Journal of Structural Biology

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Cover 2 - Editorial Board

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1









Categories: Journal Articles

Table of Contents / barcode

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1









Categories: Journal Articles

The REC domain mediated dimerization is critical for FleQ from Pseudomonas aeruginosa to function as a c-di-GMP receptor and flagella gene regulator

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Tiantian Su, Shiheng Liu, Kang Wang, Kaikai Chi, Deyu Zhu, Tiandi Wei, Yan Huang, Liming Guo, Wei Hu, Sujuan Xu, Zong Lin, Lichuan Gu

FleQ is an AAA+ ATPase enhancer-binding protein that regulates both flagella and biofilm formation in the opportunistic pathogen Pseudomonas aeruginosa. FleQ belongs to the NtrC subfamily of response regulators, but lacks the corresponding aspartic acid for phosphorylation in the REC domain (FleQR, also named FleQ domain). Here, we show that the atypical REC domain of FleQ is essential for the function of FleQ. Crystal structure of FleQR at 2.3Å reveals that the structure of FleQR is significantly different from the REC domain of NtrC1 which regulates gene expression in a phosphorylation dependent manner. FleQR forms a novel active dimer (transverse dimer), and mediates the dimerization of full-length FleQ in an unusual manner. Point mutations that affect the dimerization of FleQ lead to loss of function of the protein. Moreover, a c-di-GMP binding site deviating from the previous reported one is identified through structure analysis and point mutations.





Categories: Journal Articles

Investigating interactions of the Bacillus subtilis spore coat proteins CotY and CotZ using single molecule force spectroscopy

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Huiqing Liu, Daniela Krajcikova, Zhe Zhang, Hongda Wang, Imrich Barak, Jilin Tang

Spores formed by Bacillus subtilis are surrounded by a protective and multilayered shell, termed the coat, which grants the spores resistance to various environmental stresses and facilitates spore germination. The spore coat consists of more than seventy different proteins, arranged into at least four distinct structural layers: the undercoat, inner coat, outer coat and crust. However, how these proteins, especially the morphogenetic proteins, interact to establish the organized, functional coat layers remains poorly understood. CotY and CotZ as the components of the crust, play a morphogenetic role in the crust assembly around the spore. In this study, the single molecule force spectroscopy was used to investigate the interaction and dynamics between CotY and CotZ at the single-molecule level. The results show that homotypic interactions of CotY and CotZ and the heterotypic interaction between CotY and CotZ exist. Furthermore, the dissociation kinetics of the complexes were studied by monitoring the relationship between the unbinding forces and the loading rates at different pulling velocities. In this way, a series of kinetic parameters regarding the three different complexes were obtained. It revealed the strong interactions between CotY and CotZ, CotY and CotY, and a relatively weak interaction of CotZ and CotZ.





Categories: Journal Articles

Structure of neurotropic adeno-associated virus AAVrh.8

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Sujata Halder, Kim Van Vliet, J. Kennon Smith, Thao Thi Phuong Duong, Robert McKenna, James M. Wilson, Mavis Agbandje-McKenna

Adeno-associated virus rhesus isolate 8 (AAVrh.8) is a leading vector for the treatment of neurological diseases due to its efficient transduction of neuronal cells and reduced peripheral tissue tropism. Toward identification of the capsid determinants for these properties, the structure of AAVrh.8 was determined by X-ray crystallography to 3.5Å resolution and compared to those of other AAV isolates. The capsid viral protein (VP) structure consists of an αA helix and an eight-stranded anti-parallel β-barrel core conserved in parvoviruses, and large insertion loop regions between the β-strands form the capsid surface topology. The AAVrh.8 capsid exhibits the surface topology conserved in all AAVs: depressions at the icosahedral twofold axis and surrounding the cylindrical channel at the fivefold axis, and three protrusions around the threefold axis. A structural comparison to serotypes AAV2, AAV8, and AAV9, to which AAVrh.8 shares ∼84%, ∼91%, and ∼87% VP sequence identity, respectively, revealed differences in the surface loops known to affect receptor binding, transduction efficiency, and antigenicity. Consistent with this observation, biochemical assays showed that AAVrh.8 is unable to bind heparin and does not cross-react with conformational monoclonal antibodies and human donor serum directed against the other AAVs compared. This structure of AAVrh.8 thus identified capsid surface differences which can serve as template regions for rational design of vectors with enhanced transduction for specific tissues and escape pre-existing antibody recognition. These features are essential for the creation of an AAV vector toolkit that is amenable to personalized disease treatment.





