2015 |
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Bale, Jacob B; Park, Rachel U; Liu, Yuxi; Gonen, Shane; Gonen, Tamir; Cascio, Duilio; King, Neil P; Yeates, Todd O; Baker, David Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression Journal Article Protein Sci., 24 (10), pp. 1695–1701, 2015. @article{pmid26174163, title = {Structure of a designed tetrahedral protein assembly variant engineered to have improved soluble expression}, author = {Jacob B Bale and Rachel U Park and Yuxi Liu and Shane Gonen and Tamir Gonen and Duilio Cascio and Neil P King and Todd O Yeates and David Baker}, url = {https://cryoem.ucla.edu/wp-content/uploads/2015_bale.pdf, Main text}, doi = {10.1002/pro.2748}, year = {2015}, date = {2015-07-15}, journal = {Protein Sci.}, volume = {24}, number = {10}, pages = {1695--1701}, abstract = {We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We recently reported the development of a computational method for the design of coassembling multicomponent protein nanomaterials. While four such materials were validated at high-resolution by X-ray crystallography, low yield of soluble protein prevented X-ray structure determination of a fifth designed material, T33-09. Here we report the design and crystal structure of T33-31, a variant of T33-09 with improved soluble yield resulting from redesign efforts focused on mutating solvent-exposed side chains to charged amino acids. The structure is found to match the computational design model with atomic-level accuracy, providing further validation of the design approach and demonstrating a simple and potentially general means of improving the yield of designed protein nanomaterials. | |
Hattne, Johan; Reyes, Francis E; Nannenga, Brent L; Shi, Dan; de la Cruz, Jason M; Leslie, Andrew G W; Gonen, Tamir MicroED data collection and processing Journal Article Acta Crystallogr A Found Adv, 71 (Pt 4), pp. 353–360, 2015. @article{pmid26131894, title = {MicroED data collection and processing}, author = {Johan Hattne and Francis E Reyes and Brent L Nannenga and Dan Shi and Jason M de la Cruz and Andrew G W Leslie and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2015_hattne.pdf, Main text}, doi = {10.1107/S2053273315010669}, year = {2015}, date = {2015-07-01}, journal = {Acta Crystallogr A Found Adv}, volume = {71}, number = {Pt 4}, pages = {353--360}, abstract = {MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges.}, keywords = {}, pubstate = {published}, tppubtype = {article} } MicroED, a method at the intersection of X-ray crystallography and electron cryo-microscopy, has rapidly progressed by exploiting advances in both fields and has already been successfully employed to determine the atomic structures of several proteins from sub-micron-sized, three-dimensional crystals. A major limiting factor in X-ray crystallography is the requirement for large and well ordered crystals. By permitting electron diffraction patterns to be collected from much smaller crystals, or even single well ordered domains of large crystals composed of several small mosaic blocks, MicroED has the potential to overcome the limiting size requirement and enable structural studies on difficult-to-crystallize samples. This communication details the steps for sample preparation, data collection and reduction necessary to obtain refined, high-resolution, three-dimensional models by MicroED, and presents some of its unique challenges. | |
Gonen, Shane; DiMaio, Frank; Gonen, Tamir; Baker, David Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces Journal Article Science, 348 (6241), pp. 1365–1368, 2015. @article{pmid26089516, title = {Design of ordered two-dimensional arrays mediated by noncovalent protein-protein interfaces}, author = {Shane Gonen and Frank DiMaio and Tamir Gonen and David Baker}, url = {https://cryoem.ucla.edu/wp-content/uploads/2015_gonen.pdf, Main text}, doi = {10.1126/science.aaa9897}, year = {2015}, date = {2015-06-19}, journal = {Science}, volume = {348}, number = {6241}, pages = {1365--1368}, abstract = {We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a general approach to designing two-dimensional (2D) protein arrays mediated by noncovalent protein-protein interfaces. Protein homo-oligomers are placed into one of the seventeen 2D layer groups, the degrees of freedom of the lattice are sampled to identify configurations with shape-complementary interacting surfaces, and the interaction energy is minimized using sequence design calculations. We used the method to design proteins that self-assemble into layer groups P 3 2 1, P 4 2(1) 2, and P 6. Projection maps of micrometer-scale arrays, assembled both in vitro and in vivo, are consistent with the design models and display the target layer group symmetry. Such programmable 2D protein lattices should enable new approaches to structure determination, sensing, and nanomaterial engineering. | |
2014 |
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Huang, Po-Ssu; Oberdorfer, Gustav; Xu, Chunfu; Pei, Xue Y; Nannenga, Brent L; Rogers, Joseph M; DiMaio, Frank; Gonen, Tamir; Luisi, Ben; Baker, David High thermodynamic stability of parametrically designed helical bundles Journal Article Science, 346 (6208), pp. 481–485, 2014. @article{pmid25342806, title = {High thermodynamic stability of parametrically designed helical bundles}, author = {Po-Ssu Huang and Gustav Oberdorfer and Chunfu Xu and Xue Y Pei and Brent L Nannenga and Joseph M Rogers and Frank DiMaio and Tamir Gonen and Ben Luisi and David Baker}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_huang.pdf, Main text}, doi = {10.1126/science.1257481}, year = {2014}, date = {2014-10-24}, journal = {Science}, volume = {346}, number = {6208}, pages = {481--485}, abstract = {We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.}, keywords = {}, pubstate = {published}, tppubtype = {article} } We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications. | |
