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  • 1. Ambort, D.
    et al.
    Johansson, M.E.V.
    Gustafsson, J. K.
    Nilsson, Harriet
    KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Medicinteknik och hälsosystem, Strukturell bioteknik.
    Ermund, A.
    Johansson, B.R.
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik (Stängd 20130701).
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik (Stängd 20130701).
    Hansson, G.C.
    Calcium and pH-dependent packing and release of the gel-forming MUC2 mucin2012Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 109, nr 15, s. 5645-5650Artikkel i tidsskrift (Fagfellevurdert)
    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. Chen, Gefei
    et al.
    Abelein, Axel
    Nilsson, Harriet E.
    KTH, Skolan för teknik och hälsa (STH), Medicinsk teknik, Strukturell bioteknik.
    Leppert, Axel
    Andrade-Talavera, Yuniesky
    Tambaro, Simone
    Hemmingsson, Lovisa
    Roshan, Firoz
    Landreh, Michael
    Biverstal, Henrik
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Medicinsk teknik, Strukturell bioteknik.
    Presto, Jenny
    Hebert, Hans
    Fisahn, Andre
    Johansson, Jan
    Bri2 BRICHOS client specificity and chaperone activity are governed by assembly state2017Inngår i: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 8, artikkel-id 2081Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    . Protein misfolding and aggregation is increasingly being recognized as a cause of disease. In Alzheimer's disease the amyloid-beta peptide (A beta) misfolds into neurotoxic oligomers and assembles into amyloid fibrils. The Bri2 protein associated with Familial British and Danish dementias contains a BRICHOS domain, which reduces A beta fibrillization as well as neurotoxicity in vitro and in a Drosophila model, but also rescues proteins from irreversible nonfibrillar aggregation. How these different activities are mediated is not known. Here we show that Bri2 BRICHOS monomers potently prevent neuronal network toxicity of A beta, while dimers strongly suppress A beta fibril formation. The dimers assemble into high-molecular-weight oligomers with an apparent two-fold symmetry, which are efficient inhibitors of non-fibrillar protein aggregation. These results indicate that Bri2 BRICHOS affects qualitatively different aspects of protein misfolding and toxicity via different quaternary structures, suggesting a means to generate molecular chaperone diversity.

