2011 |
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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. | |
Andrews, Simeon ; Reichow, Steve L; Gonen, Tamir Electron Crystallography of Aquaporins Journal Article IUBMB Life, 60 (7), pp. 430–436, 2008. @article{pmid18465794, title = {Electron Crystallography of Aquaporins}, author = {Andrews, Simeon and Reichow, Steve L. and Gonen, Tamir}, url = {https://cryoem.ucla.edu/wp-content/uploads/andrews_2008.pdf, Main text}, doi = {10.1002/iub.53}, year = {2008}, date = {2008-05-08}, journal = {IUBMB Life}, volume = {60}, number = {7}, pages = {430--436}, abstract = {Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Aquaporins are a family of ubiquitous membrane proteins that form a pore for the permeation of water. Both electron and X-ray crystallography played major roles in determining the atomic structures of a number of aquaporins. This review focuses on electron crystallography, and its contribution to the field of aquaporin biology. We briefly discuss electron crystallography and the two-dimensional crystallization process. We describe features of aquaporins common to both electron and X-ray crystallographic structures; as well as some structural insights unique to electron crystallography, including aquaporin junction formation and lipid-protein interactions. | |
Engel, Andreas; Fujiyoshi, Yoshinori; Gonen, Tamir; Walz, Thomas Junction-forming aquaporins Journal Article Curr. Opin. Struct. Biol., 18 (2), pp. 229–235, 2008. @article{pmid18194855, title = {Junction-forming aquaporins}, author = {Andreas Engel and Yoshinori Fujiyoshi and Tamir Gonen and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/engel_2008.pdf, Main text}, doi = {10.1016/j.sbi.2007.11.003}, year = {2008}, date = {2008-04-01}, journal = {Curr. Opin. Struct. Biol.}, volume = {18}, number = {2}, pages = {229--235}, abstract = {Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Aquaporins (AQPs) are a family of ubiquitous membrane channels that conduct water and solutes across membranes. This review focuses on AQP0 and AQP4, which in addition to forming water channels also appear to play a role in cell adhesion. We discuss the recently determined structures of the membrane junctions mediated by these two AQPs, the mechanisms that regulate junction formation, and evidence that supports a role for AQP0 and AQP4 in cell adhesion. | |
Zheng, Hongjin; Olia, Adam S; Gonen, Melissa; Andrews, Simeon; Cingolani, Gino; Gonen, Tamir A Conformational Switch in Bacteriophage P22 Portal Protein Primes Genome Injection Journal Article Mol. Cell, 29 (3), pp. 376–383, 2008. @article{pmid18280242, title = {A Conformational Switch in Bacteriophage P22 Portal Protein Primes Genome Injection}, author = {Hongjin Zheng and Adam S Olia and Melissa Gonen and Simeon Andrews and Gino Cingolani and Tamir Gonen}, url = {https://cryoem.ucla.edu/wp-content/uploads/zheng_2008.pdf, Main text}, doi = {10.1016/j.molcel.2007.11.034}, year = {2008}, date = {2008-02-15}, journal = {Mol. Cell}, volume = {29}, number = {3}, pages = {376--383}, abstract = {Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Double-stranded DNA (dsDNA) viruses such as herpesviruses and bacteriophages infect by delivering their genetic material into cells, a task mediated by a DNA channel called "portal protein." We have used electron cryomicroscopy to determine the structure of bacteriophage P22 portal protein in both the procapsid and mature capsid conformations. We find that, just as the viral capsid undergoes major conformational changes during virus maturation, the portal protein switches conformation from a procapsid to a mature phage state upon binding of gp4, the factor that initiates tail assembly. This dramatic conformational change traverses the entire length of the DNA channel, from the outside of the virus to the inner shell, and erects a large dome domain directly above the DNA channel that binds dsDNA inside the capsid. We hypothesize that this conformational change primes dsDNA for injection and directly couples completion of virus morphogenesis to a new cycle of infection. | |
