Our laboratory studies the structures of membrane proteins. Based on structure we try to understand function and what goes wrong in disease. We focus primarily on proteins in the blood-brain barrier. The long-standing question in our laboratory is how the thousands of membrane channels and transporters that exist in the cell membrane work together to help cells maintain homeostasis. With that question in mind, we study membrane proteins that are involved in nutrient, ion and water uptake, waste removal, signaling and communication.
Our laboratory is multidisciplinary. Over the last decade we have employed structural biology techniques such as electron cryo-microscopy (cryo EM), X-ray crystallography, NMR, molecular dynamics simulations, and used membrane biochemistry and biophysics to understand the function of the proteins of interest. Within electron microscopy we have published papers using electron tomography, single particle reconstructions and electron crystallography, however our specialty lies in electron diffraction.
Part of our laboratory is also devoted to method development in cryo EM. In recent years we have developed two important methods in electron diffraction, namely the fragment based phase extension and MicroED.
Some of our recent studies are outlined below.
Lysosomal amino acid transceptor SLC38A9 and mTORC pathway activation
Recent advances in intracellular amino acid transport and mechanistic target of rapamycin complex 1 (mTORC1) signaling shed light on the solute carrier 38 family A member 9 (SLC38A9), a lysosomal transporter responsible for 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 the transmembrane helix 1 (TM1) of drSLC38A9 which was anchored at the grove between TM5 and 7. These anchoring interactions were mediated by the highly conserved motif WNTMM on TM1 and mutations in this motif abolished arginine transport by drSLC38A9. The underlying mechanism of substrate binding is critical for sensitizing mTORC1 signaling pathway to amino acids and for maintaining lysosomal amino acid homeostasis. This study offers a first glimpse into a prototypical model for SLC38 transporter
Using this initial structure we could compute models for representative SNAT proteins
@online{2018_ma_b,
title = {A conformational change in the N terminus of SLC38A9 signals mTORC1 activation},
author = {Ma, Jinming and Lei, Hsiang-Ting and Gonen, Tamir},
doi = {10.1101/339937},
year = {2018},
date = {2018-06-06},
journal = {bio},
organization = {bioRxiv},
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.
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.
@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.
@online{2018_ma,
title = {Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS},
author = {Ma, Jinming and Lei, Hsiang-Ting and Reyes, Francis E. and Sanchez-Martinez, Silvia and Sarhan, Maen and Gonen, Tamir},
doi = {10.1101/293811},
year = {2018},
date = {2018-04-03},
organization = {bioRxiv},
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 from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.
Water channels, or aquaporins, form specialized channels in membranes for water permeation. These are extremely efficient channels that allow millions of water molecules to permeate the pore per second. Because they are channels, the cell can regulate their activity dynamically to help maintain homeostasis. In the case of the eye lens water channel aquaporin-0 (AQP0), it can be regulated by at least 4 known mechanisms that we studied over the last decade. The first is irreversible and involves the cleavage of the C-terminal domain of AQP0. The cleavage results in complete pore closure and AQP0 ceases to act as a water channel. Instead it becomes an adhesive protein mediating cell-to-cell adhesive junctions (Figure 1).
Full-length AQP0 is dynamically modulated by 3 mechanisms: pH, calcium/calmodulin (Ca²⁺/CaM) and protein phosphorylation. We recently showed that the binding of Ca²⁺/CaM to AQP0 results in partial pore closure (Figure 2). The net effect is that the permeability through AQP0 halves in the presence of Ca²⁺/CaM. Conversely, we showed that phosphorylation of AQP0 by anchored PKA (AKAP2/PKA complex) abolished CaM binding, keeping AQP0 in the open conformation and functioning at maximal activity.
Our studies of channel phosphorylation led us to discover a new protein in the eye lens called AKAP2. Our biochemical and structural studies indicate that AKAPs anchor PKA onto substrate and provide the kinase a sphere of action in which the kinase could phosphorylate substrates in a cAMP independent way. This is fundamentally an exciting observation because it helps explain how fast phosphorylation can occur, as seen for example in heart cells. Moreover, we showed that inhibition of phosphorylation of AQP0 in the lens results in cataract formation. Essentially we recapitulated the lens disease ex vivo by inhibiting protein phosphorylation (Figure 3).
Membrane protein complexes
Our structure of the AQP0/CaM complex is the first for any full-length membrane channel in complex with this ubiquitous secondary messenger (Figure 4). Current efforts in the laboratory are to understand how Ca²⁺/CaM binds to and modulates the activity of other channels such as ion channels.
We are also trying to understand more about the AQP-AKAP system, in particular we are trying to assemble the AQP2-AKAP18-PKA complex and AQP0-AKAP2-PKA complex for structural studies. Intrinsically disordered regions of proteins are widespread in nature yet the mechanistic roles they play in biology are underappreciated. Such disordered segments can act simply to link functionally coupled structural domains or they can orchestrate enzymatic reactions through a variety of allosteric mechanisms. The regulatory subunits of protein kinase A provide an example of this important phenomenon where functionally defined and structurally conserved domains are connected by intrinsically disordered regions of defined length with limited sequence identity. Our studies show that this seemingly paradoxical amalgam of order and disorder permits fine-tuning of local protein phosphorylation events. The anchoring of PKA by AKAP affords the kinase a sphere of action in which multiple targets can get phosphorylated fast in a cAMP independent way (Figure 5).
Relevant papers:
Smith, Donelson F; Reichow, Steve L; Esseltine, Jessica L; Shi, Dan; Langeberg, Lorene K; Scott, John D; Gonen, Tamir
@article{pmid24192038,
title = {Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation},
author = {Donelson F Smith and Steve L Reichow and Jessica L Esseltine and Dan Shi and Lorene K Langeberg and John D Scott and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_smith.pdf, Main text},
doi = {10.7554/eLife.01319},
year = {2013},
date = {2013-11-05},
journal = {Elife},
volume = {2},
pages = {e01319},
abstract = {Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation. DOI: http://dx.doi.org/10.7554/eLife.01319.001.
