Our laboratory studies the structures of membrane proteins. Based on structure we try to understand function and what goes wrong in disease. We focus primarily on proteins in the blood-brain barrier. The long-standing question in our laboratory is how the thousands of membrane channels and transporters that exist in the cell membrane work together to help cells maintain homeostasis. With that question in mind, we study membrane proteins that are involved in nutrient, ion and water uptake, waste removal, signaling and communication.
Over the last decade we have employed structural biology techniques such as electron cryo-microscopy (cryo EM), X-ray crystallography, NMR, molecular dynamics simulations, and used membrane biochemistry and biophysics to understand the function of the proteins of interest. Within electron microscopy we have published papers using electron tomography, single particle reconstructions and electron crystallography, however our specialty lies in electron diffraction.
Part of our laboratory is also devoted to method development in cryo EM. In recent years we have developed two important methods in electron diffraction, namely the fragment based phase extension and MicroED.
Some of our recent studies are outlined below.
channels, or aquaporins, form specialized channels in membranes for
water permeation. These are extremely efficient channels that allow
millions of water molecules to permeate the pore per second. Because
they are channels, the cell can regulate their activity dynamically to
help maintain homeostasis. In the case of the eye lens water channel
aquaporin-0 (AQP0), it can be regulated by at least 4 known mechanisms
that we studied over the last decade. The first is irreversible and
involves the cleavage of the C-terminal domain of AQP0. The cleavage
results in complete pore closure and AQP0 ceases to act as a water
channel. Instead it becomes an adhesive protein mediating cell-to-cell
adhesive junctions (Figure 1).
Full-length AQP0 is dynamically modulated by 3 mechanisms: pH, calcium/calmodulin (Ca2+/CaM) and protein phosphorylation. We recently showed that the binding of Ca2+/CaM to AQP0 results in partial pore closure (Figure 2). The net effect is that the permeability through AQP0 halves in the presence of Ca2+/CaM.
Conversely, we showed that phosphorylation of AQP0 by anchored PKA
(AKAP2/PKA complex) abolished CaM binding, keeping AQP0 in the open
conformation and functioning at maximal activity.
studies of channel phosphorylation led us to discover a new protein in
the eye lens called AKAP2. Our biochemical and structural studies
indicate that AKAPs anchor PKA onto substrate and provide the kinase a
sphere of action in which the kinase could phosphorylate substrates in a
cAMP independent way. This is fundamentally an exciting observation
because it helps explain how fast phosphorylation can occur, as seen for
example in heart cells. Moreover, we showed that inhibition of
phosphorylation of AQP0 in the lens results in cataract formation.
Essentially we recapitulated the lens disease ex vivo by inhibiting
protein phosphorylation (Figure 3).
structure of the AQP0/CaM complex is the first for any full-length
membrane channel in complex with this ubiquitous secondary messenger (Figure 4). Current efforts in the laboratory are to understand how Ca2+/CaM binds to and modulates the activity of other channels such as ion channels.
are also trying to understand more about the AQP-AKAP system, in
particular we are trying to assemble the AQP2-AKAP18-PKA complex and
AQP0-AKAP2-PKA complex for structural studies. Intrinsically disordered
regions of proteins are widespread in nature yet the mechanistic roles
they play in biology are underappreciated. Such disordered segments can
act simply to link functionally coupled structural domains or they can
orchestrate enzymatic reactions through a variety of allosteric
mechanisms. The regulatory subunits of protein kinase A provide an
example of this important phenomenon where functionally defined and
structurally conserved domains are connected by intrinsically disordered
regions of defined length with limited sequence identity. Our studies
show that this seemingly paradoxical amalgam of order and disorder
permits fine-tuning of local protein phosphorylation events. The
anchoring of PKA by AKAP affords the kinase a sphere of action in which
multiple targets can get phosphorylated fast in a cAMP independent way (Figure 5).
1. Gonen T., Sliz P., Cheng Y., Kistler J. and Walz T. (2004) Aquaporin-0 membrane junctions reveal the
structure of a closed water pore. Nature. 429 : 193 – 197.
