Publications by authors named "Henning Stahlberg"

151 Publications

Single Particle Analysis for High-Resolution 2D Electron Crystallography.

Methods Mol Biol 2021 ;2215:267-284

Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Basel, Switzerland.

Electron crystallography has been used for decades to determine three-dimensional structures of membrane proteins embedded in a lipid bilayer. However, high-resolution information could only be retrieved from samples where the 2D crystals were well ordered and perfectly flat. This is rarely the case in practice. We implemented in the FOCUS package a module to export transmission electron microscopy images of 2D crystals for 3D reconstruction by single particle algorithms. This approach allows for correcting local distortions of the 2D crystals, yielding much higher resolution reconstructions than otherwise expected from the observable diffraction spots. In addition, the single particle framework enables classification of heterogeneous structures coexisting within the 2D crystals. We provide here a detailed guide on single particle analysis of 2D crystal data based on the FOCUS and FREALIGN packages.
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http://dx.doi.org/10.1007/978-1-0716-0966-8_12DOI Listing
January 2021

Alterations in Sub-Axonal Architecture Between Normal Aging and Parkinson's Diseased Human Brains Using Label-Free Cryogenic X-ray Nanotomography.

Front Neurosci 2020 25;14:570019. Epub 2020 Nov 25.

Paul Scherrer Institut, Villigen, Switzerland.

Gaining insight to pathologically relevant processes in continuous volumes of unstained brain tissue is important for a better understanding of neurological diseases. Many pathological processes in neurodegenerative disorders affect myelinated axons, which are a critical part of the neuronal circuitry. Cryo ptychographic X-ray computed tomography in the multi-keV energy range is an emerging technology providing phase contrast at high sensitivity, allowing label-free and non-destructive three dimensional imaging of large continuous volumes of tissue, currently spanning up to 400,000 μm. This aspect makes the technique especially attractive for imaging complex biological material, especially neuronal tissues, in combination with downstream optical or electron microscopy techniques. A further advantage is that dehydration, additional contrast staining, and destructive sectioning/milling are not required for imaging. We have developed a pipeline for cryo ptychographic X-ray tomography of relatively large, hydrated and unstained biological tissue volumes beyond what is typical for the X-ray imaging, using human brain tissue and combining the technique with complementary methods. We present four imaged volumes of a Parkinson's diseased human brain and five volumes from a non-diseased control human brain using cryo ptychographic X-ray tomography. In both cases, we distinguish neuromelanin-containing neurons, lipid and melanic pigment, blood vessels and red blood cells, and nuclei of other brain cells. In the diseased sample, we observed several swellings containing dense granular material resembling clustered vesicles between the myelin sheaths arising from the cytoplasm of the parent oligodendrocyte, rather than the axoplasm. We further investigated the pathological relevance of such swollen axons in adjacent tissue sections by immunofluorescence microscopy for phosphorylated alpha-synuclein combined with multispectral imaging. Since cryo ptychographic X-ray tomography is non-destructive, the large dataset volumes were used to guide further investigation of such swollen axons by correlative electron microscopy and immunogold labeling post X-ray imaging, a possibility demonstrated for the first time. Interestingly, we find that protein antigenicity and ultrastructure of the tissue are preserved after the X-ray measurement. As many pathological processes in neurodegeneration affect myelinated axons, our work sets an unprecedented foundation for studies addressing axonal integrity and disease-related changes in unstained brain tissues.
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http://dx.doi.org/10.3389/fnins.2020.570019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7724048PMC
November 2020

Author Correction: High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica.

Nat Commun 2020 Nov 12;11(1):5873. Epub 2020 Nov 12.

Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland.

A Correction to this paper has been published: https://doi.org/10.1038/s41467-020-19845-z .
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http://dx.doi.org/10.1038/s41467-020-19845-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7665023PMC
November 2020

High-resolution cryo-EM structure of urease from the pathogen Yersinia enterocolitica.

Nat Commun 2020 10 9;11(1):5101. Epub 2020 Oct 9.

Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland.

Urease converts urea into ammonia and carbon dioxide and makes urea available as a nitrogen source for all forms of life except animals. In human bacterial pathogens, ureases also aid in the invasion of acidic environments such as the stomach by raising the surrounding pH. Here, we report the structure of urease from the pathogen Yersinia enterocolitica at 2 Å resolution from cryo-electron microscopy. Y. enterocolitica urease is a dodecameric assembly of a trimer of three protein chains, ureA, ureB and ureC. The high data quality enables detailed visualization of the urease bimetal active site and of the impact of radiation damage. The obtained structure is of sufficient quality to support drug development efforts.
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http://dx.doi.org/10.1038/s41467-020-18870-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7547064PMC
October 2020

Architecture of the centriole cartwheel-containing region revealed by cryo-electron tomography.

EMBO J 2020 Nov 20;39(22):e106246. Epub 2020 Sep 20.

Department of Cell Biology, University of Geneva, Sciences III, Geneva, Switzerland.

