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Andersson, M., Jia, Q., Abella, A., Lee, X.-Y., Landreh, M., Purhonen, P., . . . Rising, A. (2017). Biomimetic spinning of artificial spider silk from a chimeric minispidroin. Nature Chemical Biology, 13(3), 262-+.
Open this publication in new window or tab >>Biomimetic spinning of artificial spider silk from a chimeric minispidroin
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2017 (English)In: Nature Chemical Biology, ISSN 1552-4450, E-ISSN 1552-4469, Vol. 13, no 3, 262-+ p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-204071 (URN)10.1038/NCHEMBIO.2269 (DOI)000394431500006 ()28068309 (PubMedID)2-s2.0-85008658159 (Scopus ID)
Note

QC 20170329

Available from: 2017-03-29 Created: 2017-03-29 Last updated: 2017-11-29Bibliographically approved
Kuang, Q., Purhonen, P., Alander, J., Svensson, R., Hoogland, V., Winerdal, J., . . . Hebert, H. (2017). Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation. Scientific Reports, 7, Article ID 7897.
Open this publication in new window or tab >>Dead-end complex, lipid interactions and catalytic mechanism of microsomal glutathione transferase 1, an electron crystallography and mutagenesis investigation
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, 7897Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Nature Publishing Group, 2017
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-214509 (URN)10.1038/s41598-017-07912-3 (DOI)000407442500037 ()28801553 (PubMedID)
Note

QC 20170929

Available from: 2017-09-29 Created: 2017-09-29 Last updated: 2017-09-29Bibliographically approved
B. Kumar, R., Zhu, L., Hebert, H. & Jegerschöld, C. (2017). Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy. Journal of Visualized Experiments (121), Article ID e55148.
Open this publication in new window or tab >>Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy
2017 (English)In: Journal of Visualized Experiments, ISSN 1940-087X, E-ISSN 1940-087X, no 121, e55148Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Journal of Visualized Experiments, 2017
Keyword
Biochemistry, Issue 121, negative staining, protein complex, TEM, lipoxygenases, visualization, non-denaturing gel electrophoresis
National Category
Other Basic Medicine
Identifiers
urn:nbn:se:kth:diva-204044 (URN)10.3791/55148 (DOI)000415744000005 ()28287545 (PubMedID)2-s2.0-85017206859 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20170327

Available from: 2017-03-23 Created: 2017-03-23 Last updated: 2018-01-13Bibliographically approved
Park, K.-H., An, Y., Jung, T., Baek, I.-Y., Noh, H., Ahn, W.-C., . . . Woo, E.-J. (2017). RNA activation-independent DNA targeting of the Type III CRISPR-Cas system by a Csm complex. EMBO Reports, 18(5), 826-840.
Open this publication in new window or tab >>RNA activation-independent DNA targeting of the Type III CRISPR-Cas system by a Csm complex
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2017 (English)In: EMBO Reports, ISSN 1469-221X, E-ISSN 1469-3178, Vol. 18, no 5, 826-840 p.Article in journal (Refereed) Published
Abstract [en]

The CRISPR-Cas system is an adaptive and heritable immune response that destroys invading foreign nucleic acids. The effector complex of the Type III CRISPR-Cas system targets RNA and DNA in a transcription-coupled manner, but the exact mechanism of DNA targeting by this complex remains elusive. In this study, an effector Csm holocomplex derived from Thermococcus onnurineus is reconstituted with a minimalistic combination of Csm1(1)2(1)3(3)4(1)5(1), and shows RNA targeting and RNA-activated single-stranded DNA (ssDNA) targeting activities. Unexpectedly, in the absence of an RNA transcript, it cleaves ssDNA containing a sequence complementary to the bound crRNA guide region in a manner dependent on the HD domain of the Csm1 subunit. This nuclease activity is blocked by a repeat tag found in the host CRISPR loci. The specific cleavage of ssDNA without a target RNA suggests a novel ssDNA targeting mechanism of the Type III system, which could facilitate the efficient and complete degradation of foreign nucleic acids.