2. Gonen T., Cheng Y., Kistler J. and Walz T. (2004) Aquaporin-0 membrane junctions form upon
proteolytic cleavage. Journal of Molecular Biology 342 : 1337 - 1345.
3. Gonen T., Cheng Y., Sliz P., Hiroaki Y., Fujiyoshi Y., Harrison SC., Walz T (2005) Lipid–protein
interactions in double layered two dimensional crystals of AQP0. Nature 438: 633 - 638.
4. Reichow L.S. and Gonen T*. (2008) Non-canonical binding of calmodulin to aquaporin-0:
implications for channel regulation. Structure. 16: 1389 – 1398.
5. Gold MG, Reichow SL., O’Neill SE, Weisbrod CR, Langeberg LK, Bruce JE, Gonen T* and Scott
JD*. (2012) AKAP2 anchors PKA with aquaporin-0 to support ocular lens transparency.
EMBO Molecular Medicine 4: 15-26.
6. Reichow S.L, Clemens D.M., Freites J.A., Németh-Cahalan K.L., Heyden M., Tobias D.J., Hall J.E*.
and Gonen T*. (2013) Allosteric mechanism of water channel gating by Ca2+–calmodulin. Nature
Structural and Molecular Biology – 20 (9): 1085 - 1092.
7. Smith FD., Reichow LS., Esseltine JL., Shi D., Langeberg LK., Scott J* and Gonen T* (2013). Intrinsic
disorder within an AKAP-Protein kinase A complex guides local substrate phosphorylation. eLife 2:e01319.
1. Gonen T*. and Walz T* (2006) The structure of Aquaporins. Quarterly Reviews in Biophysics 39 :
2. Engel, A., Fujiyoshi, Y., Gonen, T. and Walz, T. (2008) Junction forming aquaporins. Current
Opinion in Structural Biology. 18 : 229 - 235.
3. Andrews, S.A., Reichow, L.S. and Gonen T*. (2008) Electron crystallography of aquaporins.
IUBMB Life 60: 430 – 436.
4. Reichow, S.L. and Gonen T*. (2009) Lipid-protein interactions probed by electron crystallography.
Current Opinion in Structural Biology. 19: 560 - 565
5. Gold M. Gonen T and Scott J. (2013) Local cAMP signaling in disease at a glance. Journal of Cell
Science. 126: 4537 - 4543.
The major facilitator superfamily of membrane proteins is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been captured and structurally characterized in multiple transporting conformations including both the outward and inward facing states. We determined the crystal structure of XylE in a new inward-facing open conformation. Structural comparison of XylE in this conformation with its outward-facing partially occluded conformation reveals how this transporter functions through a non-symmetrical rocker switch movement of the N-domain as a rigid body and the C-domain as a flexible body. Molecular dynamics simulations were employed to help describe how XylE transitions in a lipid membrane to facilitate sugar transport. (Figure 6)
Nitrate is the preferred nitrogen source for plants on which all higher forms of life ultimately depend. Plants and microorganisms evolved to efficiently assimilate nitrate. Despite decades of effort no structure was available for any nitrate transport protein and the mechanism by which nitrate is transported remained largely obscure until our study was published. We reported the structure of a bacterial nitrate/nitrite transport protein, NarK, from Escherichia coli, with and without substrate. The structures revealed a positively charged substrate-translocation pathway lacking protonatable residues, suggesting that NarK functions as a nitrate/nitrite exchanger and that H+s are unlikely to be co-transported. Conserved arginine residues form the substrate-binding pocket, which is formed by association of helices from the two halves of NarK. Key residues that are important for substrate recognition and transport were identified and related to extensive mutagenesis and functional studies. We proposed that NarK exchanges nitrate for nitrite by a rocker-switch mechanism facilitated by inter-domain H-bond networks. (Figure 7)
1. Zheng H., Taraska J., Merz A. and Gonen T*. (2010) The prototypical H+/galactose symporter GalP
assembles into functional trimers. Journal of Molecular Biology. 396 : 593 – 601.