  • 3. Cheng, K.
    et al.
    Koeck, P.J.B.
    Idakieva, K.
    Ternström, T.
    Parvanova, K.
    Hebert, Hans
    Stainless Models of Rapana Thomasiana Hemocyanin2004Inngår i: Proc. of the 13th European Congress on Electron Microscopy, 2004, s. 22-27Konferansepaper (Fagfellevurdert)
  • 4.
    Cheng, Kimberley
    et al.
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik (Stängd 20130701).
    Karlström, M
    Purhonen, P
    Ladenstein, R.
    Herbert, Hans
    Koeck, Philip J.B.
    Low resolution structure and apparent melting temperature of the chaperonin from Pyrococcus furiosusManuskript (preprint) (Annet vitenskapelig)
  • 5.
    Cheng, Kimberley
    et al.
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Elmlund, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Idakieva, Krassimira
    Parvanova, Katja
    Schwarz, Heinz
    Ternström, Tomas
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Comparison of the two Rapana thomasiana Hemocyanin isoforms: RtH1 and RtH22006Inngår i: Proc 16. International Microscopy Conference, 2006Konferansepaper (Fagfellevurdert)
  • 6.
    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, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    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 resolution2006Inngår i: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 37, nr 6, s. 566-576Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 7.
    Elmlund, Hans
    et al.
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    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, Skolan för teknik och hälsa (STH).
    Holmberg, Steen
    Department of Genetics, Institute of Molecular Biology, Copenhagen.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Gustafsson, Claes M.
    Division of Metabolic Diseases, Karolinska Institutet.
    The cyclin-dependent kinase 8 module sterically blocks Mediator interactions with RNA polymerase II2006Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 103, nr 43, s. 15788-15793Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 8.
    Härmark, Johan
    et al.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sweden.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sweden.
    Koeck, Philip J B
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sweden.
    Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy2016Inngår i: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 85, s. 39-43Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 9.
    Härmark, Johan
    et al.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik.
    Larsson, Malin K.
    KTH, Skolan för teknik och hälsa (STH), Medicinsk teknik, Medicinsk bildteknik.
    Razuvajev, Anton
    Koeck, Philip JB
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik.
    Paradossi, Gaio
    Brodin, Lars-Åke
    KTH, Skolan för teknik och hälsa (STH), Medicinsk teknik, Medicinsk bildteknik.
    Caidahl, Kenneth
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik.
    Bjällmark, Anna
    KTH, Skolan för teknik och hälsa (STH), Medicinsk teknik, Medicinsk bildteknik.
    Investigation of the elimination process of a multimodal polymer-shelled contrast agent in rats using ultrasound and transmission electron microscopy2015Inngår i: Biomedical Spectroscopy and Imaging, ISSN 2212-8794, Vol. 4, nr 1, s. 81-93Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 10.
    Koeck, P. J. B.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik.
    Improved Zernike-type phase contrast for transmission electron microscopy2015Inngår i: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 259, nr 1, s. 74-78Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Zernike phase contrast has been recognized as a means of recording high-resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image typical phase objects such as unstained biological molecules or cryosections of biological tissue. According to the original proposal discussed in Danev and Nagayama (2001) and references therein, the Zernike phase plate applies a phase shift of /2 to all scattered electron beams outside a given scattering angle and an image is recorded at Gaussian focus or slight underfocus (below Scherzer defocus). Alternatively, a phase shift of -/2 is applied to the central beam using the Boersch phase plate. The resulting image will have an almost perfect contrast transfer function (close to 1) from a given lowest spatial frequency up to a maximum resolution determined by the wave length, the amount of defocus and the spherical aberration of the microscope. In this paper, I present theory and simulations showing that this maximum spatial frequency can be increased considerably without loss of contrast by using a Zernike or Boersch phase plate that leads to a phase shift between scattered and unscattered electrons of only /4, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies. Lay description Zernike phase contrast has been recognized as a means of recording high-resolution images with high contrast using a transmission electron microscope. This imaging mode can be used to image specimens such as unstained biological molecules or sections of biological tissue. According to the original proposal, the Zernike phase plate applies a phase shift of /2 to all scattered electron beams outside a given scattering angle and an image is recorded at or close to focus. The resulting image will be an almost perfect representation of the specimen up to a maximum resolution determined by the energy of the electrons and certain optical parameters of the microscope. In this paper, I present theory and simulations showing that this maximum resolution can be increased considerably without loss of contrast by using a Zernike phase plate that leads to a phase shift between scattered and unscattered electrons of only /4, and recording images somewhat out of focus.

  • 11.
    Koeck, P. J. B.
    et al.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik.
    Karshikoff, A.
    Limitations of the linear and the projection approximations in three-dimensional transmission electron microscopy of fully hydrated proteins2015Inngår i: Journal of Microscopy, ISSN 0022-2720, E-ISSN 1365-2818, Vol. 259, nr 3, s. 197-209Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    We establish expressions for the linear and quadratic terms in the series expansion of the phase and the phase and amplitude object description of imaging thin specimens by transmission electron microscopy. Based on these expressions we simulate the corresponding contributions to images of unstained protein complexes of varying thickness and arrive at an estimate for how much each term contributes to the contrast of the image. From this we can estimate a maximum specimen thickness for which the weak phase and the weak amplitude and phase object approximation (and therefore linear imaging) is still reasonably accurate. When discussing thick specimens it is also necessary to consider limitations due to describing the image as a filtered projection of the specimen, since the different layers of the specimen are not imaged with the same defocus value. We therefore compared simulations based on the projection approximation with the more accurate multislice model of image formation. However, we find that the errors due to nonlinear image contributions are greater than those due to the defocus gradient for the defocus values chosen for the simulations. Finally, we study how the discussed nonlinear image contributions and the defocus gradient affect the quality of three-dimensional reconstructions. We find that three-dimensional reconstructions reach high resolution when at the same time exhibiting localized systematic structural errors. Non-Technical Abstract Cryo transmission electron microscopy and three-dimensional reconstruction can be used to determine a three-dimensional model of a protein molecule. In the mathematical methods used for three-dimensional reconstruction assumptions are made about a linear relationship between the images recorded in the electron microscope and the objects being imaged. In this paper we investigate with computer simulations at what specimen thickness these assumptions start breaking down and what sort of errors can be expected in the three-dimensional reconstructions when the assumptions are not valid anymore.

