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  • 1. Ambort, D.
    et al.
    Johansson, M.E.V.
    Gustafsson, J. K.
    Nilsson, Harriet
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Ermund, A.
    Johansson, B.R.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Hansson, G.C.
    Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin2012In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, no 15, p. 5645-5650Article in journal (Refereed)
    Abstract [en]

    MUC2, the major colonic mucin, forms large polymers by N-terminal trimerization and C-terminal dimerization. Although the assembly process for MUC2 is established, it is not known how MUC2 is packed in the regulated secretory granulae of the goblet cell. When the N-terminal VWD1-D2-D'D3 domains (MUC2-N) were expressed in a goblet-like cell line, the protein was stored together with full-length MUC2. By mimicking the pH and calcium conditions of the secretory pathway we analyzed purified MUC2-N by gel filtration, density gradient centrifugation, and transmission electron microscopy. At pH 7.4 the MUC2-N trimer eluted as a single peak by gel filtration. At pH 6.2 with Ca2+ it formed large aggregates that did not enter the gel filtration column but were made visible after density gradient centrifugation. Electron microscopy studies revealed that the aggregates were composed of rings also observed in secretory granulae of colon tissue sections. TheMUC2-N aggregates were dissolved by removing Ca2+ and raising pH. After release from goblet cells, the unfolded full-length MUC2 formed stratified layers. These findings suggest a model for mucin packing in the granulae and the mechanism for mucin release, unfolding, and expansion.

  • 2. Andersson, Marlene
    et al.
    Jia, Qiupin
    Abella, Ana
    Lee, Xiau-Yeen
    Landreh, Michael
    Purhonen, Pasi
    KTH, School of Technology and Health (STH).
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Tenje, Maria
    Robinson, Carol V.
    Meng, Qing
    Plaza, Gustavo R.
    Johansson, Jan
    Rising, Anna
    Biomimetic spinning of artificial spider silk from a chimeric minispidroin2017In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 13, no 3, p. 262-+Article in journal (Refereed)
    Abstract [en]

    Herein we present a chimeric recombinant spider silk protein (spidroin) whose aqueous solubility equals that of native spider silk dope and a spinning device that is based solely on aqueous buffers, shear forces and lowered pH. The process recapitulates the complex molecular mechanisms that dictate native spider silk spinning and is highly efficient; spidroin from one liter of bacterial shake-flask culture is enough to spin a kilometer of the hitherto toughest as-spun artificial spider silk fiber.

  • 3.
    B. Kumar, Ramakrishnan
    et al.
    Karolinska institutet, Sverige.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska institutet, Sverige.
    Jegerschöld, Caroline
    Karolinska institutet, Sverige.
    Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy2017In: Journal of Visualized Experiments, E-ISSN 1940-087X, no 121, article id e55148Article in journal (Refereed)
    Abstract [en]

    Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

  • 4.
    Balakrishnan Kumar, Ramakrishnan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet, Sweden.
    Zhu, Lin
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Idborg, Helena
    Radmark, Olof
    Jakobsson, Per-Johan
    Rinaldo-Matthis, Agnes
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet,.
    Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs2016In: PLOS ONE, E-ISSN 1932-6203, Vol. 11, no 3, article id e0152116Article in journal (Refereed)
    Abstract [en]

    An important step in the production of inflammatory mediators of the leukotriene family is the Ca2+ mediated recruitment of 5 Lipoxygenase (5LO) to nuclear membranes. To study this reaction in vitro, the natural membrane mimicking environment of nanodiscs was used. Nanodiscs with 10.5 nm inner diameter were made with the lipid POPC and membrane scaffolding protein MSP1E3D1. Monomeric and dimeric 5LO were investigated. Monomeric 5LO mixed with Ca2+ and nanodiscs are shown to form stable complexes that 1) produce the expected leukotriene products from arachidonic acid and 2) can be, for the first time, visualised by native gel electrophoresis and negative stain transmission electron micros-copy and 3) show a highest ratio of two 5LO per nanodisc. We interpret this as one 5LO on each side of the disc. The dimer of 5LO is visualised by negative stain transmission electron microscopy and is shown to not bind to nanodiscs. This study shows the advantages of nanodiscs to obtain basic structural information as well as functional information of a complex between a monotopic membrane protein and the membrane.

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  • 5. Braniste, Viorica
    et al.
    Al-Asmakh, Maha
    Kowal, Czeslawa
    Anuar, Farhana
    Abbaspour, Afrouz
    Toth, Miklos
    Korecka, Agata
    Bakocevic, Nadja
    Guan, Ng Lai
    Kundu, Parag
    Gulyas, Balazs
    Halldin, Christer
    Hultenby, Kjell
    Nilsson, Harriet
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Volpe, Bruce T.
    Diamond, Betty
    Pettersson, Sven
    The gut microbiota influences blood-brain barrier permeability in mice2014In: Science Translational Medicine, ISSN 1946-6234, E-ISSN 1946-6242, Vol. 6, no 263, p. 263ra158-Article in journal (Refereed)
    Abstract [en]

    Pivotal to brain development and function is an intact blood-brain barrier (BBB), which acts as a gatekeeper to control the passage and exchange of molecules and nutrients between the circulatory system and the brain parenchyma. The BBB also ensures homeostasis of the central nervous system (CNS). We report that germ-free mice, beginning with intrauterine life, displayed increased BBB permeability compared to pathogen-free mice with a normal gut flora. The increased BBB permeability was maintained in germ-free mice after birth and during adulthood and was associated with reduced expression of the tight junction proteins occludin and claudin-5, which are known to regulate barrier function in endothelial tissues. Exposure of germ-free adult mice to a pathogen-free gut microbiota decreased BBB permeability and up-regulated the expression of tight junction proteins. Our results suggest that gut microbiota-BBB communication is initiated during gestation and propagated throughout life.

  • 6. Brismar, Torkel B.
    et al.
    Grishenkov, Dmitry
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Gustafsson, Björn
    Härmark, Johan
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Barrefelt, Åsa
    Kothapalli, Satya V. V. N.
    KTH, School of Technology and Health (STH), Medical Engineering, Neuronic Engineering.
    Margheritelli, Silvia
    Oddo, Letizia
    Caidahl, Kenneth
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Paradossi, Gaio
    Magnetite Nanoparticles Can Be Coupled to Microbubbles to Support Multimodal Imaging2012In: Biomacromolecules, ISSN 1525-7797, E-ISSN 1526-4602, Vol. 13, no 5, p. 1390-1399Article in journal (Refereed)
    Abstract [en]

    Microbubbles (MBs) are commonly used as injectable ultrasound contrast agent (UCA) in modern ultrasonography. Polymer-shelled UCAs present additional potentialities with respect to marketed lipid-shelled UCAs. They are more robust; that is, they have longer shelf and circulation life, and surface modifications are quite easily accomplished to obtain enhanced targeting and local drug delivery. The next generation of UCAs will be required to support not only ultrasound-based imaging methods but also other complementary diagnostic approaches such as magnetic resonance imaging or computer tomography. This work addresses the features of MBs that could function as contrast agents for both ultrasound and magnetic resonance imaging. The results indicate that the introduction of iron oxide nanoparticles (SPIONs) in the poly(vinyl alcohol) shell or on the external surface of the MBs does not greatly decrease the echogenicity of the host MBs compared with the unmodified one. The presence of SPIONs provides enough magnetic susceptibility to the MBs to accomplish good detectability both in vitro and in vivo. The distribution of SPIONs on the shell and their aggregation state seem to be key factors for the optimization of the transverse relaxation rate.

