils. Here we show that the VQIINK segment is the more powerful driver of tau aggregation. Two structures of this segment determined by the cryo-electron microscopy method micro-electron diffraction explain its dominant influence on tau aggregation. Of practical significance, the structures lead to the design of inhibitors that not only inhibit tau aggregation but also inhibit the ability of exogenous full-length tau fibrils to seed intracellular tau in HEK293 biosensor cells into amyloid. We also raise the possibility that the two VQIINK structures represent amyloid polymorphs of tau that may account for a subset of prion-like strains of tau.
86.
Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Yeates, Todd O.
Near-Atomic Cryo-EM Imaging of a Small Protein Displayed on a Designed Scaffolding System
bioRxiv, 2017.
@unpublished{Liu2017,
title = {Near-Atomic Cryo-EM Imaging of a Small Protein Displayed on a Designed Scaffolding System},
author = {Yuxi Liu and Shane Gonen and Tamir Gonen and Todd O. Yeates},
url = {http://biorxiv.org/lookup/doi/10.1101/212233},
doi = {10.1101/212233},
year = {2017},
date = {2017-11-01},
urldate = {2017-11-01},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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 Å 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.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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 Å 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.
85.
Vergara, Sandra; Lukes, Dylan A; Martynowycz, Michael W; Santiago, Ulises; Plascencia-Villa, Germán; Weiss, Simon C; de la Cruz, Jason M; Black, David M; Alvarez, Marcos M; López-Lozano, Xochitl; Barnes, Christopher O; Lin, Guowu; Weissker, Hans-Christian; Whetten, Robert L; Gonen, Tamir; Yacaman, Miguel Jose; Calero, Guillermo
MicroED Structure of Au₁₄₆(p-MBA)₅₇ at Subatomic Resolution Reveals a Twinned FCC Cluster
In: J Phys Chem Lett, vol. 8, no. 22, pp. 5523–5530, 2017.
@article{pmid29072840,
title = {MicroED Structure of Au₁₄₆(p-MBA)₅₇ at Subatomic Resolution Reveals a Twinned FCC Cluster},
author = {Sandra Vergara and Dylan A Lukes and Michael W Martynowycz and Ulises Santiago and Germán Plascencia-Villa and Simon C Weiss and Jason M de la Cruz and David M Black and Marcos M Alvarez and Xochitl López-Lozano and Christopher O Barnes and Guowu Lin and Hans-Christian Weissker and Robert L Whetten and Tamir Gonen and Miguel Jose Yacaman and Guillermo Calero},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_vargara.pdf},
doi = {10.1021/acs.jpclett.7b02621},
year = {2017},
date = {2017-10-26},
journal = {J Phys Chem Lett},
volume = {8},
number = {22},
pages = {5523--5530},
abstract = {Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Solving the atomic structure of metallic clusters is fundamental to understanding their optical, electronic, and chemical properties. Herein we present the structure of the largest aqueous gold cluster, Au146(p-MBA)57 (p-MBA: para-mercaptobenzoic acid), solved by electron micro-diffraction (MicroED) to subatomic resolution (0.85 Å) and by X-ray diffraction at atomic resolution (1.3 Å). 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.
84.
Gonen, Tamir; Rodriguez, Jose; Schlichting, Ilme (Ed.)
Challenges in Structural Biology
HHMI Janelia Research Campus, Janelia Research Campus, 2017, (Conference held 2017-10-15 to 2015-10-18 at Janelia Research Campus).
@conference{2017Janelia,
title = {Challenges in Structural Biology},
editor = {Tamir Gonen and Jose Rodriguez and Ilme Schlichting},
year = {2017},
date = {2017-10-13},
urldate = {2017-00-00},
publisher = {Janelia Research Campus},
address = {Janelia Research Campus},
organization = {HHMI},
note = {Conference held 2017-10-15 to 2015-10-18 at Janelia Research Campus},
keywords = {},
pubstate = {published},
tppubtype = {conference}
}
83.
Rodriguez, Jose A; Eisenberg, David S; Gonen, Tamir
In: Curr. Opin. Struct. Biol., vol. 46, pp. 79–86, 2017.
@article{pmid28648726,
title = {Taking the measure of MicroED},
author = {Jose A Rodriguez and David S Eisenberg and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_rodriguez.pdf, Main text},
doi = {10.1016/j.sbi.2017.06.004},
year = {2017},
date = {2017-10-01},
journal = {Curr. Opin. Struct. Biol.},
volume = {46},
pages = {79--86},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
82.