2. Wisedchaisri G., Dranow D.M., Lie T.J., Bonanno J.B., Patskovsky Y., Ozyurt S.A., Michael Sauder
J.M., Almo S.C., Wasserman S.R., Burley S.K., Leigh J.A. and Gonen T*. (2010) Structural
underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor
NrpR. Structure 18 1512 - 1521.
3. Zheng H, Wisedchaisri W and Gonen T*. (2013) Crystal structure of a nitrate/nitrite exchanger.
Nature – 497: 647-651.
Fragment based phase extension
In electron crystallography membrane protein structure is determined from two-dimensional crystals where the protein is embedded in a membrane. Once large and well-ordered 2D crystals are grown one of the bottlenecks in electron crystallography is the collection of image data to directly provide experimental phases to high resolution. We developed a new approach to bypass this bottleneck, eliminating the need for high-resolution imaging. We used the strengths of electron crystallography in rapidly obtaining accurate experimental phase information from low-resolution images and accurate high-resolution amplitude information from electron diffraction. The low-resolution experimental phases were used for the placement of α-helix fragments and extended to high resolution using phases from the fragments. Phases were further improved by density modifications followed by fragment expansion and structure refinement against the high-resolution diffraction data. Using this approach, structures of three membrane proteins were determined rapidly and accurately to atomic resolution without high-resolution image data. (Figure 8)
MicroED – Three dimensional electron crystallography of protein microcrystals
We demonstrated that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1 - 1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9Å resolution (Figure 9). This proof of principle paves the way for the implementation of a new technique, which we name “MicroED”, that may have wide applicability in structural biology. Current efforts include new phasing methods, automation and program development.
1. Wisedchaisri G and Gonen T*. (2011) Fragment based phase extension for membrane protein
structure determination by electron crystallography. Structure 19: 976 - 987.
2. Shi D., Nannenga B., Iadanza MG. and Gonen T* (2013). MicroED – Three dimensional electron
crystallography of protein microcrystals. eLife.
Relevant Reviews and Book Chapters:
1. Wisedchaisri W., Reichow S.L. and Gonen T*. (2011) Advances in structural and functional analysis
of membrane proteins by electron crystallography. Structure. 19:1381-93.
2. Wisedchaisri W. and Gonen T*. (2013) Phasing Electron Diffraction Data by Molecular Replacement:
Strategy for Structure Determination and Refinement. Methods in Molecular Biology 955: 243 – 272.
3. Gonen T*. (2013) The collection of high-resolution electron diffraction data. Methods in Molecular
Biology 955: 153 – 169.
4. Stokes D, Ubarretxena I, Gonen T and Engel A. (2013) High throughout methods in electron
crystallography. Methods in Molecular Biology 955: 273 – 296.
5. Nannenga B, Iadanza M, Vollmar B and Gonen T*. (2013) Electron crystallography of membrane
proteins: crystallization and screening strategies using negative stain electron microscopy. Current Protocols in Protein Science – 17 (15): 1-11.
In collaboration with David Baker (HHMI, UW) we are designing genetically encoded self assembling proteins for cellular microcircuitry.
We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. Here we use trimeric protein building blocks to design a 24-subunit, 13 nm diameter complex with octahedral symmetry and two related variants of a 12-subunit, 11 nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials. (Figure 10)
1. King NP., Sheffler W., Sawaya MR., Vollmar BS., Sumida JP., Andre I., Gonen T., Yeates TO. And Baker D. (2012) Computational design of self-assembling protein nanomaterials with atomic level accuracy. Science. 336: 1171 – 1174.
We use electrophysiology and patch clamping techniques to study the function of channels and transporters. We use the Xenopus oocyte expression system as well as whole cell patch but we also plan to record channel function from highly ordered two-dimensional crystals for a direct correlation between structure and function of target proteins as they are embedded within a biological membrane. (Figure 11)
Structure of the vibrio cholera toxin secretion channel
In collaboration with Wim Hol (UW) we studied the structure of the vibrio cholera toxin secretion channel.