@article{pmid23893133,
title = {Allosteric mechanism of water-channel gating by Ca²⁺-calmodulin},
author = {Steve L Reichow and Daniel M Clemens and Alfredo J Freites and Karin L Nemeth-Cahalan and Matthias Heyden and Douglas J Tobias and James E Hall and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_reichow.pdf, Main text},
doi = {10.1038/nsmb.2630},
year = {2013},
date = {2013-07-28},
journal = {Nat. Struct. Mol. Biol.},
volume = {20},
number = {9},
pages = {1085--1092},
abstract = {Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudoatomic structure of full-length mammalian aquaporin-0 (AQP0, Bos taurus) in complex with CaM, using EM to elucidate how this signaling protein modulates water-channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Calmodulin (CaM) is a universal regulatory protein that communicates the presence of calcium to its molecular targets and correspondingly modulates their function. This key signaling protein is important for controlling the activity of hundreds of membrane channels and transporters. However, understanding of the structural mechanisms driving CaM regulation of full-length membrane proteins has remained elusive. In this study, we determined the pseudoatomic structure of full-length mammalian aquaporin-0 (AQP0, Bos taurus) in complex with CaM, using EM to elucidate how this signaling protein modulates water-channel function. Molecular dynamics and functional mutation studies reveal how CaM binding inhibits AQP0 water permeability by allosterically closing the cytoplasmic gate of AQP0. Our mechanistic model provides new insight, only possible in the context of the fully assembled channel, into how CaM regulates multimeric channels by facilitating cooperativity between adjacent subunits.
@article{pmid22095752,
title = {AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency},
author = {Matthew G Gold and Steve L Reichow and Susan E O'Neill and Chad R Weisbrod and Lorene K Langeberg and James E Bruce and Tamir Gonen and John D Scott},
url = {https://cryoem.ucla.edu/wp-content/uploads/2012_gold.pdf, Main text},
doi = {10.1002/emmm.201100184},
year = {2011},
date = {2011-11-16},
journal = {EMBO Mol Med},
volume = {4},
number = {1},
pages = {15--26},
abstract = {A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a calmodulin (CaM)-binding domain of AQP0. The additional negative charge introduced by phosphoserine 235 perturbs electrostatic interactions between AQP0 and CaM to favour water influx through the channel. In isolated mouse lenses, displacement of PKA from the AKAP2-AQP0 channel complex promotes cortical cataracts as characterized by severe opacities and cellular damage. Thus, anchored PKA modulation of AQP0 is a homeostatic mechanism that must be physically intact to preserve lens transparency.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
A decline in ocular lens transparency known as cataract afflicts 90% of individuals by the age 70. Chronic deterioration of lens tissue occurs as a pathophysiological consequence of defective water and nutrient circulation through channel and transporter proteins. A key component is the aquaporin-0 (AQP0) water channel whose permeability is tightly regulated in healthy lenses. Using a variety of cellular and biochemical approaches we have discovered that products of the A-kinase anchoring protein 2 gene (AKAP2/AKAP-KL) form a stable complex with AQP0 to sequester protein kinase A (PKA) with the channel. This permits PKA phosphorylation of serine 235 within a calmodulin (CaM)-binding domain of AQP0. The additional negative charge introduced by phosphoserine 235 perturbs electrostatic interactions between AQP0 and CaM to favour water influx through the channel. In isolated mouse lenses, displacement of PKA from the AKAP2-AQP0 channel complex promotes cortical cataracts as characterized by severe opacities and cellular damage. Thus, anchored PKA modulation of AQP0 is a homeostatic mechanism that must be physically intact to preserve lens transparency.
@article{pmid18786401,
title = {Noncanonical Binding of Calmodulin to Aquaporin-0: Implications for Channel Regulation},
author = {Steve L Reichow and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/reichow_2008.pdf, Main text},
doi = {10.1016/j.str.2008.06.011},
year = {2008},
date = {2008-09-10},
journal = {Structure},
volume = {16},
number = {9},
pages = {1389--1398},
abstract = {Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water across cell membranes. AQPs form homotetramers containing four functional and independent water pores. Aquaporin-0 (AQP0) is expressed in the eye lens, where its water permeability is regulated by calmodulin (CaM). Here we use a combination of biochemical methods and NMR spectroscopy to probe the interaction between AQP0 and CaM. We show that CaM binds the AQP0 C-terminal domain in a calcium-dependent manner. We demonstrate that only two CaM molecules bind a single AQP0 tetramer in a noncanonical fashion, suggesting a form of cooperativity between AQP0 monomers. Based on these results, we derive a structural model of the AQP0/CaM complex, which suggests CaM may be inhibitory to channel permeability by capping the vestibules of two monomers within the AQP0 tetramer. Finally, phosphorylation within AQP0's CaM binding domain inhibits the AQP0/CaM interaction, suggesting a temporal regulatory mechanism for complex formation.
@article{pmid16319884,
title = {Lipid-protein interactions in double-layered two-dimensional AQP0 crystals},
author = {Tamir Gonen and Yifan Cheng and Piotr Sliz and Yoko Hiroaki and Yoshinori Fujiyoshi and Stephen C Harrison and Thomas Walz},
url = {https://cryoem.ucla.edu/wp-content/uploads/Gonen_2005.pdf, Main text},
doi = {10.1038/nature04321},
year = {2005},
date = {2005-12-01},
journal = {Nature},
volume = {438},
number = {7068},
pages = {633--638},
abstract = {Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.
@article{pmid15351655,
title = {Aquaporin-0 Membrane Junctions Form Upon Proteolytic Cleavage},
author = {Tamir Gonen and Yifan Cheng and Joerg Kistler and Thomas Walz},
url = {https://cryoem.ucla.edu/wp-content/uploads/Gonen_2004b.pdf, Main text},
doi = {10.1016/j.jmb.2004.07.076},
year = {2004},
date = {2004-09-24},
journal = {J. Mol. Biol.},
volume = {342},
number = {4},
pages = {1337--1345},
abstract = {Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.
@article{pmid15141214,
title = {Aquaporin-0 membrane junctions reveal the structure of a closed water pore},
author = {Tamir Gonen and Piotr Sliz and Joerg Kistler and Yifan Cheng and Thomas Walz},
url = {https://cryoem.ucla.edu/wp-content/uploads/gonen_2004a.pdf, Main text},
doi = {10.1038/nature02503},
year = {2004},
date = {2004-05-13},
journal = {Nature},
volume = {429},
number = {6988},
pages = {193--197},
abstract = {The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo. We show here that AQP0 from the lens core, containing some carboxy-terminally cleaved AQP0, forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystallography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo. We show here that AQP0 from the lens core, containing some carboxy-terminally cleaved AQP0, forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystallography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating.