Wright, Zoë V F; McCarthy, Stephen; Dickman, Rachel; Reyes, Francis E; Sanchez-Martinez, Silvia; Cryar, Adam; Kilford, Iian; Hall, Adrian; Takle, Andrew K; Topf, Maya; Gonen, Tamir; Thalassinos, Konstantinos; Tabor, Alethea B
In: J. Am. Chem. Soc., vol. 139, no. 37, pp. 13063–13075, 2017.
@article{pmid28880078,
title = {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},
author = {Zoë V F Wright and Stephen McCarthy and Rachel Dickman and Francis E Reyes and Silvia Sanchez-Martinez and Adam Cryar and Iian Kilford and Adrian Hall and Andrew K Takle and Maya Topf and Tamir Gonen and Konstantinos Thalassinos and Alethea B Tabor},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_wright.pdf, Main text},
doi = {10.1021/jacs.7b06506},
year = {2017},
date = {2017-09-07},
journal = {J. Am. Chem. Soc.},
volume = {139},
number = {37},
pages = {13063--13075},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
81.
Hughes, Michael P.; Sawaya, Michael R.; Goldschmidt, Lukasz; Rodriguez, Jose A.; Cascio, Duilio; Gonen, Tamir; Eisenberg, David S.
Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets
bioRxiv, 2017.
@unpublished{Hughes2017,
title = {Low-complexity domains adhere by reversible amyloid-like interactions between kinked β-sheets},
author = {Michael P. Hughes and Michael R. Sawaya and Lukasz Goldschmidt and Jose A. Rodriguez and Duilio Cascio and Tamir Gonen and David S. Eisenberg},
url = {http://biorxiv.org/lookup/doi/10.1101/153817},
doi = {10.1101/153817},
year = {2017},
date = {2017-06-22},
publisher = {Cold Spring Harbor Laboratory},
abstract = {Abstract 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. One Sentence Summary Atomic structures show transient membraneless organelles of cells formed by a new type of protein interaction akin to pathogenic amyloid fibrils.
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
80.
de la Cruz, M. Jason; Martynowycz, Michael; Hattne, Johan; Shi, Dan; Gonen, Tamir
bioRxiv 2017.
@online{2017_delacruz_c,
title = {A method to minimize condenser lens-induced hysteresis effects in a JEOL JEM-3200FSC microscope to enable stable cryoEM low-dose operations},
author = {de la Cruz, M. Jason and Martynowycz, Michael and Hattne, Johan and Shi, Dan and Gonen, Tamir},
doi = {10.1101/153395},
year = {2017},
date = {2017-06-21},
organization = {bioRxiv},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
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.
79.
Martynowycz, Michael W.; Glynn, Calina; Miao, Jennifer; de la Cruz, M. Jason; Hattne, Johan; Shi, Dan; Cascio, Duilio; Rodriguez, Jose; Gonen, Tamir
MicroED Structures from Micrometer Thick Protein Crystals
bioRxiv, 2017.
@unpublished{Martynowycz2017,
title = {MicroED Structures from Micrometer Thick Protein Crystals},
author = {Michael W. Martynowycz and Calina Glynn and Jennifer Miao and M. Jason de la Cruz and Johan Hattne and Dan Shi and Duilio Cascio and Jose Rodriguez and Tamir Gonen},
url = {http://biorxiv.org/lookup/doi/10.1101/152504},
doi = {10.1101/152504},
year = {2017},
date = {2017-06-20},
urldate = {2017-06-20},
publisher = {Cold Spring Harbor Laboratory},
abstract = {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.},
howpublished = {bioRxiv},
keywords = {},
pubstate = {published},
tppubtype = {unpublished}
}
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.
78.
de la Cruz, M. Jason; Hattne, Johan; Shi, Dan; Rodriguez, Jose A.; Reyes, Francis E.; Gonen, Tamir
Nature Protocol Exchange 2017.
@online{2017_delacruz_b,
title = {Micro- and nanocrystal preparation for MicroED and XFEL serial crystallography by fragmentation of imperfect crystals},
author = {de la Cruz, M. Jason and Hattne, Johan and Shi, Dan and Rodriguez, Jose A. and Reyes, Francis E. and Gonen, Tamir},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_delacruz_b.pdf},
doi = {10.1038/protex.2017.010},
year = {2017},
date = {2017-02-15},
journal = {Nature Protocol Exchange},
organization = {Nature Protocol Exchange},
abstract = {This protocol describes different techniques to fragment protein crystals for high-resolution structure determination by MicroED and serial crystallography at an X-ray free electron laser. These techniques involve agitation of the crystal of interest: 1) by vigorous pipetting, 2) by sonication, and 3) by vortexing. Aside from setup, the time needed to perform these steps ranges from seconds to minutes.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
This protocol describes different techniques to fragment protein crystals for high-resolution structure determination by MicroED and serial crystallography at an X-ray free electron laser. These techniques involve agitation of the crystal of interest: 1) by vigorous pipetting, 2) by sonication, and 3) by vortexing. Aside from setup, the time needed to perform these steps ranges from seconds to minutes.