  • 7. Busenlehner, Laura S.
    et al.
    Alander, Johan
    Jegerscohld, Caroline
    Holm, Peter J.
    Bhakat, Priyaranjan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Morgenstern, Ralf
    Armstrong, Richard N.
    Location of substrate binding sites within the integral membrane protein microsomal glutathione transferase-12007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 10, p. 2812-2822Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase-1 (MGST1) is a trimeric, membrane-bound enzyme with both glutathione (GSH) transferase and hydroperoxidase activities. As a member of the MAPEG superfamily, MGST1 aids in the detoxication of numerous xenobiotic substrates and in cellular protection from oxidative stress through the GSH-dependent reduction of phospholipid hydroperoxides. However, little is known about the location of the different substrate binding sites, including whether the transferase and peroxidase activities overlap structurally. Although molecular density attributed to GSH has been observed in the 3.2 A resolution electron crystallographic structure of MGST1, the electrophilic and phospholipid hydroperoxide substrate binding sites remain elusive. Amide H-D exchange kinetics and H-D ligand footprinting experiments indicate that GSH and hydrophobic substrates bind within similar, but distinct, regions of MGST1. Site-directed mutagenesis, guided by the H-D exchange results, demonstrates that specific residues within the GSH footprint effect transferase activity toward 1-chloro-2,4-dinitrobenzene. In addition, cytosolic residues surrounding the chemical stress sensor C49 but not modeled in the crystal structure appear to play an important role in the formation of the binding site for hydrophobic substrates. Although the fatty acid/phospholipid binding site structurally overlaps that for GSH, it does not appear to be localized to the same region as other hydrophobic substrates. Finally, H-D exchange mass spectrometry reveals a specific conformational transition that may mediate substrate binding and/or product release. Such structural changes in MGST1 are essential for activation of the enzyme and are important for its biological function.

  • 8. Busenlehner, L.S.
    et al.
    Codreanu, S.G.
    Holm, P.J.
    Bhakat, P.
    Hebert, Hans
    Karolinska Institutet.
    Morgenstern, R.
    Armstrong, R.N.
    Stress sensor triggers conformational response of the integral membrane protein microsomal glutathione transferase 12004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 35, p. 11145-11152Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione (GSH) transferase 1 (MGST1) is a trimeric, integral membrane protein involved in cellular response to chemical or oxidative stress. The cytosolic domain of MGST1 harbors the GSH binding site and a cysteine residue (C49) that acts as a sensor of oxidative and chemical stress. Spatially resolved changes in the kinetics of backbone amide H/D exchange reveal that the binding of a single molecule of GSH/trimer induces a cooperative conformational transition involving movements of the transmembrane helices and a reordering of the cytosolic domain. Alkylation of the stress sensor preorganizes the helices and facilitates the cooperative transition resulting in catalytic activation.

  • 9. Chen, G.
    et al.
    Andrade-Talavera, Y.
    Tambaro, S.
    Leppert, A.
    Nilsson, Harriet E.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Zhong, Xueying
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Landreh, M.
    Nilsson, P.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Biverstål, H.
    Fisahn, A.
    Abelein, A.
    Johansson, J.
    Augmentation of Bri2 molecular chaperone activity against amyloid-β reduces neurotoxicity in mouse hippocampus in vitro2020In: Communications Biology, E-ISSN 2399-3642, Vol. 3, no 1, article id 32Article in journal (Refereed)
    Abstract [en]

    Molecular chaperones play important roles in preventing protein misfolding and its potentially harmful consequences. Deterioration of molecular chaperone systems upon ageing are thought to underlie age-related neurodegenerative diseases, and augmenting their activities could have therapeutic potential. The dementia relevant domain BRICHOS from the Bri2 protein shows qualitatively different chaperone activities depending on quaternary structure, and assembly of monomers into high-molecular weight oligomers reduces the ability to prevent neurotoxicity induced by the Alzheimer-associated amyloid-β peptide 1-42 (Aβ42). Here we design a Bri2 BRICHOS mutant (R221E) that forms stable monomers and selectively blocks a main source of toxic species during Aβ42 aggregation. Wild type Bri2 BRICHOS oligomers are partly disassembled into monomers in the presence of the R221E mutant, which leads to potentiated ability to prevent Aβ42 toxicity to neuronal network activity. These results suggest that the activity of endogenous molecular chaperones may be modulated to enhance anti-Aβ42 neurotoxic effects.

  • 10. Chen, G.
    et al.
    Andrade-Talavera, Y.
    Zhong, Xueying
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems.
    Hassan, S.
    Biverstål, H.
    Poska, H.
    Abelein, A.
    Leppert, A.
    Kronqvist, N.
    Rising, A.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems.
    Koeck, Philip J. B.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Fisahn, A.
    Johansson, J.
    Abilities of the BRICHOS domain to prevent neurotoxicity and fibril formation are dependent on a highly conserved Asp residue2022In: RSC Chemical Biology, ISSN 2633-0679, Vol. 3, no 11, p. 1342-1358Article in journal (Refereed)
    Abstract [en]

    Proteins can self-assemble into amyloid fibrils or amorphous aggregates and thereby cause disease. Molecular chaperones can prevent both these types of protein aggregation, but to what extent the respective mechanisms are overlapping is not fully understood. The BRICHOS domain constitutes a disease-associated chaperone family, with activities against amyloid neurotoxicity, fibril formation, and amorphous protein aggregation. Here, we show that the activities of BRICHOS against amyloid-induced neurotoxicity and fibril formation, respectively, are oppositely dependent on a conserved aspartate residue, while the ability to suppress amorphous protein aggregation is unchanged by Asp to Asn mutations. The Asp is evolutionarily highly conserved in >3000 analysed BRICHOS domains but is replaced by Asn in some BRICHOS families. The conserved Asp in its ionized state promotes structural flexibility and has a pKa value between pH 6.0 and 7.0, suggesting that chaperone effects can be differently affected by physiological pH variations. 

  • 11.
    Chen, Gefei
    et al.
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Leppert, Axel
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden.;Karolinska Inst, Dept Microbiol Tumour & Cell Biol, S-17165 Solna, Sweden..
    Poska, Helen
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden.;Tallinn Univ, Sch Nat Sci & Hlth, Tallinn, Estonia..
    Nilsson, Harriet
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Alvira, Carlos Piedrafita
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Zhong, Xueying
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Koeck, Philip J. B.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Jegerschöld, Caroline
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Abelein, Axel
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Johansson, Jan
    Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Short hydrophobic loop motifs in BRICHOS domains determine chaperone activity against amorphous protein aggregation but not against amyloid formation2023In: Communications Biology, E-ISSN 2399-3642, Vol. 6, no 1, article id 497Article in journal (Refereed)
    Abstract [en]

    BRICHOS domain oligomerization exposes three short hydrophobic motifs that are necessary for efficient chaperone activity against amorphous protein aggregation. ATP-independent molecular chaperones are important for maintaining cellular fitness but the molecular determinants for preventing aggregation of partly unfolded protein substrates remain unclear, particularly regarding assembly state and basis for substrate recognition. The BRICHOS domain can perform small heat shock (sHSP)-like chaperone functions to widely different degrees depending on its assembly state and sequence. Here, we observed three hydrophobic sequence motifs in chaperone-active domains, and found that they get surface-exposed when the BRICHOS domain assembles into larger oligomers. Studies of loop-swap variants and site-specific mutants further revealed that the biological hydrophobicities of the three short motifs linearly correlate with the efficiency to prevent amorphous protein aggregation. At the same time, they do not at all correlate with the ability to prevent ordered amyloid fibril formation. The linear correlations also accurately predict activities of chimeras containing short hydrophobic sequence motifs from a sHSP that is unrelated to BRICHOS. Our data indicate that short, exposed hydrophobic motifs brought together by oligomerisation are sufficient and necessary for efficient chaperone activity against amorphous protein aggregation.

  • 12. Cheng, K.
    et al.
    Koeck, P.J.B.
    Idakieva, K.
    Ternström, T.
    Parvanova, K.
    Hebert, Hans
    Stainless Models of Rapana Thomasiana Hemocyanin2004In: Proc. of the 13th European Congress on Electron Microscopy, 2004, p. 22-27Conference paper (Refereed)
  • 13.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ivanova, Natalia
    Biomedicinskt centrum, Uppsala.
    Scheres, Sjores
    CSIC, Natl Biotechnol Ctr, Biocomp Unit, E-28049 Madrid, Spain .
    Pavlov, Michael Y
    Biomedicinskt centrum, Uppsala.
    Maria Carazo, Jose
    Lund Univ, Mol Biophys KILU.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ehrenberg, Måns
    Biomedicinskt centrum, Uppsala.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    tmRNA-SmpB complex mimics native aminoacyl-tRNAs in the A site of stalled ribosomes2010In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 169, no 3, p. 342-348Article in journal (Refereed)
    Abstract [en]

    Bacterial ribosomes stalled on faulty, often truncated, mRNAs lacking stop codons are rescued by trans-translation. It relies on an RNA molecule (tmRNA) capable of replacing the faulty mRNA with its own open reading frame (ORF). Translation of tmRNA ORF results in the tagging of faulty protein for degradation and its release from the ribosome. We used single-particle cryo-electron microscopy to visualize tmRNA together with its helper protein SmpB on the 70S Escherichia coli ribosome in states subsequent to GTP hydrolysis on elongation factor Tu (EF-Tu). Three-dimensional reconstruction and heterogeneity analysis resulted in a 15 A resolution structure of the tmRNA-SmpB complex accommodated in the A site of the ribosome, which shows that SmpB mimics the anticodon- and D-stem of native tRNAs missing in the tRNA-like domain of tmRNA. We conclude that the tmRNA-SmpB complex accommodates in the ribosomal A site very much like an aminoacyl-tRNA during protein elongation.