  • 12.
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sweden.
    Improved Hilbert phase contrast for transmission electron microscopy2015Inngår i: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 154, s. 37-41Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Hilbert phase contrast has been recognized as a means of recording high resolution images with high contrast using a transmission electron microscope. This imaging mode could be used to image typical phase objects such as unstained biological molecules or cryo sections of biological tissue. According to the original proposal by (Danev et al., 2002) the Hilbert phase plate applies a phase shift of π to approximately half the focal plane (for example the right half excluding the central beam) and an image is recorded at Gaussian focus. After correction for the inbuilt asymmetry of differential phase contrast this image will have an almost perfect contrast transfer function (close to 1) from the lowest spatial frequency up to a maximum resolution determined by the wave length and spherical aberration of the microscope. In this paper I present theory and simulations showing that this maximum spatial frequency can be increased considerably almost without loss of contrast by using a Hilbert phase plate of half the thickness, leading to a phase shift of π/2, and recording images at Scherzer defocus. The maximum resolution can be improved even more by imaging at extended Scherzer defocus, though at the cost of contrast loss at lower spatial frequencies.

  • 13.
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Medicinsk teknik, Strukturell bioteknik. KTH, Skolan för kemi, bioteknologi och hälsa (CBH). Karolinska Institutet, Department of Biosciences and Nutrition, Novum, 14183 Huddinge, Sweden.
    An aperture design for single side band imaging in the transmission electron microscope2017Inngår i: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 182, s. 81-84Artikkel i tidsskrift (Fagfellevurdert)
  • 14.
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Medicinteknik och hälsosystem, Strukturell bioteknik. Department of Biosciences and Nutrition, Novum, Huddinge, Sweden.
    Annular dark field transmission electron microscopy for protein structure determination2016Inngår i: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 161, s. 98-104Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Recently annular dark field (ADF) transmission electron microscopy (TEM) has been advocated as a means of recording images of biological specimens with better signal to noise ratio (SNR) than regular bright field images. I investigate whether and how such images could be used to determine the three-dimensional structure of proteins given that an ADF aperture with a suitable pass-band can be manufactured and used in practice. I develop an approximate theory of ADF-TEM image formation for weak amplitude and phase objects and test this theory using computer simulations. I also test whether these simulated images can be used to calculate a three-dimensional model of the protein using standard software and discuss problems and possible ways to overcome these.

  • 15.
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Medicinteknik och hälsosystem, Strukturell bioteknik.
    Design of a Charged Particle Beam Phase Plate for Transmission Electron Microscopy2019Inngår i: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 205, s. 62-69Artikkel i tidsskrift (Fagfellevurdert)
  • 16.
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH). Karolinska Institutet, Department of Biosciences and Nutrition, Novum, Huddinge, Sweden.
    Design of an Electrostatic Phase Shifting Device for Biological Transmission Electron Microscopy2018Inngår i: Ultramicroscopy, ISSN 0304-3991, E-ISSN 1879-2723, Vol. 187, s. 107-112Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    I suggest an electrostatic phase plate designed to broaden the contrast transfer function of a transmission electron microscope operated close to Scherzer defocus primarily in the low resolution direction. At higher defocus the low frequency behavior is equal to that close to Scherzer defocus, but CTF-correction becomes necessary to extend image interpretation to higher resolution. One simple realization of the phase plate consists of two ring shaped electrodes symmetrically surrounding the central beam. Since no physical components come into contact with the central beam and charge on the electrodes is controlled by an external voltage supply, problems with uncontrolled charging are expected to be reduced.

