Journal of Structural Biology

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  • An N-terminal extension to the hepatitis B virus core protein forms a poorly ordered trimeric spike in assembled virus-like particles
    [Jan 2015]

    Publication date: Available online 31 December 2014
    Source:Journal of Structural Biology

    Author(s): Richard McGonigle , Wei Boon Yap , Swee Tin Ong , Derek Gatherer , Saskia E. Bakker , Wen Siang Tan , David Bhella

    Virus-like particles composed of the core antigen of hepatitis B virus (HBcAg) have been shown to be an effective platform for the display of foreign epitopes in vaccine development. Heterologous sequences have been successfully inserted at both amino and carboxy termini as well as internally at the major immunodominant epitope. We used cryogenic electron microscopy (CryoEM) and three-dimensional image reconstruction to investigate the structure of VLPs assembled from an N-terminal extended HBcAg that contained a polyhistidine tag. The insert was seen to form a trimeric spike on the capsid surface that was poorly resolved, most likely owing to it being flexible. We hypothesise that the capacity of N-terminal inserts to form trimers may have application in the development of multivalent vaccines to trimeric antigens. Our analysis also highlights the value of tools for local resolution assessment in studies of partially disordered macromolecular assemblies by cryoEM.





    Categories: Journal Articles
  • Use of a “silver bullet” to resolve crystal lattice dislocation disorder: A cobalamin complex of Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis
    [Jan 2015]

    Publication date: Available online 31 December 2014
    Source:Journal of Structural Biology

    Author(s): Thomas Lagautriere , Ghader Bashiri , Edward N. Baker

    The use of small molecules as “silver bullets” that can bind to generate crosslinks between protein molecules has been advanced as a powerful means of enhancing success in protein crystallization (McPherson and Cudney, 2006). We have explored this approach in attempts to overcome an order–disorder phenomenon that complicated the structural analysis of the enzyme Δ1-pyrroline-5-carboxylate dehydrogenase from Mycobacterium tuberculosis (P5CDH, Mtb-PruA). Using the Silver Bullets Bio screen, we obtained new crystal packing using cobalamin as a co-crystallization agent. This crystal form did not display the order–disorder phenomenon previously encountered. Solution of the crystal structure showed that cobalamin molecules are present in the crystal contacts. Although the cobalamin binding probably does not have physiological relevance, it reflects similarities in the nucleotide-binding region of Mtb-PruA, with the nucleotide loop of cobalamin sharing the binding site for the adenine moiety of NAD+.





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  • Seeing tobacco mosaic virus through direct electron detectors
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Structural Biology

    Author(s): Simon A. Fromm , Tanmay A.M. Bharat , Arjen J. Jakobi , Wim J.H. Hagen , Carsten Sachse

    With the introduction of direct electron detectors (DED) to the field of electron cryo-microscopy, a wave of atomic-resolution structures has become available. As the new detectors still require comparative characterization, we have used tobacco mosaic virus (TMV) as a test specimen to study the quality of 3D image reconstructions from data recorded on the two direct electron detector cameras, K2 Summit and Falcon II. Using DED movie frames, we explored related image-processing aspects and compared the performance of micrograph-based and segment-based motion correction approaches. In addition, we investigated the effect of dose deposition on the atomic-resolution structure of TMV and show that radiation damage affects negative carboxyl chains first in a side-chain specific manner. Finally, using 450,000 asymmetric units and limiting the effects of radiation damage, we determined a high-resolution cryo-EM map at 3.35Å resolution. Here, we provide a comparative case study of highly ordered TMV recorded on different direct electron detectors to establish recording and processing conditions that enable structure determination up to 3.2Å in resolution using cryo-EM.





