David L. Stokes, Ph.D.

Cardiolipin Affects the Supramolecular Organization of ATP Synthase in Mitochondria
Acehan, Devrim; Malhotra, Ashim; Xu, Yang; Ren, Mindong; Stokes, David L; Schlame, Michael
2011 May 4;100(9):2184-2192, Biophysical journal
F(1)F(0) ATP synthase forms dimers that tend to assemble into large supramolecular structures. We show that the presence of cardiolipin is critical for the degree of oligomerization and the degree of order in these ATP synthase assemblies. This conclusion was drawn from the statistical analysis of cryoelectron tomograms of cristae vesicles isolated from Drosophila flight-muscle mitochondria, which are very rich in ATP synthase. Our study included a wild-type control, a cardiolipin synthase mutant with nearly complete loss of cardiolipin, and a tafazzin mutant with reduced cardiolipin levels. In the wild-type, the high-curvature edge of crista vesicles was densely populated with ATP synthase molecules that were typically organized in one or two rows of dimers. In both mutants, the density of ATP synthase was reduced at the high-curvature zone despite unchanged expression levels. Compared to the wild-type, dimer rows were less extended in the mutants and there was more scatter in the orientation of dimers. These data suggest that cardiolipin promotes the ribbonlike assembly of ATP synthase dimers and thus affects lateral organization and morphology of the crista membrane
— id: 131973, year: 2011, vol: 100, page: 2184, stat: Journal Article,

The Architecture of CopA from Archeaoglobus fulgidus Studied by Cryo-Electron Microscopy and Computational Docking
Allen, Gregory S; Wu, Chen-Chou; Cardozo, Tim; Stokes, David L
2011 Sep 7;19(9):1219-1232, Structure
CopA uses ATP to pump Cu(+) across cell membranes. X-ray crystallography has defined atomic structures of several related P-type ATPases. We have determined a structure of CopA at 10 A resolution by cryo-electron microscopy of a new crystal form and used computational molecular docking to study the interactions between the N-terminal metal-binding domain (NMBD) and other elements of the molecule. We found that the shorter-chain lipids used to produce these crystals are associated with movements of the cytoplasmic domains, with a novel dimer interface and with disordering of the NMBD, thus offering evidence for the transience of its interaction with the other cytoplasmic domains. Docking identified a binding site that matched the location of the NMBD in our previous structure by cryo-electron microscopy, allowing a more detailed view of its binding configuration and further support for its role in autoinhibition
— id: 137070, year: 2011, vol: 19, page: 1219, stat: Journal Article,

A functional, pentameric form of phospholamban is required for two-dimensional crystallization with the sarcoplasmic reticulum calcium pump
Glaves, John Paul; Trieber, C. A.; Ceholski, D. K.; Stokes, D. L.; Young, H. S.
2011 APR ;89(2):269-270, Biochemistry & cell biology
— id: 131842, year: 2011, vol: 89, page: 269, stat: Journal Article,

Phosphorylation and mutation of phospholamban alter physical interactions with the sarcoplasmic reticulum calcium pump
Glaves, John Paul; Trieber, Catharine A; Ceholski, Delaine K; Stokes, David L; Young, Howard S
2011 Jan 21;405(3):707-723, Journal of molecular biology
Phospholamban physically interacts with the sarcoplasmic reticulum calcium pump (SERCA) and regulates contractility of the heart in response to adrenergic stimuli. We studied this interaction using electron microscopy of 2D crystals of SERCA in complex with phospholamban. In earlier studies, phospholamban oligomers were found interspersed between SERCA dimer ribbons and a 3D model was constructed to show interactions with SERCA. In this study, we examined the oligomeric state of phospholamban and the effects of phosphorylation and mutation of phospholamban on the interaction with SERCA in the 2D crystals. On the basis of projection maps from negatively stained and frozen-hydrated crystals, phosphorylation of Ser16 selectively disordered the cytoplasmic domain of wild type phospholamban. This was not the case for a pentameric gain-of-function mutant (Lys27Ala), which retained inhibitory activity and remained ordered in the phosphorylated state. A partial loss-of-function mutation that altered the charge state of phospholamban (Arg14Ala) retained an ordered state, while a complete loss-of-function mutation (Asn34Ala) was also disordered. The functional state of phospholamban was correlated with an order-to-disorder transition of the phospholamban cytoplasmic domain in the 2D co-crystals. Furthermore, co-crystals of the gain-of-function mutant (Lys27Ala) facilitated data collection from frozen-hydrated crystals. An improved projection map was calculated to a resolution of 8 A, which supports the pentamer as the oligomeric state of phospholamban in the crystals. The 2D co-crystals with SERCA require a functional pentameric form of phospholamban, which physically interacts with SERCA at an accessory site distinct from that used by the phospholamban monomer for the inhibitory association
— id: 134156, year: 2011, vol: 405, page: 707, stat: Journal Article,

Toroidal surface complexes of bacteriophage varphi12 are responsible for host-cell attachment
Leo-Macias, Alejandra; Katz, Garrett; Wei, Hui; Alimova, Alexandra; Katz, A; Rice, William J; Diaz-Avalos, Ruben; Hu, Guo-Bin; Stokes, David L; Gottlieb, Paul
2011 Jun 5;414(2):103-109, Virology
Cryo-electron tomography and subtomogram averaging are utilized to determine that the bacteriophage varphi12, a member of the Cystoviridae family, contains surface complexes that are toroidal in shape, are composed of six globular domains with six-fold symmetry, and have a discrete density connecting them to the virus membrane-envelope surface. The lack of this kind of spike in a reassortant of varphi12 demonstrates that the gene for the hexameric spike is located in varphi12's medium length genome segment, likely to the P3 open reading frames which are the proteins involved in viral-host cell attachment. Based on this and on protein mass estimates derived from the obtained averaged structure, it is suggested that each of the globular domains is most likely composed of a total of four copies of P3a and/or P3c proteins. Our findings may have implications in the study of the evolution of the cystovirus species in regard to their host specificity
— id: 132579, year: 2011, vol: 414, page: 103, stat: Journal Article,

Electron tomography of paranodal septate-like junctions and the associated axonal and glial cytoskeletons in the central nervous system
Nans, Andrea; Einheber, Steven; Salzer, James L; Stokes, David L
2011 Mar;89(3):310-319, Journal of neuroscience research
The polarized domains of myelinated axons are specifically organized to maximize the efficiency of saltatory conduction. The paranodal region is directly adjacent to the node of Ranvier and contains specialized septate-like junctions that provide adhesion between axons and glial cells and that constitute a lateral diffusion barrier for nodal components. To complement and extend earlier studies on the peripheral nervous system, electron tomography was used to image paranodal regions from the central nervous system (CNS). Our three-dimensional reconstructions revealed short filamentous linkers running directly from the septate-like junctions to neurofilaments, microfilaments, and organelles within the axon. The intercellular spacing between axons and glia was measured to be 7.4 +/- 0.6 nm, over twice the value previously reported in the literature (2.5-3.0 nm). Averaging of individual junctions revealed a bifurcated structure in the intercellular space that is consistent with a dimeric complex of cell adhesion molecules composing the septate-like junction. Taken together, these findings provide new insight into the structural organization of CNS paranodes and suggest that, in addition to providing axo-glial adhesion, cytoskeletal linkage to the septate-like junctions may be required to maintain axonal domains and to regulate organelle transport in myelinated axons. (c) 2010 Wiley-Liss, Inc
— id: 121328, year: 2011, vol: 89, page: 310, stat: Journal Article,

Native ultrastructure of the red cell cytoskeleton by cryo-electron tomography
Nans, Andrea; Mohandas, Narla; Stokes, David L
2011 Nov 16;101(10):2341-2350, Biophysical journal
Erythrocytes possess a spectrin-based cytoskeleton that provides elasticity and mechanical stability necessary to survive the shear forces within the microvasculature. The architecture of this membrane skeleton and the nature of its intermolecular contacts determine the mechanical properties of the skeleton and confer the characteristic biconcave shape of red cells. We have used cryo-electron tomography to evaluate the three-dimensional topology in intact, unexpanded membrane skeletons from mouse erythrocytes frozen in physiological buffer. The tomograms reveal a complex network of spectrin filaments converging at actin-based nodes and a gradual decrease in both the density and the thickness of the network from the center to the edge of the cell. The average contour length of spectrin filaments connecting junctional complexes is 46 +/- 15 nm, indicating that the spectrin heterotetramer in the native membrane skeleton is a fraction of its fully extended length ( approximately 190 nm). Higher-order oligomers of spectrin were prevalent, with hexamers and octamers seen between virtually every junctional complex in the network. Based on comparisons with expanded skeletons, we propose that the oligomeric state of spectrin is in a dynamic equilibrium that facilitates remodeling of the network as the cell changes shape in response to shear stress
— id: 141710, year: 2011, vol: 101, page: 2341, stat: Journal Article,

Prenatal lethality and cardiac function in a mouse model of barth syndrome
Phoon C.K.L.; Acehan D.; Schlame M.; Stokes D.L.; Edelman-Novemsky I.; Yu D.; Xu Y.; Ren M.
2011 ;24(5):B27-B28, Journal of the American Society of Echocardiography
Background: Barth syndrome (BTHS) is a rare multisystem disorder caused by mutations in tafazzin that lead to cardiolipin deficiency and mitochondrial abnormalities. Patients most commonly present with early-onset cardiomyopathy, including fetal cardiomyopathy. A newly-developed transgenic mouse induces tafazzin deficiency using a doxycycline-inducible shRNA knockdown (TAZKD). Methods: TAZKD mice and wildtype controls were fed doxycycline starting in early gestation, via the mother (gestation and pre-weanling stages) or directly. 40 MHz echocardiography (axial resolution: 40 microns) with spectral and color Doppler capabilities defined in vivo cardiac function throughout fetal, newborn, and adult ages. Functional data were correlated with cardiolipin mass spectrometry, histology, and electron microscopy. Results: Abnormal cardiolipin profiles in TAZKD mice at embryonic (E13.5) and newborn stages, confirmed high-efficiency tafazzin knockdown during development. Newborn, juvenile, and adult mice did not show an obvious cardiomyopathic phenotype through 6 months of age. However, far fewer TAZKD mice were born than the expected 50:50 Mendelian ratios (4/26 TAZKD liveborn; p<0.02). We then focused on embryonic/fetal imaging of cardiovascular function at E13.5 (N=7 wildtype, N=4 TAZKD). Notably, we found a spectrum, from entirely normal function, including systolic and diastolic function, heart rate, atrioventricular conduction and rhythm, and umbilical arterial and venous flows; to a grossly abnormal embryo predicted (then confirmed) to be TAZKD based on severe bradycardia, holodiastolic aortic flow reversal, and a systolic atrial kick that suggested elevated myocardial stiffness. Echo suggested LV noncompaction in another embryo later confirmed to be TAZKD. Histology showed qualitatively thinner TAZKD ventricular myocardium with more prominent trabeculae suggestive of LV noncompaction. Electron microscopy of TAZKD embryonic hearts, similar to echocardiography, demonstrated a spectrum from normal to severely abnormal mitochondrial structures. Notably, mitochondria from TAZKD embryonic hearts with grossly abnormal hemodynamics tended to have poorly-formed lamellar cristae and disruption of the sarcomeric organization. Conclusion: A spectrum of functional and cellular cardiomyopathic abnormalities associated with prenatal lethality is seen in this novel model of human BTHS. Experiments are ongoing to better link cellular pathophysiological processes with the whole-organ/systems hemodynamics defined by in vivo embryonic mouse echocardiography
— id: 132321, year: 2011, vol: 24, page: B27, stat: Journal Article,