77.
de la Cruz, Jason M; Hattne, Johan; Shi, Dan; Seidler, Paul; Rodriguez, Jose; Reyes, Francis E; Sawaya, Michael R; Cascio, Duilio; Weiss, Simon C; Kim, Sun Kyung; Hinck, Cynthia S; Hinck, Andrew P; Calero, Guillermo; Eisenberg, David; Gonen, Tamir
Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED
In: Nat. Methods, vol. 14, no. 4, pp. 399–402, 2017.
@article{pmid28192420,
title = {Atomic-resolution structures from fragmented protein crystals with the cryoEM method MicroED},
author = {Jason M de la Cruz and Johan Hattne and Dan Shi and Paul Seidler and Jose Rodriguez and Francis E Reyes and Michael R Sawaya and Duilio Cascio and Simon C Weiss and Sun Kyung Kim and Cynthia S Hinck and Andrew P Hinck and Guillermo Calero and David Eisenberg and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_delacruz_a.pdf, Main text},
doi = {10.1038/nmeth.4178},
year = {2017},
date = {2017-02-13},
journal = {Nat. Methods},
volume = {14},
number = {4},
pages = {399--402},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
76.
Krotee, Pascal; Rodriguez, Jose A; Sawaya, Michael R; Cascio, Duilio; Reyes, Francis E; Shi, Dan; Hattne, Johan; Nannenga, Brent L; Oskarsson, Marie E; Philipp, Stephan; Griner, Sarah; Jiang, Lin; Glabe, Charles G; Westermark, Gunilla T; Gonen, Tamir; Eisenberg, David S
In: Elife, vol. 6, 2017.
@article{pmid28045370,
title = {Atomic structures of fibrillar segments of hIAPP suggest tightly mated β-sheets are important for cytotoxicity},
author = {Pascal Krotee and Jose A Rodriguez and Michael R Sawaya and Duilio Cascio and Francis E Reyes and Dan Shi and Johan Hattne and Brent L Nannenga and Marie E Oskarsson and Stephan Philipp and Sarah Griner and Lin Jiang and Charles G Glabe and Gunilla T Westermark and Tamir Gonen and David S Eisenberg},
url = {https://cryoem.ucla.edu/wp-content/uploads/2017_krotee.pdf, Main text},
doi = {10.7554/eLife.19273},
year = {2017},
date = {2017-01-03},
journal = {Elife},
volume = {6},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
2016
75.
Nannenga, Brent L; Gonen, Tamir
MicroED opens a new era for biological structure determination
In: Curr. Opin. Struct. Biol., vol. 40, pp. 128–135, 2016.
@article{pmid27701014,
title = {MicroED opens a new era for biological structure determination},
author = {Brent L Nannenga and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_nannengagonen.pdf, Main text},
doi = {10.1016/j.sbi.2016.09.007},
year = {2016},
date = {2016-10-01},
journal = {Curr. Opin. Struct. Biol.},
volume = {40},
pages = {128--135},
abstract = {In 2013 we unveiled the cryo-electron microscopy (CryoEM) method of MicroED, or three-dimensional (3D) electron diffraction of microscopic crystals. Here tiny 3D crystals of biological material are used in an electron microscope for diffraction data collection under cryogenic conditions. The data is indexed, integrated, merged and scaled using standard X-ray crystallography software to determine structures at atomic resolution. In this review we provide an overview of the MicroED method and compare it with other CryoEM methods.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In 2013 we unveiled the cryo-electron microscopy (CryoEM) method of MicroED, or three-dimensional (3D) electron diffraction of microscopic crystals. Here tiny 3D crystals of biological material are used in an electron microscope for diffraction data collection under cryogenic conditions. The data is indexed, integrated, merged and scaled using standard X-ray crystallography software to determine structures at atomic resolution. In this review we provide an overview of the MicroED method and compare it with other CryoEM methods.
74.
Sawaya, Michael R; Rodriguez, Jose; Cascio, Duilio; Collazo, Michael J; Shi, Dan; Reyes, Francis E; Hattne, Johan; Gonen, Tamir; Eisenberg, David S
Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED
In: Proc. Natl. Acad. Sci. U.S.A., vol. 113, no. 40, pp. 11232–11236, 2016.