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  • Tomo3D 2.0 – Exploitation of Advanced Vector eXtensions (AVX) for 3D reconstruction
    [Jan 2015]

    Publication date: Available online 17 December 2014
    Source:Journal of Structural Biology

    Author(s): Jose-Ignacio Agulleiro , Jose-Jesus Fernandez

    Tomo3D is a program for fast tomographic reconstruction on multicore computers. Its high speed stems from code optimization, vectorization with Streaming SIMD Extensions (SSE), multithreading and optimization of disk access. Recently, Advanced Vector eXtensions (AVX) have been introduced in the x86 processor architecture. Compared to SSE, AVX double the number of simultaneous operations, thus pointing to a potential twofold gain in speed. However, in practice, achieving this potential is extremely difficult. Here, we provide a technical description and an assessment of the optimizations included in Tomo3D to take advantage of AVX instructions. Tomo3D 2.0 allows huge reconstructions to be calculated in standard computers in a matter of minutes. Thus, it will be a valuable tool for electron tomography studies with increasing resolution needs.





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  • On the use of Legionella/Rickettsia chimeras to investigate the structure and regulation of Rickettsia effector RalF
    [Jan 2015]

    Publication date: Available online 9 December 2014
    Source:Journal of Structural Biology

    Author(s): Marcia Folly-Klan , Bastien Sancerne , Eric Alix , Craig R. Roy , Jacqueline Cherfils , Valérie Campanacci

    A convenient strategy to interrogate the biology of regulatory proteins is to replace individual domains by an equivalent domain from a related protein of the same species or from an ortholog of another species. It is generally assumed that the overall properties of the native protein are retained in the chimera, and that functional differences reflect only the specific determinants contained in the swapped domains. Here we used this strategy to circumvent the difficulty in obtaining crystals of Rickettsia prowazekii RalF, a bacterial protein that functions as a guanine nucleotide exchange factor for eukaryotic Arf GTPases. A RalF homolog is encoded by Legionella pneumophila, in which a C-terminal capping domain auto-inhibits the catalytic Sec7 domain and localizes the protein to the Legionella-containing vacuole. The crystal structures of domain-swapped chimeras were determined and used to construct a model of Legionella RalF with a RMSD of less than 1Å with the crystal structure, which validated the use of this approach to build a model of Rickettsia RalF. In the Rickettsia RalF model, sequence differences in the capping domain that target it to specific membranes are accommodated by a shift of the entire domain with respect to the Sec7 domain. However, local sequence changes also give rise to an artifactual salt bridge in one of the chimeras, which likely explains why this chimera is recalcitrant to activation. These findings highlight the structural plasticity whereby chimeras can be engineered, but also underline that unpredictable differences can modify their biochemical responses.





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  • Semi-automated selection of cryo-EM particles in RELION-1.3
    [Jan 2015]

    Publication date: Available online 6 December 2014
    Source:Journal of Structural Biology

    Author(s): Sjors H.W. Scheres

    The selection of particles suitable for high-resolution cryo-EM structure determination from noisy micrographs may represent a tedious and time-consuming step. Here, a semi-automated particle selection procedure is presented that has been implemented within the open-source software RELION. At the heart of the procedure lies a fully CTF-corrected template-based picking algorithm, which is supplemented by a fast sorting algorithm and reference-free 2D class averaging to remove false positives. With only limited user-interaction, the proposed procedure yields results that are comparable to manual particle selection. Together with an improved graphical user interface, these developments further contribute to turning RELION from a stand-alone refinement program into a convenient image processing pipeline for the entire single-particle approach.





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  • Third Harmonic Generation microscopy as a reliable diagnostic tool for evaluating lipid body modification during cell activation: The example of BV-2 microglia cells
    [Jan 2015]

    Publication date: Available online 5 December 2014
    Source:Journal of Structural Biology

    Author(s): E. Gavgiotaki , G. Filippidis , M. Kalognomou , A.A. Tsouko , I. Skordos , C. Fotakis , I. Athanassakis

    Nonlinear optical processes have found widespread applications in fields ranging from fundamental physics to biomedicine. In this study, we attempted to evaluate cell activation by using the Third Harmonic Generation (THG) imaging microscopy as a new diagnostic tool. The BV-2 microglia cell line with or without activation by lipopolysaccharide was chosen as a representative biological model. The results showed that THG imaging could discriminate between the control versus activated state of BV-2 cells not only as to THG signal intensity but also as to THG signal area, while verifying once more that the majority of the intracellular detected signal corresponds to lipid bodies. Since THG imaging is a real time, non-destructive modality and does not require any prior cell processing and staining, the results presented here provide an important tool for normal versus activated cell discrimination, which could be proved very useful in the study of inflammation.