@article{pmid24124191,
title = {Local cAMP signaling in disease at a glance},
author = {Matthew G Gold and Tamir Gonen and John D Scott},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_gold.pdf, Main text},
doi = {10.1242/jcs.133751},
year = {2013},
date = {2013-10-15},
journal = {J. Cell. Sci.},
volume = {126},
number = {Pt 20},
pages = {4537--4543},
abstract = {The second messenger cyclic AMP (cAMP) operates in discrete subcellular regions within which proteins that synthesize, break down or respond to the second messenger are precisely organized. A burgeoning knowledge of compartmentalized cAMP signaling is revealing how the local control of signaling enzyme activity impacts upon disease. The aim of this Cell Science at a Glance article and the accompanying poster is to highlight how misregulation of local cyclic AMP signaling can have pathophysiological consequences. We first introduce the core molecular machinery for cAMP signaling, which includes the cAMP-dependent protein kinase (PKA), and then consider the role of A-kinase anchoring proteins (AKAPs) in coordinating different cAMP-responsive proteins. The latter sections illustrate the emerging role of local cAMP signaling in four disease areas: cataracts, cancer, diabetes and cardiovascular diseases.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The second messenger cyclic AMP (cAMP) operates in discrete subcellular regions within which proteins that synthesize, break down or respond to the second messenger are precisely organized. A burgeoning knowledge of compartmentalized cAMP signaling is revealing how the local control of signaling enzyme activity impacts upon disease. The aim of this Cell Science at a Glance article and the accompanying poster is to highlight how misregulation of local cyclic AMP signaling can have pathophysiological consequences. We first introduce the core molecular machinery for cAMP signaling, which includes the cAMP-dependent protein kinase (PKA), and then consider the role of A-kinase anchoring proteins (AKAPs) in coordinating different cAMP-responsive proteins. The latter sections illustrate the emerging role of local cAMP signaling in four disease areas: cataracts, cancer, diabetes and cardiovascular diseases.
@article{pmid19679462,
title = {Lipid-protein interactions probed by electron crystallography},
author = {Steve L Reichow and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/reichowgonen_2009.pdf, Main text},
doi = {10.1016/j.sbi.2009.07.012},
year = {2009},
date = {2009-10-01},
journal = {Curr. Opin. Struct. Biol.},
volume = {19},
number = {5},
pages = {560--565},
abstract = {Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled, and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein's architecture where they can affect the function, structure, quaternary assembly, and the stability of the membrane protein.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Electron crystallography is arguably the only electron cryomicroscopy (cryoEM) technique able to deliver an atomic-resolution structure of membrane proteins embedded in the lipid bilayer. In the electron crystallographic structures of the light driven ion pump, bacteriorhodopsin, and the water channel, aquaporin-0, sufficiently high resolution was obtained and both lipid and protein were visualized, modeled, and described in detail. An extensive network of lipid-protein interactions mimicking native membranes is established and maintained in two-dimensional (2D) crystalline vesicles used for structural analysis by electron crystallography. Lipids are tightly integrated into the protein's architecture where they can affect the function, structure, quaternary assembly, and the stability of the membrane protein.
@article{pmid18465794,
title = {Electron Crystallography of Aquaporins},
author = {Andrews, Simeon and Reichow, Steve L. and Gonen, Tamir},
url = {https://cryoem.ucla.edu/wp-content/uploads/andrews_2008.pdf, Main text},
doi = {10.1002/iub.53},
year = {2008},
date = {2008-05-08},
journal = {IUBMB Life},
volume = {60},
number = {7},
pages = {430--436},
abstract = {Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.
@article{pmid18194855,
title = {Junction-forming aquaporins},
author = {Andreas Engel and Yoshinori Fujiyoshi and Tamir Gonen and Thomas Walz},
url = {https://cryoem.ucla.edu/wp-content/uploads/engel_2008.pdf, Main text},
doi = {10.1016/j.sbi.2007.11.003},
year = {2008},
date = {2008-04-01},
journal = {Curr. Opin. Struct. Biol.},
volume = {18},
number = {2},
pages = {229--235},
abstract = {Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.
@article{pmid17156589,
title = {The structure of aquaporins},
author = {Tamir Gonen and Thomas Walz},
url = {https://cryoem.ucla.edu/wp-content/uploads/gonenwalz_2006.pdf, Main text},
doi = {10.1017/S0033583506004458},
year = {2006},
date = {2006-11-01},
journal = {Q. Rev. Biophys.},
volume = {39},
number = {4},
pages = {361--396},
abstract = {The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.
The major facilitator superfamily of membrane proteins 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 captured and structurally characterized in multiple transporting conformations including both the outward and inward facing states. We determined the crystal structure of XylE in a new inward-facing open conformation. Structural comparison of XylE in this conformation with its outward-facing partially occluded conformation reveals how this transporter functions through a non-symmetrical rocker switch movement of the N-domain as a rigid body and the C-domain as a flexible body. Molecular dynamics simulations were employed to help describe how XylE transitions in a lipid membrane to facilitate sugar transport. (Figure 6)
Nitrogen uptake
Nitrate is the preferred nitrogen source for plants on which all higher forms of life ultimately depend. Plants and microorganisms evolved to efficiently assimilate nitrate. Despite decades of effort no structure was available for any nitrate transport protein and the mechanism by which nitrate is transported remained largely obscure until our study was published. We reported the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures revealed a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that H⁺s are unlikely to be co-transported. Conserved arginine residues form the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport were identified and related to extensive mutagenesis and functional studies. We proposed that NarK exchanges nitrate for nitrite by a rocker-switch mechanism facilitated by inter-domain H-bond networks. (Figure 7)
Amino acid uptake
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 and that can transport both D- and L- amino acids. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- and D- alanine. 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 both L- and D- type alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the transporters 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 membrane transporters.
@online{2018_ma,
title = {Structural basis for substrate binding and specificity of a sodium/alanine symporter AgcS},
author = {Ma, Jinming and Lei, Hsiang-Ting and Reyes, Francis E. and Sanchez-Martinez, Silvia and Sarhan, Maen and Gonen, Tamir},
doi = {10.1101/293811},
year = {2018},
date = {2018-04-03},
organization = {bioRxiv},
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 from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.},
keywords = {},
pubstate = {published},
tppubtype = {online}
}
The amino acid, polyamine, and organocation (APC) superfamily is the second largest superfamily of membrane proteins forming secondary transporters that move a range of organic molecules across the cell membrane. Each transporter in APC superfamily is specific for a unique sub-set of substrates, even if they possess a similar structural fold. The mechanism of substrate selectivity remains, by and large, elusive. Here we report two crystal structures of an APC member from Methanococcus maripaludis, the alanine or glycine:cation symporter (AgcS), with L- or D-alanine bound. Structural analysis combined with site-directed mutagenesis and functional studies inform on substrate binding, specificity, and modulation of the AgcS family and reveal key structural features that allow this transporter to accommodate glycine and alanine while excluding all other amino acids. Mutation of key residues in the substrate binding site expand the selectivity to include valine and leucine. Moreover, as a transporter that binds both enantiomers of alanine, the present structures provide an unprecedented opportunity to gain insights into the mechanism of stereo-selectivity in APC transporters.