Nannenga, Brent L; Shi, Dan; Hattne, Johan; Reyes, Francis E; Gonen, Tamir Structure of catalase determined by MicroED Journal Article Elife, 3 , pp. e03600, 2014. @article{pmid25303172, title = {Structure of catalase determined by MicroED}, author = {Brent L Nannenga and Dan Shi and Johan Hattne and Francis E Reyes and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_nannenga_b.pdf, Main text}, doi = {10.7554/eLife.03600}, year = {2014}, date = {2014-10-10}, journal = {Elife}, volume = {3}, pages = {e03600}, abstract = {MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.}, keywords = {}, pubstate = {published}, tppubtype = {article} } MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination. | |
Russell, Alistair B; Wexler, Aaron G; Harding, Brittany N; Whitney, John C; Bohn, Alan J; Goo, Young Ah; Tran, Bao Q; Barry, Natasha A; Zheng, Hongjin; Peterson, Brook S; Chou, Seemay; Gonen, Tamir; Goodlett, David R; Goodman, Andrew L; Mougous, Joseph D A type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism Journal Article Cell Host Microbe, 16 (2), pp. 227–236, 2014. @article{pmid25070807, title = {A type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism}, author = {Alistair B Russell and Aaron G Wexler and Brittany N Harding and John C Whitney and Alan J Bohn and Young Ah Goo and Bao Q Tran and Natasha A Barry and Hongjin Zheng and Brook S Peterson and Seemay Chou and Tamir Gonen and David R Goodlett and Andrew L Goodman and Joseph D Mougous}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_russell.pdf, Main text}, doi = {10.1016/j.chom.2014.07.007}, year = {2014}, date = {2014-08-13}, journal = {Cell Host Microbe}, volume = {16}, number = {2}, pages = {227--236}, abstract = {Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens. | |
Wisedchaisri, Goragot; Park, Min-Sun; Iadanza, Matthew G; Zheng, Hongjin; Gonen, Tamir Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE Journal Article Nat Commun, 5 , pp. 4521, 2014. @article{pmid25088546, title = {Proton-coupled sugar transport in the prototypical major facilitator superfamily protein XylE}, author = {Goragot Wisedchaisri and Min-Sun Park and Matthew G Iadanza and Hongjin Zheng and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_wisedchaisri.pdf, Main text}, doi = {10.1038/ncomms5521}, year = {2014}, date = {2014-08-04}, journal = {Nat Commun}, volume = {5}, pages = {4521}, abstract = {The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins. | |
Nannenga, Brent L; Shi, Dan; Leslie, Andrew G W; Gonen, Tamir High-resolution structure determination by continuous-rotation data collection in MicroED Journal Article Nat. Methods, 11 (9), pp. 927–930, 2014. @article{pmid25086503, title = {High-resolution structure determination by continuous-rotation data collection in MicroED}, author = {Brent L Nannenga and Dan Shi and Andrew G W Leslie and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_nannenga.pdf, Main text}, doi = {10.1038/nmeth.3043}, year = {2014}, date = {2014-08-03}, journal = {Nat. Methods}, volume = {11}, number = {9}, pages = {927--930}, abstract = {MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology.}, keywords = {}, pubstate = {published}, tppubtype = {article} } MicroED uses very small three-dimensional protein crystals and electron diffraction for structure determination. We present an improved data collection protocol for MicroED called 'continuous rotation'. Microcrystals are continuously rotated during data collection, yielding more accurate data. The method enables data processing with the crystallographic software tool MOSFLM, which resulted in improved resolution for the model protein lysozyme. These improvements are paving the way for the broad implementation and application of MicroED in structural biology. | |
Nannenga, Brent L; Gonen, Tamir Protein structure determination by MicroED Journal Article Curr. Opin. Struct. Biol., 27 , pp. 24–31, 2014. @article{pmid24709395, title = {Protein structure determination by MicroED}, author = {Brent L Nannenga and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_nannengagonen.pdf, Main text}, doi = {10.1016/j.sbi.2014.03.004}, year = {2014}, date = {2014-08-01}, journal = {Curr. Opin. Struct. Biol.}, volume = {27}, pages = {24--31}, abstract = {In this review we discuss the current advances relating to structure determination from protein microcrystals with special emphasis on the newly developed method called MicroED. This method uses a transmission electron cryo-microscope to collect electron diffraction data from extremely small 3-dimensional (3D) crystals. MicroED has been used to solve the 3D structure of the model protein lysozyme to 2.9Å resolution. As the method further matures, MicroED promises to offer a unique and widely applicable approach to protein crystallography using nanocrystals.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In this review we discuss the current advances relating to structure determination from protein microcrystals with special emphasis on the newly developed method called MicroED. This method uses a transmission electron cryo-microscope to collect electron diffraction data from extremely small 3-dimensional (3D) crystals. MicroED has been used to solve the 3D structure of the model protein lysozyme to 2.9Å resolution. As the method further matures, MicroED promises to offer a unique and widely applicable approach to protein crystallography using nanocrystals. | |
Gonen, Tamir; Waksman, Gabriel Editorial overview: Membranes: recent methods in the study of membrane protein structure Journal Article Curr. Opin. Struct. Biol., 27 , pp. iv-v, 2014. @article{pmid25242735, title = {Editorial overview: Membranes: recent methods in the study of membrane protein structure}, author = {Tamir Gonen and Gabriel Waksman}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_gonenwaksman.pdf, Main text}, doi = {10.1016/j.sbi.2014.09.002}, year = {2014}, date = {2014-08-01}, journal = {Curr. Opin. Struct. Biol.}, volume = {27}, pages = {iv-v}, keywords = {}, pubstate = {published}, tppubtype = {article} } | |