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  • Sparse and incomplete factorial matrices to screen membrane protein 2D crystallization
    [Jan 2015]

    Publication date: Available online 3 December 2014
    Source:Journal of Structural Biology

    Author(s): R. Lasala , N. Coudray , A. Abdine , Z. Zhang , M. Lopez-Redondo , R. Kirshenbaum , J. Alexopoulos , Z. Zolnai , D.L. Stokes , I. Ubarretxena-Belandia

    Electron crystallography is well suited for studying the structure of membrane proteins in their native lipid bilayer environment. This technique relies on electron cryomicroscopy of two-dimensional (2D) crystals, grown generally by reconstitution of purified membrane proteins into proteoliposomes under conditions favoring the formation of well-ordered lattices. Growing these crystals presents one of the major hurdles in the application of this technique. To identify conditions favoring crystallization a wide range of factors that can lead to a vast matrix of possible reagent combinations must be screened. However, in 2D crystallization these factors have traditionally been surveyed in a relatively limited fashion. To address this problem we carried out a detailed analysis of published 2D crystallization conditions for 12 β-barrel and 138 α-helical membrane proteins. From this analysis we identified the most successful conditions and applied them in the design of new sparse and incomplete factorial matrices to screen membrane protein 2D crystallization. Using these matrices we have run 19 crystallization screens for 16 different membrane proteins totaling over 1300 individual crystallization conditions. Six membrane proteins have yielded diffracting 2D crystals suitable for structure determination, indicating that these new matrices show promise to accelerate the success rate of membrane protein 2D crystallization.





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  • Cover 2 - Editorial Board
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3









    Categories: Journal Articles
  • Table of Contents / barcode
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3









    Categories: Journal Articles
  • Distinct structural features of Rex-family repressors to sense redox levels in anaerobes and aerobes
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Yingying Zheng , Tzu-Ping Ko , Hong Sun , Chun-Hsiang Huang , Jianjun Pei , Riyong Qiu , Andrew H.-J. Wang , Juergen Wiegel , Weilan Shao , Rey-Ting Guo

    The Rex-family repressors sense redox levels by alternative binding to NADH or NAD+. Unlike other Rex proteins that regulate aerobic respiration, RSP controls ethanol fermentation in the obligate anaerobe Thermoanaerobacter ethanolicus JW200T. It is also found in other anaerobic microorganisms. Here we present the crystal structures of apo-RSP, RSP/NADH and RSP/NAD+/DNA, which are the first structures of Rex-family members from an obligate anaerobe. RSP functions as a homodimer. It assumes an open conformation when bound to the operator DNA and a closed conformation when not DNA-bound. The DNA binds to the N-terminal winged-helix domain and the dinucleotide, either reduced or oxidized, binds to the C-terminal Rossmann-fold domain. The two distinct orientations of nicotinamide ring, anti in NADH and syn in NAD+, give rise to two sets of protein–ligand interactions. Consequently, NADH binding makes RSP into a closed conformation, which does not bind to DNA. Both the conserved residues and the DNA specificity of RSP show a number of variations from those of the aerobic Rex, reflecting different structural bases for redox-sensing by the anaerobic and aerobic Rex-family members.