@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.
@article{pmid23665960,
title = {Crystal structure of a nitrate/nitrite exchanger},
author = {Hongjin Zheng and Goragot Wisedchaisri and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_zheng.pdf, Main text},
doi = {10.1038/nature12139},
year = {2013},
date = {2013-05-30},
journal = {Nature},
volume = {497},
number = {7451},
pages = {647--651},
abstract = {Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Mineral nitrogen in nature is often found in the form of nitrate (NO3(-)). Numerous microorganisms evolved to assimilate nitrate and use it as a major source of mineral nitrogen uptake. Nitrate, which is central in nitrogen metabolism, is first reduced to nitrite (NO2(-)) through a two-electron reduction reaction. The accumulation of cellular nitrite can be harmful because nitrite can be reduced to the cytotoxic nitric oxide. Instead, nitrite is rapidly removed from the cell by channels and transporters, or reduced to ammonium or dinitrogen through the action of assimilatory enzymes. Despite decades of effort no structure is currently available for any nitrate transport protein and the mechanism by which nitrate is transported remains largely unknown. Here we report the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures reveal a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that protons are unlikely to be co-transported. Conserved arginine residues comprise the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport are identified and related to extensive mutagenesis and functional studies. We propose that NarK exchanges nitrate for nitrite by a rocker switch mechanism facilitated by inter-domain hydrogen bond networks.
Wisedchaisri, Goragot; Dranow, David M; Lie, Thomas J; Bonanno, Jeffrey B; Patskovsky, Yury; Ozyurt, Sinem A; Sauder, Michael J; Almo, Steven C; Wasserman, Stephen R; Burley, Stephen K; Leigh, John A; Gonen, Tamir
@article{pmid21070950,
title = {Structural Underpinnings of Nitrogen Regulation by the Prototypical Nitrogen-Responsive Transcriptional Factor NrpR},
author = {Goragot Wisedchaisri and David M Dranow and Thomas J Lie and Jeffrey B Bonanno and Yury Patskovsky and Sinem A Ozyurt and Michael J Sauder and Steven C Almo and Stephen R Wasserman and Stephen K Burley and John A Leigh and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2010_wisedchaisri.pdf, Main text},
doi = {10.1016/j.str.2010.08.014},
year = {2010},
date = {2010-11-10},
journal = {Structure},
volume = {18},
number = {11},
pages = {1512--1521},
abstract = {Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Plants and microorganisms reduce environmental inorganic nitrogen to ammonium, which then enters various metabolic pathways solely via conversion of 2-oxoglutarate (2OG) to glutamate and glutamine. Cellular 2OG concentrations increase during nitrogen starvation. We recently identified a family of 2OG-sensing proteins--the nitrogen regulatory protein NrpR--that bind DNA and repress transcription of nitrogen assimilation genes. We used X-ray crystallography to determine the structure of NrpR regulatory domain. We identified the NrpR 2OG-binding cleft and show that residues predicted to interact directly with 2OG are conserved among diverse classes of 2OG-binding proteins. We show that high levels of 2OG inhibit NrpRs ability to bind DNA. Electron microscopy analyses document that NrpR adopts different quaternary structures in its inhibited 2OG-bound state compared with its active apo state. Our results indicate that upon 2OG release, NrpR repositions its DNA-binding domains correctly for optimal interaction with DNA thereby enabling gene repression.
@article{pmid20006622,
title = {The prototypical H⁺/Galactose Symporter GalP Assembles into Functional Trimers},
author = {Hongjin Zheng and Justin Taraska and Alexey J Merz and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/zheng_2010.pdf, Main text},
doi = {10.1016/j.jmb.2009.12.010},
year = {2010},
date = {2010-02-26},
journal = {J. Mol. Biol.},
volume = {396},
number = {3},
pages = {593--601},
abstract = {Glucose is a primary source of energy for human cells. Glucose transporters form specialized membrane channels for the transport of sugars into and out of cells. Galactose permease (GalP) is the closest bacterial homolog of human facilitated glucose transporters. Here, we report the functional reconstitution and 2D crystallization of GalP. Single particle electron microscopy analysis of purified GalP shows that the protein assembles as an oligomer with three distinct densities. Reconstitution assays yield 2D GalP crystals that exhibit a hexagonal array having p3 symmetry. The projection structure of GalP at 18 A resolution shows that the protein is trimeric. Each monomer in the trimer forms its own channel, but an additional cavity (10 approximately 15 A in diameter) is apparent at the 3-fold axis of the oligomer. We show that the crystalline GalP is able to selectively bind substrate, suggesting that the trimeric form is biologically active.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Glucose is a primary source of energy for human cells. Glucose transporters form specialized membrane channels for the transport of sugars into and out of cells. Galactose permease (GalP) is the closest bacterial homolog of human facilitated glucose transporters. Here, we report the functional reconstitution and 2D crystallization of GalP. Single particle electron microscopy analysis of purified GalP shows that the protein assembles as an oligomer with three distinct densities. Reconstitution assays yield 2D GalP crystals that exhibit a hexagonal array having p3 symmetry. The projection structure of GalP at 18 A resolution shows that the protein is trimeric. Each monomer in the trimer forms its own channel, but an additional cavity (10 approximately 15 A in diameter) is apparent at the 3-fold axis of the oligomer. We show that the crystalline GalP is able to selectively bind substrate, suggesting that the trimeric form is biologically active.
In electron crystallography membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. We developed a new approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We used the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α-helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data. (Figure 8)
MicroED —Three‐dimensional electron crystallography of protein microcrystals
We demonstrated that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1 – 1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9Å resolution (Figure 9). This proof of principle paves the way for the implementation of a new technique, which we name “MicroED”, that may have wide applicability in structural biology. Current efforts include new phasing methods, automation and program development.
An example of lysozyme MicroED data can be viewed here.
In 2014 we further inmproved the MicroED method. Firstly, we developed an improved data collection protocol for MicroED called Continuous rotation. Microcrystals are continuously rotated during data collection yielding improved data, and allowing data processing with the crystallographic software tool MOSFLM, resulting in improved resolution for the model protein lysozyme to 2.5Å resolution. These improvements pave the way for the broad implementation and application of MicroED in structural biology. Current efforts include new phasing methods, automation and program development.