Fourier-Bessel reconstruction of helical assemblies
Diaz, Ruben; Rice, William J; Stokes, David L
2010 ;482:131-165, Methods in enzymology
Helical symmetry is commonly used for building macromolecular assemblies. Helical symmetry is naturally present in viruses and cytoskeletal filaments and also occurs during crystallization of isolated proteins, such as Ca-ATPase and the nicotinic acetyl choline receptor. Structure determination of helical assemblies by electron microscopy has a long history dating back to the original work on three-dimensional (3D) reconstruction. A helix offers distinct advantages for structure determination. Not only can one improve resolution by averaging across the constituent subunits, but each helical assembly provides multiple views of these subunits and thus provides a complete 3D data set. This review focuses on Fourier methods of helical reconstruction, covering the theoretical background, a step-by-step guide to the process, and a practical example based on previous work with Ca-ATPase. Given recent results from helical reconstructions at atomic resolution and the development of graphical user interfaces to aid in the process, these methods are likely to continue to make an important contribution to the field of structural biology
— id: 134412, year: 2010, vol: 482, page: 131, stat: Journal Article,

Automated electron microscopy for evaluating two-dimensional crystallization of membrane proteins
Hu, MH; Vink, M; Kim, C; Derr, KD; Koss, J; D'Amico, K; Cheng, AC; Pulokas, J; Ubarretxena-Belandia, I; Stokes, D
2010 JUL ;171(1):102-110, Journal of structural biology
Membrane proteins fulfill many important roles in the cell and represent the target for a large number of therapeutic drugs. Although structure determination of membrane proteins has become a major priority, it has proven to be technically challenging. Electron microscopy of two-dimensional (2D) crystals has the advantage of visualizing membrane proteins in their natural lipidic environment, but has been underutilized in recent structural genomics efforts. To improve the general applicability of electron crystallography, high-throughput methods are needed for screening large numbers of conditions for 2D crystallization, thereby increasing the chances of obtaining well ordered crystals and thus achieving atomic resolution. Previous reports describe devices for growing 2D crystals on a 96-well format. The current report describes a system for automated imaging of these screens with an electron microscope. Samples are inserted with a two-part robot: a SCARA robot for loading samples into the microscope holder, and a Cartesian robot for placing the holder into the electron microscope. A standard JEOL 1230 electron microscope was used, though a new tip was designed for the holder and a toggle switch controlling the airlock was rewired to allow robot control. A computer program for controlling the robots was integrated with the Leginon program, which provides a module for automated imaging of individual samples. The resulting images are uploaded into the Sesame laboratory information management system database where they are associated with other data relevant to the crystallization screen. (C) 2010 Elsevier Inc. All rights reserved
— id: 110440, year: 2010, vol: 171, page: 102, stat: Journal Article,

An automated pipeline to screen membrane protein 2D crystallization
Kim, Changki; Vink, Martin; Hu, Minghui; Love, James; Stokes, David L; Ubarretxena-Belandia, Iban
2010 Jun;11(2):155-166, Journal of structural & functional genomics
Electron crystallography relies on electron cryomicroscopy of two-dimensional (2D) crystals and is particularly well suited for studying the structure of membrane proteins in their native lipid bilayer environment. To obtain 2D crystals from purified membrane proteins, the detergent in a protein-lipid-detergent ternary mixture must be removed, generally by dialysis, under conditions favoring reconstitution into proteoliposomes and formation of well-ordered lattices. To identify these conditions a wide range of parameters such as pH, lipid composition, lipid-to-protein ratio, ionic strength and ligands must be screened in a procedure involving four steps: crystallization, specimen preparation for electron microscopy, image acquisition, and evaluation. Traditionally, these steps have been carried out manually and, as a result, the scope of 2D crystallization trials has been limited. We have therefore developed an automated pipeline to screen the formation of 2D crystals. We employed a 96-well dialysis block for reconstitution of the target protein over a wide range of conditions designed to promote crystallization. A 96-position magnetic platform and a liquid handling robot were used to prepare negatively stained specimens in parallel. Robotic grid insertion into the electron microscope and computerized image acquisition ensures rapid evaluation of the crystallization screen. To date, 38 2D crystallization screens have been conducted for 15 different membrane proteins, totaling over 3000 individual crystallization experiments. Three of these proteins have yielded diffracting 2D crystals. Our automated pipeline outperforms traditional 2D crystallization methods in terms of throughput and reproducibility
— id: 133797, year: 2010, vol: 11, page: 155, stat: Journal Article,

Highly compacted chromatin formed in vitro reflects the dynamics of transcription activation in vivo
Li, Guohong; Margueron, Raphael; Hu, Guobin; Stokes, David; Wang, Yuh-Hwa; Reinberg, Danny
2010 Apr 9;38(1):41-53, Molecular cell
High-order chromatin was reconstituted in vitro. This species reflects the criteria associated with transcriptional regulation in vivo. Histone H1 was determinant to formation of condensed structures, with deacetylated histones giving rise to highly compacted chromatin that approximated 30 nm fibers as evidenced by electron microscopy. Using the PEPCK promoter, we validated the integrity of these templates that were refractory to transcription by attaining transcription through the progressive action of the pertinent factors. The retinoic acid receptor binds to highly compacted chromatin, but the NF1 transcription factor binds only after histone acetylation by p300 and SWI/SNF-mediated nucleosome mobilization, reflecting the in vivo case. Mapping studies revealed the same pattern of nucleosomal repositioning on the PEPCK promoter in vitro and in vivo, correlating with NF1 binding and transcription. The reconstitution of such highly compacted '30 nm' chromatin that mimics in vivo characteristics should advance studies of its conversion to a transcriptionally active form
— id: 121297, year: 2010, vol: 38, page: 41, stat: Journal Article,

Two-dimensional crystallization of integral membrane proteins for electron crystallography
Stokes, David L; Rice, William J; Hu, Minghui; Kim, Changki; Ubarretxena-Belandia, Iban
2010 ;654:187-205, Methods in molecular biology
Although membrane proteins make up 30% of the proteome and are a common target for therapeutic drugs, determination of their atomic structure remains a technical challenge. Electron crystallography represents an alternative to the conventional methods of X-ray diffraction and NMR and relies on the formation of two-dimensional crystals. These crystals are produced by reconstituting purified, detergent-solubilized membrane proteins back into the native environment of a lipid bilayer. This chapter reviews methods for producing two-dimensional crystals and for screening them by negative stain electron microscopy. In addition, we show examples of the different morphologies that are commonly obtained and describe basic image analysis procedures that can be used to evaluate their promise for structure determination by cryoelectron microscopy
— id: 111367, year: 2010, vol: 654, page: 187, stat: Journal Article,

Membrane invagination in Rhodobacter sphaeroides is initiated at curved regions of the cytoplasmic membrane, then forms both budded and fully detached spherical vesicles
Tucker, JD; Siebert, CA; Escalante, M; Adams, PG; Olsen, JD; Otto, C; Stokes, DL; Hunter, CN
2010 MAY ;76(4):833-847, Molecular microbiology
P>The purple phototrophic bacteria synthesize an extensive system of intracytoplasmic membranes (ICM) in order to increase the surface area for absorbing and utilizing solar energy. Rhodobacter sphaeroides cells contain curved membrane invaginations. In order to study the biogenesis of ICM in this bacterium mature (ICM) and precursor (upper pigmented band - UPB) membranes were purified and compared at the single membrane level using electron, atomic force and fluorescence microscopy, revealing fundamental differences in their morphology, protein organization and function. Cryo-electron tomography demonstrates the complexity of the ICM of Rba. sphaeroides. Some ICM vesicles have no connection with other structures, others are found nearer to the cytoplasmic membrane (CM), often forming interconnected structures that retain a connection to the CM, and possibly having access to the periplasmic space. Near-spherical single invaginations are also observed, still attached to the CM by a 'neck'. Small indents of the CM are also seen, which are proposed to give rise to the UPB precursor membranes upon cell disruption. 'Free-living' ICM vesicles, which possess all the machinery for converting light energy into ATP, can be regarded as bacterial membrane organelles
— id: 109771, year: 2010, vol: 76, page: 833, stat: Journal Article,

PRESENT AND FUTURE OF MEMBRANE PROTEIN STRUCTURE DETERMINATION BY ELECTRON CRYSTALLOGRAPHY
Ubarretxena-Belandia, Iban; Stokes, David L.
2010 MAR ;81(3):33-60, Advances in Protein Chemistry & Structural Biology
Membrane proteins are critical to cell physiology, playing roles in signaling, trafficking, transport, adhesion, and recognition. Despite their relative abundance in the proteome and their prevalence as targets of therapeutic drugs, structural information about membrane proteins is in short supply. This chapter describes the use of electron crystallography as a tool for determining membrane protein structures. Electron crystallography offers distinct advantages relative to the alternatives of X-ray crystallography and NMR spectroscopy. Namely, membrane proteins are placed in their native membranous environment, which is likely to favor a native conformation and allow changes in conformation in response to physiological ligands. Nevertheless, there are significant logistical challenges in finding appropriate conditions for inducing membrane proteins to form two-dimensional arrays within the membrane and in using electron cryo-microscopy to collect the data required for structure determination. A number of developments are described for high-throughput screening of crystallization trials and for automated imaging of crystals with the electron microscope. These tools are critical for exploring the necessary range of factors governing the crystallization process. There have also been recent software developments to facilitate the process of structure determination. However, further innovations in the algorithms used for processing images and electron diffraction are necessary to improve throughput and to make electron crystallography truly viable as a method for determining atomic structures of membrane proteins
— id: 128839, year: 2010, vol: 81, page: 33, stat: Journal Article,