Categories: Journal Articles

X-ray structural and molecular dynamical studies of the globular domains of cow, deer, elk and Syrian hamster prion proteins

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Pravas Kumar Baral, Mridula Swayampakula, Adriano Aguzzi, Michael N.G. James

Misfolded prion proteins are the cause of neurodegenerative diseases that affect many mammalian species, including humans. Transmission of the prion diseases poses a considerable public-health risk as a specific prion disease such as bovine spongiform encephalopathy can be transferred to humans and other mammalian species upon contaminant exposure. The underlying mechanism of prion propagation and the species barriers that control cross species transmission has been investigated quite extensively. So far a number of prion strains have been characterized and those have been intimately linked to species-specific infectivity and other pathophysiological manifestations. These strains are encoded by a protein-only agent, and have a high degree of sequence identity across mammalian species. The molecular events that lead to strain differentiation remain elusive. In order to contribute to the understanding of strain differentiation, we have determined the crystal structures of the globular, folded domains of four prion proteins (cow, deer, elk and Syrian hamster) bound to the POM1 antibody fragment Fab. Although the overall structural folds of the mammalian prion proteins remains extremely similar, there are several local structural variations observed in the misfolding-initiator motifs. In additional molecular dynamics simulation studies on these several prion proteins reveal differences in the local fluctuations and imply that these differences have possible roles in the unfolding of the globular domains. These local variations in the structured domains perpetuate diverse patterns of prion misfolding and possibly facilitate the strain selection and adaptation.





Categories: Journal Articles

Structural characterization and modeling of the Borrelia burgdorferi hybrid histidine kinase Hk1 periplasmic sensor: A system for sensing small molecules associated with tick feeding

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): William J. Bauer, Amit Luthra, Guangyu Zhu, Justin D. Radolf, Michael G. Malkowski, Melissa J. Caimano

Two-component signal transduction systems are the primary mechanisms by which bacteria perceive and respond to changes in their environment. The Hk1/Rrp1 two-component system (TCS) in Borrelia burgdorferi consists of a hybrid histidine kinase and a response regulator with diguanylate cyclase activity, respectively. Phosphorylated Rrp1 catalyzes the synthesis of c-di-GMP, a second messenger associated with bacterial life-style control networks. Spirochetes lacking either Hk1 or Rrp1 are virulent in mice but destroyed within feeding ticks. Activation of Hk1 by exogenous stimuli represents the seminal event for c-di-GMP signaling. We reasoned that structural characterization of Hk1’s sensor would provide insights into the mechanism underlying signal transduction and aid in the identification of activating ligands. The Hk1 sensor is composed of three ligand-binding domains (D1–3), each with homology to periplasmic solute-binding proteins (PBPs) typically associated with ABC transporters. Herein, we determined the structure for D1, the most N-terminal PBP domain. As expected, D1 displays a bilobed Venus Fly Trap-fold. Similar to the prototypical sensor PBPs HK29S from Geobacter sulfurreducens and VFT2 from Bordetella pertussis, apo-D1 adopts a closed conformation. Using complementary approaches, including SAXS, we established that D1 forms a dimer in solution. The D1 structure enabled us to model the D2 and D3 domains. Differences in the ligand-binding pockets suggest that each PBP recognizes a different ligand. The ability of Hk1 to recognize multiple stimuli provides spirochetes with a means of distinguishing between the acquisition and transmission blood meals and generate a graded output response that is reflective of the perceived environmental threats.