Secondly, we used the improved MicroED protocols for data collection and analysis to determine the structure of catalase. Bovine liver catalase crystals that were only ~160nm thick were used for the structure analysis. A single crystal yielded data to 3.2Å resolution enabling structure determination rapidly.
An example of catalase MicroED data can be viewed here.
In 2015 we published the first two previously unknown structures determined by MicroED. The structures of two peptides from the toxic core of a-synuclein of Parkinsons’ Disease. The structures were determined from vanishingly small crystals, only ~200nm thick and wide, and yielded 1.4Å resolution. These structures, which are currently the highest resolution structures determined to date by any cryo EM method, show new and important structural information that could aid in the development of pharmaceuticals against this devastating neurological disease. The study, which was published by Nature also show a number of protons for the very first time.
@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.
@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.
@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.
@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.
@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.
@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.
@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.
@article{pmid24252878,
title = {Three-dimensional electron crystallography of protein microcrystals},
author = {Dan Shi and Brent L Nannenga and Matthew G Iadanza and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_shi.pdf, Main text},
doi = {10.7554/eLife.01345},
year = {2013},
date = {2013-11-19},
journal = {Elife},
volume = {2},
pages = {e01345},
abstract = {We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1-1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name 'MicroED', that may have wide applicability in structural biology. DOI: http://dx.doi.org/10.7554/eLife.01345.001.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1-1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name 'MicroED', that may have wide applicability in structural biology. DOI: http://dx.doi.org/10.7554/eLife.01345.001.
@article{pmid21742264,
title = {Fragment-Based Phase Extension for Three-Dimensional Structure Determination of Membrane Proteins by Electron Crystallography},
author = {Goragot Wisedchaisri and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2011_wisedchaisri_1.pdf, Main text},
doi = {10.1016/j.str.2011.04.008},
year = {2011},
date = {2011-07-13},
journal = {Structure},
volume = {19},
number = {7},
pages = {976--987},
abstract = {In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In electron crystallography, membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown, one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. Here, we describe an approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We use the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data.
@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.
@article{pmid23546618,
title = {Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy},
author = {Brent L Nannenga and Matthew G Iadanza and Breanna S Vollmar and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/nannenga_2013.pdf, Main text},
doi = {10.1002/0471140864.ps1715s72},
year = {2013},
date = {2013-04-01},
journal = {Curr Protoc Protein Sci},
volume = {72},
number = {1},
pages = {17.15.1--17.15.11},
chapter = {17},
abstract = {Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Electron cryomicroscopy, or cryoEM, is an emerging technique for studying the three-dimensional structures of proteins and large macromolecular machines. Electron crystallography is a branch of cryoEM in which structures of proteins can be studied at resolutions that rival those achieved by X-ray crystallography. Electron crystallography employs two-dimensional crystals of a membrane protein embedded within a lipid bilayer. The key to a successful electron crystallographic experiment is the crystallization, or reconstitution, of the protein of interest. This unit describes ways in which protein can be expressed, purified, and reconstituted into well-ordered two-dimensional crystals. A protocol is also provided for negative stain electron microscopy as a tool for screening crystallization trials. When large and well-ordered crystals are obtained, the structures of both protein and its surrounding membrane can be determined to atomic resolution.
@inbook{pmid23132065,
title = {Phasing Electron Diffraction Data by Molecular Replacement: Strategy for Structure Determination and Refinement},
author = {Goragot Wisedchaisri and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_wisedchaisrigonen.pdf, Main text},
doi = {10.1007/978-1-62703-176-9_14},
year = {2012},
date = {2012-10-07},
booktitle = {Electron Crystallography of Soluble and Membrane Proteins},
journal = {Methods Mol. Biol.},
volume = {955},
pages = {243--272},
chapter = {14},
abstract = {Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
Electron crystallography is arguably the only electron cryomicroscopy (cryo EM) technique able to deliver atomic resolution data (better then 3 Å) for membrane proteins embedded in a membrane. The progress in hardware improvements and sample preparation for diffraction analysis resulted in a number of recent examples where increasingly higher resolutions were achieved. Other chapters in this book detail the improvements in hardware and delve into the intricate art of sample preparation for microscopy and electron diffraction data collection and processing. In this chapter, we describe in detail the protocols for molecular replacement for electron diffraction studies. The use of a search model for phasing electron diffraction data essentially eliminates the need of acquiring image data rendering it immune to aberrations from drift and charging effects that effectively lower the attainable resolution.
@inbook{pmid23132060,
title = {The Collection of High-Resolution Electron Diffraction Data},
author = {Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_gonen.pdf, Main text},
doi = {10.1007/978-1-62703-176-9_9},
year = {2012},
date = {2012-10-07},
booktitle = {Electron Crystallography of Soluble and Membrane Proteins},
journal = {Methods Mol. Biol.},
volume = {955},
pages = {153--169},
chapter = {9},
abstract = {A number of atomic-resolution structures of membrane proteins (better than 3Å resolution) have been determined recently by electron crystallography. While this technique was established more than 40 years ago, it is still in its infancy with regard to the two-dimensional (2D) crystallization, data collection, data analysis, and protein structure determination. In terms of data collection, electron crystallography encompasses both image acquisition and electron diffraction data collection. Other chapters in this volume outline protocols for image collection and analysis. This chapter, however, outlines detailed protocols for data collection by electron diffraction. These include microscope setup, electron diffraction data collection, and troubleshooting.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
A number of atomic-resolution structures of membrane proteins (better than 3Å resolution) have been determined recently by electron crystallography. While this technique was established more than 40 years ago, it is still in its infancy with regard to the two-dimensional (2D) crystallization, data collection, data analysis, and protein structure determination. In terms of data collection, electron crystallography encompasses both image acquisition and electron diffraction data collection. Other chapters in this volume outline protocols for image collection and analysis. This chapter, however, outlines detailed protocols for data collection by electron diffraction. These include microscope setup, electron diffraction data collection, and troubleshooting.