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  • Biogenic nanospheres of amorphous carbonated Ca–Mg phosphate within the periostracum of the green mussel Perna viridis
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Jun Xu , Gangsheng Zhang

    Recently there is increasing evidence that the shell biomineralization proceeds via an amorphous precursor route. Therefore, the search for and investigation of amorphous biominerals in bivalve shells are of great importance and interest. Here, using a scanning electron microscope (SEM), transmission electron microscope (TEM), X-ray diffractometer (XRD), and Fourier transform infrared spectrometer (FTIR), we investigate the microstructure and mineralogy of the periostracum in Perna viridis. We find that: (1) the periostracum has three layers, of which the inner and outer layer are of proteins, while the middle layer is mineralized with nanospheres of amorphous biominerals; (2) the nanospheres are of amorphous carbonated Ca–Mg phosphate (ACCP), where the CO3 2 −/PO4 3 − weight ratio is estimated to be ∼0.3, and the Ca/P and Ca/Mg atomic ratio is ∼1.4 and 1.6, respectively; (3) the nanospheres, with a diameter of 43–106nm, are found to assemble into spherules with a diameter of 160–500nm, which are further organized into parallel microlayers separated by the proteins; and (4) the nanospheres are assumed to function as the pH stabilizer to facilitate the shell’s initial mineralization. Finally, we expect that these findings will advance our understanding of the shell’s biomineralization process.





    Categories: Journal Articles
  • Amino acid sequence homologies in the hard keratins of birds and reptiles, and their implications for molecular structure and physical properties
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): R.D. Bruce Fraser , David A.D. Parry

    Avian and reptilian epidermal appendages such as feathers, claws and scales exhibit a filament–matrix texture. Previous studies have established that both components reside within the same single-chain molecule. In the present study the homology in a wide range of aligned sequences is used to gain insights into the structure and function of the molecular segments associated with the filament and with the matrix. The notion that all molecules contain a β-rich 34-residue segment associated with the framework of the filament is reinforced by the present study. In addition, the residues involved in the polymerization of the molecules to form filaments are identified. In the Archosaurs (birds, crocodiles and turtles), and the Squamates (snakes and lizards) segments rich in glycine and tyrosine can be identified in the C-terminal domain. In Rhynocephalians (tuataras) and Squamates a similar segment is inserted at a specific point in the N-terminal domain. In some Archosaurian appendages (both avian and reptilian) segments rich in charged residues and cysteine are found in the N-terminal domain. The likely effect of these segments will be to soften the tissue without compromising its insolubility. The structure and role of the various molecular segments identified in this study and the way in which they might manifest themselves in terms of the physical properties of the particular epidermal appendage in which they appear are also discussed.





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  • Thalidomide mimics uridine binding to an aromatic cage in cereblon
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Marcus D. Hartmann , Iuliia Boichenko , Murray Coles , Fabio Zanini , Andrei N. Lupas , Birte Hernandez Alvarez

    Thalidomide and its derivatives lenalidomide and pomalidomide are important anticancer agents but can cause severe birth defects via an interaction with the protein cereblon. The ligand-binding domain of cereblon is found, with a high degree of conservation, in both bacteria and eukaryotes. Using a bacterial model system, we reveal the structural determinants of cereblon substrate recognition, based on a series of high-resolution crystal structures. For the first time, we identify a cellular ligand that is universally present: we show that thalidomide and its derivatives mimic and compete for the binding of uridine, and validate these findings in vivo. The nature of the binding pocket, an aromatic cage of three tryptophan residues, further suggests a role in the recognition of cationic ligands. Our results allow for general evaluation of pharmaceuticals for potential cereblon-dependent teratogenicity.