@article{pmid27647903,
title = {Ab initio structure determination from prion nanocrystals at atomic resolution by MicroED},
author = {Michael R Sawaya and Jose Rodriguez and Duilio Cascio and Michael J Collazo and Dan Shi and Francis E Reyes and Johan Hattne and Tamir Gonen and David S Eisenberg},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_sawaya.pdf, Main text},
doi = {10.1073/pnas.1606287113},
year = {2016},
date = {2016-09-19},
journal = {Proc. Natl. Acad. Sci. U.S.A.},
volume = {113},
number = {40},
pages = {11232--11236},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
73.
Liu, Shian; Hattne, Johan; Reyes, Francis E; Sanchez-Martinez, Silvia; de la Cruz, Jason M; Shi, Dan; Gonen, Tamir
Atomic resolution structure determination by the cryo-EM method MicroED
In: Protein Sci., vol. 26, no. 1, pp. 8–15, 2016.
@article{pmid27452773,
title = {Atomic resolution structure determination by the cryo-EM method MicroED},
author = {Shian Liu and Johan Hattne and Francis E Reyes and Silvia Sanchez-Martinez and Jason M de la Cruz and Dan Shi and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_liu.pdf, Main text},
doi = {10.1002/pro.2989},
year = {2016},
date = {2016-07-25},
urldate = {2017-07-25},
journal = {Protein Sci.},
volume = {26},
number = {1},
pages = {8--15},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
72.
Bale, Jacob B; Gonen, Shane; Liu, Yuxi; Sheffler, William; Ellis, Daniel; Thomas, Chantz; Cascio, Duilio; Yeates, Todd O; Gonen, Tamir; King, Neil P; Baker, David
Accurate design of megadalton-scale two-component icosahedral protein complexes
In: Science, vol. 353, no. 6297, pp. 389–394, 2016.
@article{pmid27463675,
title = {Accurate design of megadalton-scale two-component icosahedral protein complexes},
author = {Jacob B Bale and Shane Gonen and Yuxi Liu and William Sheffler and Daniel Ellis and Chantz Thomas and Duilio Cascio and Todd O Yeates and Tamir Gonen and Neil P King and David Baker},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_bale.pdf, Main text},
doi = {10.1126/science.aaf8818},
year = {2016},
date = {2016-07-22},
journal = {Science},
volume = {353},
number = {6297},
pages = {389--394},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
71.
Hsia, Yang; Bale, Jacob B; Gonen, Shane; Shi, Dan; Sheffler, William; Fong, Kimberly K; Nattermann, Una; Xu, Chunfu; Huang, Po‐Ssu.; Ravichandran, Rashmi; Yi, Sue; Davis, Trisha N; Gonen, Tamir; King, Neil P; Baker, David
Design of a hyperstable 60-subunit protein icosahedron
In: Nature, vol. 535, no. 7610, pp. 136–139, 2016.
@article{pmid27309817,
title = {Design of a hyperstable 60-subunit protein icosahedron},
author = {Yang Hsia and Jacob B Bale and Shane Gonen and Dan Shi and William Sheffler and Kimberly K Fong and Una Nattermann and Chunfu Xu and Po‐Ssu. Huang and Rashmi Ravichandran and Sue Yi and Trisha N Davis and Tamir Gonen and Neil P King and David Baker},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_hsia.pdf, Main text},
doi = {10.1038/nature18010},
year = {2016},
date = {2016-06-15},
journal = {Nature},
volume = {535},
number = {7610},
pages = {136--139},
abstract = {The dodecahedron [corrected] 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 dodecahedron [corrected] 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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The dodecahedron [corrected] 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 dodecahedron [corrected] 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.
70.
Hattne, Johan; Shi, Dan; de la Cruz, Jason M; Reyes, Francis E; Gonen, Tamir
Modeling truncated pixel values of faint reflections in MicroED images
In: J Appl Crystallogr, vol. 49, no. Pt 3, pp. 1029–1034, 2016.
@article{pmid27275145,
title = {Modeling truncated pixel values of faint reflections in MicroED images},
author = {Johan Hattne and Dan Shi and Jason M de la Cruz and Francis E Reyes and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/hattne_2016.pdf, Main text},
doi = {10.1107/S1600576716007196},
year = {2016},
date = {2016-06-01},
journal = {J Appl Crystallogr},
volume = {49},
number = {Pt 3},
pages = {1029--1034},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
69.