Distinct effects of tafazzin deletion in differentiated and undifferentiated mitochondria
Acehan, Devrim; Khuchua, Zaza; Houtkooper, Riekelt H; Malhotra, Ashim; Kaufman, Johanna; Vaz, Frederic M; Ren, Mindong; Rockman, Howard A; Stokes, David L; Schlame, Michael
2009 Apr;9(2):86-95, Mitochondrion
Tafazzin is a conserved mitochondrial protein that is required to maintain normal content and composition of cardiolipin. We used electron tomography to investigate the effect of tafazzin deletion on mitochondrial structure and found that cellular differentiation plays a crucial role in the manifestation of abnormalities. This conclusion was reached by comparing differentiated cardiomyocytes with embryonic stem cells from mouse and by comparing different tissues from Drosophila melanogaster. The data suggest that tafazzin deficiency affects cardiolipin in all mitochondria, but significant alterations of the ultrastructure, such as remodeling and aggregation of inner membranes, will only occur after specific differentiation
— id: 94434, year: 2009, vol: 9, page: 86, stat: Journal Article,

Biodiversity conservation in local planning
Miller, James R; Groom, Martha; Hess, George R; Steelman, Toddi; Stokes, David L; Thompson, Jan; Bowman, Troy; Fricke, Laura; King, Brandon; Marquardt, Ryan
2009 Feb;23(1):53-63, Conservation biology
Local land-use policy is increasingly being recognized as fundamental to biodiversity conservation in the United States. Many planners and conservation scientists have called for broader use of planning and regulatory tools to support the conservation of biodiversity at local scales. Yet little is known about the pervasiveness of these practices. We conducted an on-line survey of county, municipal, and tribal planning directors (n =116) in 3 geographic regions of the United States: metropolitan Seattle, Washington; metropolitan Des Moines, Iowa; and the Research Triangle, North Carolina. Our objectives were to gauge the extent to which local planning departments address biodiversity conservation and to identify factors that facilitate or hinder conservation actions in local planning. We found that biodiversity conservation was seldom a major consideration in these departments. Staff time was mainly devoted to development mandates and little time was spent on biodiversity conservation. Regulations requiring conservation actions that might benefit biodiversity were uncommon, with the exception of rules governing water quality in all 3 regions and the protection of threatened and endangered species in the Seattle region. Planning tools that could enhance habitat conservation were used infrequently. Collaboration across jurisdictions was widespread, but rarely focused on conservation. Departments with a conservation specialist on staff tended to be associated with higher levels of conservation actions. Jurisdictions in the Seattle region also reported higher levels of conservation action, largely driven by state and federal mandates. Increased funding was most frequently cited as a factor that would facilitate greater consideration of biodiversity in local planning. There are numerous opportunities for conservation biologists to play a role in improving conservation planning at local scales
— id: 94875, year: 2009, vol: 23, page: 53, stat: Journal Article,

Three-dimensional structure of the enveloped bacteriophage phi12: an incomplete T = 13 lattice is superposed on an enclosed T = 1 shell
Wei, Hui; Cheng, R Holland; Berriman, John; Rice, William J; Stokes, David L; Katz, A; Morgan, David Gene; Gottlieb, Paul
2009 ;4(9):e6850-e6850, PLoS ONE
BACKGROUND: Bacteriophage phi12 is a member of the Cystoviridae, a unique group of lipid containing membrane enveloped bacteriophages that infect the bacterial plant pathogen Pseudomonas syringae pv. phaseolicola. The genomes of the virus species contain three double-stranded (dsRNA) segments, and the virus capsid itself is organized in multiple protein shells. The segmented dsRNA genome, the multi-layered arrangement of the capsid and the overall viral replication scheme make the Cystoviridae similar to the Reoviridae. METHODOLOGY/PRINCIPAL FINDINGS: We present structural studies of cystovirus phi12 obtained using cryo-electron microscopy and image processing techniques. We have collected images of isolated phi12 virions and generated reconstructions of both the entire particles and the polymerase complex (PC). We find that in the nucleocapsid (NC), the phi12 P8 protein is organized on an incomplete T = 13 icosahedral lattice where the symmetry axes of the T = 13 layer and the enclosed T = 1 layer of the PC superpose. This is the same general protein-component organization found in phi6 NC's but the detailed structure of the entire phi12 P8 layer is distinct from that found in the best classified cystovirus species phi6. In the reconstruction of the NC, the P8 layer includes protein density surrounding the hexamers of P4 that sit at the 5-fold vertices of the icosahedral lattice. We believe these novel features correspond to dimers of protein P7. CONCLUSIONS/SIGNIFICANCE: In conclusion, we have determined that the phi12 NC surface is composed of an incomplete T = 13 P8 layer forming a net-like configuration. The significance of this finding in regard to cystovirus assembly is that vacancies in the lattice could have the potential to accommodate additional viral proteins that are required for RNA packaging and synthesis
— id: 105216, year: 2009, vol: 4, page: e6850, stat: Journal Article,

Plakoglobin is required for effective intermediate filament anchorage to desmosomes
Acehan, Devrim; Petzold, Christopher; Gumper, Iwona; Sabatini, David D; Muller, Eliane J; Cowin, Pamela; Stokes, David L
2008 Nov;128(11):2665-2675, Journal of investigative dermatology
Desmosomes are adhesive junctions that provide mechanical coupling between cells. Plakoglobin (PG) is a major component of the intracellular plaque that serves to connect transmembrane elements to the cytoskeleton. We have used electron tomography and immunolabeling to investigate the consequences of PG knockout on the molecular architecture of the intracellular plaque in cultured keratinocytes. Although knockout keratinocytes form substantial numbers of desmosome-like junctions and have a relatively normal intercellular distribution of desmosomal cadherins, their cytoplasmic plaques are sparse and anchoring of intermediate filaments is defective. In the knockout, beta-catenin appears to substitute for PG in the clustering of cadherins, but is unable to recruit normal levels of plakophilin-1 and desmoplakin to the plaque. By comparing tomograms of wild type and knockout desmosomes, we have assigned particular densities to desmoplakin and described their interaction with intermediate filaments. Desmoplakin molecules are more extended in wild type than knockout desmosomes, as if intermediate filament connections produced tension within the plaque. On the basis of our observations, we propose a particular assembly sequence, beginning with cadherin clustering within the plasma membrane, followed by recruitment of plakophilin and desmoplakin to the plaque, and ending with anchoring of intermediate filaments, which represents the key to adhesive strength
— id: 93304, year: 2008, vol: 128, page: 2665, stat: Journal Article,

Three-dimensional structure of the KdpFABC complex of Escherichia coli by electron tomography of two-dimensional crystals
Hu, Guo-Bin; Rice, William J; Drose, Stefan; Altendorf, Karlheinz; Stokes, David L
2008 Mar;161(3):411-418, Journal of structural biology
The KdpFABC complex (Kdp) functions as a K+ pump in Escherichia coli and is a member of the family of P-type ATPases. Unlike other family members, Kdp has a unique oligomeric composition and is notable for segregating K+ transport and ATP hydrolysis onto separate subunits (KdpA and KdpB, respectively). We have produced two-dimensional crystals of the KdpFABC complex within reconstituted lipid bilayers and determined its three-dimensional structure from negatively stained samples using a combination of electron tomography and real-space averaging. The resulting map is at a resolution of 2.4 nm and reveals a dimer of Kdp molecules as the asymmetric unit; however, only the cytoplasmic domains are visible due to the lack of stain penetration within the lipid bilayer. The sizes of these cytoplasmic domains are consistent with Kdp and, using a pseudo-atomic model, we have described the subunit interactions that stabilize the Kdp dimer within the larger crystallographic array. These results illustrate the utility of electron tomography in structure determination of ordered assemblies, especially when disorder is severe enough to hamper conventional crystallographic analysis
— id: 78734, year: 2008, vol: 161, page: 411, stat: Journal Article,

Electron cryo-tomographic structure of cystovirus phi 12
Hu, Guo-Bin; Wei, Hui; Rice, William J; Stokes, David L; Gottlieb, Paul
2008 Mar 1;372(1):1-9, Virology
Bacteriophage phi 12 is a member of the Cystoviridae virus family and contains a genome consisting of three segments of double-stranded RNA (dsRNA). This virus family contains eight identified members, of which four have been classified in regard to their complete genomic sequence and encoded viral proteins. A phospholipid envelope that contains the integral proteins P6, P9, P10, and P13 surrounds the viral particles. In species phi 6, host infection requires binding of a multimeric P3 complex to type IV pili. In species varphi8, phi 12, and phi 13, the attachment apparatus is a heteromeric protein assembly that utilizes the rough lipopolysaccharide (rlps) as a receptor. In phi 8 the protein components are designated P3a and P3b while in species phi 12 proteins P3a and P3c have been identified in the complex. The phospholipid envelope of the cystoviruses surrounds a nucleocapsid (NC) composed of two shells. The outer shell is composed of protein P8 with a T=13 icosahedral lattice while the primary component of the inner shell is a dodecahedral frame composed of dimeric protein P1. For the current study, the 3D architecture of the intact phi 12 virus was obtained by electron cryo-tomography. The nucleocapsid appears to be centered within the membrane envelope and possibly attached to it by bridging structures. Two types of densities were observed protruding from the membrane envelope. The densities of the first type were elongated, running parallel, and closely associated to the envelope outer surface. In contrast, the second density was positioned about 12 nm above the envelope connected to it by a flexible low-density stem. This second structure formed a torroidal structure termed 'the donut' and appears to inhibit BHT-induced viral envelope fusion
— id: 76385, year: 2008, vol: 372, page: 1, stat: Journal Article,