Centrioles are evolutionarily conserved barrels of microtubule triplets that form the core of the centrosome and the base of the cilium. While the crucial role of the proximal region in centriole biogenesis has been well documented, its native architecture and evolutionary conservation remain relatively unexplored. Here, using cryo-electron tomography of centrioles from four evolutionarily distant species, we report on the architectural diversity of the centriole's proximal cartwheel-bearing region. Our work reveals that the cartwheel central hub is constructed from a stack of paired rings with cartwheel inner densities inside. In both Paramecium and Chlamydomonas, the repeating structural unit of the cartwheel has a periodicity of 25 nm and consists of three ring pairs, with 6 radial spokes emanating and merging into a single bundle that connects to the microtubule triplet via the D2-rod and the pinhead. Finally, we identified that the cartwheel is indirectly connected to the A-C linker through the triplet base structure extending from the pinhead. Together, our work provides unprecedented evolutionary insights into the architecture of the centriole proximal region, which underlies centriole biogenesis.
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http://dx.doi.org/10.15252/embj.2020106246DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7667884PMC
November 2020

Single particle cryo-EM of the complex between interphotoreceptor retinoid-binding protein and a monoclonal antibody.

FASEB J 2020 Oct 28;34(10):13918-13934. Epub 2020 Aug 28.

Department of Ophthalmology, Gavin Herbert Eye Institute, University of California-Irvine, Irvine, CA, USA.

Interphotoreceptor retinoid-binding protein (IRBP) is a highly expressed protein secreted by rod and cone photoreceptors that has major roles in photoreceptor homeostasis as well as retinoid and polyunsaturated fatty acid transport between the neural retina and retinal pigment epithelium. Despite two crystal structures reported on fragments of IRBP and decades of research, the overall structure of IRBP and function within the visual cycle remain unsolved. Here, we studied the structure of native bovine IRBP in complex with a monoclonal antibody (mAb5) by cryo-electron microscopy, revealing the tertiary and quaternary structure at sufficient resolution to clearly identify the complex components. Complementary mass spectrometry experiments revealed the structure and locations of N-linked carbohydrate post-translational modifications. This work provides insight into the structure of IRBP, displaying an elongated, flexible three-dimensional architecture not seen among other retinoid-binding proteins. This work is the first step in elucidation of the function of this enigmatic protein.
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http://dx.doi.org/10.1096/fj.202000796RRDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7589273PMC
October 2020

Structural basis of Focal Adhesion Kinase activation on lipid membranes.

EMBO J 2020 Oct 11;39(19):e104743. Epub 2020 Aug 11.

Structural Biology Programme, Spanish National Cancer Research Centre, Madrid, Spain.

Focal adhesion kinase (FAK) is a key component of the membrane proximal signaling layer in focal adhesion complexes, regulating important cellular processes, including cell migration, proliferation, and survival. In the cytosol, FAK adopts an autoinhibited state but is activated upon recruitment into focal adhesions, yet how this occurs or what induces structural changes is unknown. Here, we employ cryo-electron microscopy to reveal how FAK associates with lipid membranes and how membrane interactions unlock FAK autoinhibition to promote activation. Intriguingly, initial binding of FAK to the membrane causes steric clashes that release the kinase domain from autoinhibition, allowing it to undergo a large conformational change and interact itself with the membrane in an orientation that places the active site toward the membrane. In this conformation, the autophosphorylation site is exposed and multiple interfaces align to promote FAK oligomerization on the membrane. We show that interfaces responsible for initial dimerization and membrane attachment are essential for FAK autophosphorylation and resulting cellular activity including cancer cell invasion, while stable FAK oligomerization appears to be needed for optimal cancer cell proliferation in an anchorage-independent manner. Together, our data provide structural details of a key membrane bound state of FAK that is primed for efficient autophosphorylation and activation, hence revealing the critical event in integrin mediated FAK activation and signaling at focal adhesions.
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http://dx.doi.org/10.15252/embj.2020104743DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7527928PMC
October 2020

Protease-activation using anti-idiotypic masks enables tumor specificity of a folate receptor 1-T cell bispecific antibody.

Nat Commun 2020 06 24;11(1):3196. Epub 2020 Jun 24.

Roche Pharma Research & Early Development, Roche Innovation Center Zurich, Wagistrasse 10, 8952, Schlieren, Switzerland.

T-cell bispecific antibodies (TCBs) crosslink tumor and T-cells to induce tumor cell killing. While TCBs are very potent, on-target off-tumor toxicity remains a challenge when selecting targets. Here, we describe a protease-activated anti-folate receptor 1 TCB (Prot-FOLR1-TCB) equipped with an anti-idiotypic anti-CD3 mask connected to the anti-CD3 Fab through a tumor protease-cleavable linker. The potency of this Prot- FOLR1-TCB is recovered following protease-cleavage of the linker releasing the anti-idiotypic anti-CD3 scFv. In vivo, the Prot-FOLR1-TCB mediates antitumor efficacy comparable to the parental FOLR1-TCB whereas a noncleavable control Prot-FOLR1-TCB is inactive. In contrast, killing of bronchial epithelial and renal cortical cells with low FOLR1 expression is prevented compared to the parental FOLR1-TCB. The findings are confirmed for mesothelin as alternative tumor antigen. Thus, masking the anti-CD3 Fab fragment with an anti-idiotypic mask and cleavage of the mask by tumor-specific proteases can be applied to enhance specificity and safety of TCBs.
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http://dx.doi.org/10.1038/s41467-020-16838-wDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7314773PMC
June 2020

The Contorsbody, an antibody format for agonism: Design, structure, and function.

