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The Resolution Revolution in CryoEM Requires High-quality Sample Preparation: a Rapid Pipeline to a High-resolution Map of Yeast Fatty Acid Synthase
Single-particle electron cryo-microscopy (cryoEM) has undergone a `resolution revolution' that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions and sample-preparation methods can result in disassembly or denaturation. The structure of the 2.6%MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. Here, a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM is reported. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, the new protocol yielded a 3.1%Å resolution map of yeast FAS from 15%000 automatically picked particles within a day. The high map quality enabled a complete atomic model of an intact fungal FAS to be built.
The Resolution Revolution in CryoEM Requires New Sample Preparation Procedures: a Rapid Pipeline to High Resolution Maps of Yeast FAS
Single-particle electron cryo-microscopy (cryoEM) has undergone a “resolution revolution” that makes it possible to characterize megadalton (MDa) complexes at atomic resolution without crystals. To fully exploit the new opportunities in molecular microscopy, new procedures for the cloning, expression and purification of macromolecular complexes need to be explored. Macromolecular assemblies are often unstable, and invasive construct design or inadequate purification conditions or sample preparation methods can result in disassembly or denaturation. The structure of the 2.6 MDa yeast fatty acid synthase (FAS) has been studied by electron microscopy since the 1960s. We report a new, streamlined protocol for the rapid production of purified yeast FAS for structure determination by high-resolution cryoEM. Together with a companion protocol for preparing cryoEM specimens on a hydrophilized graphene layer, our new protocol has yielded a 3.1 Å map of yeast FAS from 15,000 automatically picked particles within a day. The high map quality enabled us to build a complete atomic model of an intact fungal FAS.
Probing the Modularity of Megasynthases by Rational Engineering of a Fatty Acid Synthase (FAS) Type I
Modularity is an aspect of a decomposable system with a coordinating authority that acts as a glue which holds the loosely held components. These multi-component entities (“modules”) facilitate rewiring into different designs allowing for change. Such modular character is a fundamental property of many biological entities, especially the family of megasynthases such as polyketide synthases (PKSs). The ability of these PKSs to produce diverse product spectra is strongly coupled to their broad architectural modularity. Decoding the molecular basis of modularity, i.e. identifying the folds and domains that comprise the modules as well as understanding constrains of the assembly of modules, ...
Structural Insights Into the Mechanism of Archaellar Rotational Switching
Abstract: Signal transduction via phosphorylated CheY towards the flagellum and the archaellum involves a conserved mechanism of CheY phosphorylation and subsequent conformational changes within CheY. This mechanism is conserved among bacteria and archaea, despite substantial differences in the composition and architecture of archaellum and flagellum, respectively. Phosphorylated CheY has higher affinity towards the bacterial C-ring and its binding leads to conformational changes in the flagellar motor and subsequent rotational switching of the flagellum. In archaea, the adaptor protein CheF resides at the cytoplasmic face of the archaeal C-ring formed by the proteins ArlCDE and interacts wi...
Sei naiv und mach' ein Experiment: Feodor Lynen
1964 wurden Feodor Lynen und Konrad Bloch mit dem Nobelpreis für Physiologie oder Medizin ausgezeichnet. Seit Feodor Lynens Todesjahr 1979 erhalten jährlich bis zu 150 Nachwuchswissenschaftler ein Feodor-Lynen-Stipendium für Forschung im Ausland von der Alexander von Humboldt-Stiftung (AvH)und seit 1982 gibt es die Feodor Lynen Lecture für die erfolgreichsten Biochemiker der Welt. Heike Will verfasste nun die erste Biographie in engem Austausch mit vielen Zeitzeugen über Feodor Lynen als Mensch - bayerisches Urgestein -, Wissenschaftler und diplomatischen Gestalter. Sie beschreibt die Evolution der Biochemie von den dreißiger bis zu den achtziger Jahren des 20. Jahrhunderts: Wie Feodor...
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