Nanoscale increases in CD2-CD48-mediated intermembrane spacing decrease adhesion and reorganize the immunological synapse
Milstein, Oren; Tseng, Su-Yi; Starr, Toby; Llodra, Jaime; Nans, Andrea; Liu, Mengling; Wild, Martin K; van der Merwe, P Anton; Stokes, David L; Reisner, Yair; Dustin, Michael L
2008 Dec 5;283(49):34414-34422, Journal of biological chemistry
The relationship between intermembrane spacing, adhesion efficiency, and lateral organization of adhesion receptors has not been established for any adhesion system. We have utilized the CD2 ligand CD48 with two (wild type CD48 (CD48-WT)), four (CD48-CD2), or five (CD48-CD22) Ig-like domains. CD48-WT was 10-fold more efficient in mediating adhesion than CD48-CD2 or CD48-CD22. Electron tomography of contact areas with planar bilayers demonstrated average intermembrane spacing of 12.8 nm with CD48-WT, 14.7 nm with CD48-CD2, and 15.6 nm with CD48-CD22. Both CD48-CD2 and CD48-CD22 chimeras segregated completely from CD48-WT in mixed contact areas. In contrast, CD48-CD2 and CD48-CD22 co-localized when mixed contacts were formed. Confocal imaging of immunological synapses formed between primary T lymphocytes and Chinese hamster ovary cells presenting major histocompatibility complex-peptide complexes, and different forms of CD48 demonstrated that CD48-CD2 and CD48-CD22 induce an eccentric CD2/T cell antigen receptor cluster. We propose that this reorganization of the immunological synapse sequesters the T cell antigen receptor in a location where it cannot interact with its ligand and dramatically reduces T cell sensitivity
— id: 92165, year: 2008, vol: 283, page: 34414, stat: Journal Article,

Cryoelectron tomography of isolated desmosomes
Owen, Gethin Rh; Acehan, Devrim; Derr, K D; Rice, William J; Stokes, David L
2008 Apr;36(Pt 2):173-179, Transactions (Biochemical Society (Great Britain))
Desmosomes are a complex assembly of protein molecules that form at the cell surface and mediate cell-cell adhesion. Much is known about the composition of desmosomes and there is an established consensus for the location of and interactions between constituent proteins within the assembly. Furthermore, X-ray crystallography has determined atomic structures of isolated domains from several constituent proteins. Nevertheless, there is a lack of understanding about the architecture of the intact assembly and the physical principles behind the adhesive strength of desmosomes therefore remain vague. We have used electron tomography to address this problem. In previous work, we investigated the in situ structure of desmosomes from newborn mouse skin preserved by freeze-substitution and imaged in resin-embedded thin sections. In our present work, we have isolated desmosomes from cow snout and imaged them in the frozen unstained state. Although not definitive, the resulting images provide support for the irregular groupings of cadherin molecules seen previously in mouse skin
— id: 94877, year: 2008, vol: 36, page: 173, stat: Journal Article,

Structure of a copper pump suggests a regulatory role for its metal-binding domain
Wu, Chen-Chou; Rice, William J; Stokes, David L
2008 Jun;16(6):976-985, Structure
P-type ATPases play an important role in Cu homeostasis, which provides sufficient Cu for metalloenzyme biosynthesis but prevents oxidative damage of free Cu to the cell. The P(IB) group of P-type ATPases includes ATP-dependent pumps of Cu and other transition metal ions, and it is distinguished from other family members by the presence of N-terminal metal-binding domains (MBD). We have determined structures of two constructs of a Cu pump from Archaeoglobus fulgidus (CopA) by cryoelectron microscopy of tubular crystals, which reveal the overall architecture and domain organization of the molecule. By comparing these structures, we localized its N-terminal MBD within the cytoplasmic domains that use ATP hydrolysis to drive the transport cycle. We have built a pseudoatomic model by fitting existing crystallographic structures into the cryoelectron microscopy maps for CopA, which suggest a Cu-dependent regulatory role for the MBD
— id: 94876, year: 2008, vol: 16, page: 976, stat: Journal Article,

Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography
Acehan, Devrim; Xu, Yang; Stokes, David L; Schlame, Michael
2007 Jan;87(1):40-48, Laboratory investigation
Barth syndrome (BTHS) is a mitochondrial disorder that is caused by mutations in the tafazzin gene, which affects phospholipid composition. To determine whether this defect leads to alterations in the internal three-dimensional organization of mitochondrial membranes, we applied electron microscopic tomography to lymphoblast mitochondria from BTHS patients and controls. Tomograms were formed from 50 and 150 nm sections of chemically fixed lymphoblasts and the data were used to manually segment volumes of relevant structural details. Normal lymphoblast mitochondria contained well-aligned, lamellar cristae with slot-like junctions to the inner boundary membrane. In BTHS, mitochondrial size was more variable and the total mitochondrial volume per cell increased mainly due to clusters of fragmented mitochondria inside nuclear invaginations. However, mitochondria showed reduced cristae density, less cristae alignment, and inhomogeneous cristae distribution. Three-dimensional reconstruction of BTHS mitochondria revealed zones of adhesion of the opposing inner membranes, causing obliteration of the intracrista space. We found small isolated patches of adhesion as well as extended adhesion zones, resulting in sheets of collapsed cristae packaged in multiple concentric layers. We also found large tubular structures (diameter 30-150 nm) that appeared to be derivatives of the adhesion zones. The data suggest that mitochondrial abnormalities of BTHS involve adhesions of inner mitochondrial membranes with subsequent collapse of the intracristae space
— id: 71408, year: 2007, vol: 87, page: 40, stat: Journal Article,

Application of the iterative helical real-space reconstruction method to large membranous tubular crystals of P-type ATPases
Pomfret, Andrew J; Rice, William J; Stokes, David L
2007 Jan;157(1):106-116, Journal of structural biology
Since the development of three-dimensional helical reconstruction methods in the 1960's, advances in Fourier-Bessel methods have facilitated structure determination to near-atomic resolution. A recently developed iterative helical real-space reconstruction (IHRSR) method provides an alternative that uses single-particle analysis in conjunction with the imposition of helical symmetry. In this work, we have adapted the IHRSR algorithm to work with frozen-hydrated tubular crystals of P-type ATPases. In particular, we have implemented layer-line filtering to improve the signal-to-noise ratio, Wiener-filtering to compensate for the contrast transfer function, solvent flattening to improve reference reconstructions, out-of-plane tilt compensation to deal with flexibility in three dimensions, systematic calculation of Fourier shell correlations to track the progress of the refinement, and tools to control parameters as the refinement progresses. We have tested this procedure on datasets from Na(+)/K(+)-ATPase, rabbit skeletal Ca(2+)-ATPase and scallop Ca(2+)-ATPase in order to evaluate the potential for sub-nanometer resolution as well as the robustness in the presence of disorder. We found that Fourier-Bessel methods perform better for well-ordered samples of skeletal Ca(2+)-ATPase and Na(+)/K(+)-ATPase, although improvements to IHRSR are discussed that should reduce this disparity. On the other hand, IHRSR was very effective for scallop Ca(2+)-ATPase, which was too disordered to analyze by Fourier-Bessel methods
— id: 71141, year: 2007, vol: 157, page: 106, stat: Journal Article,

Desmosomes from a structural perspective
Stokes, David L
2007 Oct;19(5):565-571, Current opinion in cell biology
Desmosomes are cell-cell junctions responsible for maintaining the structural integrity of tissues by resisting shear forces. Defects result in diseases of mechanically challenged tissues such as skin and heart. The architectural design represents the key to understanding the strength and durability inherent to desmosomes. A number of different proteins contribute to this architecture, and X-ray crystallography has made considerable progress in defining the atomic structure of various isolated domains. Electron tomography has been used to determine the three-dimensional structure of intact desmosomes in situ. By combining information from X-ray crystallography, cell and molecular biology and electron tomography, it should ultimately be possible to deduce the specific protein interactions that define the mechanical properties of this important adhesive junction
— id: 75667, year: 2007, vol: 19, page: 565, stat: Journal Article,

A high-throughput strategy to screen 2D crystallization trials of membrane proteins
Vink, Martin; Derr, Kd; Love, James; Stokes, David L; Ubarretxena-Belandia, Iban
2007 Dec;160(3):295-304, Journal of structural biology
Electron microscopy of two-dimensional (2D) crystals has demonstrated potential for structure determination of membrane proteins. Technical limitations in large-scale crystallization screens have, however, prevented a major breakthrough in the routine application of this technology. Dialysis is generally used for detergent removal and reconstitution of the protein into a lipid bilayer, and devices for testing numerous conditions in parallel are not readily available. Furthermore, the small size of resulting 2D crystals requires electron microscopy to evaluate the results and automation of the necessary steps is essential to achieve a reasonable throughput. We have designed a crystallization block, using standard microplate dimensions, by which 96 unique samples can be dialyzed simultaneously against 96 different buffers and have demonstrated that the rate of detergent dialysis is comparable to those obtained with conventional dialysis devices. A liquid-handling robot was employed to set up 2D crystallization trials with the membrane proteins CopA from Archaeoglobus fulgidus and light-harvesting complex II (LH2) from Rhodobacter sphaeroides. For CopA, 1 week of dialysis yielded tubular crystals and, for LH2, large and well-ordered vesicular 2D crystals were obtained after 24 h, illustrating the feasibility of this approach. Combined with a high-throughput procedure for preparation of EM-grids and automation of the subsequent negative staining step, the crystallization block offers a novel pipeline that promises to speed up large-scale screening of 2D crystallization and to increase the likelihood of producing well-ordered crystals for analysis by electron crystallography
— id: 94878, year: 2007, vol: 160, page: 295, stat: Journal Article,

Reconstitution and crystallization of CopA from archaeoglolms fulgidis
Wu, CC; Stokes, DL
2007 JAN ;108(11):7A-7A, Biophysical journal
— id: 71386, year: 2007, vol: 108, page: 7A, stat: Journal Article,

Cross-linking of C-terminal residues of phospholamban to the Ca2+ pump of cardiac sarcoplasmic reticulum to probe spatial and functional interactions within the transmembrane domain
Chen, Zhenhui; Akin, Brandy L; Stokes, David L; Jones, Larry R
2006 May 19;281(20):14163-14172, Journal of biological chemistry
Interactions between the transmembrane domains of phospholamban (PLB) and the cardiac Ca2+ pump (SERCA2a) have been investigated by chemical cross-linking. Specifically, C-terminal, transmembrane residues 45-52 of PLB were individually mutated to Cys, then cross-linked to V89C in the M2 helix of SERCA2a with the thiol-specific cross-linking reagents Cu2+-phenanthroline, dibromobimane, and bismaleimidohexane. V49C-, M50C-, and L52C-PLB all cross-linked strongly to V89C-SERCA2a, coupling to 70-100% of SERCA2a molecules. Residues 45-48 and 51 of PLB also cross-linked to V89C of SERCA2a, but more weakly. Evidence for the mechanism of PLB regulation of SERCA2a was provided by the conformational dependence of cross-linking. In particular, the required absence of Ca2+ for cross-linking implicated the E2 conformation of SERCA2a, and its enhancement by ATP confirmed E2 x ATP as the conformation with the highest affinity for PLB. In contrast, E2 phosphorylated with inorganic phosphate (E2P) and E2 inhibited by thapsigargin (E2 x TG) both failed to cross-link to PLB. These results with transmembrane PLB residues are completely consistent with cytoplasmic PLB residues studied previously, suggesting that the dissociation of PLB from the Ca2+ pump is complete, not partial, when the pump binds Ca2+ (E1 x Ca2) or adopts the E2P or E2 x TG conformations. V49C of PLB cross-linked to 100% of SERCA2a molecules, suggesting that this residue might have functional importance for regulation. Indeed, we found that mutation of Val49 to smaller side-chained residues V49A or V49G augmented PLB inhibition, whereas mutation to the larger hydrophobic residue, V49L, prevented PLB inhibition. A model for the interaction of PLB with SERCA2a is presented, showing that Val49 fits into a constriction at the lumenal end of the M2 helix of SERCA, possibly controlling access of PLB to its binding site on SERCA
— id: 94879, year: 2006, vol: 281, page: 14163, stat: Journal Article,