@inbook{pmid23132066,
title = {High-Throughput Methods for Electron Crystallography},
author = {David L Stokes and Iban Ubarretxena-Belandia and Tamir Gonen and Andreas Engel},
url = {https://cryoem.ucla.edu/wp-content/uploads/2013_stokes.pdf, Main text},
doi = {10.1007/978-1-62703-176-9_15},
year = {2012},
date = {2012-10-07},
booktitle = {Electron Crystallography of Soluble and Membrane Proteins},
journal = {Methods Mol. Biol.},
volume = {955},
pages = {273--296},
chapter = {15},
abstract = {Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.},
keywords = {},
pubstate = {published},
tppubtype = {inbook}
}
Membrane proteins play a tremendously important role in cell physiology and serve as a target for an increasing number of drugs. Structural information is key to understanding their function and for developing new strategies for combating disease. However, the complex physical chemistry associated with membrane proteins has made them more difficult to study than their soluble cousins. Electron crystallography has historically been a successful method for solving membrane protein structures and has the advantage of providing a native lipid environment for these proteins. Specifically, when membrane proteins form two-dimensional arrays within a lipid bilayer, electron microscopy can be used to collect images and diffraction and the corresponding data can be combined to produce a three-dimensional reconstruction, which under favorable conditions can extend to atomic resolution. Like X-ray crystallography, the quality of the structures are very much dependent on the order and size of the crystals. However, unlike X-ray crystallography, high-throughput methods for screening crystallization trials for electron crystallography are not in general use. In this chapter, we describe two alternative methods for high-throughput screening of membrane protein crystallization within the lipid bilayer. The first method relies on the conventional use of dialysis for removing detergent and thus reconstituting the bilayer; an array of dialysis wells in the standard 96-well format allows the use of a liquid-handling robot and greatly increases throughput. The second method relies on titration of cyclodextrin as a chelating agent for detergent; a specialized pipetting robot has been designed not only to add cyclodextrin in a systematic way, but to use light scattering to monitor the reconstitution process. In addition, the use of liquid-handling robots for making negatively stained grids and methods for automatically imaging samples in the electron microscope are described.
@article{pmid22000511,
title = {Advances in Structural and Functional Analysis of Membrane Proteins by Electron Crystallography},
author = {Goragot Wisedchaisri and Steve L Reichow and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2011_wisedchaisri.pdf, Main text},
doi = {10.1016/j.str.2011.09.001},
year = {2011},
date = {2011-10-12},
journal = {Structure},
volume = {19},
number = {10},
pages = {1381--1393},
abstract = {Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Electron crystallography is a powerful technique for the study of membrane protein structure and function in the lipid environment. When well-ordered two-dimensional crystals are obtained the structure of both protein and lipid can be determined and lipid-protein interactions analyzed. Protons and ionic charges can be visualized by electron crystallography and the protein of interest can be captured for structural analysis in a variety of physiologically distinct states. This review highlights the strengths of electron crystallography and the momentum that is building up in automation and the development of high throughput tools and methods for structural and functional analysis of membrane proteins by electron crystallography.
Computational design of genetically encoded self‐assembling proteins
In collaboration with David Baker (HHMI, UW) we are designing genetically encoded self assembling proteins for cellular microcircuitry.
We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. Here we use trimeric protein building blocks to design a 24-subunit, 13 nm diameter complex with octahedral symmetry and two related variants of a 12-subunit, 11 nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials. (Figure 10)
Relevant papers
Bale, Jacob B; Park, Rachel U; Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Cascio, Duilio; King, Neil P; Yeates, Todd O; Baker, David
@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.
@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.
@article{pmid24870237,
title = {Accurate design of co-assembling multi-component protein nanomaterials},
author = {Neil P King and Jacob B Bale and William Sheffler and Dan E McNamara and Shane Gonen and Tamir Gonen and Todd O Yeates and David Baker},
url = {https://cryoem.ucla.edu/wp-content/uploads/2014_king.pdf, Main text},
doi = {10.1038/nature13404},
year = {2014},
date = {2014-06-05},
journal = {Nature},
volume = {510},
number = {7503},
pages = {103--108},
abstract = {The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The self-assembly of proteins into highly ordered nanoscale architectures is a hallmark of biological systems. The sophisticated functions of these molecular machines have inspired the development of methods to engineer self-assembling protein nanostructures; however, the design of multi-component protein nanomaterials with high accuracy remains an outstanding challenge. Here we report a computational method for designing protein nanomaterials in which multiple copies of two distinct subunits co-assemble into a specific architecture. We use the method to design five 24-subunit cage-like protein nanomaterials in two distinct symmetric architectures and experimentally demonstrate that their structures are in close agreement with the computational design models. The accuracy of the method and the number and variety of two-component materials that it makes accessible suggest a route to the construction of functional protein nanomaterials tailored to specific applications.
@article{pmid22654060,
title = {Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy},
author = {Neil P King and William Sheffler and Michael R Sawaya and Breanna S Vollmar and John P Sumida and Ingemar André and Tamir Gonen and Todd O Yeates and David Baker},
url = {https://cryoem.ucla.edu/wp-content/uploads/2012_king.pdf, Main text},
doi = {10.1126/science.1219364},
year = {2012},
date = {2012-06-01},
journal = {Science},
volume = {336},
number = {6085},
pages = {1171--1174},
abstract = {We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.
We use electrophysiology and patch clamping techniques to study the function of channels and transporters. We use the Xenopus oocyte expression system as well as whole cell patch but we also plan to record channel function from highly ordered two-dimensional crystals for a direct correlation between structure and function of target proteins as they are embedded within a biological membrane.
(Figure 11)
Other notable studies (not currently active in the lab)
Structure of the vibrio cholera toxin secretion channel
In collaboration with Wim Hol (UW) we studied the structure of the vibrio cholera toxin secretion channel.