Categories: Journal Articles

PAPP-A affects tendon structure and mechanical properties

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Tai-Hua Yang, Andrew R. Thoreson, Kai-Nan An, Chunfeng Zhao, Cheryl A. Conover, Peter C. Amadio

Pregnancy-associated plasma protein-A (PAPP-A) serves to increase local insulin-like growth factor (IGF) stimulation of proliferation and differentiation in many tissues through proteolysis of inhibitory IGF-binding proteins. The purpose of this study was to investigate the effects of PAPP-A on tendon structure and mechanical properties. A total of 30 tails from 6-month-old mice were tested with 10 tails in each of following groups: PAPP-A knockout (KO), skeletal-specific PAPP-A overexpressing transgenic (Tg) and wild type (WT). Morphologically, the total tail cross-sectional area (CSA), individual tissue CSAs of bone, muscle and tendon, and fascicle diameter were measured. A fascicle pullout test was performed to assess stiffness and strength of interfascicular structures. Fascicles were mechanically characterized through low and high displacement rate uniaxial tension tests providing modulus at each rate, hysteresis area and stress relaxation ratio. The KO mice had a smaller total tail CSA (p <0.05), fascicle diameter (p <0.05), absolute tendon CSA (p <0.05), fast and slow stiffness (p <0.05 for both) and larger hysteresis area (p <0.05) compared to WT and Tg mice. On the other hand, the Tg mice had a larger fascicle diameter (p <0.05), absolute tendon CSA (p <0.05), higher interfascicular strength and stiffness (p <0.05) and lower fascicular modulus at low displacement rates (p <0.05) compared to WT and KO mice. Tg mice also had larger total tail CSA area (p <0.05) and smaller hysteresis area (p <0.05) than KO mice, and larger normalized tendon CSA (p <0.05) than WT mice. Based on these data, we conclude that PAPP-A affects fascicle structure, thereby affecting tendon phenotype.





Categories: Journal Articles

Absolute polarity determination of teeth cementum by phase sensitive second harmonic generation microscopy

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Hanane Aboulfadl, Jürg Hulliger

The absolute sign of local polarity in relation to the biological growth direction has been investigated for teeth cementum using phase sensitive second harmonic generation microscopy (PS-SHGM) and a crystal of 2-cyclooctylamino-5-nitropyridine (COANP) as a nonlinear optic (NLO) reference material. A second harmonic generation (SHG) response was found in two directions of cementum: radial (acellular extrinsic fibers that are oriented more or less perpendicular to the root surface) and circumferential (cellular intrinsic fibers that are oriented more or less parallel to the surface). A mono-polar state was demonstrated for acellular extrinsic cementum. However, along the different parts of cementum in circumferential direction, two corresponding domains were observed featuring an opposite sign of polarity indicative for a bi-polar microscopic state of cellular intrinsic cementum. The phase information showed that the orientation of radial collagen fibrils of cementum is regularly organized with the donor (D) groups pointing to the surface. Circumferential collagen molecules feature orientational disorder and are oriented up and down in random manner showing acceptor or donor groups at the surface of cementum. Considering that the cementum continues to grow in thickness throughout life, we can conclude that the cementum is growing circumferentially in two opposite directions and radially in one direction. A Markov chain type model for polarity formation in the direction of growth predicts D-groups preferably appearing at the fiber front.





Categories: Journal Articles

Structural and computational dissection of the catalytic mechanism of the inorganic pyrophosphatase from Mycobacterium tuberculosis

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Andrew C. Pratt, Sajeewa W. Dewage, Allan H. Pang, Tapan Biswas, Sandra Barnard-Britson, G. Andrés Cisneros, Oleg V. Tsodikov