  • 17.
    Koeck, Philip J. B.
    Karolinska Institute.
    Missing Data in Image and Signal Processing: The case of binary objects2004Inngår i: Optik (Stuttgart), ISSN 0030-4026, E-ISSN 1618-1336, Vol. 115, nr 10, s. 459-472Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    I investigated which portions of the Fourier transform of binary signals, images and three-dimensional objects are necessary to correctly identify an object in the presence of noise. This is practically possible for very small binary data sets since the total number of possible objects is then very limited. There are for example 512 different binary images with 9 pixels. It is easy to see that this number soon becomes impractically large for bigger images or if one allows more than two possible pixel values. It turns out that even in the presence of large amounts of noise a relatively small portion of the Fourier transform is essential for deciding which of all possible binary objects the Fourier transform belongs to. These ‘decision experiments’ can be used as a standard for how well algorithms for retrieval of missing Fourier components perform. In another set of computer experiments I investigate the possibility of retrieving various missing Fourier components algorithmically. The main finding of this second set of computer experiments is that the simple retrieval algorithm (a limited form of ‘projection onto convex sets’) used falls very much short of what one might expect from the ‘decision experiments’.

    I conclude with a discussion what this discrepancy might be due to and some suggestions how to improve the performance of retrieval algorithms for binary objects.

  • 18.
    Koeck, Philip J. B.
    et al.
    Karolinska Institutet.
    Purhonen, P.
    Alvang, R.
    Grundberg, B.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    3D-correlation-averaging for membrane-protein-crystals2008Inngår i: EMC 2008 14th European Microscopy Congress, 2008, s. 55-56Konferansepaper (Fagfellevurdert)
    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].

  • 19. Koeck, Philip J. B.
    et al.
    Purhonen, P
    Alvang, R
    Grundberg, B
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Single-particle refinement in electron crystallography of membrane-proteins2007Konferansepaper (Fagfellevurdert)
  • 20.
    Koeck, Philip J. B.
    et al.
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik (Stängd 20130701). KTH, Skolan för kemi, bioteknologi och hälsa (CBH), Medicinteknik och hälsosystem, Strukturell bioteknik.
    Purhonen, Pasi
    Alvang, Ronny
    KTH, Skolan för teknik och hälsa (STH).
    Grundberg, Björn
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik (Stängd 20130701).
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik (Stängd 20130701).
    Single particle refinement in electron crystallography: A pilot study2007Inngår i: Journal of Structural Biology, ISSN 1047-8477, E-ISSN 1095-8657, Vol. 160, nr 3, s. 344-352Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 21.
    Kuang, Qie
    et al.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sweden.
    Purhonen, Pasi
    Jegerschöld, Caroline
    Köck, Philip
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sweden.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sweden.
    Free RCK Arrangement in Kch, a Putative Escherichia coli Potassium Channel, as Suggested by Electron Crystallography2015Inngår i: Structure, ISSN 0969-2126, E-ISSN 1878-4186, Vol. 23, nr 1, s. 199-205Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 22.
    Kuang, Qie
    et al.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Purhonen, Pasi
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Pattipaka, Thirupathi
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Ayele, Yohannes H
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    Köck, Philip J B
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet,Department of Biosciences and Nutrition, Sweden.
    A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy2015Inngår i: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 21, nr 4, s. 876-85Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 23. 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 Hsp212011Inngår i: Protein Science, ISSN 0961-8368, E-ISSN 1469-896X, Vol. 20, nr 2, s. 291-301Artikkel i tidsskrift (Fagfellevurdert)
    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.