Gonen, Tamir; Hite, Richard K; Cheng, Yifan; Petre, Benjamin M; Kistler, Joerg; Walz, Thomas Polymorphic Assemblies and Crystalline Arrays of Lens Tetraspanin MP20 Journal Article J. Mol. Biol., 376 (2), pp. 380–392, 2008. @article{pmid18166196, title = {Polymorphic Assemblies and Crystalline Arrays of Lens Tetraspanin MP20}, author = {Tamir Gonen and Richard K Hite and Yifan Cheng and Benjamin M Petre and Joerg Kistler and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/gonen_2008.pdf, Main text}, doi = {10.1016/j.jmb.2007.09.001}, year = {2008}, date = {2008-02-15}, journal = {J. Mol. Biol.}, volume = {376}, number = {2}, pages = {380--392}, abstract = {Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 A resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 2D crystals, reconstitution of MP20 with native lipids also produced a variety of large protein-lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Members of the tetraspanin superfamily function as transmembrane scaffold proteins that mediate the assembly of membrane proteins into specific signaling complexes. Tetraspanins also interact with each other and concentrate membrane proteins into tetraspanin-enriched microdomains (TEMs). Here we report that lens-specific tetraspanin MP20 can form multiple types of higher-order assemblies and we present crystalline arrays of MP20. When isolated in the absence of divalent cations, MP20 is solubilized predominantly in tetrameric form, whereas the presence of divalent cations during solubilization promotes the association of MP20 tetramers into higher-order species. This effect only occurs when divalent cations are present during solubilization but not when divalent cations are added to solubilized tetrameric MP20, suggesting that other factors may also be involved. When purified MP20 tetramers are reconstituted with native lens lipids in the presence of magnesium, MP20 forms two-dimensional (2D) crystals. A projection map at 18 A resolution calculated from negatively stained 2D crystals showed that the building block of the crystal is an octamer consisting of two tetramers related to each other by 2-fold symmetry. In addition to 2D crystals, reconstitution of MP20 with native lipids also produced a variety of large protein-lipid complexes, and we present three-dimensional (3D) reconstructions of the four most abundant of these complexes in negative stain. The various complexes formed by MP20 most likely reflect the many ways in which tetraspanins can interact with each other to allow formation of TEMs. | |
2007 |
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Franck, Andrew D; Powers, Andrew F; Gestaut, Daniel R; Gonen, Tamir; Davis, Trisha N; Asbury, Charles L Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis Journal Article Nat. Cell Biol., 9 (7), pp. 832–837, 2007. @article{pmid17572669, title = {Tension applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis}, author = {Andrew D Franck and Andrew F Powers and Daniel R Gestaut and Tamir Gonen and Trisha N Davis and Charles L Asbury}, url = {https://cryoem.ucla.edu/wp-content/uploads/franck2007.pdf, Main text}, doi = {10.1038/ncb1609}, year = {2007}, date = {2007-06-17}, journal = {Nat. Cell Biol.}, volume = {9}, number = {7}, pages = {832--837}, abstract = {In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis.}, keywords = {}, pubstate = {published}, tppubtype = {article} } In dividing cells, kinetochores couple chromosomes to the tips of growing and shortening microtubule fibres and tension at the kinetochore-microtubule interface promotes fibre elongation. Tension-dependent microtubule fibre elongation is thought to be essential for coordinating chromosome alignment and separation, but the mechanism underlying this effect is unknown. Using optical tweezers, we applied tension to a model of the kinetochore-microtubule interface composed of the yeast Dam1 complex bound to individual dynamic microtubule tips. Higher tension decreased the likelihood that growing tips would begin to shorten, slowed shortening, and increased the likelihood that shortening tips would resume growth. These effects are similar to the effects of tension on kinetochore-attached microtubule fibres in many cell types, suggesting that we have reconstituted a direct mechanism for microtubule-length control in mitosis. | |