The type II secretion system (T2SS) is a macromolecular complex spanning the bacterial inner and outer membranes of Gram-negative bacteria, including many pathogenic bacteria such as Vibrio cholerae and enterotoxigenic Escherichia coli. The T2SS secretes folded proteins including cholera toxin and heat-labile enterotoxin. The major outer membrane T2SS protein is the “secretin” GspD. Electron cryomicroscopy (cryoEM) reconstruction of the V. cholerae secretin at 19 Å resolution reveals a dodecameric structure reminiscent of a barrel with a large channel at its center that appears to be in a closed state. On the periplasmic side of the channel vestibule contains both a constriction and a gate. On the extracellular side a large chamber is enclosed by a cap structure. By combining our results with structural data on a large exoprotein and the dimensions of the tip of the pseudopilus of the T2SS, we provide a structural basis for a possible secretion mechanism of exoproteins by the T2SS in which the constriction site plays a critical role. (Figure 12)
Relevant papers:
Reichow, Steve L; Korotkov, Konstantin V; Gonen, Melissa; Sun, Ji; Delarosa, Jaclyn R; Hol, Wim G J; Gonen, Tamir
@article{pmid21406971,
title = {The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel},
author = {Steve L Reichow and Konstantin V Korotkov and Melissa Gonen and Ji Sun and Jaclyn R Delarosa and Wim G J Hol and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2011_reichow.pdf, Main text},
doi = {10.4161/chan.5.3.15268},
year = {2011},
date = {2011-06-30},
journal = {Channels (Austin)},
volume = {5},
number = {3},
pages = {215--218},
abstract = {The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB5 heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The type II secretion system (T2SS) is a large macromolecular complex spanning the inner and outer membranes of many gram-negative bacteria. The T2SS is responsible for the secretion of virulence factors such as cholera toxin (CT) and heat-labile enterotoxin (LT) from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. CT and LT are closely related AB5 heterohexamers, composed of one A subunit and a B-pentamer. Both CT and LT are translocated, as folded protein complexes, from the periplasm across the outer membrane through the type II secretion channel, the secretin GspD. We recently published the 19 Å structure of the V. cholerae secretin (VcGspD) in its closed state and showed by SPR measurements that the periplasmic domain of GspD interacts with the B-pentamer complex. Here we extend these studies by characterizing the binding of the cholera toxin B-pentamer to VcGspD using electron microscopy of negatively stained preparations. Our studies indicate that the pentamer is captured within the large periplasmic vestibule of VcGspD. These new results agree well with our previously published studies and are in accord with a piston-driven type II secretion mechanism.
@article{pmid20852644,
title = {Structure of the cholera toxin secretion channel in its closed state},
author = {Steve L Reichow and Konstantin V Korotkov and Wim G J Hol and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/reichow_2010.pdf, Main text},
doi = {10.1038/nsmb.1910},
year = {2010},
date = {2010-09-19},
journal = {Nat. Struct. Mol. Biol.},
volume = {17},
number = {10},
pages = {1226--1232},
abstract = {The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the 'secretin' GspD. Cryo-EM reconstruction of the V. cholerae secretin at 19-Å resolution reveals a dodecameric structure reminiscent of a barrel, with a large channel at its center that contains a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus tip, we provide a structural basis for a possible secretion mechanism of the T2SS.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The type II secretion system (T2SS) is a macromolecular complex spanning the inner and outer membranes of Gram-negative bacteria. Remarkably, the T2SS secretes folded proteins, including multimeric assemblies such as cholera toxin and heat-labile enterotoxin from Vibrio cholerae and enterotoxigenic Escherichia coli, respectively. The major outer membrane T2SS protein is the 'secretin' GspD. Cryo-EM reconstruction of the V. cholerae secretin at 19-Å resolution reveals a dodecameric structure reminiscent of a barrel, with a large channel at its center that contains a closed periplasmic gate. The GspD periplasmic domain forms a vestibule with a conserved constriction, and it binds to a pentameric exoprotein and to the trimeric tip of the T2SS pseudopilus. By combining our results with structures of the cholera toxin and T2SS pseudopilus tip, we provide a structural basis for a possible secretion mechanism of the T2SS.
@article{pmid21565514,
title = {Secretins: dynamic channels for protein transport across membranes},
author = {Konstantin V Korotkov and Tamir Gonen and Wim G J Hol},
url = {https://cryoem.ucla.edu/wp-content/uploads/2011_korotkov.pdf, Main text},
doi = {10.1016/j.tibs.2011.04.002},
year = {2011},
date = {2011-08-01},
journal = {Trends Biochem. Sci.},
volume = {36},
number = {8},
pages = {433--443},
abstract = {Secretins form megadalton bacterial-membrane channels in at least four sophisticated multiprotein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion-system partner proteins, and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and it is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other. Secretins might also play a key role in the biogenesis of their cognate secretion systems.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Secretins form megadalton bacterial-membrane channels in at least four sophisticated multiprotein systems that are crucial for translocation of proteins and assembled fibers across the outer membrane of many species of bacteria. Secretin subunits contain multiple domains, which interact with numerous other proteins, including pilotins, secretion-system partner proteins, and exoproteins. Our understanding of the structure of secretins is rapidly progressing, and it is now recognized that features common to all secretins include a cylindrical arrangement of 12-15 subunits, a large periplasmic vestibule with a wide opening at one end and a periplasmic gate at the other. Secretins might also play a key role in the biogenesis of their cognate secretion systems.
Structure and function of the yeast kinetochore and microtubule dynamics
In collaboration with Sue Biggins (FHCRC) we studied the structure of the yeast kinetochore by electron tomography. In collaboration with Trisha Davis and Chip Asbury (UW) we studied microtubule dynamics and microtubule binding proteins.
Chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are macromolecular machines that move chromosomes by maintaining load-bearing attachments to the assembling and disassembling tips of spindle microtubules. The mechanism by which kinetochores attach to microtubules is still not clear although a number of models have been proposed. Sues laboratory previously developed an assay to purify functional native budding yeast kinetochore particles that contain the majority of core structural components and can maintain attachments to microtubules under force. We presented the structure of these isolated kinetochore particles as visualized by electron microscopy (EM) and electron tomography of negatively stained preparations. The budding yeast kinetochore appeared as a ~126 nm particle having a large central hub attached to multiple outer globular domains. Microtubule binding experiments indicated that the globular domains are important for microtubule attachments both in the presence or absence of a ring encircling the microtubule. Our data showed that kinetochores bind to microtubules via multivalent attachments, consistent with a biased diffusion mechanism where multiple kinetochore components cooperate to form a strong yet dynamic linkage to the microtubule. Although rings are not required for lateral binding, they likely maintain processive attachments to the ends of dynamic microtubules. These studies lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division. (Figure 13)
Relevant papers
Umbreit, Neil T.; Gestaut, Daniel R.; Tien, Jerry F.; Vollmar, Breanna S.; Gonen, Tamir; Asbury, Charles L.; Davis, Trisha N.