Role of metal-binding domains of the copper pump from Archaeoglobus fulgidus
Rice, William J; Kovalishin, Aleksandra; Stokes, David L
2006 Sep 15;348(1):124-131, Biochemical & biophysical research communications
CopA from the extreme thermophile Archaeoglobus fulgidus is a P-type ATPase that transports Cu(+) and Ag(+) and has individual metal-binding domains (MBDs) at both N- and C-termini. We expressed and purified full-length CopA as well as constructs with MBDs deleted either individually or collectively. Cu(+) and Ag(+)-dependent ATPase assays showed that full-length CopA had submicromolar affinity for both ions, but was inhibited by concentrations above 1muM. Deletion of both MBDs had no effect on affinity but resulted in loss of this inhibition. Individual deletions implicated the N-terminal MBD in causing the inhibition at concentrations >1muM. Rates of phosphoenzyme decay indicated that neither the dephosphorylation step, nor the E1P-E2P equilibrium accounted for this inhibition, suggesting the involvement of a different catalytic step. Alternative hypotheses are discussed by which the N-terminal MBD could influence the catalytic activity of CopA
— id: 69062, year: 2006, vol: 348, page: 124, stat: Journal Article,

Interactions between Ca2+-ATPase and the pentameric form of phospholamban in two-dimensional co-crystals
Stokes, David L; Pomfret, Andrew J; Rice, William J; Glaves, John Paul; Young, Howard S
2006 Jun 1;90(11):4213-4223, Biophysical journal
Phospholamban (PLB) physically interacts with Ca(2+)-ATPase and regulates contractility of the heart. We have studied this interaction using electron microscopy of large two-dimensional co-crystals of Ca(2+)-ATPase and the I40A mutant of PLB. Crystallization conditions were derived from those previously used for thin, helical crystals, but the addition of a 10-fold higher concentration of magnesium had a dramatic effect on the crystal morphology and packing. Two types of crystals were observed, and were characterized both by standard crystallographic methods and by electron tomography. The two crystal types had the same underlying lattice, which comprised antiparallel dimer ribbons of Ca(2+)-ATPase molecules previously seen in thin, helical crystals, but packed into a novel lattice with p22(1)2(1) symmetry. One crystal type was single-layered, whereas the other was a flattened tube and therefore double-layered. Additional features were observed between the dimer ribbons, which were substantially farther apart than in previous helical crystals. We attributed these additional densities to PLB, and built a three-dimensional model to show potential interactions with Ca(2+)-ATPase. These densities are most consistent with the pentameric form of PLB, despite the use of the presumed monomeric I40A mutant. Furthermore, our results indicate that this pentameric form of PLB is capable of a direct interaction with Ca(2+)-ATPase
— id: 66999, year: 2006, vol: 90, page: 4213, stat: Journal Article,

Role of leucine 31 of phospholamban in structural and functional interactions with the Ca2+ pump of cardiac sarcoplasmic reticulum
Chen, Zhenhui; Stokes, David L; Jones, Larry R
2005 Mar 18;280(11):10530-10539, Journal of biological chemistry
The ability of two loss-of-function mutants, L31A and L31C, of phospholamban (PLB) to bind to and inhibit the Ca(2+) pump of cardiac sarcoplasmic reticulum (SERCA2a) was investigated using a molecular cross-linking approach. Leu(31) of PLB, located at the cytoplasmic membrane boundary, is a critical amino acid shown previously to be essential for Ca(2+)-ATPase inhibition. We observed that L31A or L31C mutations of PLB prevented the inhibition of Ca(2+)-ATPase activity and disabled the cross-linking of N27C and N30C of PLB to Lys(328) and Cys(318) of SERCA2a. Although L31C-PLB failed to cross-link to any Cys or Lys residue of wild-type SERCA2a, L31C did cross-link with high efficiency to T317C of SERCA2a with use of the homobifunctional sulfhydryl cross-linking reagent, 1,6-bismaleimidohexane. This places Leu(31) of PLB within 10 angstroms of Thr(317) of SERCA2a in the M4 helix. Thus, contrary to previous suggestions, PLB with loss-of-function mutations at Leu(31) retains the ability to bind to SERCA2a, despite losing inhibitory activity. Cross-linking of L31C-PLB to T317C-SERCA2a occurred only in the absence of Ca(2+) and in the presence of nucleotide and was prevented by thapsigargin and by anti-PLB antibody, demonstrating for a fourth cross-linking pair that PLB interacts near M4 only when the Ca(2+) pump is in the Ca(2+)-free, nucleotide-bound E2 conformation, but not in the E2 state inhibited by thapsigargin. L31I-PLB retained full functional and cross-linking activity, suggesting that a bulky hydrophobic residue at position 31 of PLB is essential for productive interaction with SERCA2a. A model for the three-dimensional structure of the interaction site is proposed
— id: 94881, year: 2005, vol: 280, page: 10530, stat: Journal Article,

Normal mode-based fitting of atomic structure into electron density maps: application to sarcoplasmic reticulum Ca-ATPase
Hinsen, Konrad; Reuter, Nathalie; Navaza, Jorge; Stokes, David L; Lacapere, Jean-Jacques
2005 Feb;88(2):818-827, Biophysical journal
A method for the flexible docking of high-resolution atomic structures into lower resolution densities derived from electron microscopy is presented. The atomic structure is deformed by an iterative process using combinations of normal modes to obtain the best fit of the electron microscopical density. The quality of the computed structures has been evaluated by several techniques borrowed from crystallography. Two atomic structures of the SERCA1 Ca-ATPase corresponding to different conformations were used as a starting point to fit the electron density corresponding to a different conformation. The fitted models have been compared to published models obtained by rigid domain docking, and their relation to the known crystallographic structures are explored by normal mode analysis. We find that only a few number of modes contribute significantly to the transition. The associated motions involve almost exclusively rotation and translation of the cytoplasmic domains as well as displacement of cytoplasmic loops. We suggest that the movements of the cytoplasmic domains are driven by the conformational change that occurs between nonphosphorylated and phosphorylated intermediate, the latter being mimicked by the presence of vanadate at the phosphorylation site in the electron microscopy structure
— id: 94883, year: 2005, vol: 88, page: 818, stat: Journal Article,

Plasmonics-based nanostructures for surface-enhanced Raman scattering bioanalysis
Vo-Dinh, Tuan; Yan, Fei; Stokes, David L
2005 ;300:255-283, Methods in molecular biology
Surface-enhanced Raman scattering (SERS) spectroscopy is a plasmonics-based spectroscopic technique that combines modern laser spectroscopy with unique optical properties of metallic nanostructures, resulting in strongly increased Raman signals when molecules are adsorbed on or near nanometer-size structures of special metals such as gold, silver, and transition metals. This chapter provides a synopsis of the development and application of SERS-active metallic nanostructures, especially for the analysis of biologically relevant compounds. Some highlights of this chapter include reports of SERS as an immunoassay readout method, SERS gene nanoprobes, near-field scanning optical microscopy SERS probes, SERS as a tool for single-molecule detection, and SERS nanoprobes for cellular studies
— id: 94880, year: 2005, vol: 300, page: 255, stat: Journal Article,

A hyperspectral imaging system for in vivo optical diagnostics. Hyperspectral imaging basic principles, instrumental systems, and applications of biomedical interest
Vo-Dinh, Tuan; Stokes, David L; Wabuyele, Musundi B; Martin, Matt E; Song, Joon Myong; Jagannathan, Ramesh; Michaud, Edward; Lee, Robert J; Pan, Xiaogang
2004 Sep-Oct;23(5):40-49, IEEE Engineering in Medicine & Biology Magazine
— id: 94882, year: 2004, vol: 23, page: 40, stat: Journal Article,

Untangling desmosomal knots with electron tomography
He, Wanzhong; Cowin, Pamela; Stokes, David L
2003 Oct 3;302(5642):109-113, Science
Cell adhesion by adherens junctions and desmosomes relies on interactions between cadherin molecules. However, the molecular interfaces that define molecular specificity and that mediate adhesion remain controversial. We used electron tomography of plastic sections from neonatal mouse skin to visualize the organization of desmosomes in situ. The resulting three-dimensional maps reveal individual cadherin molecules forming discrete groups and interacting through their tips. Fitting of an x-ray crystal structure for C-cadherin to these maps is consistent with a flexible intermolecular interface mediated by an exchange of amino-terminal tryptophans. This flexibility suggests a novel mechanism for generating both cis and trans interactions and for propagating these adhesive interactions along the junction
— id: 38124, year: 2003, vol: 302, page: 109, stat: Journal Article,

Structure and function of the calcium pump
Stokes, David L; Green, N Michael
2003 ;32:445-468, Annual review of biophysics & biomolecular structure
Active transport of cations is achieved by a large family of ATP-dependent ion pumps, known as P-type ATPases. Various members of this family have been targets of structural and functional investigations for over four decades. Recently, atomic structures have been determined for Ca2+-ATPase by X-ray crystallography, which not only reveal the architecture of these molecules but also offer the opportunity to understand the structural mechanisms by which the energy of ATP is coupled to calcium transport across the membrane. This energy coupling is accomplished by large-scale conformational changes. The transmembrane domain undergoes plastic deformations under the influence of calcium binding at the transport site. Cytoplasmic domains undergo dramatic rigid-body movements that deliver substrates to the catalytic site and that establish new domain interfaces. By comparing various structures and correlating functional data, we can now begin to associate the chemical changes constituting the reaction cycle with structural changes in these domains
— id: 48169, year: 2003, vol: 32, page: 445, stat: Journal Article,