  • 14.
    Cheng, Kimberley
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Elmlund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Idakieva, Krassimira
    Parvanova, Katja
    Schwarz, Heinz
    Ternström, Tomas
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Comparison of the two Rapana thomasiana Hemocyanin isoforms: RtH1 and RtH22006In: Proc 16. International Microscopy Conference, 2006Conference paper (Refereed)
  • 15.
    Cheng, Kimberley
    et al.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Koeck, Philip J. B.
    Department of Biosciences at NOVUM, Karolinska Institutet and School of Technology and Health, Royal Institute of Technology, S-141 57 Huddinge, Sweden.
    Elmund, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Ternström, Tomas
    Schwarz, Heinz
    Idakieva, Krassimira
    Parvanova, Katja
    Rapana thomasiana hemocyanin (RtH): Comparison of the two isoforms, RtH1 and RtH2, at 19 Å and 16 Å resolution2006In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 37, no 6, p. 566-576Article in journal (Refereed)
    Abstract [en]

    Three-dimensional (3D) reconstructions of the two 8.4 MDa Rapana thomasiana hemocyanin isoforms, RtH1 and RtH2, have been obtained by cryoelectron microscopy of molecules embedded in vitreous ice and single particle image processing. The final 3D structures of the RtH1 and RtH2 didecamers at 19 angstrom and 16 angstrom resolution, respectively, are very similar to earlier reconstructions of gastropodan hemocyanins, revealing structural features such as the obliquely oriented subunits, the five- and two-fold symmetrical axes. Three new interactions are defined; two of them connecting the arch and the wall while the third is formed between the collar and the wall. The collar-wall connection and one of the arch-wall connections are positioned between two individual subunit dimers, while the second arch-wall connection is located between two subunits within the subunit dimer. All three interactions establish connections to the first tier of the wall. Furthermore, for each interaction we have allocated two first tier functional units most likely involved in forming the connections.

  • 16.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Baraznenok, Vera
    Division of Metabolic Diseases, Karolinska Institutet.
    Lindahl, Martin
    Department of Molecular Biophysics, Lund University.
    Samuelsen, Camilla O.
    Department of Genetics, Institute of Molecular Biology, Copenhagen.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH).
    Holmberg, Steen
    Department of Genetics, Institute of Molecular Biology, Copenhagen.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Division of Metabolic Diseases, Karolinska Institutet.
    The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II2006In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, no 43, p. 15788-15793Article in journal (Refereed)
    Abstract [en]

    CDK8 (cyclin-dependent kinase 8), along with CycC, Med12, and Med13, form a repressive module (the Cdk8 module) that prevents RNA polymerase II (pol II) interactions with Mediator. Here, we report that the ability of the Cdk8 module to prevent pol II interactions is independent of the Cdk8-dependent kinase activity. We use electron microscopy and single-particle reconstruction to demonstrate that the Cdk8 module forms a distinct structural entity that binds to the head and middle region of Mediator, thereby sterically blocking interactions with pol II.

  • 17.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Baraznenok, Vera
    Division of Metabolic Diseases, Karolinska Institutet.
    Linder, Tomas
    Division of Metabolic Diseases, Karolinska Institutet.
    Rofougaran, Reza
    Dept. of Medical Biochemistry and Biophysics, Umeå University.
    Hofer, Anders
    Dept. of Medical Biochemistry and Biophysics, Umeå University.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Dept. of Medical Biochemistry and Cell Biology, Göteborg University.
    Visualization of a massive TBP-binding coupled histone-fold domain rearrangement within the general transcription factor IIDArticle in journal (Other academic)
  • 18. Elmlund, Hans
    et al.
    Baraznenok, Vera
    Linder, Tomas
    Szilagyi, Zsolt
    Rofougaran, Reza
    Hofer, Anders
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin Joakim
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gustafsson, Claes M.
    Cryo-EM Reveals Promoter DNA Binding and Conformational Flexibility of the General Transcription Factor TFIID2009In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 17, no 11, p. 1442-1452Article in journal (Refereed)
    Abstract [en]

    The general transcription factor IID (TFIID) is required for initiation of RNA polymerase II-dependent transcription at many eukaryotic promoters. TFIID comprises the TATA-binding protein (TBP) and several conserved TBP-associated factors (TAFs). Recognition of the core promoter by TFIID assists assembly of the preinitiation complex. Using cryo-electron microscopy in combination with methods for ab initio single-particle reconstruction and heterogeneity analysis, we have produced density maps of two conformational states of Schizosaccharomyces pombe TFIID, containing and lacking TBP. We report that TBP-binding is coupled to a massive histone-fold domain rearrangement. Moreover, docking of the TBP-TAF1(N-terminus) atomic structure to the THID map and reconstruction of a TAF-promoter DNA complex helps to account for TAF-dependent regulation of promoter-TBP and promoter-TAF interactions.

  • 19.
    Elmlund, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lundqvist, Joakirn
    Lund Univ, Dept Mol Biophys.
    Al-Karadaghi, Salam
    Lund Univ, Dept Mol Biophys.
    Hansson, Mats
    Lund Univ, Dept Biochem.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Lindahl, Martin
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    A new cryo-EM single-particle ab initio reconstruction method visualizes secondary structure elements in an ATP-fueled AAA+ motor2008In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 375, no 4, p. 934-947Article in journal (Refereed)
    Abstract [en]

    The generation of ab initio three-dimensional (3D) models is a bottleneck in the studies of large macromolecular assemblies by single-particle cryo-electron microscopy. We describe here a novel method, in which established methods for two-dimensional image processing are combined with newly developed programs for joint rotational 3D alignment of a large number of class averages (RAD) and calculation of 3D volumes from aligned projections (VolRec). We demonstrate the power of the method by reconstructing an similar to 660-kDa ATP-fueled AAA+ motor to 7.5 angstrom resolution, with secondary structure elements identified throughout the structure. We propose the method as a generally applicable automated strategy to obtain 3D reconstructions from unstained single particles imaged in vitreous ice.

  • 20. Ermund, Anna
    et al.
    Meiss, Lauren N.
    Rodriguez-Pineiro, Ana M.
    Baehr, Andrea
    Nilsson, Harriet E.
    KTH, School of Technology and Health (STH), Medical Engineering. Department of Medical Biochemistry, University of Gothenburg, SE-405 30 Gothenburg, Sweden; Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.
    Trillo-Muyo, Sergio
    Ridley, Caroline
    Thornton, David J.
    Wine, Jeffrey J.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet, and School of Technology and Health, KTH Royal Institute of Technology, Novum, SE-141 57 Huddinge, Sweden.
    Klymiuk, Nikolai
    Hansson, Gunnar C.
    The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin2017In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 492, no 3, p. 331-337Article in journal (Refereed)
    Abstract [en]

    To understand the mucociliary clearance system, mucins were visualized by light, confocal and electron microscopy, and mucus was stained by Alcian blue and tracked by video microscopy on tracheal explants of newborn piglets. We observed long linear mucus bundles that appeared at the submucosal gland openings and were transported cephalically. The mucus bundles were shown by mass spectrometry and immunostaining to have a core made of MUC5B mucin and were coated with MUC5AC mucin produced by surface goblet cells. The transport speed of the bundles was slower than the airway surface liquid flow. We suggest that the goblet cell MUC5AC mucin anchors the mucus bundles and thus controls their transport. Normal clearance of the respiratory tree of pigs and humans, both rich in submucosal glands, is performed by thick and long mucus bundles. 