Family I inorganic pyrophosphatases (PPiases) are ubiquitous enzymes that are critical for phosphate metabolism in all domains of life. The detailed catalytic mechanism of these enzymes, including the identity of the general base, is not fully understood. We determined a series of crystal structures of the PPiase from Mycobacterium tuberculosis (Mtb PPiase) bound to catalytic metals, inorganic pyrophosphate (PPi; the reaction substrate) and to one or two inorganic phosphate ions (Pi; the reaction product), ranging in resolution from 1.85 to 3.30Å. These structures represent a set of major kinetic intermediates in the catalytic turnover pathway for this enzyme and suggest an order of association and dissociation of the divalent metals, the substrate and the two products during the catalytic turnover. The active site of Mtb PPiase exhibits significant structural differences from the well characterized Escherichia coli PPiase in the vicinity of the bound PPi substrate. Prompted by these differences, quantum mechanics/molecular mechanics (QM/MM) analysis yielded an atomic description of the hydrolysis step for Mtb PPiase and, unexpectedly, indicated that Asp89, rather than Asp54 that was proposed for E. coli PPiase, can abstract a proton from a water molecule to activate it for a nucleophilic attack on the PPi substrate. Mutagenesis studies of the key Asp residues of Mtb PPiase supported this mechanism. This combination of structural and computational analyses clarifies our understanding of the mechanism of family I PPiases and has potential utility for rational development of drugs targeting this enzyme.





Categories: Journal Articles

Polyhedra structures and the evolution of the insect viruses

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Xiaoyun Ji, Danny Axford, Robin Owen, Gwyndaf Evans, Helen M. Ginn, Geoff Sutton, David I. Stuart

Polyhedra represent an ancient system used by a number of insect viruses to protect virions during long periods of environmental exposure. We present high resolution crystal structures of polyhedra for seven previously uncharacterised types of cypoviruses, four using ab initio selenomethionine phasing (two of these required over 100 selenomethionine crystals each). Approximately 80% of residues are structurally equivalent between all polyhedrins (pairwise rmsd ⩽1.5Å), whilst pairwise sequence identities, based on structural alignment, are as little as 12%. These structures illustrate the effect of 400million years of evolution on a system where the crystal lattice is the functionally conserved feature in the face of massive sequence variability. The conservation of crystal contacts is maintained across most of the molecular surface, except for a dispensable virus recognition domain. By spreading the contacts over so much of the protein surface the lattice remains robust in the face of many individual changes. Overall these unusual structural constraints seem to have skewed the molecule’s evolution so that surface residues are almost as conserved as the internal residues.





Categories: Journal Articles

Structural and biochemical characterization of GTP cyclohydrolase II from Helicobacter pylori reveals its redox dependent catalytic activity

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Savita Yadav, Subramanian Karthikeyan

GTP cyclohydrolase II (GCHII), catalyzes the conversion of GTP to 2,5-diamino-6-β-ribosyl-4(3H)-pyrimidinone-5′-phosphate and has been shown to be essential for pathogens. Here we describe the biochemical, kinetic and structural characterization of GCHII from Helicobacter pylori (hGCHII). The crystal structure of hGCHII, unlike other GCHII structures, revealed that cysteines at the active site existed in oxidized state forming two disulfide bonds and lacked Zn2+ that was shown to be indispensable for catalytic activity in other species. However, incubation of hGCHII with hydrogen peroxide, an oxidizing agent, followed by PAR-assay showed that Zn2+ was intrinsically present, indicating that all cysteines at the catalytic site remained in reduced state. Moreover, site directed mutagenesis of catalytic site cysteines revealed that only three, out of four cysteines were essential for hGCHII activity. These results, though, indicated that hGCHII crystallized in oxidized form, the expulsion of Zn2+ upon oxidation of catalytic cysteines revealed its ability to act in response to the redox environment. Exploring further, incubation of hGCHII with reversible thiol modifying agent S-methyl-methane-thiosulfonate resulted in loss of GCHII activity due to oxidation of its cysteine residues as revealed by mass spectrometry studies. However, addition of reducing agent DTT partially restored the hGCHII catalytic activity. Taken together, these results demonstrate that hGCHII can regulate its catalytic activity depending on the redox environment, a function hitherto unknown for GCHII.