Viadiu, Hector; Gonen, Tamir; Walz, Thomas Projection Map of Aquaporin-9 at 7 Å Resolution Journal Article J. Mol. Biol., 367 (1), pp. 80–88, 2007. @article{pmid17239399, title = {Projection Map of Aquaporin-9 at 7 Å Resolution}, author = {Hector Viadiu and Tamir Gonen and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/Viadiu_2006.pdf, Main text}, doi = {10.1016/j.jmb.2006.12.042}, year = {2007}, date = {2007-03-16}, journal = {J. Mol. Biol.}, volume = {367}, number = {1}, pages = {80--88}, abstract = {Aquaporin-9, an aquaglyceroporin present in diverse tissues, is unique among aquaporins because it is not only permeable to water, urea and glycerol, but also allows passage of larger uncharged solutes. Single particle analysis of negatively stained recombinant rat aquaporin-9 revealed a particle size characteristic of the tetrameric organization of all members of the aquaporin family. Reconstitution of aquaporin-9 into two-dimensional crystals enabled us to calculate a projection map at 7 A resolution. The projection structure indicates a tetrameric structure, similar to GlpF, with each square-like monomer forming a pore. A comparison of the pore-lining residues between the crystal structure of GlpF and a homology model of aquaporin-9 locates substitutions in these residues predominantly to the hydrophobic edge of the tripathic pore of GlpF, providing first insights into the structural basis for the broader substrate specificity of aquaporin-9.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Aquaporin-9, an aquaglyceroporin present in diverse tissues, is unique among aquaporins because it is not only permeable to water, urea and glycerol, but also allows passage of larger uncharged solutes. Single particle analysis of negatively stained recombinant rat aquaporin-9 revealed a particle size characteristic of the tetrameric organization of all members of the aquaporin family. Reconstitution of aquaporin-9 into two-dimensional crystals enabled us to calculate a projection map at 7 A resolution. The projection structure indicates a tetrameric structure, similar to GlpF, with each square-like monomer forming a pore. A comparison of the pore-lining residues between the crystal structure of GlpF and a homology model of aquaporin-9 locates substitutions in these residues predominantly to the hydrophobic edge of the tripathic pore of GlpF, providing first insights into the structural basis for the broader substrate specificity of aquaporin-9. | |
2006 |
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Gonen, Tamir; Walz, Thomas The structure of aquaporins Journal Article Q. Rev. Biophys., 39 (4), pp. 361–396, 2006. @article{pmid17156589, title = {The structure of aquaporins}, author = {Tamir Gonen and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/gonenwalz_2006.pdf, Main text}, doi = {10.1017/S0033583506004458}, year = {2006}, date = {2006-11-01}, journal = {Q. Rev. Biophys.}, volume = {39}, number = {4}, pages = {361--396}, abstract = {The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The ubiquitous members of the aquaporin (AQP) family form transmembrane pores that are either exclusive for water (aquaporins) or are also permeable for other small neutral solutes such as glycerol (aquaglyceroporins). The purpose of this review is to provide an overview of our current knowledge of AQP structures and to describe the structural features that define the function of these membrane pores. The review will discuss the mechanisms governing water conduction, proton exclusion and substrate specificity, and how the pore permeability is regulated in different members of the AQP family. | |
2005 |