King, Neil P; Bale, Jacob B; Sheffler, William; McNamara, Dan E; Gonen, Shane; Gonen, Tamir; Yeates, Todd O; Baker, David Accurate design of co-assembling multi-component protein nanomaterials Journal Article Nature, 510 (7503), pp. 103–108, 2014. @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. | |
Iadanza, Matthew G; Gonen, Tamir A suite of software for processing MicroED data of extremely small protein crystals Journal Article J Appl Crystallogr, 47 (Pt 3), pp. 1140–1145, 2014. @article{pmid24904248, title = {A suite of software for processing MicroED data of extremely small protein crystals}, author = {Matthew G Iadanza and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_iadanzagonen.pdf, Main text}, doi = {10.1107/S1600576714008073}, year = {2014}, date = {2014-06-01}, journal = {J Appl Crystallogr}, volume = {47}, number = {Pt 3}, pages = {1140--1145}, abstract = {Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. MicroED patterns, which are collected in a transmission electron microscope, were initially not amenable to indexing and intensity extraction by standard software, which necessitated the development of a suite of programs for data processing. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination. This ad hoc solution has been modified for more general use to provide a means for processing MicroED data until the technique can be fully implemented into existing crystallographic software packages. The suite is written in Python and the source code is available under a GNU General Public License.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. MicroED patterns, which are collected in a transmission electron microscope, were initially not amenable to indexing and intensity extraction by standard software, which necessitated the development of a suite of programs for data processing. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination. This ad hoc solution has been modified for more general use to provide a means for processing MicroED data until the technique can be fully implemented into existing crystallographic software packages. The suite is written in Python and the source code is available under a GNU General Public License. | |
Anderson, Thomas M; Clay, Mary C; Cioffi, Alexander G; Diaz, Katrina A; Hisao, Grant S; Tuttle, Marcus D; Nieuwkoop, Andrew J; Comellas, Gemma ; Maryum, Nashrah ; Wang, Shu ; Uno, Brice E; Wildeman, Erin L; Gonen, Tamir ; Rienstra, Chad M; Burke, Martin D Amphotericin forms an extramembranous and fungicidal sterol sponge Journal Article Nat. Chem. Biol., 10 (5), pp. 400–406, 2014. @article{pmid24681535, title = {Amphotericin forms an extramembranous and fungicidal sterol sponge}, author = {Anderson, Thomas M. and Clay, Mary C. and Cioffi, Alexander G. and Diaz, Katrina A. and Hisao, Grant S. and Tuttle, Marcus D. and Nieuwkoop, Andrew J. and Comellas, Gemma and Maryum, Nashrah and Wang, Shu and Uno, Brice E. and Wildeman, Erin L. and Gonen, Tamir and Rienstra, Chad M. and Burke, Martin D.}, url = {https://cryoem.ucla.edu/wp-content/uploads/2014_anderson.pdf, Full text}, doi = {10.1038/nchembio.1496}, year = {2014}, date = {2014-03-30}, journal = {Nat. Chem. Biol.}, volume = {10}, number = {5}, pages = {400--406}, abstract = {For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells.}, keywords = {}, pubstate = {published}, tppubtype = {article} } For over 50 years, amphotericin has remained the powerful but highly toxic last line of defense in treating life-threatening fungal infections in humans with minimal development of microbial resistance. Understanding how this small molecule kills yeast is thus critical for guiding development of derivatives with an improved therapeutic index and other resistance-refractory antimicrobial agents. In the widely accepted ion channel model for its mechanism of cytocidal action, amphotericin forms aggregates inside lipid bilayers that permeabilize and kill cells. In contrast, we report that amphotericin exists primarily in the form of large, extramembranous aggregates that kill yeast by extracting ergosterol from lipid bilayers. These findings reveal that extraction of a polyfunctional lipid underlies the resistance-refractory antimicrobial action of amphotericin and suggests a roadmap for separating its cytocidal and membrane-permeabilizing activities. This new mechanistic understanding is also guiding development of what are to our knowledge the first derivatives of amphotericin that kill yeast but not human cells. | |
2013 |
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Shi, Dan; Nannenga, Brent L; Iadanza, Matthew G; Gonen, Tamir Three-dimensional electron crystallography of protein microcrystals Journal Article Elife, 2 , pp. e01345, 2013. @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. | |
Smith, Donelson F; Reichow, Steve L; Esseltine, Jessica L; Shi, Dan; Langeberg, Lorene K; Scott, John D; Gonen, Tamir Intrinsic disorder within an AKAP-protein kinase A complex guides local substrate phosphorylation Journal Article Elife, 2 , pp. e01319, 2013. @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. | |
Gold, Matthew G; Gonen, Tamir; Scott, John D Local cAMP signaling in disease at a glance Journal Article J. Cell. Sci., 126 (Pt 20), pp. 4537–4543, 2013. @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. | |
Reichow, Stephen ; Gonen, Tamir Dynamic Modulation of Water Permeability in the Lens Aquaporin-0 Inproceedings Proceedings of Microscopy & Microanalysis, pp. 50–51, 2013. @inproceedings{reichow_2013, title = {Dynamic Modulation of Water Permeability in the Lens Aquaporin-0}, author = {Reichow, Stephen and Gonen, Tamir}, doi = {10.1017/S1431927613002249}, year = {2013}, date = {2013-10-09}, booktitle = {Proceedings of Microscopy & Microanalysis}, journal = {Proceedings of Microscopy & Microanlysis}, volume = {19}, number = {S2}, pages = {50--51}, keywords = {}, pubstate = {published}, tppubtype = {inproceedings} } | |
