2014
54.
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
In: Nature, vol. 510, no. 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 = {2014-05-25},
urldate = {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.
53.
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
In: Nat. Chem. Biol., vol. 10, no. 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
52.
Shi, Dan; Nannenga, Brent L; Iadanza, Matthew G; Gonen, Tamir
Three-dimensional electron crystallography of protein microcrystals
In: Elife, vol. 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.
51.
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
In: Elife, vol. 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.
50.
Gold, Matthew G; Gonen, Tamir; Scott, John D
Local cAMP signaling in disease at a glance
In: J. Cell. Sci., vol. 126, no. 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.
49.
Reichow, Stephen; Gonen, Tamir
Dynamic Modulation of Water Permeability in the Lens Aquaporin-0
In: 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}
}
48.
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
In: Mol. Cell, vol. 51, no. 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.
47.
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
In: Nat. Struct. Mol. Biol., vol. 20, no. 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.
46.
Zheng, Hongjin; Wisedchaisri, Goragot; Gonen, Tamir
Crystal structure of a nitrate/nitrite exchanger
In: Nature, vol. 497, no. 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.
45.
Nannenga, Brent L; Iadanza, Matthew G; Vollmar, Breanna S; Gonen, Tamir
In: Curr Protoc Protein Sci, vol. 72, no. 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.
44.
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
In: Proc. Natl. Acad. Sci. U.S.A., vol. 110, no. 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
43.
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
In: Nat. Struct. Mol. Biol., vol. 20, no. 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.
42.
Wisedchaisri, Goragot; Gonen, Tamir
In: Electron Crystallography of Soluble and Membrane Proteins, vol. 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.
41.
Gonen, Tamir
The Collection of High-Resolution Electron Diffraction Data
In: Electron Crystallography of Soluble and Membrane Proteins, vol. 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.
40.
Stokes, David L; Ubarretxena-Belandia, Iban; Gonen, Tamir; Engel, Andreas
High-Throughput Methods for Electron Crystallography
In: Electron Crystallography of Soluble and Membrane Proteins, vol. 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.
39.
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
In: Proc. Natl. Acad. Sci. U.S.A., vol. 109, no. 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.
38.
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
In: Nat. Struct. Mol. Biol., vol. 19, no. 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.
37.
Gonen, Tamir; Waksman, Gabriel
Recent progress in membrane protein structures and investigation methods
In: Curr. Opin. Struct. Biol., vol. 22, no. 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}
}
36.
Jiang, Qiu-Xing; Gonen, Tamir
The influence of lipids on voltage-gated ion channels
In: Curr. Opin. Struct. Biol., vol. 22, no. 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.
35.
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
In: Science, vol. 336, no. 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
34.
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
In: Nat. Struct. Mol. Biol., vol. 19, no. 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.
33.
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
In: EMBO Mol Med, vol. 4, no. 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.
32.
Wisedchaisri, Goragot; Reichow, Steve L; Gonen, Tamir
Advances in Structural and Functional Analysis of Membrane Proteins by Electron Crystallography
In: Structure, vol. 19, no. 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.
31.
Korotkov, Konstantin V; Gonen, Tamir; Hol, Wim G J
Secretins: dynamic channels for protein transport across membranes
In: Trends Biochem. Sci., vol. 36, no. 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.
30.
Wisedchaisri, Goragot; Gonen, Tamir
In: Structure, vol. 19, no. 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.
29.
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
In: Channels (Austin), vol. 5, no. 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.
28.
Jehle, Stefan; Vollmar, Breanna S; Bardiaux, Benjamin; Dove, Katja K; Rajagopal, Ponni; Gonen, Tamir; Oschkinat, Hartmut; Klevit, Rachel E
In: Proc. Natl. Acad. Sci. U.S.A., vol. 108, no. 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.
27.
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
In: ACS Chem Neurosci, vol. 2, no. 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
26.
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
In: Nature, vol. 468, no. 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.
25.
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
In: Structure, vol. 18, no. 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.
24.