  • 24.
    Nilsson, Harriet E.
    et al.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik.
    Ambort, Daniel
    Bäckström, Malin
    Thomsson, Elisabeth
    Koeck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Department of Biosciences and Nutrition, Karolinska Institutet.
    Hansson, Gunnar C.
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. epartment of Biosciences and Nutrition, Karolinska Institutet.
    Intestinal MUC2 Mucin Supramolecular Topology by Packing and Release Resting on D3 Domain Assembly2014Inngår i: Journal of Molecular Biology, ISSN 0022-2836, E-ISSN 1089-8638, Vol. 426, nr 14, s. 2567-2579Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    MUC2 is the major gel-forming mucin of the colon forming a protective gel barrier organized into an inner stratified and an outer loose layer. The MUC2 N-terminus (D1-D2-D'D3 domains) has a dual function in building a net-like structure by disulfide-bonded trimerization and packing the MUC2 polymer into an N-terminal concatenated polygonal platform with the C-termini extending perpendicularly by pH- and calcium-dependent interactions. We studied the N-terminal D'D3 domain by producing three recombinant variants, with or without Myc tag and GFP (green fluorescent protein), and analyzed these by gel filtration, electron microscopy and single particle image processing. The three variants were all trimers when analyzed upon denaturing conditions but eluted as hexanners upon gel filtration under native conditions. Studies by electron microscopy and three-dimensional maps revealed cage-like structures with 2- and 3-fold symmetries. The structure of the MUC2 D3 domain confirms that the MUC2 mucin forms branched net-like structures. This suggests that the MUC2 mucin is stored with two N-terminal concatenated ring platforms turned by 180 against each other, implicating that every second unfolded MUC2 net in mature mucus is turned upside down.

  • 25. Purhonen, P.
    et al.
    Grundberg, B.
    Alvang, R.
    Koeck, Philip J. B.
    Thomsen, K.
    Hebert, Hans
    Maunsbach, A.B.
    Renal Na, K-ATPase Structure from Cryo-Electron Microscopy with 2-D Crystals Showing Structural Variability2004Inngår i: Proc. of the 13th European Congress on Electron Microscopy, 2004Konferansepaper (Fagfellevurdert)
  • 26. Purhonen, P
    et al.
    Koeck, Philip J. B.
    Thomsen, K
    Maunsbach, AB
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Cryo-EM studies of renal Na,K-ATPase in native membranes2008Inngår i: Proc of the 12th International ATPase Conference. Na,K-ATPase and related transport ATPases of P-type, 2008Konferansepaper (Fagfellevurdert)
  • 27. Purhonen, P
    et al.
    Thomsen, K
    Koeck, Philip J. B.
    Maunsbach, AB
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Strukturell bioteknik.
    Three-dimensional structure of renal Na,K-ATPase as determined by cryo-electron microscopy2007Konferansepaper (Fagfellevurdert)
  • 28. Rutsdottir, Gudrun
    et al.
    Härmark, Johan
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin. Karolinska Institutet, Sverige.
    Weide, Yoran
    Hebert, Hans
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sverige.
    Rasmussen, Morten I.
    Wernersson, Sven
    Respondek, Michal
    Akke, Mikael
    Højrup, Peter
    Köck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH), Naturvetenskap och biomedicin, Strukturell bioteknik. Karolinska Institutet, Sverige.
    Söderberg, Christopher A. G.
    Emanuelsson, Cecilia
    Structural model of dodecameric heat-shock protein Hsp21: Flexible N-terminal arms interact with client proteins while C-terminal tails maintain the dodecamer and chaperone activity2017Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 292, nr 19, s. 8103-8121Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Small heat-shock proteins (sHsps) prevent aggregation of thermosensitive client proteins in a first line of defense against cellular stress. The mechanisms by which they perform this function have been hard to define due to limited structural information; currently, there is only one high-resolution structure of a plant sHsp published, that of the cytosolic Hsp16.9. We took interest in Hsp21, a chloroplast-localized sHsp crucial for plant stress resistance, which has even longer N-terminal arms than Hsp16.9, with a functionally important and conserved methionine-rich motif. To provide a framework for investigating structure-function relationships of Hsp21 and understanding these sequence variations, we developed a structural model of Hsp21 based on homology modeling, cryo-EM, cross-linking mass spectrometry, NMR, and small-angle X-ray scattering. Our data suggest a dodecameric arrangement of two trimer-of-dimer discs stabilized by the C-terminal tails, possibly through tail-to-tail interactions between the discs, mediated through extended IXVXI motifs. Our model further suggests that six N-terminal arms are located on the outside of the dodecamer, accessible for interaction with client proteins, and distinct from previous undefined or inwardly facing arms. To test the importance of the IXVXI motif, we created the point mutant V181A, which, as expected, disrupts the Hsp21 dodecamer and decreases chaperone activity. Finally, our data emphasize that sHsp chaperone efficiency depends on oligomerization and that client interactions can occur both with and without oligomer dissociation. These results provide a generalizable workflow to explore sHsps, expand our understanding of sHsp structural motifs, and provide a testable Hsp21 structure model to inform future investigations.