Silverman, Julie M; Agnello, Danielle M; Zheng, Hongjin; Andrews, Benjamin T; Li, Mo; Catalano, Carlos E; Gonen, Tamir; Mougous, Joseph D Haemolysin Coregulated Protein Is an Exported Receptor and Chaperone of Type VI Secretion Substrates Journal Article Mol. Cell, 51 (5), pp. 584–593, 2013. @article{pmid23954347, title = {Haemolysin Coregulated Protein Is an Exported Receptor and Chaperone of Type VI Secretion Substrates}, author = {Julie M Silverman and Danielle M Agnello and Hongjin Zheng and Benjamin T Andrews and Mo Li and Carlos E Catalano and Tamir Gonen and Joseph D Mougous}, url = {https://cryoem.ucla.edu/wp-content/uploads/2013_silverman.pdf, Main text}, doi = {10.1016/j.molcel.2013.07.025}, year = {2013}, date = {2013-09-12}, journal = {Mol. Cell}, volume = {51}, number = {5}, pages = {584--593}, abstract = {Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp-effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Secretion systems require high-fidelity mechanisms to discriminate substrates among the vast cytoplasmic pool of proteins. Factors mediating substrate recognition by the type VI secretion system (T6SS) of Gram-negative bacteria, a widespread pathway that translocates effector proteins into target bacterial cells, have not been defined. We report that haemolysin coregulated protein (Hcp), a ring-shaped hexamer secreted by all characterized T6SSs, binds specifically to cognate effector molecules. Electron microscopy analysis of an Hcp-effector complex from Pseudomonas aeruginosa revealed the effector bound to the inner surface of Hcp. Further studies demonstrated that interaction with the Hcp pore is a general requirement for secretion of diverse effectors encompassing several enzymatic classes. Though previous models depict Hcp as a static conduit, our data indicate it is a chaperone and receptor of substrates. These unique functions of a secreted protein highlight fundamental differences between the export mechanism of T6 and other characterized secretory pathways. | |
Reichow, Steve L; Clemens, Daniel M; Freites, Alfredo J; Nemeth-Cahalan, Karin L; Heyden, Matthias; Tobias, Douglas J; Hall, James E; Gonen, Tamir Allosteric mechanism of water-channel gating by Ca²⁺-calmodulin Journal Article Nat. Struct. Mol. Biol., 20 (9), pp. 1085–1092, 2013. @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. | |
Zheng, Hongjin; Wisedchaisri, Goragot; Gonen, Tamir Crystal structure of a nitrate/nitrite exchanger Journal Article Nature, 497 (7451), pp. 647–651, 2013. @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. | |
Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy Journal Article Curr Protoc Protein Sci, 72 (1), pp. 17.15.1–17.15.11, 2013. @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. | |
Shi, Liang; Zheng, Hongjin; Zheng, Hui; Borkowski, Brian A; Shi, Dan; Gonen, Tamir; Jiang, Qiu-Xing Voltage sensor ring in a native structure of a membrane-embedded potassium channel Journal Article Proc. Natl. Acad. Sci. U.S.A., 110 (9), pp. 3369–3374, 2013, (Retracted). @article{pmid23401554, title = {Voltage sensor ring in a native structure of a membrane-embedded potassium channel}, author = {Liang Shi and Hongjin Zheng and Hui Zheng and Brian A Borkowski and Dan Shi and Tamir Gonen and Qiu-Xing Jiang}, doi = {10.1073/pnas.1218203110}, year = {2013}, date = {2013-02-26}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {110}, number = {9}, pages = {3369--3374}, abstract = {Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid-protein interactions in general and for the mechanism of voltage sensing in particular.}, note = {Retracted}, keywords = {}, pubstate = {published}, tppubtype = {article} } Voltage-gated ion channels support electrochemical activity in cells and are largely responsible for information flow throughout the nervous systems. The voltage sensor domains in these channels sense changes in transmembrane potential and control ion flux across membranes. The X-ray structures of a few voltage-gated ion channels in detergents have been determined and have revealed clear structural variations among their respective voltage sensor domains. More recent studies demonstrated that lipids around a voltage-gated channel could directly alter its conformational state in membrane. Because of these disparities, the structural basis for voltage sensing in native membranes remains elusive. Here, through electron-crystallographic analysis of membrane-embedded proteins, we present the detailed view of a voltage-gated potassium channel in its inactivated state. Contrary to all known structures of voltage-gated ion channels in detergents, our data revealed a unique conformation in which the four voltage sensor domains of a voltage-gated potassium channel from Aeropyrum pernix (KvAP) form a ring structure that completely surrounds the pore domain of the channel. Such a structure is named the voltage sensor ring. Our biochemical and electrophysiological studies support that the voltage sensor ring represents a physiological conformation. These data together suggest that lipids exert strong effects on the channel structure and that these effects may be changed upon membrane disruption. Our results have wide implications for lipid-protein interactions in general and for the mechanism of voltage sensing in particular. | |
2012 |