Shi, Dan; Nannenga, Brent L; de la Cruz, Jason M; Liu, Jinyang; Sawtelle, Steven; Calero, Guillermo; Reyes, Francis E; Hattne, Johan; Gonen, Tamir
The collection of MicroED data for macromolecular crystallography
In: Nat Protoc, vol. 11, no. 5, pp. 895–904, 2016.
@article{pmid27077331,
title = {The collection of MicroED data for macromolecular crystallography},
author = {Dan Shi and Brent L Nannenga and Jason M de la Cruz and Jinyang Liu and Steven Sawtelle and Guillermo Calero and Francis E Reyes and Johan Hattne and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_shi.pdf, Main text},
doi = {10.1038/nprot.2016.046},
year = {2016},
date = {2016-04-14},
journal = {Nat Protoc},
volume = {11},
number = {5},
pages = {895--904},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
68.
Meyer, Peter A; Socias, Stephanie; Key, Jason; Ransey, Elizabeth; Tjon, Emily C; Buschiazzo, Alejandro; Lei, Ming; Botka, Chris; Withrow, James; Neau, David; Rajashankar, Kanagalaghatta; Anderson, Karen S; Baxter, Richard H; Blacklow, Stephen C; Boggon, Titus J; Bonvin, Alexandre M J J; Borek, Dominika; Brett, Tom J; Caflisch, Amedeo; Chang, Chung-I; Chazin, Walter J; Corbett, Kevin D; Cosgrove, Michael S; Crosson, Sean; Dhe‐Paganon, Sirano; Cera, Enrico Di; Drennan, Catherine L; Eck, Michael J; Eichman, Brandt F; Fan, Qing R; Ferré‐D’Ámaré, Adrian R; Fromme, Christopher J; Garcia, Christopher K; Gaudet, Rachelle; Gong, Peng; Harrison, Stephen C; Heldwein, Ekaterina E; Jia, Zongchao; Keenan, Robert J; Kruse, Andrew C; Kvansakul, Marc; McLellan, Jason S; Modis, Yorgo; Nam, Yunsun; Otwinowski, Zbyszek; Pai, Emil F; Pereira, Pedro José Barbosa; Petosa, Carlo; Raman, C S; Rapoport, Tom A; Roll‐Mecak, Antonina; Rosen, Michael K; Rudenko, Gabby; Schlessinger, Joseph; Schwartz, Thomas U; Shamoo, Yousif; Sondermann, Holger; Tao, Yizhi J; Tolia, Niraj H; Tsodikov, Oleg V; Westover, Kenneth D; Wu, Hao; Foster, Ian; Fraser, James S; Maia, Felipe R N C; Gonen, Tamir; Kirchhausen, Tom; Diederichs, Kay; Crosas, Mercè; Sliz, Piotr
Data publication with the structural biology data grid supports live analysis
In: Nat Commun, vol. 7, pp. 10882, 2016.
@article{pmid26947396,
title = {Data publication with the structural biology data grid supports live analysis},
author = {Peter A Meyer and Stephanie Socias and Jason Key and Elizabeth Ransey and Emily C Tjon and Alejandro Buschiazzo and Ming Lei and Chris Botka and James Withrow and David Neau and Kanagalaghatta Rajashankar and Karen S Anderson and Richard H Baxter and Stephen C Blacklow and Titus J Boggon and Alexandre M J J Bonvin and Dominika Borek and Tom J Brett and Amedeo Caflisch and Chung-I Chang and Walter J Chazin and Kevin D Corbett and Michael S Cosgrove and Sean Crosson and Sirano Dhe‐Paganon and Enrico Di Cera and Catherine L Drennan and Michael J Eck and Brandt F Eichman and Qing R Fan and Adrian R Ferré‐D’Ámaré and Christopher J Fromme and Christopher K Garcia and Rachelle Gaudet and Peng Gong and Stephen C Harrison and Ekaterina E Heldwein and Zongchao Jia and Robert J Keenan and Andrew C Kruse and Marc Kvansakul and Jason S McLellan and Yorgo Modis and Yunsun Nam and Zbyszek Otwinowski and Emil F Pai and Pedro José Barbosa Pereira and Carlo Petosa and C S Raman and Tom A Rapoport and Antonina Roll‐Mecak and Michael K Rosen and Gabby Rudenko and Joseph Schlessinger and Thomas U Schwartz and Yousif Shamoo and Holger Sondermann and Yizhi J Tao and Niraj H Tolia and Oleg V Tsodikov and Kenneth D Westover and Hao Wu and Ian Foster and James S Fraser and Felipe R N C Maia and Tamir Gonen and Tom Kirchhausen and Kay Diederichs and Mercè Crosas and Piotr Sliz},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_meyer.pdf, Main text},
doi = {10.1038/ncomms10882},
year = {2016},
date = {2016-03-07},
journal = {Nat Commun},
volume = {7},
pages = {10882},
abstract = {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.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
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.