A structural model for the catalytic cycle of Ca(2+)-ATPase
Xu, Chen; Rice, William J; He, Wanzhong; Stokes, David L
2002 Feb 8;316(1):201-211, Journal of molecular biology
Ca(2+)-ATPase is responsible for active transport of calcium ions across the sarcoplasmic reticulum membrane. This coupling involves an ordered sequence of reversible reactions occurring alternately at the ATP site within the cytoplasmic domains, or at the calcium transport sites within the transmembrane domain. These two sites are separated by a large distance and conformational changes have long been postulated to play an important role in their coordination. To characterize the nature of these conformational changes, we have built atomic models for two reaction intermediates and postulated the mechanisms governing the large structural changes. One model is based on fitting the X-ray crystallographic structure of Ca(2+)-ATPase in the E1 state to a new 6 A structure by cryoelectron microscopy in the E2 state. This fit indicates that calcium binding induces enormous movements of all three cytoplasmic domains as well as significant changes in several transmembrane helices. We found that fluorescein isothiocyanate displaced a decavanadate molecule normally located at the intersection of the three cytoplasmic domains, but did not affect their juxtaposition; this result indicates that our model likely reflects a native E2 conformation and not an artifact of decavanadate binding. To explain the dramatic structural effect of calcium binding, we propose that M4 and M5 transmembrane helices are responsive to calcium binding and directly induce rotation of the phosphorylation domain. Furthermore, we hypothesize that both the nucleotide-binding and beta-sheet domains are highly mobile and driven by Brownian motion to elicit phosphoenzyme formation and calcium transport, respectively. If so, the reaction cycle of Ca(2+)-ATPase would have elements of a Brownian ratchet, where the chemical reactions of ATP hydrolysis are used to direct the random thermal oscillations of an innately flexible molecule
— id: 39718, year: 2002, vol: 316, page: 201, stat: Journal Article,

Structure of Na+,K+-ATPase at 11-A resolution: comparison with Ca2+-ATPase in E1 and E2 states
Rice WJ; Young HS; Martin DW; Sachs JR; Stokes DL
2001 May;80(5):2187-2197, Biophysical journal
Na+,K+-ATPase is a heterodimer of alpha and beta subunits and a member of the P-type ATPase family of ion pumps. Here we present an 11-A structure of the heterodimer determined from electron micrographs of unstained frozen-hydrated tubular crystals. For this reconstruction, the enzyme was isolated from supraorbital glands of salt-adapted ducks and was crystallized within the native membranes. Crystallization conditions fixed Na+,K+-ATPase in the vanadate-inhibited E2 conformation, and the crystals had p1 symmetry. A large number of helical symmetries were observed, so a three-dimensional structure was calculated by averaging both Fourier-Bessel coefficients and real-space structures of data from the different symmetries. The resulting structure clearly reveals cytoplasmic, transmembrane, and extracellular regions of the molecule with densities separately attributable to alpha and beta subunits. The overall shape bears a remarkable resemblance to the E2 structure of rabbit sarcoplasmic reticulum Ca2+-ATPase. After aligning these two structures, atomic coordinates for Ca2+-ATPase were fit to Na+,K+-ATPase, and several flexible surface loops, which fit the map poorly, were associated with sequences that differ in the two pumps. Nevertheless, cytoplasmic domains were very similarly arranged, suggesting that the E2-to-E1 conformational change postulated for Ca2+-ATPase probably applies to Na+,K+-ATPase as well as other P-type ATPases
— id: 20699, year: 2001, vol: 80, page: 2187, stat: Journal Article,

Locating phospholamban in co-crystals with ca(2+)-atpase by cryoelectron microscopy
Young HS; Jones LR; Stokes DL
2001 Aug;81(2):884-894, Biophysical journal
Phospholamban (PLB) is responsible for regulating Ca(2+) transport by Ca(2+)-ATPase across the sarcoplasmic reticulum of cardiac and smooth muscle. This regulation is coupled to beta-adrenergic stimulation, and dysfunction has been associated with end-stage heart failure. PLB appears to directly bind to Ca(2+)-ATPase, thus slowing certain steps in the Ca(2+) transport cycle. We have determined 3D structures from co-crystals of PLB with Ca(2+)-ATPase by cryoelectron microscopy of tubular co-crystals at 8-10 A resolution. Specifically, we have used wild-type PLB, a monomeric PLB mutant (L37A), and a pentameric PLB mutant (N27A) for co-reconstitution and have compared resulting structures with three control structures of Ca(2+)-ATPase alone. The overall molecular shape of Ca(2+)-ATPase was indistinguishable in the various reconstructions, indicating that PLB did not have any global effects on Ca(2+)-ATPase conformation. Difference maps reveal densities which we attributed to the cytoplasmic domain of PLB, though no difference densities were seen for PLB's transmembrane helix. Based on these difference maps, we propose that a single PLB molecule interacts with two Ca(2+)-ATPase molecules. Our model suggests that PLB may resist the large domain movements associated with the catalytic cycle, thus inhibiting turnover
— id: 21107, year: 2001, vol: 81, page: 884, stat: Journal Article,

Locating the thapsigargin-binding site on Ca(2+)-ATPase by cryoelectron microscopy
Young HS; Xu C; Zhang P; Stokes DL
2001 Apr 27;308(2):231-240, Journal of molecular biology
Thapsigargin (TG) is a potent inhibitor of Ca(2+)-ATPase from sarcoplasmic and endoplasmic reticula. Previous enzymatic studies have concluded that Ca(2+)-ATPase is locked in a dead-end complex upon binding TG with an affinity of <1 nM and that this complex closely resembles the E(2) enzymatic state. We have studied the structural effects of TG binding by cryoelectron microscopy of tubular crystals, which have previously been shown to comprise Ca(2+)-ATPase molecules in the E(2) conformation. In particular, we have compared 3D reconstructions of Ca(2+)-ATPase in the absence and presence of either TG or its dansylated derivative. The overall molecular shape of Ca(2+)-ATPase in the reconstructions is very similar, demonstrating that the TG/Ca(2+)-ATPase complex does indeed physically resemble the E(2) conformation, in contrast to massive domain movements that appear to be induced by Ca(2+) binding. Difference maps reveal a consistent difference on the lumenal side of the membrane, which we conclude corresponds to the thapsigargin-binding site. Modeling the atomic structure for Ca(2+)-ATPase into our density maps reveals that this binding site is composed of the loops between transmembrane segments M3/M4 and M7/M8. Indirect effects are proposed to explain the effects of the S3 stalk segment on thapsigargin affinity as well as thapsigargin-induced changes in ATP affinity. Indeed, a second difference density was observed at the decavanadate-binding site within the three cytoplasmic domains, which we believe reflects an altered affinity as a result of the long-range conformational coupling that drives the reaction cycle of this family of ATP-dependent ion pumps.
— id: 20697, year: 2001, vol: 308, page: 231, stat: Journal Article,

Modeling a dehalogenase fold into the 8-A density map for Ca(2+)-ATPase defines a new domain structure
Stokes DL; Green NM
2000 Apr;78(4):1765-1776, Biophysical journal
Members of the large family of P-type pumps use active transport to maintain gradients of a wide variety of cations across cellular membranes. Recent structures of two P-type pumps at 8-A resolution have revealed the arrangement of transmembrane helices but were insufficient to reveal the architecture of the cytoplasmic domains. However, recent proposals of a structural homology with a superfamily of hydrolases offer a new basis for modeling these domains. In the current work, we have extended the sequence comparison for the superfamily and delineated domains in the 8-A density map of Ca(2+)-ATPase. The homology suggests a new domain structure for Ca(2+)-ATPase and, specifically, that the phosphorylation domain adopts a Rossman fold. Accordingly, the atomic structure of L-2 haloacid dehalogenase has been fitted into the relevant domain of Ca(2+)-ATPase. The resulting model suggests the existence of two ATP sites at the interface between two domains. Based on this new model, we are able to reconcile numerous results of mutagenesis and chemical cross-linking within the catalytic domains. Furthermore, we have used the model to predict the configuration of Mg.ATP at its binding site. Based on this prediction, we propose a mechanism, involving a change in Mg(2+) liganding, for initiating the domain movements that couple sites of ion transport to ATP hydrolysis
— id: 11787, year: 2000, vol: 78, page: 1765, stat: Journal Article,

Calcium transport across the sarcoplasmic reticulum structure and function of Ca2+-ATPase and the ryanodine receptor [In Process Citation]
Stokes DL; Wagenknecht T
2000 Sep;167(17):5274-5279, European journal of biochemistry
Contraction of striated muscle results from a rise in cytoplasmic calcium concentration in a process termed excitation/contraction coupling. Most of this calcium moves back and forth across the sarcoplasmic-reticulum membrane in cycles of contraction and relaxation. The channel responsible for release from the sarcoplasmic reticulum is the ryanodine receptor, whereas Ca2+-ATPase effects reuptake in an ATP-dependent manner. The structures of these two molecules have been studied by cryoelectron microscopy, with helical crystals in the case of Ca2+-ATPase and as isolated tetramers in the case of ryanodine receptor. Structures of Ca2+-ATPase at 8-A resolution reveal the packing of transmembrane helices and have allowed fitting of a putative ATP-binding domain among the cytoplasmic densities. Comparison of ATPases in different conformations gives hints about the conformational changes that accompany the reaction cycle. Structures of ryanodine receptor at 30-A resolution reveal a multitude of isolated domains in the cytoplasmic portion, as well as a distinct transmembrane assembly. Binding sites for various protein ligands have been determined and conformational changes induced by ATP, calcium and ryanodine have been characterized. Both molecules appear to use large conformational changes to couple interactions in their cytoplasmic domains with calcium transport through their membrane domains, and future studies at higher resolution will focus on the mechanisms for this coupling
— id: 11538, year: 2000, vol: 167, page: 5274, stat: Journal Article,

Averaging data derived from images of helical structures with different symmetries
DeRosier D; Stokes DL; Darst SA
1999 May 28;289(1):159-165, Journal of molecular biology
There are many examples of macromolecules that form helical tubes or crystals, which are useful for structure determination by electron microscopy and image processing. Helical crystals can be thought of as two-dimensional crystals that have been rolled into a cylinder such that two lattice points are superimposed. In many real cases, helical crystals of a particular macromolecule derive from an identical two-dimensional lattice but have different lattice points superimposed, thus producing different helical symmetries which cannot be simply averaged in Fourier-space. When confronted with this situation, one can select images corresponding to one of the observed symmetries at the expense of reducing the number of images that can be used for data collection and averaging, or one can calculate separate density maps from each symmetry, then align and average them together in real-space. Here, we present a third alternative, which is based on averaging of the Fourier-Bessel coefficients, gn,l(r), and which allows the inclusion of data from all symmetry groups derived from a common two-dimensional lattice. The method is straightforward and simple in practice and is shown, through a specific example with real data, to give results comparable to real-space averaging
— id: 20736, year: 1999, vol: 289, page: 159, stat: Journal Article,