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Filbin, Megan E; Vollmar, Breanna S; Shi, Dan; Gonen, Tamir; Kieft, Jeffrey S HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation Journal Article Nat. Struct. Mol. Biol., 20 (2), pp. 150–158, 2012. @article{pmid23262488, title = {HCV IRES manipulates the ribosome to promote the switch from translation initiation to elongation}, author = {Megan E Filbin and Breanna S Vollmar and Dan Shi and Tamir Gonen and Jeffrey S Kieft}, url = {https://cryoem.ucla.edu/wp-content/uploads/2013_filbin.pdf, Main text}, doi = {10.1038/nsmb.2465}, year = {2012}, date = {2012-12-23}, journal = {Nat. Struct. Mol. Biol.}, volume = {20}, number = {2}, pages = {150--158}, abstract = {The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The internal ribosome entry site (IRES) of the hepatitis C virus (HCV) drives noncanonical initiation of protein synthesis necessary for viral replication. Functional studies of the HCV IRES have focused on 80S ribosome formation but have not explored its role after the 80S ribosome is poised at the start codon. Here, we report that mutations of an IRES domain that docks in the 40S subunit's decoding groove cause only a local perturbation in IRES structure and result in conformational changes in the IRES-rabbit 40S subunit complex. Functionally, the mutations decrease IRES activity by inhibiting the first ribosomal translocation event, and modeling results suggest that this effect occurs through an interaction with a single ribosomal protein. The ability of the HCV IRES to manipulate the ribosome provides insight into how the ribosome's structure and function can be altered by bound RNAs, including those derived from cellular invaders. | |
Wisedchaisri, Goragot; Gonen, Tamir Phasing Electron Diffraction Data by Molecular Replacement: Strategy for Structure Determination and Refinement Book Chapter Electron Crystallography of Soluble and Membrane Proteins, 955 , Chapter 14, pp. 243–272, 2012. @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. | |
Gonen, Tamir The Collection of High-Resolution Electron Diffraction Data Book Chapter Electron Crystallography of Soluble and Membrane Proteins, 955 , Chapter 9, pp. 153–169, 2012. @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. | |
Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas High-Throughput Methods for Electron Crystallography Book Chapter Electron Crystallography of Soluble and Membrane Proteins, 955 , Chapter 15, pp. 273–296, 2012. @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. | |
Umbreit, Neil T; Gestaut, Daniel R; Tien, Jerry F; Vollmar, Breanna S; Gonen, Tamir ; Asbury, Charles L; Davis, Trisha N The Ndc80 kinetochore complex directly modulates microtubule dynamics Journal Article Proc. Natl. Acad. Sci. U.S.A., 109 (40), pp. 16113–16118, 2012. @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. | |
Gonen, Shane; Akiyoshi, Bungo; Iadanza, Matthew G; Shi, Dan; Duggan, Nicole; Biggins, Sue; Gonen, Tamir The structure of purified kinetochores reveals multiple microtubule-attachment sites Journal Article Nat. Struct. Mol. Biol., 19 (9), pp. 925–929, 2012. @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. | |
Gonen, Tamir; Waksman, Gabriel Recent progress in membrane protein structures and investigation methods Journal Article Curr. Opin. Struct. Biol., 22 (4), pp. 467–468, 2012. @article{pmid22819667, title = {Recent progress in membrane protein structures and investigation methods}, author = {Tamir Gonen and Gabriel Waksman}, url = {https://cryoem.ucla.edu/wp-content/uploads/2012_gonenwaksman.pdf, Main text}, doi = {10.1016/j.sbi.2012.07.002}, year = {2012}, date = {2012-08-01}, journal = {Curr. Opin. Struct. Biol.}, volume = {22}, number = {4}, pages = {467--468}, keywords = {}, pubstate = {published}, tppubtype = {article} } | |
Jiang, Qiu-Xing; Gonen, Tamir The influence of lipids on voltage-gated ion channels Journal Article Curr. Opin. Struct. Biol., 22 (4), pp. 529–536, 2012. @article{pmid22483432, title = {The influence of lipids on voltage-gated ion channels}, author = {Qiu-Xing Jiang and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/2012_jianggonen.pdf, Main text}, doi = {10.1016/j.sbi.2012.03.009}, year = {2012}, date = {2012-08-01}, journal = {Curr. Opin. Struct. Biol.}, volume = {22}, number = {4}, pages = {529--536}, abstract = {Voltage-gated ion channels are responsible for transmitting electrochemical signals in both excitable and non-excitable cells. Structural studies of voltage-gated potassium and sodium channels by X-ray crystallography have revealed atomic details on their voltage-sensor domains (VSDs) and pore domains, and were put in context of disparate mechanistic views on the voltage-driven conformational changes in these proteins. Functional investigation of voltage-gated channels in membranes, however, showcased a mechanism of lipid-dependent gating for voltage-gated channels, suggesting that the lipids play an indispensible and critical role in the proper gating of many of these channels. Structure determination of membrane-embedded voltage-gated ion channels appears to be the next frontier in fully addressing the mechanism by which the VSDs control channel opening. Currently electron crystallography is the only structural biology method in which a membrane protein of interest is crystallized within a complete lipid-bilayer mimicking the native environment of a biological membrane. At a sufficiently high resolution, an electron crystallographic structure could reveal lipids, the channel and their mutual interactions at the atomic level. Electron crystallography is therefore a promising avenue toward understanding how lipids modulate channel activation through close association with the VSDs.