@article{pmid22908300,
title = {The Ndc80 kinetochore complex directly modulates microtubule dynamics},
author = {Umbreit, Neil T. and Gestaut, Daniel R. and Tien, Jerry F. and Vollmar, Breanna S. and Gonen, Tamir and Asbury, Charles L. and Davis, Trisha N. },
url = {https://cryoem.ucla.edu/wp-content/uploads/2012_umbreit.pdf, Main text},
doi = {10.1073/pnas.1209615109},
year = {2012},
date = {2012-10-02},
journal = {Proc. Natl. Acad. Sci. U.S.A.},
volume = {109},
number = {40},
pages = {16113--16118},
abstract = {The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The conserved Ndc80 complex is an essential microtubule-binding component of the kinetochore. Recent findings suggest that the Ndc80 complex influences microtubule dynamics at kinetochores in vivo. However, it was unclear if the Ndc80 complex mediates these effects directly, or by affecting other factors localized at the kinetochore. Using a reconstituted system in vitro, we show that the human Ndc80 complex directly stabilizes the tips of disassembling microtubules and promotes rescue (the transition from microtubule shortening to growth). In vivo, an N-terminal domain in the Ndc80 complex is phosphorylated by the Aurora B kinase. Mutations that mimic phosphorylation of the Ndc80 complex prevent stable kinetochore-microtubule attachment, and mutations that block phosphorylation damp kinetochore oscillations. We find that the Ndc80 complex with Aurora B phosphomimetic mutations is defective at promoting microtubule rescue, even when robustly coupled to disassembling microtubule tips. This impaired ability to affect dynamics is not simply because of weakened microtubule binding, as an N-terminally truncated complex with similar binding affinity is able to promote rescue. Taken together, these results suggest that in addition to regulating attachment stability, Aurora B controls microtubule dynamics through phosphorylation of the Ndc80 complex.
@article{pmid22885327,
title = {The structure of purified kinetochores reveals multiple microtubule-attachment sites},
author = {Shane Gonen and Bungo Akiyoshi and Matthew G Iadanza and Dan Shi and Nicole Duggan and Sue Biggins and Tamir Gonen},
url = {https://cryoem.ucla.edu/wp-content/uploads/2012_gonen.pdf, Main text},
doi = {10.1038/nsmb.2358},
year = {2012},
date = {2012-08-12},
journal = {Nat. Struct. Mol. Biol.},
volume = {19},
number = {9},
pages = {925--929},
abstract = {Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Chromosomes must be accurately partitioned to daughter cells to prevent aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are the macromolecular machines that segregate chromosomes by maintaining load-bearing attachments to the dynamic tips of microtubules. Here, we present the structure of isolated budding-yeast kinetochore particles, as visualized by EM and electron tomography of negatively stained preparations. The kinetochore appears as an ~126-nm particle containing a large central hub surrounded by multiple outer globular domains. In the presence of microtubules, some particles also have a ring that encircles the microtubule. Our data, showing that kinetochores bind to microtubules via multivalent attachments, lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division.
@article{pmid21107429,
title = {Tension directly stabilizes reconstituted kinetochore-microtubule attachments},
author = {Akiyoshi, Bungo and Sarangapani, Krishna K. and Powers, Andrew F. and Nelson, Christian R. and Reichow, Steve L. and Arellano-Santoyo, Hugo and Gonen, Tamir and Ranish, Jeffrey A. and Asbury, Charles L. and Biggins, Sue},
url = {https://cryoem.ucla.edu/wp-content/uploads/2010_akiyoshi.pdf, Main text},
doi = {10.1038/nature09594},
year = {2010},
date = {2010-11-24},
journal = {Nature},
volume = {468},
number = {7323},
pages = {576--579},
abstract = {Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
Kinetochores are macromolecular machines that couple chromosomes to dynamic microtubule tips during cell division, thereby generating force to segregate the chromosomes. Accurate segregation depends on selective stabilization of correct 'bi-oriented' kinetochore-microtubule attachments, which come under tension as the result of opposing forces exerted by microtubules. Tension is thought to stabilize these bi-oriented attachments indirectly, by suppressing the destabilizing activity of a kinase, Aurora B. However, a complete mechanistic understanding of the role of tension requires reconstitution of kinetochore-microtubule attachments for biochemical and biophysical analyses in vitro. Here we show that native kinetochore particles retaining the majority of kinetochore proteins can be purified from budding yeast and used to reconstitute dynamic microtubule attachments. Individual kinetochore particles maintain load-bearing associations with assembling and disassembling ends of single microtubules for >30 min, providing a close match to the persistent coupling seen in vivo between budding yeast kinetochores and single microtubules. Moreover, tension increases the lifetimes of the reconstituted attachments directly, through a catch bond-like mechanism that does not require Aurora B. On the basis of these findings, we propose that tension selectively stabilizes proper kinetochore-microtubule attachments in vivo through a combination of direct mechanical stabilization and tension-dependent phosphoregulation.
@article{pmid20479468,
title = {Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B},
author = {Jerry F Tien and Neil T Umbreit and Daniel R Gestaut and Andrew D Franck and Jeremy Cooper and Linda Wordeman and Tamir Gonen and Charles L Asbury and Trisha N Davis},
url = {https://cryoem.ucla.edu/wp-content/uploads/Tien_2010.pdf, Main text},
doi = {10.1083/jcb.200910142},
year = {2010},
date = {2010-05-17},
journal = {J. Cell Biol.},
volume = {189},
number = {4},
pages = {713--723},
abstract = {The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore-microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore-microtubule attachment is regulated by conserved signals for error correction.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
The coupling of kinetochores to dynamic spindle microtubules is crucial for chromosome positioning and segregation, error correction, and cell cycle progression. How these fundamental attachments are made and persist under tensile forces from the spindle remain important questions. As microtubule-binding elements, the budding yeast Ndc80 and Dam1 kinetochore complexes are essential and not redundant, but their distinct contributions are unknown. In this study, we show that the Dam1 complex is a processivity factor for the Ndc80 complex, enhancing the ability of the Ndc80 complex to form load-bearing attachments to and track with dynamic microtubule tips in vitro. Moreover, the interaction between the Ndc80 and Dam1 complexes is abolished when the Dam1 complex is phosphorylated by the yeast aurora B kinase Ipl1. This provides evidence for a mechanism by which aurora B resets aberrant kinetochore-microtubule attachments. We propose that the action of the Dam1 complex as a processivity factor in kinetochore-microtubule attachment is regulated by conserved signals for error correction.
@article{pmid17572669,
title = {Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis},
author = {Andrew D Franck and Andrew F Powers and Daniel R Gestaut and Tamir Gonen and Trisha N Davis and Charles L Asbury},
url = {https://cryoem.ucla.edu/wp-content/uploads/franck2007.pdf, Main text},
doi = {10.1038/ncb1609},
year = {2007},
date = {2007-06-17},
journal = {Nat. Cell Biol.},
volume = {9},
number = {7},
pages = {832--837},
abstract = {In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.},
keywords = {},
pubstate = {published},
tppubtype = {article}
}
In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.