The type II secretion system (T2SS) is a macromolecular complex spanning the bacterial inner and outer membranes of Gram-negative bacteria, including many pathogenic bacteria such as Vibrio cholerae and enterotoxigenic Escherichia coli. The T2SS secretes folded proteins including cholera toxin and heat-labile enterotoxin. The major outer membrane T2SS protein is the “secretin” GspD. Electron cryomicroscopy (cryoEM) reconstruction of the V. cholerae secretin at 19 Å resolution reveals a dodecameric structure reminiscent of a barrel with a large channel at its center that appears to be in a closed state. On the periplasmic side the channel vestibule contains both a constriction and a gate. On the extracellular side a large chamber is enclosed by a cap structure. By combining our results with structural data on a large exoprotein and the dimensions of the tip of the pseudopilus of the T2SS, we provide a structural basis for a possible secretion mechanism of exoproteins by the T2SS in which the constriction site plays a critical role. (Figure 12)
1. Reichow S.L., Korotkov K.V., Hol W.G.J*. and Gonen T*. (2010) Structure of the cholera toxin
secretion channel in its closed state. Nature Structural & Molecular Biology. 17 : 1226 - 1232.
2. Reichow SL., Korotkov KV., Gonen M., Sun J., Delarosa J., Hol WGJ* and Gonen T* (2011) The
binding of cholera toxin to the periplasmic vestibule of the type II secretion channel. Channels 5 (3): 215 – 218.
1. Korotkov KV., Gonen T and Hol WGJ. Secretins: dynamic channels for protein transport across
membranes. Trends in Biochemical Sciences 36 (8) : 433 - 443 (2011).
Structure and function of the yeast kinetochore and microtubule dynamics
In collaboration with Sue Biggins (FHCRC) we studied the structure of the yeast kinetochore by electron tomography. In collaboration with Trisha Davis and Chip Asbury (UW) we studied microtubule dynamics and microtubule binding proteins.
Chromosomes must be accurately partitioned to daughter cells to prevent genomic instability and aneuploidy, a hallmark of many tumors and birth defects. Kinetochores are macromolecular machines that move chromosomes by maintaining load-bearing attachments to the assembling and disassembling tips of spindle microtubules. The mechanism by which kinetochores attach to microtubules is still not clear although a number of models have been proposed. Sues laboratory previously developed an assay to purify functional native budding yeast kinetochore particles that contain the majority of core structural components and can maintain attachments to microtubules under force. We presented the structure of these isolated kinetochore particles as visualized by electron microscopy (EM) and electron tomography of negatively stained preparations. The budding yeast kinetochore appeared as a ~126 nm particle having a large central hub attached to multiple outer globular domains. Microtubule binding experiments indicated that the globular domains are important for microtubule attachments both in the presence or absence of a ring encircling the microtubule. Our data showed that kinetochores bind to microtubules via multivalent attachments, consistent with a biased diffusion mechanism where multiple kinetochore components cooperate to form a strong yet dynamic linkage to the microtubule. Although rings are not required for lateral binding, they likely maintain processive attachments to the ends of dynamic microtubules. These studies lay the foundation to uncover the key mechanical and regulatory mechanisms by which kinetochores control chromosome segregation and cell division. (Figure 13)
1. Franck, A.D., Powers, A.F., Gestaut, D.R., Gonen, T., Davis, T.N. and Asbury, C.L. (2007) Tension
applied through the Dam1 complex promotes microtubule elongation providing a direct mechanism for length control in mitosis. Nature Cell Biology. 9 : 832 - 837.
2. Tien J.F., Umbreit N.T., Gestaut D.R., Franck A.D., Cooper J., Wordeman L., Gonen T., Asbury C.L.
and Davis T.N. (2010) Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances processive microtubule coupling and is regulated by Aurora B. Journal of Cell Biology. 189 : 713 – 723.
3. Akiyoshi B., Sarangapani K.K., Powers A.F., Nelson C.R., Reichow S.L., Arellano-Santoyo H.,
Gonen T., Ranish J.A., Asbury C.L., and Biggins S. (2010) Tension directly stabilizes reconstituted kinetochore-microtubule attachments. Nature 468 : 576 - 581.