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Voltage-gated ion channels are responsible for transmitting electrochemical signals in both excitable and non-excitable cells. Structural studies of voltage-gated potassium and sodium channels by X-ray crystallography have revealed atomic details on their voltage-sensor domains (VSDs) and pore domains, and were put in context of disparate mechanistic views on the voltage-driven conformational changes in these proteins. Functional investigation of voltage-gated channels in membranes, however, showcased a mechanism of lipid-dependent gating for voltage-gated channels, suggesting that the lipids play an indispensible and critical role in the proper gating of many of these channels. Structure determination of membrane-embedded voltage-gated ion channels appears to be the next frontier in fully addressing the mechanism by which the VSDs control channel opening. Currently electron crystallography is the only structural biology method in which a membrane protein of interest is crystallized within a complete lipid-bilayer mimicking the native environment of a biological membrane. At a sufficiently high resolution, an electron crystallographic structure could reveal lipids, the channel and their mutual interactions at the atomic level. Electron crystallography is therefore a promising avenue toward understanding how lipids modulate channel activation through close association with the VSDs. | |
King, Neil P; Sheffler, William; Sawaya, Michael R; Vollmar, Breanna S; Sumida, John P; André, Ingemar; Gonen, Tamir; Yeates, Todd O; Baker, David Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy Journal Article Science, 336 (6085), pp. 1171–1174, 2012. @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. | |
2011 |
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Lo, Sheng-Ying; Brett, Christopher L; Plemel, Rachael L; Vignali, Marissa; Fields, Stanley; Gonen, Tamir; Merz, Alexey J Intrinsic tethering activity of endosomal Rab proteins Journal Article Nat. Struct. Mol. Biol., 19 (1), pp. 40–47, 2011. @article{pmid22157956, title = {Intrinsic tethering activity of endosomal Rab proteins}, author = {Sheng-Ying Lo and Christopher L Brett and Rachael L Plemel and Marissa Vignali and Stanley Fields and Tamir Gonen and Alexey J Merz}, url = {https://cryoem.ucla.edu/wp-content/uploads/2012_lo.pdf, Main text}, doi = {10.1038/nsmb.2162}, year = {2011}, date = {2011-12-01}, journal = {Nat. Struct. Mol. Biol.}, volume = {19}, number = {1}, pages = {40--47}, abstract = {Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with conventional effectors and SNAREs to drive efficient docking and fusion.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Rab small G proteins control membrane trafficking events required for many processes including secretion, lipid metabolism, antigen presentation and growth factor signaling. Rabs recruit effectors that mediate diverse functions including vesicle tethering and fusion. However, many mechanistic questions about Rab-regulated vesicle tethering are unresolved. Using chemically defined reaction systems, we discovered that Vps21, a Saccharomyces cerevisiae ortholog of mammalian endosomal Rab5, functions in trans with itself and with at least two other endosomal Rabs to directly mediate GTP-dependent tethering. Vps21-mediated tethering was stringently and reversibly regulated by an upstream activator, Vps9, and an inhibitor, Gyp1, which were sufficient to drive dynamic cycles of tethering and detethering. These experiments reveal a previously undescribed mode of tethering by endocytic Rabs. In our working model, the intrinsic tethering capacity Vps21 operates in concert with conventional effectors and SNAREs to drive efficient docking and fusion. | |
Gold, Matthew G; Reichow, Steve L; O'Neill, Susan E; Weisbrod, Chad R; Langeberg, Lorene K; Bruce, James E; Gonen, Tamir; Scott, John D AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency Journal Article EMBO Mol Med, 4 (1), pp. 15–26, 2011. @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. | |
Wisedchaisri, Goragot; Reichow, Steve L; Gonen, Tamir Advances in Structural and Functional Analysis of Membrane Proteins by Electron Crystallography Journal Article Structure, 19 (10), pp. 1381–1393, 2011. @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. | |
Korotkov, Konstantin V; Gonen, Tamir; Hol, Wim G J Secretins: dynamic channels for protein transport across membranes Journal Article Trends Biochem. Sci., 36 (8), pp. 433–443, 2011. @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. | |
Wisedchaisri, Goragot; Gonen, Tamir Fragment-Based Phase Extension for Three-Dimensional Structure Determination of Membrane Proteins by Electron Crystallography Journal Article Structure, 19 (7), pp. 976–987, 2011. @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. | |
Reichow, Steve L; Korotkov, Konstantin V; Gonen, Melissa; Sun, Ji; Delarosa, Jaclyn R; Hol, Wim G J; Gonen, Tamir The binding of cholera toxin to the periplasmic vestibule of the type II secretion channel Journal Article Channels (Austin), 5 (3), pp. 215–218, 2011. @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. | |
Jehle, Stefan; Vollmar, Breanna S; Bardiaux, Benjamin; Dove, Katja K; Rajagopal, Ponni; Gonen, Tamir; Oschkinat, Hartmut; Klevit, Rachel E N-terminal domain of αB-crystallin provides a conformational switch for multimerization and structural heterogeneity Journal Article Proc. Natl. Acad. Sci. U.S.A., 108 (16), pp. 6409–6414, 2011. @article{pmid21464278, title = {N-terminal domain of αB-crystallin provides a conformational switch for multimerization and structural heterogeneity}, author = {Stefan Jehle and Breanna S Vollmar and Benjamin Bardiaux and Katja K Dove and Ponni Rajagopal and Tamir Gonen and Hartmut Oschkinat and Rachel E Klevit}, url = {https://cryoem.ucla.edu/wp-content/uploads/2011_jehle.pdf, Main text}, doi = {10.1073/pnas.1014656108}, year = {2011}, date = {2011-04-19}, journal = {Proc. Natl. Acad. Sci. U.S.A.