  • 21.
    Gustafsson, JK
    et al.
    Department of Medical Biochemistry and 2Department of Internal Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
    Ermund, Anna
    Department of Medical Biochemistry and 2Department of Internal Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
    Ambort, Daniel
    Department of Medical Biochemistry and 2Department of Internal Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
    Johansson, MEV
    Nilsson, Harriet
    Karolinska Institutet, Sweden .
    Thorell, K
    Department of Medical Biochemistry and 2Department of Internal Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
    Hebert, Hans
    Karolinska Institutet, Sweden .
    Sjövall, H
    Department of Medical Biochemistry and 2Department of Internal Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
    Hansson, Gunnar
    Department of Medical Biochemistry and 2Department of Internal Medicine, University of Gothenburg, 405 30 Gothenburg, Sweden.
    Bicarbonate and functional CFTR channel are required for proper mucin secretion and link cystic fibrosis with its mucus phenotype2012In: Journal of Experimental Medicine, ISSN 0022-1007, E-ISSN 1540-9538, Vol. 209, no 7, p. 1263-1272Article in journal (Refereed)
    Abstract [en]

    Cystic fibrosis (CF) is caused by a nonfunctional chloride and bicarbonate ion channel (CF transmembrane regulator [CFTR]), but the link to the phenomenon of stagnant mucus is not well understood. Mice lacking functional CFTR (CftrΔ508) have no lung phenotype but show similar ileal problems to humans. We show that the ileal mucosa in CF have a mucus that adhered to the epithelium, was denser, and was less penetrable than that of wild-type mice. The properties of the ileal mucus of CF mice were normalized by secretion into a high concentration sodium bicarbonate buffer (~100 mM). In addition, bicarbonate added to already formed CF mucus almost completely restored the mucus properties. This knowledge may provide novel therapeutic options for CF.

  • 22.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    CryoEM: a crystals to single particles round-trip2019In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 58, p. 59-67Article in journal (Refereed)
    Abstract [en]

    In the era of intense and steadily increasing attention to cryo electron microscopy (cryoEM) as a powerful tool in structural biology, particularly with regard to randomly oriented biological macromolecules, studies of 2D and small 3D crystals using cryoEM provide added value for addressing-specific questions. Size and shape demands are not as restrictive as for single particle specimens. Crystallization may stabilize whole or partly flexible molecules. Resolutions beyond 2 A, for 3D crystals even sub-angstrom ngstrom structures, can be obtained allowing studies of chemical properties in detail. The electron dose can be kept low and reduce radiation damage for sensitive specimens. In contrast to X-ray crystallography, scattering of electrons will be directly related to the Coulomb potential and thus give information about charge distribution in biomolecules.

  • 23.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,.
    Two-dimensional crystallization of biological macromolecules2009In: Molecules: Nucleation, Aggregation and Crystallization: Beyond Medical and Other Implications, World Scientific Publishing Co. , 2009, p. 95-111Chapter in book (Other academic)
    Abstract [en]

    Biological macromolecules can be arranged periodically in single layers as twodimensional (2D) crystals. This enables crystallographic structure analysis using transmission electron microscopy. Periodic repeat of a large number of unit cells contributes to significant information in images or diffraction patterns from unstained specimens. Structural details at a resolution of a few Ångströms in all directions can be obtained. Such three-dimensional maps are used for building atomic models. The techniques have been used to determine structure and function relationships for membrane proteins. The dense packing obtained in 2D crystals can also be used for constructing devices at the molecular level.

  • 24.
    Hebert, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    The structure of membrane associated proteins in eicosanoid and glutathione metabolism as determined by electron crystallography2007In: Current opinion in structural biology, ISSN 0959-440X, E-ISSN 1879-033X, Vol. 17, no 4, p. 396-404Article, review/survey (Refereed)
    Abstract [en]

    Membrane associated proteins in eicosanoid and glutathione metabolism (MAPEG) are involved in biosynthesis of arachidonic-derived mediators of pain, fever, and inflammation as well as in biotransformation and detoxification of electrophilic substances. Structure determination of microsomal glutathione transferase 1 using electron crystallography has provided the first atomic model of an MAPEG member. The homotrimer consists of three repeats of a four-helix transmembrane bundle with the largest extramembranous domain connecting the first and second helix and with a short proline rich loop on the same side between helices three and four. Residues of importance for intramolecular or intermolecular contacts as well as for stabilizing the active site have been identified and the results can be applied for interpreting structure-function relationship for similar MAPEG members.

  • 25.
    Hebert, Hans
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH). Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Purhonen, Pasi
    KTH, School of Technology and Health (STH). Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.;Royal Inst Technol, Sch Technol & Hlth, S-14157 Huddinge, Sweden..
    Bhakat, Priya
    KTH, School of Technology and Health (STH). Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Gheorghe, Karina
    KTH, School of Technology and Health (STH). Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Gyobu, Nobuhiko
    Natl Inst Adv Ind Sci & Technol, Biol Informat Res Ctr, Koto Ku, Aomi 2-41-6, Tokyo 1350064, Japan..
    Mitsuoka, Kaoru
    Natl Inst Adv Ind Sci & Technol, Biol Informat Res Ctr, Koto Ku, Aomi 2-41-6, Tokyo 1350064, Japan..
    Pawelzik, Sven
    Karolinska Inst, Dept Med, Rheumatol Unit, S-17177 Stockholm, Sweden.;Karolinska Inst, Karolinska Biom Ctr, S-17177 Stockholm, Sweden..
    Jakobsson, Per-Johan
    Karolinska Inst, Dept Med, Rheumatol Unit, S-17177 Stockholm, Sweden.;Karolinska Inst, Karolinska Biom Ctr, S-17177 Stockholm, Sweden..
    Morgenstern, Ralf
    Karolinska Inst, Inst Environm Med, SE-17177 Stockholm, Sweden..
    Structure of Microsomal Prostaglandin E Synthase 1 as Determined by Electron Crystallography2009In: Acta Crystallographica Section A: Foundations and Advances, E-ISSN 2053-2733, Vol. 65, p. S77-S77Article in journal (Other academic)
  • 26. Holm, Peter J.
    et al.
    Bhakat, Priyaranjan
    Jegerschold, Caroline
    Gyobu, Nobuhiko
    Mitsuoka, Kaoru
    Fujiyoshi, Yoshinori
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Structural basis for detoxification and oxidative stress protection in membranes2006In: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 360, no 5, p. 934-945Article in journal (Refereed)
    Abstract [en]

    Synthesis of mediators of fever, pain and inflammation as well as protection against reactive molecules and oxidative stress is a hallmark of the MAPEG superfamily (membrane associated proteins in eicosanoid and glutathione metabolism). The structure of a MAPEG member, rat mictosomal glutathione transferase 1, at 3.2 angstrom resolution, solved here in complex with glutathione by electron crystallography, defines the active site location and a cytosolic domain involved in enzyme activation. The glutathione binding site is found to be different from that of the canonical soluble glutathione transferases. The architecture of the homotrimer supports a catalytic mechanism involving subunit interactions and reveals both cytosolic and membraneous substrate entry sites, providing a rationale for the membrane location of the enzyme.

  • 27.
    Härmark, Johan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Koeck, Philip J B
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy2016In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 85, p. 39-43Article in journal (Refereed)
    Abstract [en]

    Intravenously injected microbubbles (MBs) can be utilized as ultrasound contrast agent (CA) resulting in enhanced image quality. A novel CA, consisting of air filled MBs stabilized with a shell of polyvinyl alcohol (PVA) has been developed. These spherical MBs have been decorated with superparamagnetic iron oxide nanoparticles (SPIONs) in order to serve as both ultrasound and magnetic resonance imaging (MRI) CA. In this study, a mathematical model was introduced that determined the shell thickness of two types of SPIONs decorated MBs (Type A and Type B). The shell thickness of MBs is important to determine, as it affects the acoustical properties. In order to investigate the shell thickness, thin sections of plastic embedded MBs were prepared and imaged using transmission electron microscopy (TEM). However, the sections were cut at random distances from the MB center, which affected the observed shell thickness. Hence, the model determined the average shell thickness of the MBs from corrected mean values of the outer and inner radii observed in the TEM sections. The model was validated using simulated slices of MBs with known shell thickness and radius. The average shell thickness of Type A and Type B MBs were 651nm and 637nm, respectively.

  • 28.
    Härmark, Johan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Larsson, Malin K.
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Razuvajev, Anton
    Koeck, Philip JB
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Paradossi, Gaio
    Brodin, Lars-Åke
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Caidahl, Kenneth
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Bjällmark, Anna
    KTH, School of Technology and Health (STH), Medical Engineering, Medical Imaging.
    Investigation of the elimination process of a multimodal polymer-shelled contrast agent in rats using ultrasound and transmission electron microscopy2015In: Biomedical Spectroscopy and Imaging, ISSN 2212-8794, Vol. 4, no 1, p. 81-93Article in journal (Refereed)
    Abstract [en]

    BACKGROUND: A novel polymer-shelled contrast agent (CA) with multimodal imaging and target specific potential was developed recently and tested for its acoustical properties using different in-vitro setups.