Place, publisher, year, edition, pages
Wiley-VCH Verlagsgesellschaft, 2017
Keyword
CRISPR, Csm complex, DNase, RNase, Thermococcus onnurineus
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-207890 (URN)10.15252/embr.201643700 (DOI)000400446100016 ()28364023 (PubMedID)2-s2.0-85017406426 (Scopus ID)
Note

QC 20170530

Available from: 2017-05-30 Created: 2017-05-30 Last updated: 2017-05-30Bibliographically approved
Zhu, L., Petrlova, J., Gysbers, P., Hebert, H., Wallin, S., Jegerschöld, C. & Lagerstedt, J. O. (2017). Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations. Biochimica et Biophysica Acta - General Subjects, 1861(11), 2726-2738.
Open this publication in new window or tab >>Structures of apolipoprotein A-I in high density lipoprotein generated by electron microscopy and biased simulations
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2017 (English)In: Biochimica et Biophysica Acta - General Subjects, ISSN 0304-4165, E-ISSN 1872-8006, Vol. 1861, no 11, 2726-2738 p.Article in journal (Refereed) Published
Abstract [en]

Background: Apolipoprotein A-I (apoA-I) in high-density lipoprotein (HDL) is a key protein for the transport of cholesterol from the vascular wall to the liver. The formation and structure of nascent HDL, composed of apoA-I and phospholipids, is critical to this process. Methods: The HDL was assembled in vitro from apoA-I, cholesterol and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at a 1:4:50 molar ratio. The structure of HDL was investigated in vitreous samples, frozen at cryogenic temperatures, as well as in negatively stained samples by transmission electron microscopy. Low resolution electron density maps were next used as restraints in biased Monte Carlo simulations of apolipoprotein A-I dimers, with an initial structure derived from atomic resolution X-ray structures. Results: Two final apoA-I structure models for the full-length structure of apoA-I dimer in the lipid bound conformation were generated, showing a nearly circular, flat particle with an uneven particle thickness. Conclusions: The generated structures provide evidence for the discoidal, antiparallel arrangement of apoA-I in nascent HDL, and propose two preferred conformations of the flexible N-termini.

Place, publisher, year, edition, pages
Elsevier, 2017
Keyword
Apolipoprotein A-I, Biased simulations, Cryo-EM, HDL, Negative stain electron microscopy, 2 oleoyl 1 palmitoylphosphatidylcholine, apolipoprotein A1, cholesterol, dimer, high density lipoprotein, amino terminal sequence, Article, disorders of lipoprotein metabolism, electron, electron microscopy, image analysis, in vitro study, priority journal, protein analysis, protein lipid interaction, protein modification, protein structure, simulation, temperature, transmission electron microscopy
National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-218631 (URN)10.1016/j.bbagen.2017.07.017 (DOI)000415768500022 ()28754383 (PubMedID)2-s2.0-85026642644 (Scopus ID)
Funder
Swedish Research Council, K2014-54X-22426-01-3The Wenner-Gren Foundation
Note

QC 20171130

Available from: 2017-11-30 Created: 2017-11-30 Last updated: 2017-12-07Bibliographically approved
Ermund, A., Meiss, L. N., Rodriguez-Pineiro, A. M., Baehr, A., Nilsson, H. E., Trillo-Muyo, S., . . . Hansson, G. C. (2017). The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin. Biochemical and Biophysical Research Communications - BBRC, 492(3), 331-337.
Open this publication in new window or tab >>The normal trachea is cleaned by MUC5B mucin bundles from the submucosal glands coated with the MUC5AC mucin
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2017 (English)In: Biochemical and Biophysical Research Communications - BBRC, ISSN 0006-291X, E-ISSN 1090-2104, Vol. 492, no 3, 331-337 p.Article in journal (Refereed) Published
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. 

Place, publisher, year, edition, pages
ACADEMIC PRESS INC ELSEVIER SCIENCE, 2017
Keyword
Respiratory tract, Mucus, MUC5AC, Airway surface liquid, Lung
National Category
Medical Biotechnology
Identifiers
urn:nbn:se:kth:diva-215783 (URN)10.1016/j.bbrc.2017.08.113 (DOI)000411424300008 ()28859985 (PubMedID)2-s2.0-85028533537 (Scopus ID)
Note

QC 20171023

Available from: 2017-10-23 Created: 2017-10-23 Last updated: 2017-10-23Bibliographically approved
Vijayvargia, R., Epand, R., Leitner, A., Jung, T., Shin, B., Jung, R., . . . Seong, I. S. (2016). Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function. eLIFE, 5, Article ID e11184.
Open this publication in new window or tab >>Huntingtin's spherical solenoid structure enables polyglutamine tract-dependent modulation of its structure and function
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2016 (English)In: eLIFE, E-ISSN 2050-084X, Vol. 5, e11184Article in journal (Refereed) Published
Abstract [en]