67.
Rodriguez, J A; Gonen, T
High-Resolution Macromolecular Structure Determination by MicroED, a cryo-EM Method
In: Methods in Enzymology, vol. 579, Chapter 14, pp. 369–392, 2016.
@inbook{pmid27572734,
title = {High-Resolution Macromolecular Structure Determination by MicroED, a cryo-EM Method},
author = {J A Rodriguez and T Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2016_rodriguezgonen.pdf, Main text},
doi = {10.1016/bs.mie.2016.04.017},
year = {2016},
date = {2016-01-01},
booktitle = {Methods in Enzymology},
issuetitle = {579},
journal = {Meth. Enzymol.},
volume = {579},
pages = {369--392},
chapter = {14},
abstract = {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., 2015; Shi et al., 2016; Shi, Nannenga, Iadanza, & Gonen, 2013), we aim to provide context and highlight important features that should be considered when performing a MicroED experiment.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
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., 2015; Shi et al., 2016; Shi, Nannenga, Iadanza, & Gonen, 2013), we aim to provide context and highlight important features that should be considered when performing a MicroED experiment.
2015
66.
Rodriguez, Jose A; Ivanova, Magdalena I; Sawaya, Michael R; Cascio, Duilio; Reyes, Francis E; Shi, Dan; Sangwan, Smriti; Guenther, Elizabeth L; Johnson, Lisa M; Zhang, Meng; Jiang, Lin; Arbing, Mark A; Nannenga, Brent L; Hattne, Johan; Whitelegge, Julian; Brewster, Aaron S; Messerschmidt, Marc; Boutet, Sébastien; Sauter, Nicholas K; Gonen, Tamir; Eisenberg, David S
Structure of the toxic core of α-synuclein from invisible crystals
In: Nature, vol. 525, no. 7570, pp. 486–490, 2015.
@article{pmid26352473,
title = {Structure of the toxic core of α-synuclein from invisible crystals},
author = {Jose A Rodriguez and Magdalena I Ivanova and Michael R Sawaya and Duilio Cascio and Francis E Reyes and Dan Shi and Smriti Sangwan and Elizabeth L Guenther and Lisa M Johnson and Meng Zhang and Lin Jiang and Mark A Arbing and Brent L Nannenga and Johan Hattne and Julian Whitelegge and Aaron S Brewster and Marc Messerschmidt and Sébastien Boutet and Nicholas K Sauter and Tamir Gonen and David S Eisenberg},
url = {https://cryoem.ucla.edu/wp-content/uploads/2015rodriguez.pdf, Main text},
doi = {10.1038/nature15368},
year = {2015},
date = {2015-09-09},
journal = {Nature},
volume = {525},
number = {7570},
pages = {486--490},
abstract = {The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The protein α-synuclein is the main component of Lewy bodies, the neuron-associated aggregates seen in Parkinson disease and other neurodegenerative pathologies. An 11-residue segment, which we term NACore, appears to be responsible for amyloid formation and cytotoxicity of human α-synuclein. Here we describe crystals of NACore that have dimensions smaller than the wavelength of visible light and thus are invisible by optical microscopy. As the crystals are thousands of times too small for structure determination by synchrotron X-ray diffraction, we use micro-electron diffraction to determine the structure at atomic resolution. The 1.4 Å resolution structure demonstrates that this method can determine previously unknown protein structures and here yields, to our knowledge, the highest resolution achieved by any cryo-electron microscopy method to date. The structure exhibits protofibrils built of pairs of face-to-face β-sheets. X-ray fibre diffraction patterns show the similarity of NACore to toxic fibrils of full-length α-synuclein. The NACore structure, together with that of a second segment, inspires a model for most of the ordered portion of the toxic, full-length α-synuclein fibril, presenting opportunities for the design of inhibitors of α-synuclein fibrils.
65.
Bale, Jacob B; Park, Rachel U; Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Cascio, Duilio; King, Neil P; Yeates, Todd O; Baker, David
In: Protein Sci., vol. 24, no. 10, pp. 1695–1701, 2015.