    Categories: Journal Articles
  • Crystal structure of Legionella pneumophila dephospho-CoA kinase reveals a non-canonical conformation of P-loop
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Xiaojian Gong , Xiaofang Chen , Dongmin Yu , Nannan Zhang , Zhongliang Zhu , Liwen Niu , Yuxin Mao , Honghua Ge

    Dephospho-CoA kinase (DPCK; EC 2.7.1.24) catalyzes the final step in the coenzyme A biosynthetic pathway. DPCK transfers a phosphate group from ATP to the 3-hydroxyl group of the ribose of dephosphocoenzyme A (dCoA) to yield CoA and ADP. Upon the binding of ligands, large conformational changes is induced in DPCKs, as well as in many other kinases, to shield the bound ATP in their catalytic site from the futile hydrolysis by bulk water molecules. To investigate the molecular mechanisms underlying the phosphoryl transfer during DPCK catalytic cycle, we determined the crystal structures of the Legionella pneumophila DPCK (LpDPCK) both in its apo-form and in complex with ATP. The structures reveal that LpDPCK comprises of three domains, the classical core domain, the CoA domain, and the LID domain, which are packed together to create a central cavity for substrate-binding and enzymatic catalysis. The binding of ATP induces large conformational changes, including a hinge-bending motion of the CoA binding domain and the “helix to loop” conformational change of the P-loop. Finally, modeling of a dCoA molecule to the enzyme provides insights into the catalytic mechanism of DPCK.





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  • Dark-field transmission electron microscopy of cortical bone reveals details of extrafibrillar crystals
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Henry P. Schwarcz , Elizabeth A. McNally , Gianluigi A. Botton

    In a previous study we showed that most of the mineral in bone is present in the form of “mineral structures”, 5–6nm-thick, elongated plates which surround and are oriented parallel to collagen fibrils. Using dark-field transmission electron microscopy, we viewed mineral structures in ion-milled sections of cortical human bone cut parallel to the collagen fibrils. Within the mineral structures we observe single crystals of apatite averaging 5.8±2.7nm in width and 28±19nm in length, their long axes oriented parallel to the fibril axis. Some appear to be composite, co-aligned crystals as thin as 2nm. From their similarity to TEM images of crystals liberated from deproteinated bone we infer that we are viewing sections through platy crystals of apatite that are assembled together to form the mineral structures.





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  • Crystal structure of the VapBC-15 complex from Mycobacterium tuberculosis reveals a two-metal ion dependent PIN-domain ribonuclease and a variable mode of toxin–antitoxin assembly
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Uddipan Das , Vivian Pogenberg , Udaya Kumar Tiruttani Subhramanyam , Matthias Wilmanns , Samudrala Gourinath , Alagiri Srinivasan

    Although PIN (PilT N-terminal)-domain proteins are known to have ribonuclease activity, their specific mechanism of action remains unknown. VapCs form a family of ribonucleases that possess a PIN-domain assembly and are known as toxins. The activities of VapCs are impaired by VapB antitoxins. Here we present the crystal structure of the VapBC-15 toxin–antitoxin complex from Mycobacterium tuberculosis determined to 2.1Å resolution. The VapB-15 and VapC-15 components assemble into one heterotetramer (VapB2C2) and two heterotrimers (VapBC2) in each asymmetric unit of the crystal. The active site of VapC-15 toxin consists of a cluster of acidic amino acid residues and two divalent metal ions, forming a well organised ribonuclease active site. The distribution of the catalytic-site residues of the VapC-15 toxin is similar to that of T4 RNase H and of Methanococcus jannaschii FEN-1, providing strong evidence that these three proteins share a similar mechanism of activity. The presence of both VapB2C2 and VapBC2 emphasizes the fact that the same antitoxin can bind the toxin in 1:1 and 1:2 ratios. The crystal structure determination of the VapBC-15 complex reveals for the first time a PIN-domain ribonuclease protein that shows two metal ions at the active site and a variable mode of toxin–antitoxin assembly. The structure further shows that VapB-15 antitoxin binds to the same groove meant for the binding of putative substrate (RNA), resulting in the inhibition of VapC-15’s toxicity.