  • 29.
    Trillo-Muyo, Sergio
    et al.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Nilsson, Harriet E.
    KTH, Skolan för teknik och hälsa (STH). Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden.;Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Recktenwald, Christian V.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Ermund, Anna
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Ridley, Caroline
    Univ Manchester, Manchester Acad Hlth Sci Ctr, Wellcome Trust Ctr Cell Matrix Res, Fac Biol Med & Hlth, Manchester M13 9PT, Lancs, England..
    Meiss, Lauren N.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Baehr, Andrea
    Ludwig Maximilians Univ Munchen, Inst Mol Anim Breeding & Biotechnol, Gene Ctr, Hackerstr 27, D-85764 Oberschleissheim, Germany..
    Klymiuk, Nikolai
    Ludwig Maximilians Univ Munchen, Inst Mol Anim Breeding & Biotechnol, Gene Ctr, Hackerstr 27, D-85764 Oberschleissheim, Germany..
    Wine, Jeffrey J.
    Stanford Univ, Cyst Fibrosis Res Lab, Stanford, CA 94305 USA..
    Köck, Philip J. B.
    KTH, Skolan för teknik och hälsa (STH). Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.
    Thornton, David J.
    Univ Manchester, Manchester Acad Hlth Sci Ctr, Wellcome Trust Ctr Cell Matrix Res, Fac Biol Med & Hlth, Manchester M13 9PT, Lancs, England..
    Hebert, Hans
    Karolinska Inst, Dept Biosci & Nutr, S-14157 Huddinge, Sweden.;KTH Royal Inst Technol, Sch Technol & Hlth, S-14157 Huddinge, Sweden..
    Hansson, Gunnar C.
    Univ Gothenburg, Dept Med Biochem, Box 440, S-40530 Gothenburg, Sweden..
    Granule-stored MUC5B mucins are packed by the non-covalent formation of N-terminal head-to-head tetramers2018Inngår i: Journal of Biological Chemistry, ISSN 0021-9258, E-ISSN 1083-351X, Vol. 293, nr 15, s. 5746-5754Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    Most MUC5B mucin polymers in the upper airways of humans and pigs are produced by submucosal glands. MUC5B forms N-terminal covalent dimers that are further packed into larger assemblies because of low pH and high Ca2+ in the secretory granule of the mucin-producing cell. We purified the recombinant MUC5B N-terminal covalent dimer and used single-particle electron microscopy to study its structure under intracellular conditions. We found that, at intragranular pH, the dimeric MUC5B organized into head-to-head noncovalent tetramers where the von Willebrand D1-D2 domains hooked into each other. These N-terminal tetramers further formed long linear complexes from which, we suggest, the mucin domains and their C termini project radially outwards. Using conventional and video microscopy, we observed that, upon secretion into the submucosal gland ducts, a flow of bicarbonate-rich fluid passes the mucin-secreting cells. We suggest that this unfolds and pulls out the MUC5B assemblies into long linear threads. These further assemble into thicker mucin bundles in the glandular ducts before emerging at the gland duct opening. We conclude that the combination of intracellular packing of the MUC5B mucin and the submucosal gland morphology creates an efficient machine for producing linear mucin bundles.