The polyglutamine expansion in huntingtin protein causes Huntington's disease. Here, we investigated structural and biochemical properties of huntingtin and the effect of the polyglutamine expansion using various biophysical experiments including circular dichroism, single particle electron microscopy and cross-linking mass spectrometry. Huntingtin is likely composed of five distinct domains and adopts a spherical alpha-helical solenoid where the amino-terminal and carboxyl-terminal regions fold to contain a circumscribed central cavity. Interestingly, we showed that the polyglutamine expansion increases alpha-helical properties of huntingtin and affects the intramolecular interactions among the domains. Our work delineates the structural characteristics of full-length huntingtin, which are affected by the polyglutamine expansion, and provides an elegant solution to the apparent conundrum of how the extreme amino-terminal polyglutamine tract confers a novel property on huntingtin, causing the disease.

Place, publisher, year, edition, pages
ELIFE SCIENCES PUBLICATIONS, 2016
National Category
Structural Biology
Identifiers
urn:nbn:se:kth:diva-185632 (URN)10.7554/eLife.11184 (DOI)000373331100001 ()2-s2.0-84964285135 (Scopus ID)
Note

QC 20160429

Available from: 2016-04-29 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved
Härmark, J., Hebert, H. & Koeck, P. J. (2016). Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy. Micron, 85, 39-43.
Open this publication in new window or tab >>Shell thickness determination of polymer-shelled microbubbles using transmission electron microscopy
2016 (English)In: Micron, ISSN 0968-4328, E-ISSN 1878-4291, Vol. 85, 39-43 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Elsevier, 2016
Keyword
Shell thickness, Contrast agent, Transmission electron microscopy
National Category
Medical Engineering
Research subject
Medical Technology
Identifiers
urn:nbn:se:kth:diva-186876 (URN)10.1016/j.micron.2016.03.009 (DOI)000376217400006 ()27077316 (PubMedID)2-s2.0-84962835270 (Scopus ID)
Funder
EU, FP7, Seventh Framework Programme, 245572
Note

QC 20160516

Available from: 2016-05-16 Created: 2016-05-16 Last updated: 2017-11-30Bibliographically approved
Balakrishnan Kumar, R., Zhu, L., Idborg, H., Radmark, O., Jakobsson, P.-J., Rinaldo-Matthis, A., . . . Jegerschöld, C. (2016). Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs. PLoS ONE, 11(3), Article ID e0152116.
Open this publication in new window or tab >>Structural and Functional Analysis of Calcium Ion Mediated Binding of 5-Lipoxygenase to Nanodiscs
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2016 (English)In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 11, no 3, e0152116Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Public Library of Science, 2016
National Category
Biophysics
Identifiers
urn:nbn:se:kth:diva-185630 (URN)10.1371/journal.pone.0152116 (DOI)000372708000092 ()27010627 (PubMedID)2-s2.0-84962094377 (Scopus ID)
Funder
Swedish Research CouncilStockholm County Council
Note

QC 20160428

Available from: 2016-04-28 Created: 2016-04-25 Last updated: 2017-11-30Bibliographically approved
Kuang, Q., Purhonen, P., Pattipaka, T., Ayele, Y. H., Hebert, H. & Köck, P. J. (2015). A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy. Microscopy and Microanalysis, 21(4), 876-85.
Open this publication in new window or tab >>A Refined Single-Particle Reconstruction Procedure to Process Two-Dimensional Crystal Images from Transmission Electron Microscopy
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2015 (English)In: Microscopy and Microanalysis, ISSN 1431-9276, E-ISSN 1435-8115, Vol. 21, no 4, 876-85 p.Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
Cambridge University Press, 2015
Keyword
cryo-EM, electron crystallography, melibiose permease, membrane proteins, single-particle reconstruction
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-171876 (URN)10.1017/S1431927615000616 (DOI)000358834600009 ()25990985 (PubMedID)2-s2.0-84938421884 (Scopus ID)
Funder
Swedish Research Council
Note

QC 20150817

Available from: 2015-08-17 Created: 2015-08-10 Last updated: 2017-12-04Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-3220-9402

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