    Categories: Journal Articles
  • Separation of replication and transcription domains in nucleoli
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): E. Smirnov , J. Borkovec , L. Kováčik , S. Svidenská , A. Schröfel , M. Skalníková , Z. Švindrych , P. Křížek , M. Ovesný , G.M. Hagen , P. Juda , K. Michalová , M.V. Cardoso , D. Cmarko , I. Raška

    In mammalian cells, active ribosomal genes produce the 18S, 5.8S and 28S RNAs of ribosomal particles. Transcription levels of these genes are very high throughout interphase, and the cell needs a special strategy to avoid collision of the DNA polymerase and RNA polymerase machineries. To investigate this problem, we measured the correlation of various replication and transcription signals in the nucleoli of HeLa, HT-1080 and NIH 3T3 cells using a specially devised software for analysis of confocal images. Additionally, to follow the relationship between nucleolar replication and transcription in living cells, we produced a stable cell line expressing GFP-RPA43 (subunit of RNA polymerase I, pol I) and RFP-PCNA (the sliding clamp protein) based on human fibrosarcoma HT-1080 cells. We found that replication and transcription signals are more efficiently separated in nucleoli than in the nucleoplasm. In the course of S phase, separation of PCNA and pol I signals gradually increased. During the same period, separation of pol I and incorporated Cy5-dUTP signals decreased. Analysis of single molecule localization microscopy (SMLM) images indicated that transcriptionally active FC/DFC units (i.e. fibrillar centers with adjacent dense fibrillar components) did not incorporate DNA nucleotides. Taken together, our data show that replication of the ribosomal genes is spatially separated from their transcription, and FC/DFC units may provide a structural basis for that separation.





    Categories: Journal Articles
  • Particle migration analysis in iterative classification of cryo-EM single-particle data
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): Bo Chen , Bingxin Shen , Joachim Frank

    Recently developed classification methods have enabled resolving multiple biological structures from cryo-EM data collected on heterogeneous biological samples. However, there remains the problem of how to base the decisions in the classification on the statistics of the cryo-EM data, to reduce the subjectivity in the process. Here, we propose a quantitative analysis to determine the iteration of convergence and the number of distinguishable classes, based on the statistics of the single particles in an iterative classification scheme. We start the classification with more number of classes than anticipated based on prior knowledge, and then combine the classes that yield similar reconstructions. The classes yielding similar reconstructions can be identified from the migrating particles (jumpers) during consecutive iterations after the iteration of convergence. We therefore termed the method “jumper analysis”, and applied it to the output of RELION 3D classification of a benchmark experimental dataset. This work is a step forward toward fully automated single-particle reconstruction and classification of cryo-EM data.





    Categories: Journal Articles
  • Crystal structure of the essential Mycobacterium tuberculosis phosphopantetheinyl transferase PptT, solved as a fusion protein with maltose binding protein
    [Jan 2015]

    Publication date: December 2014
    Source:Journal of Structural Biology, Volume 188, Issue 3

    Author(s): James Jung , Ghader Bashiri , Jodie M. Johnston , Alistair S. Brown , David F. Ackerley , Edward N. Baker

    Phosphopantetheinyl transferases (PPTases) are key enzymes in the assembly-line production of complex molecules such as fatty acids, polyketides and polypeptides, where they activate acyl or peptidyl carrier proteins, transferring a 4′-phosphopantetheinyl moiety from coenzyme A (CoA) to a reactive serine residue on the carrier protein. The human pathogen Mycobacterium tuberculosis encodes two PPTases, both essential and therefore attractive drug targets. We report the structure of the type-II PPTase PptT, obtained from crystals of a fusion protein with maltose binding protein. The structure, at 1.75Å resolution (R =0.156, R free =0.191), reveals an α/β fold broadly similar to other type-II PPTases, but with differences in peripheral structural elements. A bound CoA is clearly defined with its pantetheinyl arm tucked into a hydrophobic pocket. Interactions involving the CoA diphosphate, bound Mg2+ and three active site acidic side chains suggest a plausible pathway for proton transfer during catalysis.





    Categories: Journal Articles