@article{pmid26174163,
title = {Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression},
author = {Jacob B Bale and Rachel U Park and Yuxi Liu and Shane Gonen and Tamir Gonen and Duilio Cascio and Neil P King and Todd O Yeates and David Baker},
url = {https://cryoem.ucla.edu/wp-content/uploads/2015_bale.pdf, Main text},
doi = {10.1002/pro.2748},
year = {2015},
date = {2015-07-15},
journal = {Protein Sci.},
volume = {24},
number = {10},
pages = {1695--1701},
abstract = {We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.
64.
Hattne, Johan; Reyes, Francis E; Nannenga, Brent L; Shi, Dan; de la Cruz, Jason M; Leslie, Andrew G W; Gonen, Tamir
MicroED data collection and processing
In: Acta Crystallogr A Found Adv, vol. 71, no. Pt 4, pp. 353–360, 2015.
@article{pmid26131894,
title = {MicroED data collection and processing},
author = {Johan Hattne and Francis E Reyes and Brent L Nannenga and Dan Shi and Jason M de la Cruz and Andrew G W Leslie and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2015_hattne.pdf, Main text},
doi = {10.1107/S2053273315010669},
year = {2015},
date = {2015-07-01},
journal = {Acta Crystallogr A Found Adv},
volume = {71},
number = {Pt 4},
pages = {353--360},
abstract = {MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.
63.
Gonen, Shane; DiMaio, Frank; Gonen, Tamir; Baker, David
Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces
In: Science, vol. 348, no. 6241, pp. 1365–1368, 2015.
@article{pmid26089516,
title = {Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces},
author = {Shane Gonen and Frank DiMaio and Tamir Gonen and David Baker},
url = {https://cryoem.ucla.edu/wp-content/uploads/2015_gonen.pdf, Main text},
doi = {10.1126/science.aaa9897},
year = {2015},
date = {2015-06-19},
journal = {Science},
volume = {348},
number = {6241},
pages = {1365--1368},
abstract = {We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.
2014
62.
Huang, Po-Ssu; Oberdorfer, Gustav; Xu, Chunfu; Pei, Xue Y; Nannenga, Brent L; Rogers, Joseph M; DiMaio, Frank; Gonen, Tamir; Luisi, Ben; Baker, David
High thermodynamic stability of parametrically designed helical bundles
In: Science, vol. 346, no. 6208, pp. 481–485, 2014.
@article{pmid25342806,
title = {High thermodynamic stability of parametrically designed helical bundles},
author = {Po-Ssu Huang and Gustav Oberdorfer and Chunfu Xu and Xue Y Pei and Brent L Nannenga and Joseph M Rogers and Frank DiMaio and Tamir Gonen and Ben Luisi and David Baker},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_huang.pdf, Main text},
doi = {10.1126/science.1257481},
year = {2014},
date = {2014-10-24},
journal = {Science},
volume = {346},
number = {6208},
pages = {481--485},
abstract = {We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.
61.
Nannenga, Brent L; Shi, Dan; Hattne, Johan; Reyes, Francis E; Gonen, Tamir
Structure of catalase determined by MicroED
In: Elife, vol. 3, pp. e03600, 2014.
@article{pmid25303172,
title = {Structure of catalase determined by MicroED},
author = {Brent L Nannenga and Dan Shi and Johan Hattne and Francis E Reyes and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_nannenga_b.pdf, Main text},
doi = {10.7554/eLife.03600},
year = {2014},
date = {2014-10-10},
journal = {Elife},
volume = {3},
pages = {e03600},
abstract = {MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.
60.
Russell, Alistair B; Wexler, Aaron G; Harding, Brittany N; Whitney, John C; Bohn, Alan J; Goo, Young Ah; Tran, Bao Q; Barry, Natasha A; Zheng, Hongjin; Peterson, Brook S; Chou, Seemay; Gonen, Tamir; Goodlett, David R; Goodman, Andrew L; Mougous, Joseph D
A type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism
In: Cell Host Microbe, vol. 16, no. 2, pp. 227–236, 2014.
@article{pmid25070807,
title = {A type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism},
author = {Alistair B Russell and Aaron G Wexler and Brittany N Harding and John C Whitney and Alan J Bohn and Young Ah Goo and Bao Q Tran and Natasha A Barry and Hongjin Zheng and Brook S Peterson and Seemay Chou and Tamir Gonen and David R Goodlett and Andrew L Goodman and Joseph D Mougous},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_russell.pdf, Main text},
doi = {10.1016/j.chom.2014.07.007},
year = {2014},
date = {2014-08-13},
journal = {Cell Host Microbe},
volume = {16},
number = {2},
pages = {227--236},
abstract = {Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.
59.
Wisedchaisri, Goragot; Park, Min-Sun; Iadanza, Matthew G; Zheng, Hongjin; Gonen, Tamir
Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE
In: Nat Commun, vol. 5, pp. 4521, 2014.