    OBJECTIVE: The aim of this study was to investigate the elimination of three types of the novel polymer-shelled CA, one unmodified and two shell modified versions, in rats.

    METHODS: The blood elimination time was estimated by measuring the image intensity, from ultrasound images of the common carotid artery, over time after a bolus injection of the three types of the novel CA. The commercially available CA SonoVue was used as a reference. The subcellular localization of the three CAs was investigated using transmission electron microscopy.

    RESULTS: The ultrasound measurements indicated a blood half-life of 17–85 s for the different types of the novel CA, which was significant longer than the blood half-life time for SonoVue. Additionally, CAs were exclusively found in the circulatory system, either taken up by, or found in the vicinity of macrophages.

    CONCLUSIONS: Compared to the commercially available CA SonoVue, the blood circulation times for the three types of the novel polymer-shelled CA were prolonged. Moreover, macrophages were suggested to be responsible for the elimination of the CA.

  • 29.
    Härmark, Johan
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Månsson, Cecilia
    Rasmussen, Morten
    Höjrup, Peter
    Al-Karadaghi, Salam
    Söderberg, Christopher G
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Emanuelsson, Cecilia
    Structural information on the oligomeric human molecular chaperone DNAJB6Manuscript (preprint) (Other academic)
  • 30.
    Jang, Seongmin
    et al.
    Korea Adv Inst Sci & Technol, Dept Biol Sci, Daejeon 34141, South Korea..
    Kang, Chanshin
    Seoul Natl Univ, Dept Phys & Astron, Seoul 08826, South Korea..
    Yang, Han-Sol
    Sungkyunkwan Univ, Sch Pharm, Suwon 16419, South Korea..
    Jung, Taeyang
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, S-14152 Huddinge, Sweden..
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, S-14152 Huddinge, Sweden..
    Chung, Ka Young
    Sungkyunkwan Univ, Sch Pharm, Suwon 16419, South Korea..
    Kim, Seung Joong
    Korea Adv Inst Sci & Technol, Dept Phys, Daejeon 34141, South Korea..
    Hohng, Sungchul
    Seoul Natl Univ, Dept Phys & Astron, Seoul 08826, South Korea..
    Song, Ji-Joon
    Korea Adv Inst Sci & Technol, Dept Biol Sci, Daejeon 34141, South Korea..
    Structural basis of recognition and destabilization of the histone H2B ubiquitinated nucleosome by the DOT1L histone H3 Lys79 methyltransferase2019In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 33, no 11-12, p. 620-625Article in journal (Refereed)
    Abstract [en]

    DOT1L is a histone H3 Lys79 methyltransferase whose activity is stimulated by histone H2B Lys120 ubiquitination, suggesting cross-talk between histone H3 methylation and H2B ubiquitination. Here, we present cryo-EM structures of DOT1L complexes with unmodified or H2B ubiquitinated nucleosomes, showing that DOT1L recognizes H2B ubiquitin and the H2A/H2B acidic patch through a C-terminal hydrophobic helix and an arginine anchor in DOT1L, respectively. Furthermore, the structures combined with single-molecule FRET experiments show that H2B ubiquitination enhances a noncatalytic function of the DOT1L-destabilizing nucleosome. These results establish the molecular basis of the cross-talk between H2B ubiquitination and H3 Lys79 methylation as well as nucleosome destabilization by DOT1L.

  • 31.
    Jegerschöld, Caroline
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Paweizik, Sven-Christian
    Purhonen, Pasi
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Bhakat, Priyaranian
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gheorghe, Karina Roxana
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Gyobu, Nobuhiko
    Mitsuoka, Kaoru
    Morgenstern, Ralf
    Jakobsson, Per-Johan
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Structural basis for induced formation of the inflammatory mediator prostaglandin E-22008In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 105, no 32, p. 11110-11115Article in journal (Refereed)
    Abstract [en]

    Prostaglandins (PG) are bioactive lipids produced from arachidonic acid via the action of cyclooxygenases and terminal PG synthases. Microsomal prostaglandin E synthase 1 (MPGES1) constitutes an inducible glutathione-dependent integral membrane protein that catalyzes the oxidoreduction of cyclooxygenase derived PGH(2) into PGE(2). MPGES1 has been implicated in a number of human diseases or pathological conditions, such as rheumatoid arthritis, fever, and pain, and is therefore regarded as a primary target for development of novel antiinflammatory drugs. To provide a structural basis for insight in the catalytic mechanism, we determined the structure of MPGES1 in complex with glutathione by electron crystallography from 2D crystals induced in the presence of phospholipids. Together with results from site-directed mutagenesis and activity measurements, we can thereby demonstrate the role of specific amino acid residues. Glutathione is found to bind in a U-shaped conformation at the interface between subunits in the protein trimer. It is exposed to a site facing the lipid bilayer, which forms the specific environment for the oxidoreduction of PGH(2) to PGE(2) after displacement of the cytoplasmic half of the IN-terminal transmembrane helix. Hence, insight into the dynamic behavior of MPGES1 and homologous membrane proteins in inflammation and detoxification is provided.

  • 32.
    Jung, Taeyang
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Korea Adv Inst Sci & Technol KAIST, Dept Biol Sci, KI BioCentury, Daejeon 34141, South Korea.;Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Shin, Baehyun
    Massachusetts Gen Hosp, Ctr Genom Med, Boston, MA 02114 USA.;Harvard Med Sch, Dept Neurol, Boston, MA 02114 USA..
    Tamo, Giorgio
    Ecole Polytech Fed Lausanne EPFL, Inst Bioengn, Sch Life Sci, CH-1015 Lausanne, Switzerland..
    Kim, Hyeongju
    Korea Adv Inst Sci & Technol KAIST, Dept Biol Sci, KI BioCentury, Daejeon 34141, South Korea..
    Vijayvargia, Ravi
    Massachusetts Gen Hosp, Ctr Genom Med, Boston, MA 02114 USA.;Harvard Med Sch, Dept Neurol, Boston, MA 02114 USA.;Maharaja Sayajirao Univ Baroda, Dept Biochem, Vadodara 390002, Gujarat, India..
    Leitner, Alexander
    Swiss Fed Inst Technol, Dept Biol, Inst Mol Syst Biol, CH-8093 Zurich, Switzerland..
    Marcaida, Maria J.
    Ecole Polytech Fed Lausanne EPFL, Inst Bioengn, Sch Life Sci, CH-1015 Lausanne, Switzerland..
    Astorga-Wells, Juan
    Karolinska Inst, Dept Med Biochem & Biophys, S-17165 Solna, Sweden.;HDxperts AB, S-18348 Taby, Sweden..
    Jung, Roy
    Massachusetts Gen Hosp, Ctr Genom Med, Boston, MA 02114 USA.;Harvard Med Sch, Dept Neurol, Boston, MA 02114 USA..
    Aebersold, Ruedi
    Swiss Fed Inst Technol, Dept Biol, Inst Mol Syst Biol, CH-8093 Zurich, Switzerland.;Univ Zurich, Fac Sci, Zurich, Switzerland..
    Dal Peraro, Matteo
    Ecole Polytech Fed Lausanne EPFL, Inst Bioengn, Sch Life Sci, CH-1015 Lausanne, Switzerland..
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Seong, Ihn Sik
    Massachusetts Gen Hosp, Ctr Genom Med, Boston, MA 02114 USA.;Harvard Med Sch, Dept Neurol, Boston, MA 02114 USA..
    Song, Ji-Joon
    Korea Adv Inst Sci & Technol KAIST, Dept Biol Sci, KI BioCentury, Daejeon 34141, South Korea..
    The Polyglutamine Expansion at the N-Terminal of Huntingtin Protein Modulates the Dynamic Configuration and Phosphorylation of the C-Terminal HEAT Domain2020In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 28, no 9, p. 1035-1050.e8Article in journal (Refereed)
    Abstract [en]

    The polyQ expansion in huntingtin protein (HTT) is the prime cause of Huntington's disease (HD). The recent cryoelectron microscopy (cryo-EM) structure of HTT-HAP40 complex provided the structural information on its HEAT-repeat domains. Here, we present analyses of the impact of polyQ length on the structure and function of HTT via an integrative structural and biochemical approach. The cryo-EM analysis of normal (Q23) and disease (Q78) type HTTs shows that the structures of apo HTTs significantly differ from the structure of HTT in a HAP40 complex and that the polyQ expansion induces global structural changes in the relative movements among the HTT domains. In addition, we show that the polyQ expansion alters the phosphorylation pattern across HTT and that Ser2116 phosphorylation in turn affects the global structure and function of HTT These results provide a molecular basis for the effect of the polyQ segment on HTT structure and activity, which may be important for HTT pathology.