Categories: Journal Articles

Multi-scale simulation of plant stem reinforcement by brachysclereids: A case study in apple fruit peduncles

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Melanie Horbens, Dominik Branke, Roland Gärtner, Axel Voigt, Florian Stenger, Christoph Neinhuis

Sclereid formation in addition to or in gaps of fragmented fibre rings is common in dicotyledonous plant stems. Whether this sclereid formation is force-triggered remains open so far. In fruit peduncles of several Malus species as modified plant stems, for example, the persistent fibre ring is displaced to the centre by formation of cortex parenchyma during growth. Parenchyma cells subsequently differentiate into an additional layer of brachysclereids, previously interpreted as an adaptation to continuously rising fruit loads. The present study pursues a multi-scale numerical modelling approach, to verify the important effect for different cellular architectures in both sclerenchyma categories on the stiffness of these tissues and the entire peduncle. First, different material properties are simulated analogue to plant tissues on the basis of three cell types. A regular three-dimensional and a random Voronoi microstructure combined with various mechanical cell wall parameters are applied. Using homogenisation simulations based on HILL’s principle, numerical calculations predict a lower effective homogenised tissue stiffness of isodiametric brachysclereids compared to those of fibres, confirming experimentally obtained data from Malus fruit peduncles. Furthermore, a curved peduncle model with a complex arrangement of different material layers is generated. Diverse material sets are tested under three representative loadings, using an adaptive diffuse domain approach (AMDiS). The model explains the function of sclereids as considerable contributors to the stiffness against bending and tensile deformations, as well as torsion, especially in consequence of superimposed load conditions in the case of a curved plant stem.





Categories: Journal Articles

Local amino acid sequence patterns dominate the heterogeneous phenotype for the collagen connective tissue disease Osteogenesis Imperfecta resulting from Gly mutations

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Jianxi Xiao, Zhangfu Yang, Xiuxia Sun, Rayna Addabbo, Jean Baum

Osteogenesis Imperfecta (OI), a hereditary connective tissue disease in collagen that arises from a single Gly→X mutation in the collagen chain, varies widely in phenotype from perinatal lethal to mild. It is unclear why there is such a large variation in the severity of the disease considering the repeating (Gly-X-Y)n sequence and the uniform rod-like structure of collagen. We systematically evaluate the effect of local (Gly-X-Y)n sequence around the mutation site on OI phenotype using integrated bio-statistical approaches, including odds ratio analysis and decision tree modeling. We show that different Gly→X mutations have different local sequence patterns that are correlated with lethal and nonlethal phenotypes providing a mechanism for understanding the sensitivity of local context in defining lethal and non-lethal OI. A number of important trends about which factors are related to OI phenotypes are revealed by the bio-statistical analyses; most striking is the complementary relationship between the placement of Pro residues and small residues and their correlation to OI phenotype. When Pro is present or small flexible residues are absent nearby a mutation site, the OI case tends to be lethal; when Pro is present or small flexible residues are absent further away from the mutation site, the OI case tends to be nonlethal. The analysis also reveals the dominant role of local sequence around mutation sites in the Major Ligand Binding Regions that are primarily responsible for collagen binding to its receptors and shows that non-lethal mutations are highly predicted by local sequence considerations alone whereas lethal mutations are not as easily predicted and may be a result of more complex interactions. Understanding the sequence determinants of OI mutations will enhance genetic counseling and help establish which steps in the collagen hierarchy to target for drug therapy.





Categories: Journal Articles

Structural insights on the catalytic site protection of human carbonyl reductase 1 by glutathione

Sun, 10/11/2015 - 00:04
Publication date: October 2015
Source:Journal of Structural Biology, Volume 192, Issue 1

Author(s): Qingnan Liang, Rui Liu, Shuqi Du, Yu Ding

The NADPH-dependent human carbonyl reductase 1 (hCBR1), a member of the short-chain dehydrogenase/reductase protein family, plays an important role in the ubiquitous metabolism of endogenous and xenobiotic carbonyl containing compounds. Glutathione (GSH) is also a cofactor of hCBR1, however, its role in the carbonyl reductase function of the enzyme is still unclear. In this study, we presented the crystal structure of hCBR1 in complex with GSH, in the absence of its substrates or inhibitors. Interestingly, we found that the GSH molecule presents in a configuration quite different from that was previously reported when substrate is binding to hCBR1. Our structure indicates that GSH contributes to the substrate selectivity of hCBR1 and protects the catalytic center of hCBR1 through a switch-like mechanism. The isothermal titration calorimetry and enzymology data shows that GSH directly binding with hCBR1 when there’s no substrate exist. The enzymology data also shows GSH protects NADPH being attacked by oxidative small molecules. This is the first time that GSH is found to demonstrate such functions as a co-enzyme. Our crystal structure succeeds in providing critical insights into the substrate selectivity of hCBR1 and the interaction between hCBR1 and GSH.