@article{pmid25088546,
title = {Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE},
author = {Goragot Wisedchaisri and Min-Sun Park and Matthew G Iadanza and Hongjin Zheng and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_wisedchaisri.pdf, Main text},
doi = {10.1038/ncomms5521},
year = {2014},
date = {2014-08-04},
journal = {Nat Commun},
volume = {5},
pages = {4521},
abstract = {The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.
58.
Nannenga, Brent L; Shi, Dan; Leslie, Andrew G W; Gonen, Tamir
High-resolution structure determination by continuous-rotation data collection in MicroED
In: Nat. Methods, vol. 11, no. 9, pp. 927–930, 2014.
@article{pmid25086503,
title = {High-resolution structure determination by continuous-rotation data collection in MicroED},
author = {Brent L Nannenga and Dan Shi and Andrew G W Leslie and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_nannenga.pdf, Main text},
doi = {10.1038/nmeth.3043},
year = {2014},
date = {2014-08-03},
journal = {Nat. Methods},
volume = {11},
number = {9},
pages = {927--930},
abstract = {MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.
57.
Nannenga, Brent L; Gonen, Tamir
Protein structure determination by MicroED
In: Curr. Opin. Struct. Biol., vol. 27, pp. 24–31, 2014.
@article{pmid24709395,
title = {Protein structure determination by MicroED},
author = {Brent L Nannenga and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_nannengagonen.pdf, Main text},
doi = {10.1016/j.sbi.2014.03.004},
year = {2014},
date = {2014-08-01},
journal = {Curr. Opin. Struct. Biol.},
volume = {27},
pages = {24--31},
abstract = {In this review we discuss the current advances relating to structure determination from protein microcrystals with special emphasis on the newly developed method called MicroED. This method uses a transmission electron cryo-microscope to collect electron diffraction data from extremely small 3-dimensional (3D) crystals. MicroED has been used to solve the 3D structure of the model protein lysozyme to 2.9Å resolution. As the method further matures, MicroED promises to offer a unique and widely applicable approach to protein crystallography using nanocrystals.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In this review we discuss the current advances relating to structure determination from protein microcrystals with special emphasis on the newly developed method called MicroED. This method uses a transmission electron cryo-microscope to collect electron diffraction data from extremely small 3-dimensional (3D) crystals. MicroED has been used to solve the 3D structure of the model protein lysozyme to 2.9Å resolution. As the method further matures, MicroED promises to offer a unique and widely applicable approach to protein crystallography using nanocrystals.
56.
Gonen, Tamir; Waksman, Gabriel
Editorial overview: Membranes: recent methods in the study of membrane protein structure
In: Curr. Opin. Struct. Biol., vol. 27, pp. iv-v, 2014.
@article{pmid25242735,
title = {Editorial overview: Membranes: recent methods in the study of membrane protein structure},
author = {Tamir Gonen and Gabriel Waksman},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_gonenwaksman.pdf, Main text},
doi = {10.1016/j.sbi.2014.09.002},
year = {2014},
date = {2014-08-01},
journal = {Curr. Opin. Struct. Biol.},
volume = {27},
pages = {iv-v},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
55.
Iadanza, Matthew G; Gonen, Tamir
A suite of software for processing MicroED data of extremely small protein crystals
In: J Appl Crystallogr, vol. 47, no. Pt 3, pp. 1140–1145, 2014.
@article{pmid24904248,
title = {A suite of software for processing MicroED data of extremely small protein crystals},
author = {Matthew G Iadanza and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_iadanzagonen.pdf, Main text},
doi = {10.1107/S1600576714008073},
year = {2014},
date = {2014-06-01},
journal = {J Appl Crystallogr},
volume = {47},
number = {Pt 3},
pages = {1140--1145},
abstract = {Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. MicroED patterns, which are collected in a transmission electron microscope, were initially not amenable to indexing and intensity extraction by standard software, which necessitated the development of a suite of programs for data processing. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination. This ad hoc solution has been modified for more general use to provide a means for processing MicroED data until the technique can be fully implemented into existing crystallographic software packages. The suite is written in Python and the source code is available under a GNU General Public License.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. MicroED patterns, which are collected in a transmission electron microscope, were initially not amenable to indexing and intensity extraction by standard software, which necessitated the development of a suite of programs for data processing. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination. This ad hoc solution has been modified for more general use to provide a means for processing MicroED data until the technique can be fully implemented into existing crystallographic software packages. The suite is written in Python and the source code is available under a GNU General Public License.