}, volume = {108}, number = {16}, pages = {6409--6414}, abstract = {The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The small heat shock protein (sHSP) αB-crystallin (αB) plays a key role in the cellular protection system against stress. For decades, high-resolution structural studies on heterogeneous sHSPs have been confounded by the polydisperse nature of αB oligomers. We present an atomic-level model of full-length αB as a symmetric 24-subunit multimer based on solid-state NMR, small-angle X-ray scattering (SAXS), and EM data. The model builds on our recently reported structure of the homodimeric α-crystallin domain (ACD) and C-terminal IXI motif in the context of the multimer. A hierarchy of interactions contributes to build multimers of varying sizes: Interactions between two ACDs define a dimer, three dimers connected by their C-terminal regions define a hexameric unit, and variable interactions involving the N-terminal region define higher-order multimers. Within a multimer, N-terminal regions exist in multiple environments, contributing to the heterogeneity observed by NMR. Analysis of SAXS data allows determination of a heterogeneity parameter for this type of system. A mechanism of multimerization into higher-order asymmetric oligomers via the addition of up to six dimeric units to a 24-mer is proposed. The proposed asymmetric multimers explain the homogeneous appearance of αB in negative-stain EM images and the known dynamic exchange of αB subunits. The model of αB provides a structural basis for understanding known disease-associated missense mutations and makes predictions concerning substrate binding and the reported fibrilogenesis of αB. | |
Budzinski, Kristi L; Sgro, Allyson E; Fujimoto, Bryant S; Gadd, Jennifer C; Shuart, Noah G; Gonen, Tamir; Bajjaleih, Sandra M; Chiu, Daniel T Synaptosomes as a Platform for Loading Nanoparticles into Synaptic Vesicles Journal Article ACS Chem Neurosci, 2 (5), pp. 236–241, 2011. @article{pmid21666849, title = {Synaptosomes as a Platform for Loading Nanoparticles into Synaptic Vesicles}, author = {Kristi L Budzinski and Allyson E Sgro and Bryant S Fujimoto and Jennifer C Gadd and Noah G Shuart and Tamir Gonen and Sandra M Bajjaleih and Daniel T Chiu}, url = {https://cryoem.ucla.edu/wp-content/uploads/2011_budzinski.pdf, Main text}, doi = {10.1021/cn200009n}, year = {2011}, date = {2011-03-08}, journal = {ACS Chem Neurosci}, volume = {2}, number = {5}, pages = {236--241}, abstract = {Synaptosomes are intact, isolated nerve terminals that contain the necessary machinery to recycle synaptic vesicles via endocytosis and exocytosis upon stimulation. Here we use this property of synaptosomes to load quantum dots into synaptic vesicles. Vesicles are then isolated from the synaptosomes, providing a method to probe isolated, individual synaptic vesicles where each vesicle contains a single, encapsulated nanoparticle. This technique provided an encapsulation efficiency of ~16%, that is, ~16% of the vesicles contained a single quantum dot while the remaining vesicles were empty. The ability to load single nanoparticles into synaptic vesicles opens new opportunity for employing various nanoparticle-based sensors to study the dynamics of vesicular transporters.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Synaptosomes are intact, isolated nerve terminals that contain the necessary machinery to recycle synaptic vesicles via endocytosis and exocytosis upon stimulation. Here we use this property of synaptosomes to load quantum dots into synaptic vesicles. Vesicles are then isolated from the synaptosomes, providing a method to probe isolated, individual synaptic vesicles where each vesicle contains a single, encapsulated nanoparticle. This technique provided an encapsulation efficiency of ~16%, that is, ~16% of the vesicles contained a single quantum dot while the remaining vesicles were empty. The ability to load single nanoparticles into synaptic vesicles opens new opportunity for employing various nanoparticle-based sensors to study the dynamics of vesicular transporters. | |
2010 |
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Akiyoshi, Bungo ; Sarangapani, Krishna K; Powers, Andrew F; Nelson, Christian R; Reichow, Steve L; Arellano-Santoyo, Hugo ; Gonen, Tamir ; Ranish, Jeffrey A; Asbury, Charles L; Biggins, Sue Tension directly stabilizes reconstituted kinetochore-microtubule attachments Journal Article Nature, 468 (7323), pp. 576–579, 2010. @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. | |
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 Structural Underpinnings of Nitrogen Regulation by the Prototypical Nitrogen-Responsive Transcriptional Factor NrpR Journal Article Structure, 18 (11), pp. 1512–1521, 2010. @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. | |
Reichow, Steve L; Korotkov, Konstantin V; Hol, Wim G J; Gonen, Tamir Structure of the cholera toxin secretion channel in its closed state Journal Article Nat. Struct. Mol. Biol., 17 (10), pp. 1226–1232, 2010. @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. | |
Sedlak, Ruth Hall; Hnilova, Marketa; Gachelet, Eliora; Przybyla, Lyralynne; Dranow, David; Gonen, Tamir; Sarikaya, Mehmet; Tamerler, Candan; Traxler, Beth An Engineered DNA-binding Protein Self-assembles Metallic Nanostructures Journal Article Chembiochem, 11 (15), pp. 2108–2112, 2010. @article{pmid20827792, title = {An Engineered DNA-binding Protein Self-assembles Metallic Nanostructures}, author = {Ruth Hall Sedlak and Marketa Hnilova and Eliora Gachelet and Lyralynne Przybyla and David Dranow and Tamir Gonen and Mehmet Sarikaya and Candan Tamerler and Beth Traxler}, url = {https://cryoem.ucla.edu/wp-content/uploads/hall_2010.pdf, Main text}, doi = {10.1002/cbic.201000407}, year = {2010}, date = {2010-09-09}, journal = {Chembiochem}, volume = {11}, number = {15}, pages = {2108--2112}, keywords = {}, pubstate = {published}, tppubtype = {article} } | |