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Gonen, Tamir; Cheng, Yifan; Sliz, Piotr; Hiroaki, Yoko; Fujiyoshi, Yoshinori; Harrison, Stephen C; Walz, Thomas Lipid-protein interactions in double-layered two-dimensional AQP0 crystals Journal Article Nature, 438 (7068), pp. 633–638, 2005. @article{pmid16319884, title = {Lipid-protein interactions in double-layered two-dimensional AQP0 crystals}, author = {Tamir Gonen and Yifan Cheng and Piotr Sliz and Yoko Hiroaki and Yoshinori Fujiyoshi and Stephen C Harrison and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/Gonen_2005.pdf, Main text}, doi = {10.1038/nature04321}, year = {2005}, date = {2005-12-01}, journal = {Nature}, volume = {438}, number = {7068}, pages = {633--638}, abstract = {Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Lens-specific aquaporin-0 (AQP0) functions as a specific water pore and forms the thin junctions between fibre cells. Here we describe a 1.9 A resolution structure of junctional AQP0, determined by electron crystallography of double-layered two-dimensional crystals. Comparison of junctional and non-junctional AQP0 structures shows that junction formation depends on a conformational switch in an extracellular loop, which may result from cleavage of the cytoplasmic amino and carboxy termini. In the centre of the water pathway, the closed pore in junctional AQP0 retains only three water molecules, which are too widely spaced to form hydrogen bonds with each other. Packing interactions between AQP0 tetramers in the crystalline array are mediated by lipid molecules, which assume preferred conformations. We were therefore able to build an atomic model for the lipid bilayer surrounding the AQP0 tetramers, and we describe lipid-protein interactions. | |
2004 |
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Gonen, Tamir; Cheng, Yifan; Kistler, Joerg; Walz, Thomas Aquaporin-0 Membrane Junctions Form Upon Proteolytic Cleavage Journal Article J. Mol. Biol., 342 (4), pp. 1337–1345, 2004. @article{pmid15351655, title = {Aquaporin-0 Membrane Junctions Form Upon Proteolytic Cleavage}, author = {Tamir Gonen and Yifan Cheng and Joerg Kistler and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/Gonen_2004b.pdf, Main text}, doi = {10.1016/j.jmb.2004.07.076}, year = {2004}, date = {2004-09-24}, journal = {J. Mol. Biol.}, volume = {342}, number = {4}, pages = {1337--1345}, abstract = {Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Aquaporin-0 (AQP0), previously known as major intrinsic protein (MIP), is the only water pore protein expressed in lens fiber cells. AQP0 is highly specific to lens fiber cells and constitutes the most abundant intrinsic membrane protein in these cells. The protein is initially expressed as a full-length protein in young fiber cells in the lens cortex, but becomes increasingly cleaved in the lens core region. Reconstitution of AQP0 isolated from the core of sheep lenses containing a proportion of truncated protein, produced double-layered two-dimensional (2D) crystals, which displayed the same dimensions as the thin 11 nm lens fiber cell junctions, which are prominent in the lens core. In contrast reconstitution of full-length AQP0 isolated from the lens cortex reproducibly yielded single-layered 2D crystals. We present electron diffraction patterns and projection maps of both crystal types. We show that cleavage of the intracellular C terminus enhances the adhesive properties of the extracellular surface of AQP0, indicating a conformational change in the molecule. This change of function of AQP0 from a water pore in the cortex to an adhesion molecule in the lens core constitutes another manifestation of the gene sharing concept originally proposed on the basis of the dual function of crystallins. | |
Gonen, Tamir; Sliz, Piotr; Kistler, Joerg; Cheng, Yifan; Walz, Thomas Aquaporin-0 membrane junctions reveal the structure of a closed water pore Journal Article Nature, 429 (6988), pp. 193–197, 2004. @article{pmid15141214, title = {Aquaporin-0 membrane junctions reveal the structure of a closed water pore}, author = {Tamir Gonen and Piotr Sliz and Joerg Kistler and Yifan Cheng and Thomas Walz}, url = {https://cryoem.ucla.edu/wp-content/uploads/gonen_2004a.pdf, Main text}, doi = {10.1038/nature02503}, year = {2004}, date = {2004-05-13}, journal = {Nature}, volume = {429}, number = {6988}, pages = {193--197}, abstract = {The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo. We show here that AQP0 from the lens core, containing some carboxy-terminally cleaved AQP0, forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystallography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating.}, keywords = {}, pubstate = {published}, tppubtype = {article} } The lens-specific water pore aquaporin-0 (AQP0) is the only aquaporin known to form membrane junctions in vivo. We show here that AQP0 from the lens core, containing some carboxy-terminally cleaved AQP0, forms double-layered crystals that recapitulate in vivo junctions. We present the structure of the AQP0 membrane junction as determined by electron crystallography. The junction is formed by three localized interactions between AQP0 molecules in adjoining membranes, mainly mediated by proline residues conserved in AQP0s from different species but not present in most other aquaporins. Whereas all previously determined aquaporin structures show the pore in an open conformation, the water pore is closed in AQP0 junctions. The water pathway in AQP0 also contains an additional pore constriction, not seen in other known aquaporin structures, which may be responsible for pore gating. | |
2003 |
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Grey, Angus C; Jacobs, Marc D; Gonen, Tamir; Kistler, Joerg; Donaldson, Paul J Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier Journal Article Exp. Eye Res., 77 (5), pp. 567–574, 2003. @article{pmid14550398, title = {Insertion of MP20 into lens fibre cell plasma membranes correlates with the formation of an extracellular diffusion barrier}, author = {Angus C Grey and Marc D Jacobs and Tamir Gonen and Joerg Kistler and Paul J Donaldson}, url = {https://cryoem.ucla.edu/wp-content/uploads/grey_2003.pdf, Main text}, doi = {10.1016/s0014-4835(03)00192-1}, year = {2003}, date = {2003-11-01}, journal = {Exp. Eye Res.}, volume = {77}, number = {5}, pages = {567--574}, abstract = {It is known that during lens differentiation a number of fibre cell specific membrane proteins change their expression profiles. In this study we have investigated how the profiles of the two most abundant fibre cell membrane proteins AQP0 (formerly known as Major Intrinsic Protein, MIP) and MP20 change as a function of fibre cell differentiation. While AQP0 was always found associated with fibre cell membranes, MP20 was initially found in the cytoplasm of peripheral fibre cells before becoming inserted into the membranes of deeper fibre cells. To determine at what stage in fibre cell differentiation MP20 becomes inserted into the membrane, sections were double-labelled with an antibody against MP20, and propidium iodide, a marker of cell nuclei. This showed that membrane insertion of MP20 occurs in a discrete transition zone that coincided with the degradation of cell nuclei. To test the significance of the membrane insertion of MP20 to overall lens function, whole lenses were incubated for varying times in a solution containing either Texas Red-dextran or Lucifer yellow as markers of extracellular space. Lenses were fixed and then processed for immunocytochemistry. Analysis of these sections showed that both tracer dyes were excluded from the extracellular space in an area that coincided with insertion of MP20 into the plasma membrane. Our results suggest that the insertion of MP20 into fibre cell membranes coincides with the creation of a barrier that restricts the diffusion of molecules into the lens core via the extracellular space.}, keywords = {}, pubstate = {published}, tppubtype = {article} } It is known that during lens differentiation a number of fibre cell specific membrane proteins change their expression profiles. In this study we have investigated how the profiles of the two most abundant fibre cell membrane proteins AQP0 (formerly known as Major Intrinsic Protein, MIP) and MP20 change as a function of fibre cell differentiation. While AQP0 was always found associated with fibre cell membranes, MP20 was initially found in the cytoplasm of peripheral fibre cells before becoming inserted into the membranes of deeper fibre cells. To determine at what stage in fibre cell differentiation MP20 becomes inserted into the membrane, sections were double-labelled with an antibody against MP20, and propidium iodide, a marker of cell nuclei. This showed that membrane insertion of MP20 occurs in a discrete transition zone that coincided