Comparison of H+-ATPase and Ca2+-ATPase suggests that a large conformational change initiates P-type ion pump reaction cycles
Stokes DL; Auer M; Zhang P; Kuhlbrandt W
1999 Jul 1;9(13):672-679, Current biology. CB
BACKGROUND: Structures have recently been solved at 8 A resolution for both Ca2+-ATPase from rabbit sarcoplasmic reticulum and H+-ATPase from Neurospora crassa. These cation pumps are two distantly related members of the family of P-type ATPases, which are thought to use similar mechanisms to generate ATP-dependent ion gradients across a variety of cellular membranes. We have undertaken a detailed comparison of the two structures in order to describe their similarities and differences as they bear on their mechanism of active transport. RESULTS: Our first important finding was that the arrangement of 10 transmembrane helices was remarkably similar in the two molecules. This structural homology strongly supports the notion that these pumps use the same basic mechanism to transport their respective ions. Despite this similarity in the membrane-spanning region, the cytoplasmic regions of the two molecules were very different, both in their disposition relative to the membrane and in the juxtaposition of their various subdomains. CONCLUSIONS: On the basis of the crystallization conditions, we propose that these two crystal structures represent different intermediates in the transport cycle, distinguished by whether cations are bound to their transport sites. Furthermore, we propose that the corresponding conformational change (E2 to E1 ) has two components: the first is an inclination of the main cytoplasmic mass by 20 degrees relative to the membrane-spanning domain; the second is a rearrangement of the domains comprising the cytoplasmic part of the molecules. Accordingly, we present a rough model for this important conformational change, which relays the effects of cation binding within the membrane-spanning domain to the nucleotide-binding site, thus initiating the transport cycle
— id: 6157, year: 1999, vol: 9, page: 672, stat: Journal Article,

Two-dimensional crystallization of Ca-ATPase by detergent removal
Lacapere JJ; Stokes DL; Olofsson A; Rigaud JL
1998 Sep;75(3):1319-1329, Biophysical journal
By using Bio-Beads as a detergent-removing agent, it has been possible to produce detergent-depleted two-dimensional crystals of purified Ca-ATPase. The crystallinity and morphology of these different crystals were analyzed by electron microscopy under different experimental conditions. A lipid-to-protein ratio below 0.4 w/w was required for crystal formation. The rate of detergent removal critically affected crystal morphology, and large multilamellar crystalline sheets or wide unilamellar tubes were generated upon slow or fast detergent removal, respectively. Electron crystallographic analysis indicated unit cell parameters of a = 159 A, b = 54 A, and gamma = 90 degrees for both types of crystals, and projection maps at 15-A resolution were consistent with Ca-ATPase molecules alternately facing the two sides of the membrane. Crystal formation was also affected by the protein conformation. Indeed, tubular and multilamellar crystals both required the presence of Ca2+; the presence of ADP gave rise to another type of packing within the unit cell (a = 86 A, b = 77 A, and gamma = 90 degrees), while maintaining a bipolar orientation of the molecules within the bilayer. All of the results are discussed in terms of nucleation and crystal growth, and a model of crystallogenesis is proposed that may be generally true for asymmetrical proteins with a large hydrophilic cytoplasmic domain
— id: 7642, year: 1998, vol: 75, page: 1319, stat: Journal Article,

Structure of the Ca2+ pump of sarcoplasmic reticulum: a view along the lipid bilayer at 9-A resolution
Ogawa H; Stokes DL; Sasabe H; Toyoshima C
1998 Jul;75(1):41-52, Biophysical journal
We have used multilamellar crystals of the ATP-driven calcium pump from sarcoplasmic reticulum to address the structural effects of calcium binding to the enzyme. They are stacks of disk-shaped two-dimensional crystals. A density map projected along the lipid bilayer was obtained at 9-A resolution by frozen-hydrated electron microscopy. Although only in projection, much more details of the structure were revealed than previously available, especially in the transmembrane region. Quantitative comparison was made with the model obtained from the tubular crystals of this enzyme formed in the absence of calcium. Unexpectedly large differences in conformation were found, particularly in the cytoplasmic domain
— id: 20737, year: 1998, vol: 75, page: 41, stat: Journal Article,

Three-dimensional crystals of Ca2+-ATPase from sarcoplasmic reticulum: merging electron diffraction tilt series and imaging the (h, k, 0) projection
Shi D; Lewis MR; Young HS; Stokes DL
1998 Dec 18;284(5):1547-1564, Journal of molecular biology
Electron crystallography offers an increasingly viable alternative to X-ray crystallography for structure determination, especially for membrane proteins. The methodology has been developed and successfully applied to 2D crystals; however, well-ordered thin, 3D crystals are often produced during crystallization trials and generally discarded due to complexities in structure analysis. To cope with these complexities, we have developed a general method for determining unit cell geometry and for merging electron diffraction data from tilt series. We have applied this method to thin, monoclinic crystals of Ca2+-ATPase from sarcoplasmic reticulum, thus characterizing the unit cell and generating a 3D set of electron diffraction amplitudes to 8 A resolution with tilt angles up to 30 degrees. The indexing of data from the tilt series has been verified by an analysis of Laue zones near the (h, k, 0) projection and the unit cell geometry is consistent with low-angle X-ray scattering from these crystals. Based on this unit cell geometry, we have systematically tilted crystals to record images of the (h, k, 0) projection. After averaging the corresponding phases to 8 A resolution, an (h, k, 0) projection map has been calculated by combining image phases with electron diffraction amplitudes. This map contains discrete densities that most likely correspond to Ca2+-ATPase dimers, unlike previous maps of untilted crystals in which molecules from successive layers are not aligned. Comparison with a projection structure from tubular crystals reveals differences that are likely due to the conformational change accompanying calcium binding to Ca2+-ATPase.
— id: 6044, year: 1998, vol: 284, page: 1547, stat: Journal Article,

Electron crystallography of multilamellar Ca-ATPase crystals: Space group, lattice parameter and merging of electron diffraction
Shi, D; Lewis, M R; Stokes, D L
1998 Feb 22-26;74(2 PART 2):A182-A182, Biophysical journal
— id: 15936, year: 1998, vol: 74, page: A182, stat: Journal Article,

Cryoelectron microscopy of the calcium pump from sarcoplasmic reticulum: two crystal forms reveal two different conformations
Stokes DL; Zhang P; Toyoshima C; Yonekura K; Ogawa H; Lewis MR; Shi D
1998 Aug;643:35-43, Acta physiologica Scandinavica. Supplementum
— id: 7454, year: 1998, vol: 643, page: 35, stat: Journal Article,

Co-reconstitution and co-crystallization of phospholamban and Ca(2+)-ATPase
Young HS; Reddy LG; Jones LR; Stokes DL
1998 Sep 16;853:103-115, Annals of the New York Academy of Sciences
Significant advances have recently been made in understanding the regulation of Ca(2+)-ATPase by phospholamban and in modeling their structures. However, these insights would be furthered by determining the 3-D structure of both proteins within the membrane, thus revealing the structural basis for their interaction. To this end, we have developed methods for reconstituting purified Ca(2+)-ATPase with recombinant phospholamban. After reconstitution at high lipid-to-protein ratios, we have verified their functional association by measuring calcium transport and ATPase activity. Furthermore, we have grown co-crystals after reconstitution at low lipid-to-protein ratios. The structure of Ca(2+)-ATPase has recently been solved by cryoelectron microscopy at 8-A resolution, thus revealing transmembrane alpha-helices. Using a variety of constraints, we have associated these helices with the predicted transmembrane sequences to produce a detailed model for the packing of transmembrane helices. Structure determination of the co-crystals is currently underway, which we hope will eventually reveal the interaction of phospholamban with Ca(2+)-ATPase at a similar level of detail
— id: 11897, year: 1998, vol: 853, page: 103, stat: Journal Article,

3 dimensional structure of the phospholamban-calcium pump complex by cryoelectron microscopy
Young, HS; Jones, LR; Stokes, DL
1998 FEB ;74(2):A356-A356, Biophysical journal
— id: 53441, year: 1998, vol: 74, page: A356, stat: Journal Article,

Structure of the calcium pump from sarcoplasmic reticulum at 8-A resolution
Zhang P; Toyoshima C; Yonekura K; Green NM; Stokes DL
1998 Apr 23;392(6678):835-839, Nature
The calcium pump from sarcoplasmic reticulum (Ca2+-ATPase) is typical of the large family of P-type cation pumps. These couple ATP hydrolysis with cation transport, generating cation gradients across membranes. Ca2+-ATPase specifically maintains the low cytoplasmic calcium concentration of resting muscle by pumping calcium into the sarcoplasmic reticulum; subsequent release is used to initiate contraction. No high-resolution structure of a P-type pump has yet been determined, although a 14-A structure of Ca2+-ATPase, obtained by electron microscopy of frozen-hydrated, tubular crystals, showed a large cytoplasmic head connected to the transmembrane domain by a narrow stalk. We have now improved the resolution to 8A and can discern ten transmembrane alpha-helices, four of which continue into the stalk On the basis of constraints from transmembrane topology, site-directed mutagenesis and disulphide crosslinking, we have made tentative assignments for these alpha-helices within the amino-acid sequence. A distinct cavity leads to the putative calcium-binding site, providing a plausible path for calcium release to the lumen of the sarcoplasmic reticulum
— id: 7981, year: 1998, vol: 392, page: 835, stat: Journal Article,

Two-dimensional crystal formation from solubilized membrane proteins using Bio-Beads to remove detergent
Lacapere JJ; Stokes DL; Mosser G; Ranck JL; Leblanc G; Rigaud JL
1997 Nov 3;834(1):9-18, Annals of the New York Academy of Sciences
— id: 20738, year: 1997, vol: 834, page: 9, stat: Journal Article,