  • 33.
    Kim, Gijeong
    et al.
    Korea Adv Inst Sci & Technol, Dept Biol Sci, Daejeon 34141, South Korea..
    Azmi, Liyana
    Univ Glasgow, Inst Infect Immun & Inflammat, Glasgow G12 8QQ, Lanark, Scotland..
    Jang, Seongmin
    Korea Adv Inst Sci & Technol, Dept Biol Sci, Daejeon 34141, South Korea..
    Jung, Taeyang
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Korea Adv Inst Sci & Technol, Dept Biol Sci, Daejeon 34141, South Korea..
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, S-14183 Huddinge, Sweden..
    Roe, Andrew J.
    Univ Glasgow, Inst Infect Immun & Inflammat, Glasgow G12 8QQ, Lanark, Scotland..
    Byron, Olwyn
    Univ Glasgow, Sch Life Sci, Glasgow G12 8QQ, Lanark, Scotland..
    Song, Ji-Joon
    Korea Adv Inst Sci & Technol, Dept Biol Sci, Daejeon 34141, South Korea..
    Aldehyde-alcohol dehydrogenase forms a high-order spirosome architecture critical for its activity2019In: Nature Communications, E-ISSN 2041-1723, Vol. 10, no 1, article id 4527Article in journal (Refereed)
    Abstract [en]

    Aldehyde-alcohol dehydrogenase (AdhE) is a key enzyme in bacterial fermentation, converting acetyl-CoA to ethanol, via two consecutive catalytic reactions. Here, we present a 3.5 angstrom resolution cryo-EM structure of full-length AdhE revealing a high-order spirosome architecture. The structure shows that the aldehyde dehydrogenase (ALDH) and alcohol dehydrogenase (ADH) active sites reside at the outer surface and the inner surface of the spirosome respectively, thus topologically separating these two activities. Furthermore, mutations disrupting the helical structure abrogate enzymatic activity, implying that formation of the spirosome structure is critical for AdhE activity. In addition, we show that this spirosome structure undergoes conformational change in the presence of cofactors. This work presents the atomic resolution structure of AdhE and suggests that the high-order helical structure regulates its enzymatic activity.

  • 34.
    Kim, Hyeongju
    et al.
    Korea Adv Inst Sci & Technol KAIST, Dept Biol Sci, KI BioCentury, Daejeon, South Korea..
    Lenoir, Sophie
    Univ Grenoble Alpes, CHU Grenoble Alpes, INSERM, U1216,Grenoble Inst Neurosci, Grenoble, France..
    Helfricht, Angela
    ProQR Therapeut NV, Leiden, Netherlands..
    Jung, Taeyang
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Korea Adv Inst Sci & Technol KAIST, Dept Biol Sci, KI BioCentury, Daejeon, South Korea..
    Karneva, Zhana K.
    ProQR Therapeut NV, Leiden, Netherlands..
    Lee, Yejin
    Korea Adv Inst Sci & Technol KAIST, Dept Biol Sci, KI BioCentury, Daejeon, South Korea..
    Beumer, Wouter
    ProQR Therapeut NV, Leiden, Netherlands..
    Horst, Geert B. van der
    ProQR Therapeut NV, Leiden, Netherlands..
    Anthonijsz, Herma
    ProQR Therapeut NV, Leiden, Netherlands..
    Buil, Levi C. M.
    ProQR Therapeut NV, Leiden, Netherlands..
    Ham, Frits van der
    ProQR Therapeut NV, Leiden, Netherlands..
    Platenburg, Gerard J.
    ProQR Therapeut NV, Leiden, Netherlands..
    Purhonen, Pasi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. KTH Roual Inst Technol, Dept Biomed Engn & Hlth Syst, S-14152 Huddinge, Sweden..
    Humbert, Sandrine
    Univ Grenoble Alpes, CHU Grenoble Alpes, INSERM, U1216,Grenoble Inst Neurosci, Grenoble, France.;Univ Grenoble Alpes, Grenoble Inst Neurosci, INSERM, U1216,CHU Grenoble Alpes,, F-38000 Grenoble, France..
    Saudou, Frederic
    Univ Grenoble Alpes, CHU Grenoble Alpes, INSERM, U1216,Grenoble Inst Neurosci, Grenoble, France.;Univ Grenoble Alpes, Grenoble Inst Neurosci, INSERM, U1216,CHU Grenoble Alpes,, F-38000 Grenoble, France..
    Klein, Pontus
    ProQR Therapeut NV, Leiden, Netherlands.;ProQR Therapeut NV, NL-2333 CK Leiden, Netherlands..
    Song, Ji-Joon
    Korea Adv Inst Sci & Technol KAIST, Dept Biol Sci, KI BioCentury, Daejeon, South Korea.;291 Daehakro, Daejeon 34141, South Korea..
    A pathogenic proteolysis-resistant huntingtin isoform induced by an antisense oligonucleotide maintains huntingtin function2022In: JCI Insight, ISSN 2379-3708, Vol. 7, no 17, article id e154108Article in journal (Refereed)
    Abstract [en]

    Huntington's disease (HD) is a late-onset neurological disorder for which therapeutics are not available. Its key pathological mechanism involves the proteolysis of polyglutamine-expanded (polyQ-expanded) mutant huntingtin (mHTT), which generates N-terminal fragments containing polyQ, a key contributor to HD pathogenesis. Interestingly, a naturally occurring spliced form of HTT mRNA with truncated exon 12 encodes an HTT (HTT & UDelta;12) with a deletion near the caspase-6 cleavage site. In this study, we used a multidisciplinary approach to characterize the therapeutic potential of targeting HTT exon 12. We show that HTT & UDelta;12 was resistant to caspase-6 cleavage in both cell-free and tissue lysate assays. However, HTT & UDelta;12 retained overall biochemical and structural properties similar to those of wt-HTT. We generated mice in which HTT exon 12 was truncated and found that the canonical exon 12 was dispensable for the main physiological functions of HTT, including embryonic development and intracellular trafficking. Finally, we pharmacologically induced HTT & UDelta;12 using the antisense oligonucleotide (ASO) QRX-704. QRX-704 showed predictable pharmacology and efficient biodistribution. In addition, it was stable for several months and inhibited pathogenic proteolysis. Furthermore, QRX-704 treatments resulted in a reduction of HTT aggregation and an increase in dendritic spine count. Thus, ASO-induced HTT exon 12 splice switching from HTT may provide an alternative therapeutic strategy for HD.

  • 35.
    Koeck, Philip J. B.
    et al.
    Karolinska Institutet.
    Purhonen, P.
    Alvang, R.
    Grundberg, B.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    3D-correlation-averaging for membrane-protein-crystals2008In: EMC 2008 14th European Microscopy Congress, 2008, p. 55-56Conference paper (Refereed)
    Abstract [en]

    Few 2-dimensional protein crystals can be used to determine high-resolution structures, whereas most electron crystallography projects remain at a resolution around 10 Ångström. This might be partly due to lack of flatness of many two-dimensional crystals [1]. We have investigated this problem and suggest single particle projection matching (3D-correlation averaging) of locally averaged unit cells to improve the quality of three-dimensional maps. Theoretical considerations and tests on simulated data demonstrate the feasibility of this refinement method [2].

  • 36. Koeck, Philip J. B.
    et al.
    Purhonen, P
    Alvang, R
    Grundberg, B
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Single-particle refinement in electron crystallography of membrane-proteins2007Conference paper (Refereed)
  • 37.
    Koeck, Philip J. B.
    et al.
    KTH, School of Technology and Health (STH), Structural Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Purhonen, Pasi
    Alvang, Ronny
    KTH, School of Technology and Health (STH).
    Grundberg, Björn
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Single particle refinement in electron crystallography: A pilot study2007In: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 160, no 3, p. 344-352Article in journal (Refereed)
    Abstract [en]

    Electron crystallography can be used to determine the structures of membrane proteins at near-atomic resolution in some cases. However, most electron crystallography projects remain at a resolution around 10 angstrom. This might be partly due to lack of flatness of many two-dimensional crystals. We have investigated this problem and suggest single particle processing of locally averaged unit cells to improve the quality and possibly the resolution of three-dimensional maps. Applying this method to the secondary transporter melibiose permease we have calculated a three-dimensional map that is clearer and easier to interpret than the map derived using purely electron-crystallographic methods.