Categories: Journal Articles

A novel fully automatic scheme for fiducial marker-based alignment in electron tomography

Sun, 10/11/2015 - 00:04
Publication date: Available online 1 October 2015
Source:Journal of Structural Biology

Author(s): Renmin Han, Liansan Wang, Zhiyong Liu, Fei Sun, Fa Zhang

Although the topic of fiducial marker-based alignment in electron tomography (ET) has been widely discussed for decades, alignment without human intervention remains a difficult problem. Specifically, the emergence of subtomogram averaging has increased the demand for batch processing during tomographic reconstruction; fully automatic fiducial marker-based alignment is the main technique in this process. However, the lack of an accurate method for detecting and tracking fiducial markers precludes fully automatic alignment. In this paper, we present a novel, fully automatic alignment scheme for ET. Our scheme has two main contributions: First, we present a series of algorithms to ensure a high recognition rate and precise localization during the detection of fiducial markers. Our proposed solution reduces fiducial marker detection to a sampling and classification problem and further introduces an algorithm to solve the parameter dependence of marker diameter and marker number. Second, we propose a novel algorithm to solve the tracking of fiducial markers by reducing the tracking problem to an incomplete point set registration problem. Because a global optimization of a point set registration occurs, the result of our tracking is independent of the initial image position in the tilt series, allowing for the robust tracking of fiducial markers without pre-alignment. The experimental results indicate that our method can achieve an accurate tracking, almost identical to the current best one in IMOD with half automatic scheme. Furthermore, our scheme is fully automatic, depends on fewer parameters (only requires a gross value of the marker diameter) and does not require any manual interaction, providing the possibility of automatic batch processing of electron tomographic reconstruction.





Categories: Journal Articles

Protein domain mapping by internal labeling and single particle electron microscopy

Sun, 10/11/2015 - 00:04
Publication date: Available online 30 September 2015
Source:Journal of Structural Biology

Author(s): Claudio Ciferri, Gabriel C. Lander, Eva Nogales

In recent years, electron microscopy (EM) and single particle analysis have emerged as essential tools for investigating the architecture of large biological complexes. When high resolution is achievable, crystal structure docking and de-novo modeling allows for precise assignment of individual protein domain sequences. However, the achievable resolution may limit the ability to do so, especially when small or flexible complexes are under study. In such cases, protein labeling has emerged as an important complementary tool to characterize domain architecture and elucidate functional mechanistic details. All labeling strategies proposed to date are either focused on the identification of the position of protein termini or require multi-step labeling strategies, potentially interfering with the final labeling efficiency. Here we describe a strategy for determining the position of internal protein domains within EM maps using a recombinant one-step labeling approach named Efficient Mapping by Internal Labeling (EMIL). EMIL takes advantage of the close spatial proximity of the GFP’s N- and C-termini to generate protein chimeras containing an internal GFP at desired locations along the main protein chain. We apply this method to characterize the subunit domain localization of the human Polycomb Repressive Complex 2.