with the degradation of cell nuclei. To test the significance of the membrane insertion of MP20 to overall lens function, whole lenses were incubated for varying times in a solution containing either Texas Red-dextran or Lucifer yellow as markers of extracellular space. Lenses were fixed and then processed for immunocytochemistry. Analysis of these sections showed that both tracer dyes were excluded from the extracellular space in an area that coincided with insertion of MP20 into the plasma membrane. Our results suggest that the insertion of MP20 into fibre cell membranes coincides with the creation of a barrier that restricts the diffusion of molecules into the lens core via the extracellular space. | |
2001 |
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Gonen, Tamir; Grey, Angus C; Jacobs, Marc D; Donaldson, Paul J; Kistler, Joerg MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3 Journal Article BMC Cell Biol., 2 (17), 2001. @article{pmid11532191, title = {MP20, the second most abundant lens membrane protein and member of the tetraspanin superfamily, joins the list of ligands of galectin-3}, author = {Tamir Gonen and Angus C Grey and Marc D Jacobs and Paul J Donaldson and Joerg Kistler}, url = {https://cryoem.ucla.edu/wp-content/uploads/gonen_2001.pdf, Main text}, year = {2001}, date = {2001-08-14}, journal = {BMC Cell Biol.}, volume = {2}, number = {17}, abstract = {Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator. MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction. MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity.}, keywords = {}, pubstate = {published}, tppubtype = {article} } Although MP20 is the second most highly expressed membrane protein in the lens its function remains an enigma. Putative functions for MP20 have recently been inferred from its assignment to the tetraspanin superfamily of integral membrane proteins. Members of this family have been shown to be involved in cellular proliferation, differentiation, migration, and adhesion. In this study, we show that MP20 associates with galectin-3, a known adhesion modulator. MP20 and galectin-3 co-localized in selected areas of the lens fiber cell plasma membrane. Individually, these proteins purified with apparent molecular masses of 60 kDa and 22 kDa, respectively. A 104 kDa complex was formed in vitro upon mixing the purified proteins. A 102 kDa complex of MP20 and galectin-3 could also be isolated from detergent-solubilized native fiber cell membranes. Binding between MP20 and galectin-3 was disrupted by lactose suggesting the lectin site was involved in the interaction. MP20 adds to a growing list of ligands of galectin-3 and appears to be the first representative of the tetraspanin superfamily identified to possess this specificity. | |
2000 |
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Gonen, Tamir; Donaldson, Paul; Kistler, Joerg Galectin-3 Is Associated with the Plasma Membrane of Lens Fiber Cells Journal Article Investigative Ophthalmology and Visual Science, 41 (1), pp. 199–203, 2000. @article{gonen_2000, title = {Galectin-3 Is Associated with the Plasma Membrane of Lens Fiber Cells}, author = {Tamir Gonen and Paul Donaldson and Joerg Kistler}, url = {https://cryoem.ucla.edu/wp-content/uploads/gonen_2000.pdf, Main text}, year = {2000}, date = {2000-01-01}, journal = {Investigative Ophthalmology and Visual Science}, volume = {41}, number = {1}, pages = {199--203}, abstract = {PURPOSE: To discover proteins that have the potential to contribute to the tight packing of fiber cells in the lens. METHODS: Crude fiber cell membranes were isolated from ovine lens cortex. Proteins were separated by two-dimensional gel electrophoresis, and selected protein spots identified by micro-sequencing. The identification of galectin-3 was confirmed by immunoblotting with a specific antibody. The association of galectin-3 with the fiber cell plasma membrane was investigated using immunofluorescence microscopy, solubilization trials with selected reagents, and immunoprecipitation to identify candidate ligands. RESULTS: A cluster of three protein spots with an apparent molecular weight of 31,000 and isoelectric points ranging between 7 