4. Umbreita NT., Gestaut DR., Tien JF., Vollmar BS., Gonen T, Asbury CL and Davis TN. (2012) The
Ndc80 kinetochore complex directly modulates microtubule dynamics. Proceedings of the National Academy of Sciences 109 (40): 16113 - 16118.
5. Gonen S., Akiyoshi B., Iadanza MG., Shi D., Duggan N., Biggins S*. and Gonen T*. (2012) The
structure of purified kinetochores reveals multiple microtubule-attachment sites. Nature Structural and Molecular Biology – 19 (9): 925 – 930.
Tamir Gonen Group Leader
Johan Hattne Research Staff
Angela Lei Postdoctoral Associate
Jinming Ma Postdoctoral Associate
Brent Nannenga Postdoctoral Associate
Francis Reyes Research Staff
Silvia Sanchez-Martinez Research Staff
Dan Shi Senior Scientist
In this review we discuss the current advances relating to structure determination from protein microcrystals with special emphasis on the newly developed method called MicroED. This method uses a transmission electron cryo-microscope to collect electron diffraction data from extremely small 3-dimensional (3D) crystals. MicroED has been used to solve the 3D structure of the model protein lysozyme to 2.9Å resolution. As the method further matures, MicroED promises to offer a unique and widely applicable approach to protein crystallography using nanocrystals.
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.
We describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil-generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔGfold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.
Amphotericin forms an extramembranous and fungicidal sterol sponge.Nature chemical biology 2014
T. M. Anderson, M. C. Clay, A. G. Cioffi, K. A. Diaz, G. S. Hisao, M. D. Tuttle, A. J. Nieuwkoop, G. Comellas, N. Maryum, S. Wang, B. E. Uno, E. L. Wildeman, T. Gonen, C. M. Rienstra, and M. D. Burke Nature chemical biology, (2014)
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.
Electron diffraction of extremely small three-dimensional crystals (MicroED) allows for structure determination from crystals orders of magnitude smaller than those used for X-ray crystallography. MicroED patterns, which are collected in a transmission electron microscope, were initially not amenable to indexing and intensity extraction by standard software, which necessitated the development of a suite of programs for data processing. The MicroED suite was developed to accomplish the tasks of unit-cell determination, indexing, background subtraction, intensity measurement and merging, resulting in data that can be carried forward to molecular replacement and structure determination. This ad hoc solution has been modified for more general use to provide a means for processing MicroED data until the technique can be fully implemented into existing crystallographic software packages. The suite is written in Python and the source code is available under a GNU General Public License.
A Type VI Secretion-Related Pathway in Bacteroidetes Mediates Interbacterial Antagonism.Cell host & microbe 2014
A. B. Russell, A. G. Wexler, B. N. Harding, J. C. Whitney, A. J. Bohn, Y. Goo, B. Q. Tran, N. A. Barry, H. Zheng, B. S. Peterson, S. Chou, T. Gonen, D. R. Goodlett, A. L. Goodman, and J. D. Mougous Cell host & microbe, 16:227-36 (2014)
Bacteroidetes are a phylum of Gram-negative bacteria abundant in mammalian-associated polymicrobial communities, where they impact digestion, immunity, and resistance to infection. Despite the extensive competition at high cell density that occurs in these settings, cell contact-dependent mechanisms of interbacterial antagonism, such as the type VI secretion system (T6SS), have not been defined in this group of organisms. Herein we report the bioinformatic and functional characterization of a T6SS-like pathway in diverse Bacteroidetes. Using prominent human gut commensal and soil-associated species, we demonstrate that these systems localize dynamically within the cell, export antibacterial proteins, and target competitor bacteria. The Bacteroidetes system is a distinct pathway with marked differences in gene content and high evolutionary divergence from the canonical T6S pathway. Our findings offer a potential molecular explanation for the abundance of Bacteroidetes in polymicrobial environments, the observed stability of Bacteroidetes in healthy humans, and the barrier presented by the microbiota against pathogens.
