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Göttingen, Germany

Stark H.,MPI for Biophysical Chemistry | Stark H.,University of Gottingen
Methods in Enzymology | Year: 2010

Here, we review the GraFix (Gradient Fixation) method to purify and stabilize macromolecular complexes for single particle cryo-electron microscopy (cryo-EM). During GraFix, macromolecules undergo a weak, intramolecular chemical cross-linking while being purified by density gradient ultracentrifugation. GraFix-stabilized particles can be used directly for negative-stain cryo-EM or, after a brief buffer-exchange step, for unstained cryo-EM. This highly reproducible method has proved to dramatically reduce problems in heterogeneity due to particle dissociation during EM grid preparation. Additionally, there is often an appreciable increase in particles binding to the carbon support film. This and the fact that binding times can be drastically increased, with no apparent disruption of the native structures of the macromolecules, makes GraFix a method of choice when preparing low-abundance complexes for cryo-EM. The higher sample quality following GraFix purification is evident when examining raw images, which usually present a low background of fragmented particles, good particle dispersion, and high-contrast, well-defined particles. Setting up the GraFix method is straightforward, and the resulting improvement in sample homogeneity has been beneficial in successfully obtaining the 3D structures of numerous macromolecular complexes by cryo-EM in the past few years. © 2010 Elsevier Inc. Source

Ardenkjaer-Larsen J.-H.,General Electric | Ardenkjaer-Larsen J.-H.,Technical University of Denmark | Boebinger G.S.,Florida State University | Comment A.,Ecole Polytechnique Federale de Lausanne | And 16 more authors.
Angewandte Chemie - International Edition | Year: 2015

In the Spring of 2013, NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments, particularly when applied in biomolecular settings. This multi-author interdisciplinary Review presents a state-of-the-art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. All of these are orthogonal efforts, whose gains could multiply and thereby enhance the sensitivity of solid- and liquid-state experiments. While substantial advances have been made in all these areas, numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges, and the ways by which scientists and engineers are striving to solve them, are also addressed. A new spin on bio-NMR: This Review presents a state-of-the-art description of the leading approaches being considered today to improve the sensitivity of NMR spectroscopy, particularly as applied in biomolecular settings. The focus is on the future of ultrahigh-field NMR systems, emerging NMR detection technologies, new approaches to nuclear hyperpolarization, and progress in sample preparation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Boesler C.,MPI for Biophysical Chemistry | Boesler C.,Targos Molecular Pathology GmbH | Rigo N.,MPI for Biophysical Chemistry | Anokhina M.M.,MPI for Biophysical Chemistry | And 8 more authors.
Nature Communications | Year: 2016

The precise role of the spliceosomal DEAD-box protein Prp28 in higher eukaryotes remains unclear. We show that stable tri-snRNP association during pre-catalytic spliceosomal B complex formation is blocked by a dominant-negative hPrp28 mutant lacking ATPase activity. Complexes formed in the presence of ATPase-deficient hPrp28 represent a novel assembly intermediate, the pre-B complex, that contains U1, U2 and loosely associated tri-snRNP and is stalled before disruption of the U1/5′ss base pairing interaction, consistent with a role for hPrp28 in the latter. Pre-B and B complexes differ structurally, indicating that stable tri-snRNP integration is accompanied by substantial rearrangements in the spliceosome. Disruption of the U1/5′ss interaction alone is not sufficient to bypass the block by ATPase-deficient hPrp28, suggesting hPrp28 has an additional function at this stage of splicing. Our data provide new insights into the function of Prp28 in higher eukaryotes, and the requirements for stable tri-snRNP binding during B complex formation. Source

Westphal V.,MPI for Biophysical Chemistry | Hell S.W.,MPI for Biophysical Chemistry
Optics InfoBase Conference Papers | Year: 2011

Diffraction-unlimited imaging is one of the emerging fields in microscopy. In all of these techniques, fluorophore switching is key. The first technique developed is STED, recent advances will be shown. © 2011 OSA. Source

Samwer M.,MPI for Biophysical Chemistry | Dehne H.-J.,MPI for Biophysical Chemistry | Spira F.,Austrian Academy of Sciences | Kollmar M.,Systems Biology of Motor Proteins Group | And 3 more authors.
EMBO Journal | Year: 2013

Nuclei of Xenopus laevis oocytes grow 100 000-fold larger in volume than a typical somatic nucleus and require an unusual intranuclear F-actin scaffold for mechanical stability. We now developed a method for mapping F-actin interactomes and identified a comprehensive set of F-actin binders from the oocyte nuclei. Unexpectedly, the most prominent interactor was a novel kinesin termed NabKin (Nuclear and meiotic actin-bundling Kinesin). NabKin not only binds microtubules but also F-actin structures, such as the intranuclear actin bundles in prophase and the contractile actomyosin ring during cytokinesis. The interaction between NabKin and F-actin is negatively regulated by Importin-β and is responsive to spatial information provided by RanGTP. Disconnecting NabKin from F-actin during meiosis caused cytokinesis failure and egg polyploidy. We also found actin-bundling activity in Nabkin's somatic paralogue KIF14, which was previously shown to be essential for somatic cell division. Our data are consistent with the notion that NabKin/KIF14 directly link microtubules with F-actin and that such link is essential for cytokinesis. © 2013 European Molecular Biology Organization. Source

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