  • 38.
    Koulakiotis, Nikolaos Stavros
    et al.
    GAIA Research Center, Bioanalytical Department, The Goulandris Natural History Museum, 14562 Kifissia, Greece.
    Purhonen, Pasi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Gikas, Evangelos
    Department of Pharmacy, National and Kapodistrian University of Athens, 15771 Athens, Greece.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology.
    Tsarbopoulos, Anthony
    GAIA Research Center, Bioanalytical Department, The Goulandris Natural History Museum, 14562, Kifissia, Greece and Department of Pharmacology, National and Kapodistrian University of Athens Medical School, 75 Mikras Asias Street, 11527, Athens, Greece. .
    Crocus-derived compounds alter the aggregation pathway of Alzheimer's Disease: associated beta amyloid protein2020In: Scientific Reports, ISSN 2045-2322, article id 74770Article in journal (Refereed)
    Abstract [en]

    Natural products have played a dominant role in the discovery of lead compounds for the development of drugs aimed at the treatment of human diseases. This electrospray ionization-ion mobility spectrometry-mass spectrometry (ESI-IMS-MS)—based study demonstrates that dietary antioxidants, isolated components from the stigmas of saffron (Crocus sativus L.) may be effective in inhibiting Aβ fibrillogenesis, a neuropathological hallmark of Alzheimer’s Disease (AD). This study reveals a substantial alteration in the monomer/oligomer distribution of Aβ1-40, concomitant with re-direction of fibril formation, induced by the natural product interaction. These alterations on the Aβ1-40 aggregation pathway are most prominent for trans-crocin-4 (TC4). Use of ESI-IMS-MS, electron microscopy alongside Thioflavin-T kinetics, and the interpretation of 3-dimensional Driftscope plots indicate a correlation of these monomer/oligomer distribution changes with alterations to Aβ1-40 amyloid formation. The latter could prove instrumental in the development of novel aggregation inhibitors for the prevention, or treatment of AD.

  • 39. Kronqvist, Nina
    et al.
    Sarr, Medoune
    Lindqvist, Anton
    Nordling, Kerstin
    Otikovs, Martins
    Venturi, Luca
    Pioselli, Barbara
    Purhonen, Pasi
    Landreh, Michael
    Biverstal, Henrik
    Toleikis, Zigmantas
    Sjöberg, Lisa
    Robinson, Carol V.
    Pelizzi, Nicola
    Jornvall, Hans
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sverige.
    Jaudzems, Kristaps
    Curstedt, Tore
    Rising, Anna
    Johansson, Jan
    Efficient protein production inspired by how spiders make silk2017In: Nature Communications, E-ISSN 2041-1723, Vol. 8, article id 15504Article in journal (Refereed)
    Abstract [en]

    Membrane proteins are targets of most available pharmaceuticals, but they are difficult to produce recombinantly, like many other aggregation-prone proteins. Spiders can produce silk proteins at huge concentrations by sequestering their aggregation-prone regions in micellar structures, where the very soluble N-terminal domain (NT) forms the shell. We hypothesize that fusion to NT could similarly solubilize non-spidroin proteins, and design a charge-reversed mutant (NT star) that is pH insensitive, stabilized and hypersoluble compared to wildtype NT. NT star-transmembrane protein fusions yield up to eight times more of soluble protein in Escherichia coli than fusions with several conventional tags. NT star enables transmembrane peptide purification to homogeneity without chromatography and manufacture of low-cost synthetic lung surfactant that works in an animal model of respiratory disease. NT star also allows efficient expression and purification of non-transmembrane proteins, which are otherwise refractory to recombinant production, and offers a new tool for reluctant proteins in general.

  • 40.
    Kuang, Qie
    et al.
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Purhonen, Pasi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Alander, Johan
    Karolinska Institutet.
    Svensson, Richard
    Karolinska Institutet.
    Hoogland, Veronika
    Karolinska Institutet.
    Winerdal, Jens
    Karolinska Institutet.
    Spahiu, Linda
    Karolinska institutet.
    Ottosson-Wadlund, Astrid
    Karolinska Insitutet.
    Jegerschöld, Caroline
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Department of Biosciences and Nutrition, Karolinska Institutet.
    Morgenstern, Ralf
    Karolinska Institutet.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Medical Engineering, Structural Biotechnology. Department of Biosciences and Nutrition, Karolinska Institutet.
    Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation2017In: Scientific Reports, E-ISSN 2045-2322, Vol. 7, article id 7897Article in journal (Refereed)
    Abstract [en]

    Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two aliphatic chains and one glutathione (GSH) molecule. The functional unit is a homotrimer centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer supported by new in vitro mutagenesis data. Mutation of Arginine 130 to alanine resulted in complete loss of activity consistent with a role for Arginine 130 in stabilizing the strongly nucleophilic GSH thiolate required for catalysis. Based on the new model and an electron diffraction data set from crystals soaked with trinitrobenzene, that forms a dead-end Meisenheimer complex with GSH, a difference map was calculated. The map reveals side chain movements opening a cavity that defines the second substrate site.

  • 41.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH). Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Structure of potassium channels2015In: Cellular and Molecular Life Sciences (CMLS), ISSN 1420-682X, E-ISSN 1420-9071, Vol. 17, no 19, p. 3677-3693Article, review/survey (Refereed)
    Abstract [en]

    Potassium channels ubiquitously exist in nearly all kingdoms of life and perform diverse but important functions. Since the first atomic structure of a prokaryotic potassium channel (KcsA, a channel from Streptomyces lividans) was determined, tremendous progress has been made in understanding the mechanism of potassium channels and channels conducting other ions. In this review, we discuss the structure of various kinds of potassium channels, including the potassium channel with the pore-forming domain only (KcsA), voltage-gated, inwardly rectifying, tandem pore domain, and ligand-gated ones. The general properties shared by all potassium channels are introduced first, followed by specific features in each class. Our purpose is to help readers to grasp the basic concepts, to be familiar with the property of the different domains, and to understand the structure and function of the potassium channels better.

  • 42.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Two-Dimensional Crystallization Procedure, from Protein Expression to Sample Preparation2015In: BioMed Research International, ISSN 2314-6133, E-ISSN 2314-6141, article id 693869Article, review/survey (Refereed)
    Abstract [en]

    Membrane proteins play important roles for living cells. Structural studies of membrane proteins provide deeper understanding of their mechanisms and further aid in drug design. As compared to other methods, electron microscopy is uniquely suitable for analysis of a broad range of specimens, from small proteins to large complexes. Of various electron microscopic methods, electron crystallography is particularly well-suited to study membrane proteins which are reconstituted into two-dimensional crystals in lipid environments. In this review, we discuss the steps and parameters for obtaining large and well-ordered twodimensional crystals. A general description of the principle in each step is provided since this information can also be applied to other biochemical and biophysical methods. The examples are taken from our own studies and published results with related proteins. Our purpose is to give readers a more general idea of electron crystallography and to share our experiences in obtaining suitable crystals for data collection.

  • 43. Kuang, Qie
    et al.
    Purhonen, Pasi
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    The projection structure of Kch, a putative potassium channel in Escherichia coli, by electron crystallography2014In: Biochimica et Biophysica Acta - Biomembranes, ISSN 0005-2736, E-ISSN 1879-2642, Vol. 1838, no 1, p. 237-243Article in journal (Refereed)
    Abstract [en]

    The kch gene, the only potassium channel gene in Escherichia coil, has the property to express both full-length Kch and its cytosolic domain (RCK) due to a methionine at position 240. The RCK domains are believed to form an octameric ring structure and regulate the gating of the potassium channels after having bound certain ligands. Several different gating ring structures have been reported for the soluble RCK domains, however, these were studied isolated from their transmembrane parts. We previously reported an octameric structure of Kch in solution by electron microscopy and single particle reconstruction, composed of two tetrameric full-length proteins through RCK interaction. To exclude the effect of the detergent, we have now performed an electron crystallographic study of the full-length Kch in membrane bound form. Well-ordered two-dimensional crystals were grown in a natural phospholipid environment. A projection map merged from the fifteen best images extended to 6 angstrom resolution. The c12 two-sided plane group of the two-dimensional crystals showed that Kch crystallized as two symmetrically related overlapping layers. The arrangement suggests that the two layers of RCK domains are shifted with respect to each other and the RCK octameric gating ring of Kch does not form under the crystallization condition.