Reichow, Steve L; Korotkov, Konstantin V; Hol, Wim G J; Gonen, Tamir
Structure of the cholera toxin secretion channel in its closed state
In: Nat. Struct. Mol. Biol., vol. 17, no. 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.
23.
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
In: Chembiochem, vol. 11, no. 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}
}
22.
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
In: MBio, vol. 1, no. 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.
21.
Tien, Jerry F; Umbreit, Neil T; Gestaut, Daniel R; Franck, Andrew D; Cooper, Jeremy; Wordeman, Linda; Gonen, Tamir; Asbury, Charles L; Davis, Trisha N
In: J. Cell Biol., vol. 189, no. 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.
20.
Zheng, Hongjin; Taraska, Justin; Merz, Alexey J; Gonen, Tamir
The prototypical H⁺/Galactose Symporter GalP Assembles into Functional Trimers
In: J. Mol. Biol., vol. 396, no. 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
19.
Reichow, Steve L; Gonen, Tamir
Lipid-protein interactions probed by electron crystallography
In: Curr. Opin. Struct. Biol., vol. 19, no. 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
18.
Reichow, Steve L; Gonen, Tamir
Noncanonical Binding of Calmodulin to Aquaporin-0: Implications for Channel Regulation
In: Structure, vol. 16, no. 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.
17.
Zheng, Hongjin; Wisedchaisri, Goragot; Gonen, Tamir
Single Particle Electron Cryomicroscopy of Bacteriophage P22 Portal Protein Complexes
In: Microscopy and Microanalysis, vol. 14, no. 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}
}
16.
Hite, Richard K; Gonen, Tamir; Harrison, Stephen C; Walz, Thomas
Interactions of lipids with aquaporin-0 and other membrane proteins
In: Pflugers Arch., vol. 456, no. 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.
15.
Andrews, Simeon; Reichow, Steve L.; Gonen, Tamir
Electron Crystallography of Aquaporins
In: IUBMB Life, vol. 60, no. 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.
14.
Engel, Andreas; Fujiyoshi, Yoshinori; Gonen, Tamir; Walz, Thomas
In: Curr. Opin. Struct. Biol., vol. 18, no. 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.
13.
Zheng, Hongjin; Olia, Adam S; Gonen, Melissa; Andrews, Simeon; Cingolani, Gino; Gonen, Tamir
A Conformational Switch in Bacteriophage P22 Portal Protein Primes Genome Injection
In: Mol. Cell, vol. 29, no. 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.
12.
Gonen, Tamir; Hite, Richard K; Cheng, Yifan; Petre, Benjamin M; Kistler, Joerg; Walz, Thomas
Polymorphic Assemblies and Crystalline Arrays of Lens Tetraspanin MP20
In: J. Mol. Biol., vol. 376, no. 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
11.
Franck, Andrew D; Powers, Andrew F; Gestaut, Daniel R; Gonen, Tamir; Davis, Trisha N; Asbury, Charles L
In: Nat. Cell Biol., vol. 9, no. 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.
10.
Viadiu, Hector; Gonen, Tamir; Walz, Thomas
Projection Map of Aquaporin-9 at 7 Å Resolution
In: J. Mol. Biol., vol. 367, no. 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
9.
Gonen, Tamir; Walz, Thomas
In: Q. Rev. Biophys., vol. 39, no. 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
8.
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
In: Nature, vol. 438, no. 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
7.
Gonen, Tamir; Cheng, Yifan; Kistler, Joerg; Walz, Thomas
Aquaporin-0 Membrane Junctions Form Upon Proteolytic Cleavage
In: J. Mol. Biol., vol. 342, no. 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.
6.
Gonen, Tamir; Sliz, Piotr; Kistler, Joerg; Cheng, Yifan; Walz, Thomas
Aquaporin-0 membrane junctions reveal the structure of a closed water pore
In: Nature, vol. 429, no. 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
5.
Grey, Angus C; Jacobs, Marc D; Gonen, Tamir; Kistler, Joerg; Donaldson, Paul J
In: Exp. Eye Res., vol. 77, no. 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.