Categories: Journal Articles

Numerical Geometry of Map and Model Assessment

Sun, 10/11/2015 - 00:04
Publication date: Available online 28 September 2015
Source:Journal of Structural Biology

Author(s): Willy Wriggers, Jing He

We are describing best practices and assessment strategies for the atomic interpretation of cryo-Electron Microscopy (cryo-EM) maps. Multiscale numerical geometry strategies in the Situs package and in secondary structure detection software are currently evolving due to the recent increases in cryo-EM resolution. Criteria that aim to predict the accuracy of fitted atomic models at low (worse than 8 Å) and medium (4-8 Å) resolution remain challenging. However, a high level of confidence in atomic models can be achieved by combining such criteria. The observed errors are due to map-model discrepancies and due to the effect of imperfect global docking strategies. Extending the earlier motion capture approach developed for flexible fitting, we use simulated fiducials (pseudoatoms) at varying levels of coarse graining to track the local drift of structural features. We compare three tracking approaches: naïve vector quantization, a smoothly deformable model, and a tessellation of the structure into rigid Voronoi cells which are fitted using a multi-fragment refinement approach. The lowest error is an upper bound for the (small) discrepancy between crystal structure and EM map due to different conditions in their structure determination. When internal features such as secondary structures are visible in medium-resolution EM maps, it is possible to extend the idea of point-based fiducials to more complex geometric representations such as helical axes, strands, and skeletons. We propose a quantitative strategies to assess map-model pairs when such secondary structure patterns are prominent.





Categories: Journal Articles

Structure of Liver Receptor Homolog-1 (NR5A2) with PIP3 hormone bound in the ligand binding pocket

Sun, 10/11/2015 - 00:04
Publication date: Available online 28 September 2015
Source:Journal of Structural Biology

Author(s): Elena P. Sablin, Raymond D. Blind, Rubatharshini Uthayaruban, H.J. Chiu, Ashley M. Deacon, Debanu Das, Holly A. Ingraham, Robert J. Fletterick

The nuclear receptor LRH-1 (Liver Receptor Homolog-1, NR5A2) is a transcription factor that regulates gene expression programs critical for many aspects of metabolism and reproduction. Although LRH-1 is able to bind phospholipids, it is still considered an orphan nuclear receptor (NR) with an unknown regulatory hormone. Our prior cellular and structural studies demonstrated that the signaling phosphatidylinositols PI(4,5)P2 (PIP2) and PI(3,4,5)P3 (PIP3) bind and regulate SF-1 (Steroidogenic Factor-1, NR5A1), a close homolog of LRH-1. Here, we describe the crystal structure of human LRH-1 ligand binding domain (LBD) bound by PIP3 - the first phospholipid with a head group endogenous to mammals. We show that the phospholipid hormone binds LRH-1 with high affinity, stabilizing the receptor LBD. While the hydrophobic PIP3 tails (C16/C16) are buried inside the LRH-1 ligand binding pocket, the negatively charged PIP3 head group is presented on the receptor surface, similar to the phosphatidylinositol binding mode observed in the PIP3-SF-1 structure. Thus, data presented in this work reinforce our earlier findings demonstrating that signaling phosphatidylinositols regulate the NR5A receptors LRH-1 and SF-1.





Categories: Journal Articles

Crystal structure of halogenase PltA from the pyoluteorin biosynthetic pathway

Sun, 10/11/2015 - 00:04
Publication date: Available online 28 September 2015
Source:Journal of Structural Biology

Author(s): Allan H. Pang, Sylvie Garneau-Tsodikova, Oleg V. Tsodikov

Pyoluteorin is an antifungal agent composed of a 4,5-dichlorinated pyrrole group linked to a resorcinol moiety. The pyoluteorin biosynthetic gene cluster in Pseudomonas fluorescens Pf-5 encodes the halogenase PltA, which has been previously demonstrated to perform both chlorinations in vitro. PltA selectively accepts as a substrate a pyrrole moiety covalently tethered to a nonribosomal peptide thiolation domain PltL (pyrrolyl-S-PltL) for FAD-dependent di-chlorination, yielding 4,5-dichloropyrrolyl-S-PltL. We report a 2.75Å-resolution crystal structure of PltA in complex with FAD and chloride. PltA is a dimeric enzyme, containing a flavin-binding fold conserved in flavin-dependent halogenases and monooxygenases, and an additional unique helical region at the C-terminus. This C-terminal region blocks a putative substrate-binding cleft, suggesting that a conformational change involving repositioning of this region is necessary to allow binding of the pyrrolyl-S-PltL substrate for its dichlorination by PltA.





Categories: Journal Articles