  • 44.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Jegerschöld, Caroline
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Institutet, Sweden.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Free RCK Arrangement in Kch, a Putative Escherichia coli Potassium Channel, as Suggested by Electron Crystallography2015In: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 23, no 1, p. 199-205Article in journal (Refereed)
    Abstract [en]

    The ligand-gated potassium channels are stimulated by various kinds of messengers. Previous studies showed that ligand-gated potassium channels containing RCK domains (the regulator of the conductance of potassium ion) form a dimer of tetramer structure through the RCK octameric gating ring in the presence of detergent. Here, we have analyzed the structure of Kch, a channel of this type from Escherichia coli, in a lipid environment using electron crystallography. By combining information from the 3D map of the transmembrane part of the protein and docking of an atomic model of a potassium channel, we conclude that the RCK domains face the solution and that an RCK octameric gating ring arrangement does not form under our crystallization condition. Our findings may be applied to other potassium channels that have an RCK gating ring arrangement.

  • 45.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Purhonen, Pasi
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Pattipaka, Thirupathi
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Ayele, Yohannes H
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Koeck, Philip J. B.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy2015In: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 21, no 4, p. 876-85Article in journal (Refereed)
    Abstract [en]

    Single-particle reconstruction (SPR) and electron crystallography (EC), two major applications in electron microscopy, can be used to determine the structure of membrane proteins. The three-dimensional (3D) map is obtained from separated particles in conventional SPR, but from periodic unit cells in EC. Here, we report a refined SPR procedure for processing 2D crystal images. The method is applied to 2D crystals of melibiose permease, a secondary transporter in Escherichia coli. The current procedure is improved from our previously published one in several aspects. The "gold standard Fourier shell correlation" resolution of our final reconstruction reaches 13 A, which is significantly better than the previously obtained 17 A resolution. The choices of different refinement parameters for reconstruction are discussed. Our refined SPR procedure could be applied to determine the structure of other membrane proteins in small or locally distorted 2D crystals, which are not ideal for EC.

  • 46.
    Kuang, Qie
    et al.
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Ålander, Johan
    Svensson, Richard
    Hoogland, Veronika
    Winerdal, Jens
    Spahiu, Linda
    Ottosson-Wadlund, Astrid
    Armstrong, Richard
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    Morgenstern, Ralf
    Hebert, Hans
    KTH, School of Technology and Health (STH), Basic Science and Biomedicine, Structural Biotechnology.
    A refined atomic model for microsomal glutathione transferase 1 from electron crystallographyManuscript (preprint) (Other academic)
    Abstract [en]

    Microsomal glutathione transferase 1 (MGST1) is a detoxification enzyme belonging to the Membrane Associated Proteins in Eicosanoid and Glutathione Metabolism (MAPEG) superfamily. Here we have used electron crystallography of two-dimensional (2D) crystals in order to determine an atomic model of rat MGST1 in a lipid environment. The 2D crystals were of the p6 two-sided plane group symmetry. For the refinement, information to 3.5 Å resolution from 225 electron diffraction patterns recorded from specimens at tilt angles up to 66° was used. The model comprises 123 of the 155 amino acid residues, two structured phospholipid molecules, two hydrocarbon chains, and one glutathione (GSH) molecule. Interactions between subunits form trimers centered on the crystallographic three-fold axes of the unit cell. The GSH substrate binds in an extended conformation at the interface between two subunits of the trimer. The location of GSH is supported by mutagenesis data in vitro.

  • 47. Kumar, Ramakrishnan B.
    et al.
    Hebert, Hans
    KTH, School of Technology and Health (STH), Structural Biotechnology.
    Jegerschold, Caroline
    Deciphering the Interaction of FLAP and 5LO2013In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 104, no 2, p. 540A-540AArticle in journal (Other academic)
  • 48.
    Kumar, Ramakrishnan B.
    et al.
    Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.;Vironova AB, Stockholm, Sweden..
    Purhonen, Pasi
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.
    Jegerschöld, Caroline
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH), Biomedical Engineering and Health Systems, Structural Biotechnology. Karolinska Inst, Dept Biosci & Nutr, Stockholm, Sweden.
    Arachidonic acid promotes the binding of 5-lipoxygenase on nanodiscs containing 5-lipoxygenase activating protein in the absence of calcium-ions2020In: PLOS ONE, E-ISSN 1932-6203, Vol. 15, no 7, article id e0228607Article in journal (Refereed)
    Abstract [en]

    Among the first steps in inflammation is the conversion of arachidonic acid (AA) stored in the cell membranes into leukotrienes. This occurs mainly in leukocytes and depends on the interaction of two proteins: 5-lipoxygenase (5LO), stored away from the nuclear membranes until use and 5-lipoxygenase activating protein (FLAP), a transmembrane, homotrimeric protein, constitutively present in nuclear membrane. We could earlier visualize the binding of 5LO to nanodiscs in the presence of Ca2+-ions by the use of transmission electron microscopy (TEM) on samples negatively stained by sodium phosphotungstate. In the absence of Ca2+-ions 5LO did not bind to the membrane. In the present communication, FLAP reconstituted in the nanodiscs which could be purified if the His-tag was located on the FLAP C-terminus but not the N-terminus. Our aim was to find out if 1) 5LO would bind in a Ca2+-dependent manner also when FLAP is present? 2) Would the substrate (AA) have effects on 5LO binding to FLAP-nanodiscs? TEM was used to assess the complex formation between 5LO and FLAP-nanodiscs along with, sucrose gradient purification, gel-electrophoresis and mass spectrometry. It was found that presence of AA by itself induces complex formation in the absence of added calcium. This finding corroborates that AA is necessary for the complex formation and that a Ca2+-flush is mainly needed for the recruitment of 5LO to the membrane. Our results also showed that the addition of Ca2+-ions promoted binding of 5LO on the FLAP-nanodiscs as was also the case for nanodiscs without FLAP incorporated. In the absence of added substances no 5LO-FLAP complex was formed. Another finding is that the formation of a 5LO-FLAP complex appears to induce fragmentation of 5LOin vitro.

  • 49.
    Kumar, Ramakrishnan
    et al.
    Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Hebert, Hans
    KTH, School of Engineering Sciences in Chemistry, Biotechnology and Health (CBH). Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Jegerschöld, Caroline
    KTH, School of Technology and Health (STH). Karolinska Inst, Dept Biosci & Nutr, Huddinge, Sweden.
    Elucidating the Interaction of 5-Lipoxygenase and FLAP2015In: Biophysical Journal, ISSN 0006-3495, E-ISSN 1542-0086, Vol. 108, no 2, p. 499A-499AArticle in journal (Other academic)
  • 50. Lambert, W
    et al.
    Koeck, Philip J. B.
    Department of Bioscience, Karolinska Institutet, Novum, Sweden.
    Ahrman, E
    Purhonen, P
    Cheng, K
    Elmlund, D
    Hebert, Hans
    Department of Bioscience, Karolinska Institutet, Novum, Sweden.
    Emanuelsson, C
    Subunit arrangement in the dodecameric chloroplast small heat shock protein Hsp212011In: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, no 2, p. 291-301Article in journal (Refereed)
    Abstract [en]

    Unfolding proteins are prevented from irreversible aggregation by small heat shock proteins (sHsps) through interactions that depend on a dynamic equilibrium between sHsp subunits and sHsp oligomers. A chloroplast-localized sHsp, Hsp21, provides protection to client proteins to increase plant stress resistance. Structural information is lacking concerning the oligomeric conformation of this sHsp. We here present a structure model of Arabidopsis thaliana Hsp21, obtained by homology modeling, single-particle electron microscopy, and lysine-specific chemical crosslinking. The model shows that the Hsp21 subunits are arranged in two hexameric discs, similar to a cytosolic plant sHsp homolog that has been structurally determined after crystallization. However, the two hexameric discs of Hsp21 are rotated by 25 degrees in relation to each other, suggesting a role for global dynamics in dodecamer function.

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