and 8.5 were resolved and identified as galectin-3. This protein was associated peripherally with the fiber cell plasma membrane and interacted with MP20, an abundant intrinsic membrane protein that had been identified previously as a component of membrane junctions between fiber cells. CONCLUSIONS: The detection of galectin-3 in the lens is a novel result and adds to the growing list of lens proteins with adhesive properties. Its location at the fiber cell membrane and its association with the junction-forming MP20 is consistent with a potential role in the development or maintenance of the tightly packed lens tissue architecture. (Invest Ophthalmol Vis Sci. 2000;41:199 –203)}, keywords = {}, pubstate = {published}, tppubtype = {article} } PURPOSE: To discover proteins that have the potential to contribute to the tight packing of fiber cells in the lens. METHODS: Crude fiber cell membranes were isolated from ovine lens cortex. Proteins were separated by two-dimensional gel electrophoresis, and selected protein spots identified by micro-sequencing. The identification of galectin-3 was confirmed by immunoblotting with a specific antibody. The association of galectin-3 with the fiber cell plasma membrane was investigated using immunofluorescence microscopy, solubilization trials with selected reagents, and immunoprecipitation to identify candidate ligands. RESULTS: A cluster of three protein spots with an apparent molecular weight of 31,000 and isoelectric points ranging between 7 and 8.5 were resolved and identified as galectin-3. This protein was associated peripherally with the fiber cell plasma membrane and interacted with MP20, an abundant intrinsic membrane protein that had been identified previously as a component of membrane junctions between fiber cells. CONCLUSIONS: The detection of galectin-3 in the lens is a novel result and adds to the growing list of lens proteins with adhesive properties. Its location at the fiber cell membrane and its association with the junction-forming MP20 is consistent with a potential role in the development or maintenance of the tightly packed lens tissue architecture. (Invest Ophthalmol Vis Sci. 2000;41:199 –203) | |
1999 |
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Kistler, Joerg; Merriman-Smith, Rachelle; Young, Miriam; Gonen, Tamir; Cowan, Dougal; Chee, Kaa-Sandra; Lin, Jun Sheng; Green, Colin; Hasler, Lorenz; Engel, Andreas; Donaldson, Paul Molecular Solutions To Tissue Transparency Journal Article N.Z. Biosciences, pp. 35–37, 1999. @article{kistler_1999, title = {Molecular Solutions To Tissue Transparency}, author = {Joerg Kistler and Rachelle Merriman-Smith and Miriam Young and Tamir Gonen and Dougal Cowan and Kaa-Sandra Chee and Jun Sheng Lin and Colin Green and Lorenz Hasler and Andreas Engel and Paul Donaldson}, url = {https://cryoem.ucla.edu/wp-content/uploads/kistler_1999.pdf, Main text}, year = {1999}, date = {1999-08-01}, journal = {N.Z. Biosciences}, pages = {35--37}, keywords = {}, pubstate = {published}, tppubtype = {article} } | |
Baker, Ted; Metcalf, Peter; Smith, Clyde; Arcus, Vic; Ashton, Rachael; Baker, Heather; Banfield, Mark; Cross, Jenny; Drew, David; Goldstone, David; Gonen, Tamir; Haebel, Peter; Holliss, Caroline; Ivanovich, Ivan; Kagawa, Todd; Kidd, Richard; Koon, Nayden; Leydier, Sabine; Lott, Shaun; McCarthy, Andrew; Nurizzo, Didier; Shewry, Steve; Sigrell, Jill; Sun, Xiaolin More Than Just A Pretty Picture Journal Article N.Z. Biosciences, pp. 32–35, 1999. @article{baker_1999, title = {More Than Just A Pretty Picture}, author = {Ted Baker and Peter Metcalf and Clyde Smith and Vic Arcus and Rachael Ashton and Heather Baker and Mark Banfield and Jenny Cross and David Drew and David Goldstone and Tamir Gonen and Peter Haebel and Caroline Holliss and Ivan Ivanovich and Todd Kagawa and Richard Kidd and Nayden Koon and Sabine Leydier and Shaun Lott and Andrew McCarthy and Didier Nurizzo and Steve Shewry and Jill Sigrell and Xiaolin Sun}, url = {https://cryoem.ucla.edu/wp-content/uploads/baker_1999.pdf, Main text}, year = {1999}, date = {1999-08-01}, journal = {N.Z. Biosciences}, pages = {32--35}, keywords = {}, pubstate = {published}, tppubtype = {article} } | |