Sanowar, Sarah; Singh, Pragya; Pfuetzner, Richard A; André, Ingemar; Zheng, Hongjin; Spreter, Thomas; Strynadka, Natalie C J; Gonen, Tamir; Baker, David; Goodlett, David R; Miller, Samuel I Interactions of the Transmembrane Polymeric Rings of the Salmonella enterica Serovar Typhimurium type III Secretion System Journal Article MBio, 1 (3), pp. e00158-10, 2010. @article{pmid20824104, title = {Interactions of the Transmembrane Polymeric Rings of the Salmonella enterica Serovar Typhimurium type III Secretion System}, author = {Sarah Sanowar and Pragya Singh and Richard A Pfuetzner and Ingemar André and Hongjin Zheng and Thomas Spreter and Natalie C J Strynadka and Tamir Gonen and David Baker and David R Goodlett and Samuel I Miller}, url = {https://cryoem.ucla.edu/wp-content/uploads/sanowar_2010.pdf, Main text}, doi = {10.1128/mBio.00158-10}, year = {2010}, date = {2010-08-03}, journal = {MBio}, volume = {1}, number = {3}, pages = {e00158-10}, abstract = {The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The type III secretion system (T3SS) is an interspecies protein transport machine that plays a major role in interactions of Gram-negative bacteria with animals and plants by delivering bacterial effector proteins into host cells. T3SSs span both membranes of Gram-negative bacteria by forming a structure of connected oligomeric rings termed the needle complex (NC). Here, the localization of subunits in the Salmonella enterica serovar Typhimurium T3SS NC were probed via mass spectrometry-assisted identification of chemical cross-links in intact NC preparations. Cross-links between amino acids near the amino terminus of the outer membrane ring component InvG and the carboxyl terminus of the inner membrane ring component PrgH and between the two inner membrane components PrgH and PrgK allowed for spatial localization of the three ring components within the electron density map structures of NCs. Mutational and biochemical analysis demonstrated that the amino terminus of InvG and the carboxyl terminus of PrgH play a critical role in the assembly and function of the T3SS apparatus. Analysis of an InvG mutant indicates that the structure of the InvG oligomer can affect the switching of the T3SS substrate to translocon and effector components. This study provides insights into how structural organization of needle complex base components promotes T3SS assembly and function. | |
Tien, Jerry F; Umbreit, Neil T; Gestaut, Daniel R; Franck, Andrew D; Cooper, Jeremy; Wordeman, Linda; Gonen, Tamir; Asbury, Charles L; Davis, Trisha N Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B Journal Article J. Cell Biol., 189 (4), pp. 713–723, 2010. @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. | |
Zheng, Hongjin; Taraska, Justin; Merz, Alexey J; Gonen, Tamir The prototypical H⁺/Galactose Symporter GalP Assembles into Functional Trimers Journal Article J. Mol. Biol., 396 (3), pp. 593–601, 2010. @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. | |
2009 |
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Reichow, Steve L; Gonen, Tamir Lipid-protein interactions probed by electron crystallography Journal Article Curr. Opin. Struct. Biol., 19 (5), pp. 560–565, 2009. @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. | |
2008 |
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Reichow, Steve L; Gonen, Tamir Noncanonical Binding of Calmodulin to Aquaporin-0: Implications for Channel Regulation Journal Article Structure, 16 (9), pp. 1389–1398, 2008. @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. | |
Zheng, Hongjin; Wisedchaisri, Goragot; Gonen, Tamir Single Particle Electron Cryomicroscopy of Bacteriophage P22 Portal Protein Complexes Journal Article Microscopy and Microanalysis, 14 (S2), pp. 1572–1573, 2008. @article{zheng_2008, title = {Single Particle Electron Cryomicroscopy of Bacteriophage P22 Portal Protein Complexes}, author = {Hongjin Zheng and Goragot Wisedchaisri and Tamir Gonen}, doi = {10.1017/S1431927608088661}, year = {2008}, date = {2008-08-03}, journal = {Microscopy and Microanalysis}, volume = {14}, number = {S2}, pages = {1572--1573}, keywords = {}, pubstate = {published}, tppubtype = {article} } | |
Hite, Richard K; Gonen, Tamir; Harrison, Stephen C; Walz, Thomas Interactions of lipids with aquaporin-0 and other membrane proteins Journal Article Pflugers Arch., 456 (4), pp. 651–661, 2008. @article{pmid17932686, title = {Interactions of lipids with aquaporin-0 and other membrane proteins}, author = {Richard K Hite and Tamir Gonen and Stephen C Harrison and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/hite_2008.pdf, Main text}, doi = {10.1007/s00424-007-0353-9}, year = {2008}, date = {2008-07-01}, journal = {Pflugers Arch.}, volume = {456}, number = {4}, pages = {651--661}, abstract = {The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The structure of aquaporin-0 (AQP0) has recently been determined by electron crystallography of two-dimensional (2D) crystals and by X-ray crystallography of three-dimensional (3D) crystals. The electron crystallographic structure revealed nine lipids per AQP0 monomer, which form an almost complete bilayer. The lipids adopt a wide variety of conformations and tightly fill the space between adjacent AQP0 tetramers. The conformations of the lipid acyl chains appear to be determined not only by the protein surface but also by the acyl chains of adjacent lipid molecules. In the X-ray structure, the hydrophobic region of the protein is surrounded by a detergent micelle, with two ordered detergent molecules per AQP0 monomer. Despite the different environments, the electron crystallographic and X-ray structures of AQP0 are virtually identical, but they differ in the temperature factors of the atoms that either contact the lipids in the 2D crystals or are exposed to detergents in the 3D crystals. The temperature factors are higher in the X-ray structure, suggesting that the detergent-exposed AQP0 residues are less ordered than the corresponding ones contacting lipids in the 2D crystals. An examination of ordered detergent molecules in crystal structures of other aquaporins and of lipid molecules in 2D and 3D crystals of bacteriorhodopsin suggests that the increased conformational variability of detergent-exposed residues compared to lipid-contacting residues is a general feature. | |