Keeping calcium in its place: Ca(2+)-ATPase and phospholamban
Stokes DL
1997 Aug;7(4):550-556, Current opinion in structural biology
Electron microscopy is gradually revealing more and more about the structure of the calcium pump from the sarcoplasmic reticulum, Ca(2+)-ATPase. The most recent result reveals the ATP-binding site, and two different avenues are being pursued towards achieving a higher resolution structure. Although no such structures are currently available for phospholamban, various spectroscopies and site-directed mutagenesis have been combined to produce a compelling structural model for its regulation of Ca(2+)-ATPase
— id: 7956, year: 1997, vol: 7, page: 550, stat: Journal Article,

The ATP-binding site of Ca(2+)-ATPase revealed by electron image analysis
Yonekura K; Stokes DL; Sasabe H; Toyoshima C
1997 Mar;72(3):997-1005, Biophysical journal
The location of the ATP-binding site of a P-type ion pump, Ca(2+)-ATPase from rabbit sarcoplasmic reticulum, was examined by cryoelectron microscopy. A nonhydrolyzable analog of ATP, beta, gamma-bidentate chromium (III) complex of ATP (CrATP), was used to stabilize the enzyme in the Ca(2+)-occluded state. Tubular crystals were then induced by vanadate in the presence of EGTA, keeping CrATP bound to the enzyme. The three-dimensional structures of the crystals were determined at 14 A resolution by cryoelectron microscopy and helical image analysis. Statistical comparison of the structures with and without CrATP showed clear and significant differences at the groove proposed previously as the ATP-binding pocket
— id: 20739, year: 1997, vol: 72, page: 997, stat: Journal Article,

How to make tubular crystals by reconstitution of detergent-solubilized Ca2(+)-ATPase
Young HS; Rigaud JL; Lacapere JJ; Reddy LG; Stokes DL
1997 Jun;72(6):2545-2558, Biophysical journal
In an attempt to better define the parameters governing reconstitution and two-dimensional crystallization of membrane proteins, we have studied Ca2(+)-ATPase from rabbit sarcoplasmic reticulum. This ion pump forms vanadate-induced crystals in its native membrane and has previously been reconstituted at high lipid-to-protein ratios for functional studies. We have characterized the reconstitution of purified Ca2(+)-ATPase at low lipid-to-protein ratios and discovered procedures that produce long, tubular crystals suitable for helical reconstruction. C12E8 (n-dodecyl-octaethylene-glycol monoether) was used to fully solubilize various mixtures of lipid and purified Ca2(+)-ATPase, and BioBeads were then used to remove the C12E8. Slow removal resulted in two populations of vesicles, and the proteoliposome population was separated from the liposome population on a sucrose density gradient. These proteoliposomes had a lipid-to-protein ratio of 1:2, and virtually 100% of molecules faced the outside of vesicles, as determined by fluorescein isothiocyanate labeling. Cycles of freeze-thaw caused considerable aggregation of these proteoliposomes, and, if phosphatidyl ethanolamine and phosphatidic acid were included, or if the bilayers were doped with small amounts of C12E8, vanadate-induced tubular crystals grew from the aggregates. Thus our procedure comprised two steps-reconstitution followed by crystallization-allowing us to consider mechanisms of bilayer formation separately from those of crystallization and tube formation
— id: 7288, year: 1997, vol: 72, page: 2545, stat: Journal Article,

Lamellar stacking in three-dimensional crystals of Ca(2+)-ATPase from sarcoplasmic reticulum
Cheong GW; Young HS; Ogawa H; Toyoshima C; Stokes DL
1996 Apr;70(4):1689-1699, Biophysical journal
Electron microscopy of multilamellar crystals of CA(2+)-ATPase currently offers the best opportunity for obtaining a high-resolution structure of this ATP-driven ion pump. Under certain conditions small, wormlike crystals are formed and provide views parallel to the lamellar plane, from which parameters of lamellar stacking can be directly measured. Assuming that molecular packing is the same, data from these views could supplement those obtained by tilting large, thin platelike crystals. However, we were surprised to discover that the lamellar spacing was variable and depended on the amount of glycerol present during crystallization (20% versus 5%). Projection maps (h,0,l) from these womklike crystals suggest different molecular contacts that give rise to the different lamellar spacings. Based on an orthogonal projection map (h,k,0) from collapsed, wormlike crystals and on x-ray powder patterns, we conclude that molecular packing within the lamellar plane is the same as that in thin, platelike crystals and is unaffected by glycerol. Finally, the orientation of molecules in the lamellar plane was characterized from freeze-dried, shadowed crystals. Comparing the profile of molecules in these multilamellar crystals with that previously observed in helical tubes induced by vanadate gives structural evidence of the conformational change that accompanies binding of calcium of Ca(2+)-ATPase
— id: 7933, year: 1996, vol: 70, page: 1689, stat: Journal Article,

Purified, reconstituted cardiac Ca2+-ATPase is regulated by phospholamban but not by direct phosphorylation with Ca2+/calmodulin-dependent protein kinase
Reddy LG; Jones LR; Pace RC; Stokes DL
1996 Jun 21;271(25):14964-14970, Journal of biological chemistry
Regulation of calcium transport by sarcoplasmic reticulum provides increased cardiac contractility in response to beta-adrenergic stimulation. This is due to phosphorylation of phospholamban by cAMP-dependent protein kinase or by calcium/calmodulin-dependent protein kinase, which activates the calcium pump (Ca2+-ATPase). Recently, direct phosphorylation of Ca2+-ATPase by calcium/calmodulin-dependent protein kinase has been proposed to provide additional regulation. To investigate these effects in detail, we have purified Ca2+-ATPase from cardiac sarcoplasmic reticulum using affinity chromatography and reconstituted it with purified, recombinant phospholamban. The resulting proteoliposomes had high rates of calcium transport, which was tightly coupled to ATP hydrolysis (approximately 1.7 calcium ions transported per ATP molecule hydrolyzed). Co-reconstitution with phospholamban suppressed both calcium uptake and ATPase activities by approximately 50%, and this suppression was fully relieved by a phospholamban monoclonal antibody or by phosphorylation either with cAMP-dependent protein kinase or with calcium/calmodulin-dependent protein kinase. These effects were consistent with a change in the apparent calcium affinity of Ca2+-ATPase and not with a change in Vmax. Neither the purified, reconstituted cardiac Ca2+-ATPase nor the Ca2+-ATPase in longitudinal cardiac sarcoplasmic reticulum vesicles was a substrate for calcium/calmodulin-dependent protein kinase, and accordingly, we found no effect of calcium/calmodulin-dependent protein kinase phosphorylation on Vmax for calcium transport
— id: 7035, year: 1996, vol: 271, page: 14964, stat: Journal Article,

Functional reconstitution of recombinant phospholamban with rabbit skeletal Ca(2+)-ATPase
Reddy LG; Jones LR; Cala SE; O'Brian JJ; Tatulian SA; Stokes DL
1995 Apr 21;270(16):9390-9397, Journal of biological chemistry
Phospholamban (PLB) is a small, transmembrane protein that resides in the cardiac sarcoplasmic reticulum (SR) and regulates the activity of Ca(2+)-ATPase in response to beta-adrenergic stimulation. We have used the baculovirus expression system in Sf21 cells to express milligram quantities of wild-type PLB. After purification by antibody affinity chromatography, the function of this recombinant PLB was tested by reconstitution with Ca(2+)-ATPase purified from skeletal SR. The results obtained with recombinant PLB were indistinguishable from those obtained with purified, canine cardiac PLB. In particular, PLB reduced the apparent calcium affinity of Ca(2+)-ATPase but had no effect on Vmax. At pCa 6.8, PLB inhibited both calcium uptake and ATPase activity of Ca(2+)-ATPase by 50%. This inhibition was fully reversed by addition of a monoclonal antibody to PLB, which mimics the physiological effects of PLB phosphorylation. Maximal PLB regulatory effects occurred at a molar stoichiometry of approximately 3:1, PLB/Ca(2+)-ATPase. We also investigated peptides corresponding to the two main domains of PLB. The membrane-spanning domain, PLB26-52, appeared to uncouple ATPase hydrolysis from calcium transport, even though the permeability of the reconstituted vesicles was not altered. The cytoplasmic peptide, PLB1-31, had little effect, even at a 300:1 molar excess over Ca(2+)-ATPase
— id: 18158, year: 1995, vol: 270, page: 9390, stat: Journal Article,

Preparation and analysis of large, flat crystals of Ca(2+)-ATPase for electron crystallography
Shi D; Hsiung HH; Pace RC; Stokes DL
1995 Mar;68(3):1152-1162, Biophysical journal
Obtaining large, flat, well ordered crystals represents the key to structure determination by electron crystallography. Multilamellar crystals of Ca(2+)-ATPase are a good candidate for this methodology, and we have optimized methods of crystallization and of preparation for cryoelectron microscopy. In particular, high concentrations of glycerol were found to prevent nucleation and to reduce stacking; thus, by seeding solutions containing 40% glycerol, we obtained thin crystals that were 5-30 microns in diameter and 2-10 unit cells thick. We found that removing vesicles and minimizing concentrations of divalent cations were critical to preparing flat crystals in the frozen-hydrated state. Finally, we developed two methods for determining the number of lamellae composing individual crystals, information that is required for structure determination of this crystal form. The first method, using low magnification images of freeze-dried crystals, is more practical in our case. Nevertheless, the alternative method, involving analysis of Laue zones from electron diffraction patterns of slightly tilted crystals, may be of general use in structure determination from thin, three-dimensional crystals
— id: 8406, year: 1995, vol: 68, page: 1152, stat: Journal Article,

Three-dimensional cryo-electron microscopy of the calcium ion pump in the sarcoplasmic reticulum membrane [published erratum appears in Nature 1993 May 20;363(6426):286]
Toyoshima C; Sasabe H; Stokes DL
1993 Apr 1;362(6419):467-471, Nature
The ATP-driven calcium pump (Ca(2+)-ATPase) is an integral membrane protein (M(r) 110K) which relaxes striated muscle by pumping calcium out of the cytoplasm into the sarcoplasmic reticulum against a large concentration gradient. Recent efforts have attempted to relate the sequence of Ca(2+)-ATPase to its structure and function. In particular, site-directed mutagenesis has identified critical amino-acid residues, and its predicted secondary structure, which includes ten transmembrane helices, has gained experimental support. But direct visualization of the molecule has so far been limited to the cytoplasmic domains at low resolution. We present here the three-dimensional structure of Ca(2+)-ATPase in the native sarcoplasmic reticulum membrane at 14 A resolution, determined by cryo-electron microscopy and helical image analysis. The structure shows an unexpected transmembrane organization, consisting of three distinct segments, one of which is highly inclined. These features can be related to earlier predictions of secondary structure
— id: 8405, year: 1993, vol: 362, page: 467, stat: Journal Article,