  • 30. Wu, S.R.
    et al.
    Löving, R.
    Lindqvist, B.
    Hebert, Hans
    Department of Biosciences and Nutrition, Karolinska Institute.
    Koeck, Philip J. B.
    Sjöberg, M.
    Garoff, H.
    Single-particle cryoelectron microscopy analysis reveals the HIV-1 spike as a tripod structure2010Inngår i: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 107, nr 44, s. 18844-18849Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The HIV-1 spike is a trimer of the transmembrane gp41 and the peripheral gp120 subunit pair. It is activated for virus-cell membrane fusion by binding sequentially to CD4 and to a chemokine receptor. Here we have studied the structural transition of the trimeric spike during the activation process. We solubilized and isolated unliganded and CD4-bound spikes from virus-like particles and used cryoelectron microscopy to reconstruct their 3D structures. In order to increase the yield and stability of the spike, we used an endodomain deleted and gp120-gp41 disulfide-linked variant. The unliganded spike displayed a hollow cage-like structure where the gp120-gp41 protomeric units formed a roof and bottom, and separated lobes and legs on the sides. The tripod structure was verified by fitting the recent atomic core structure of gp120 with intact N- and C-terminal ends into the spike density map. This defined the lobe as gp120 core, showed that the legs contained the polypeptide termini, and suggested the deleted variable loops V1/V2 and V3 to occupy the roof and gp41 the bottom. CD4 binding shifted the roof density peripherally and condensed the bottom density centrally. Fitting with a V3 containing gp120 core suggested that the V1/V2 loops in the roof were displaced laterally and the V3 lifted up, while the core and leg were kept in place. The loop displacements probably prepared the spike for coreceptor interaction and roof opening so that a new fusion-active gp41 structure, assembled at the center of the cage bottom, could reach the target membrane.

  • 31. Wu, S.R.
    et al.
    Sjöberg, M.
    Wallin, M.
    Lindqvist, B.
    Hebert, Hans
    Department of Biosciences and Nutrition, Karolinska Institute.
    Koeck, Philip J. B.
    Garoff, H.
    Turning of the receptor binding domains opens up the murine leukaemia virus Env for membrane fusion2008Inngår i: EMBO Journal, ISSN 0261-4189, E-ISSN 1460-2075, Vol. 27, nr 20, s. 2799-2808Artikkel i tidsskrift (Fagfellevurdert)
    Abstract [en]

    The activity of the membrane fusion protein Env of Moloney mouse leukaemia virus is controlled by isomerization of the disulphide that couples its transmembrane (TM) and surface (SU) subunits. We have arrested Env activation at a stage prior to isomerization by alkylating the active thiol in SU and compared the structure of isomerization-arrested Env with that of native Env. Env trimers of respective form were isolated from solubilized particles by sedimentation and their structures were reconstructed from electron microscopic images of both vitrified and negatively stained samples. We found that the protomeric unit of both trimers formed three protrusions, a top, middle and a lower one. The atomic structure of the receptor-binding domain of SU fitted into the upper protrusion. This was formed similar to a bent finger. Significantly, in native Env the tips of the fingers were directed against each other enclosing a cavity below, whereas they had turned outward in isomerization-arrested Env transforming the cavity into an open well. This might subsequently guide the fusion peptides in extended TM subunits into the target membrane.

1 - 31 of 31
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