MicroED is a recently developed method that uses electron diffraction for structure determination from very small three-dimensional crystals of biological material. Previously we used a series of still diffraction patterns to determine the structure of lysozyme at 2.9 Å resolution with MicroED (Shi et al., 2013). Here we present the structure of bovine liver catalase determined from a single crystal at 3.2 Å resolution by MicroED. The data were collected by continuous rotation of the sample under constant exposure and were processed and refined using standard programs for X-ray crystallography. The ability of MicroED to determine the structure of bovine liver catalase, a protein that has long resisted atomic analysis by traditional electron crystallography, demonstrates the potential of this method for structure determination.
The major facilitator superfamily (MFS) is the largest collection of structurally related membrane proteins that transport a wide array of substrates. The proton-coupled sugar transporter XylE is the first member of the MFS that has been structurally characterized in multiple transporting conformations, including both the outward and inward-facing states. Here we report the crystal structure of XylE in a new inward-facing open conformation, allowing us to visualize the rocker-switch movement of the N-domain against the C-domain during the transport cycle. Using molecular dynamics simulation, and functional transport assays, we describe the movement of XylE that facilitates sugar translocation across a lipid membrane and identify the likely candidate proton-coupling residues as the conserved Asp27 and Arg133. This study addresses the structural basis for proton-coupled substrate transport and release mechanism for the sugar porter family of proteins.
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.
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.
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.
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.
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.
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.
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.
Overview of electron crystallography of membrane proteins: crystallization and screening strategies using negative stain electron microscopy.Current protocols in protein science / editorial board, John E. Coligan ... [et al.] 2013
B. L. Nannenga, M. G. Iadanza, B. S. Vollmar, and T. Gonen Current protocols in protein science / editorial board, John E. Coligan ... [et al.], Chapter 17:Unit17.15 (2013)
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.
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.
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.
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.
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.
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.
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.
Prior Publications (14 of 35)
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.
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.
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.
The Ndc80 kinetochore complex directly modulates microtubule dynamics.Proceedings of the National Academy of Sciences of the United States of America 2012
N. T. Umbreit, D. R. Gestaut, J. F. Tien, B. S. Vollmar, T. Gonen, C. L. Asbury, and T. N. Davis Proceedings of the National Academy of Sciences of the United States of America, (2012)
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.
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.
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.
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.
Structural underpinnings of nitrogen regulation by the prototypical nitrogen-responsive transcriptional factor NrpR.Structure (London, England : 1993) 2010
G. Wisedchaisri, D. M. Dranow, T. J. Lie, J. B. Bonanno, Y. Patskovsky, S. A. Ozyurt, M. J. Sauder, S. C. Almo, S. R. Wasserman, S. K. Burley, J. A. Leigh, and T. Gonen Structure (London, England : 1993), 18:1512-21 (2010)
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.
Interactions of the transmembrane polymeric rings of the Salmonella enterica serovar Typhimurium type III secretion system.mBio 2010
S. Sanowar, P. Singh, R. A. Pfuetzner, I. André, H. Zheng, T. Spreter, N. C J. Strynadka, T. Gonen, D. Baker, D. R. Goodlett, and S. I. Miller mBio, 1 (2010)
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.
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.
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.
An engineered DNA-binding protein self-assembles metallic nanostructures.Chembiochem: A European Journal of Chemical Biology 2010
R. Hall Sedlak, M. Hnilova, E. Gachelet, L. Przybyla, D. Dranow, T. Gonen, M. Sarikaya, C. Tamerler, and B. Traxler Chembiochem: A European Journal of Chemical Biology, 11:2108-12 (2010)
Cooperation of the Dam1 and Ndc80 kinetochore complexes enhances microtubule coupling and is regulated by aurora B.The Journal of Cell Biology 2010
J. F. Tien, N. T. Umbreit, D. R. Gestaut, A. D. Franck, J. Cooper, L. Wordeman, T. Gonen, C. L. Asbury, and T. N. Davis The Journal of Cell Biology, 189:713-23 (2010)
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.
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.