Comput Struct Biotechnol J 2020 14;18:1210-1220. Epub 2020 May 14.

Roche Pharmaceutical Research and Early Development, Large Molecule Research, Roche Innovation Center Munich, Nonnenwald 2, 82377 Penzberg, Germany.

The careful design of the antibody architecture is becoming more and more important, especially when the purpose is agonism. We present the design of a novel antibody format that is able to promote receptor dimerization and induce signal transduction resulting in cell proliferation. Mono-specific bivalent Y-shape IgGs made of two light chains and two heavy chains are engineered into single chain dimers of two modified heavy chains, resulting in the fixation of the two Fab fragments along the Fc dimerizing moiety. By this, an antagonist of the Her-receptor family, Trastuzumab, is re-formatted into an agonist by simply incorporating the original binding motif into a different geometrically and sterically constrained conformation. This novel format, named Contorsbody, retains antigen binding properties of the parental IgGs and can be produced by standard technologies established for recombinant IgGs. Structural analyses using molecular dynamics and electron microscopy are described to guide the initial design and to confirm the Contorsbody as a very compact molecule, respectively. Contorsbodies show increased rigidity compared to IgGs and their Fab moieties are positioned parallel and adjacent to each other. This geometry has an increased potential to trigger cell surface antigen or receptor 'cis'-dimerization without 'trans'-bridging of cells or mere receptor blockade.
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http://dx.doi.org/10.1016/j.csbj.2020.05.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7283085PMC
May 2020

Cryo-EM, X-ray diffraction, and atomistic simulations reveal determinants for the formation of a supramolecular myelin-like proteolipid lattice.

J Biol Chem 2020 06 7;295(26):8692-8705. Epub 2020 Apr 7.

Faculty of Biochemistry and Molecular Medicine, University of Oulu, 90014 Oulu, Finland

Myelin protein P2 is a peripheral membrane protein of the fatty acid-binding protein family that functions in the formation and maintenance of the peripheral nerve myelin sheath. Several P2 gene mutations cause human Charcot-Marie-Tooth neuropathy, but the mature myelin sheath assembly mechanism is unclear. Here, cryo-EM of myelin-like proteolipid multilayers revealed an ordered three-dimensional (3D) lattice of P2 molecules between stacked lipid bilayers, visualizing supramolecular assembly at the myelin major dense line. The data disclosed that a single P2 layer is inserted between two bilayers in a tight intermembrane space of ∼3 nm, implying direct interactions between P2 and two membrane surfaces. X-ray diffraction from P2-stacked bicelle multilayers revealed lateral protein organization, and surface mutagenesis of P2 coupled with structure-function experiments revealed a role for both the portal region of P2 and its opposite face in membrane interactions. Atomistic molecular dynamics simulations of P2 on model membrane surfaces suggested that Arg-88 is critical for P2-membrane interactions, in addition to the helical lid domain. Negatively charged lipid headgroups stably anchored P2 on the myelin-like bilayer surface. Membrane binding may be accompanied by opening of the P2 β-barrel structure and ligand exchange with the apposing bilayer. Our results provide an unprecedented view into an ordered, multilayered biomolecular membrane system induced by the presence of a peripheral membrane protein from human myelin. This is an important step toward deciphering the 3D assembly of a mature myelin sheath at the molecular level.
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http://dx.doi.org/10.1074/jbc.RA120.013087DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7324508PMC
June 2020

New insights on the structure of alpha-synuclein fibrils using cryo-electron microscopy.

Curr Opin Neurobiol 2020 04 26;61:89-95. Epub 2020 Feb 26.

Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Mattenstrasse 26, 4058 Basel, Switzerland. Electronic address:

Fibrils of alpha-synuclein are significant components of cellular inclusions associated with several neuropathological disorders including Parkinson's disease, multiple system atrophy and dementia with Lewy bodies. In recent years, technological advances in the field of transmission electron microscopy and image processing have made it possible to solve the structure of alpha-synuclein fibrils at high resolution. This review discusses the results of structural studies using cryo-electron microscopy, which revealed that in-vitro produced fibrils vary in diameter from 5nm for single-protofilament fibrils, to 10nm for two-protofilament fibrils. In addition, the atomic models hint at contributions of the familial Parkinson's disease mutation sites to inter-protofilament interaction and the locations where post-translational modifications take place. Here, we propose a nomenclature system that allows identifying the existing alpha-synuclein polymorphs and that will allow to incorporate additional high-resolution structures determined in the future.
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http://dx.doi.org/10.1016/j.conb.2020.01.014DOI Listing
April 2020

A helical inner scaffold provides a structural basis for centriole cohesion.

Sci Adv 2020 02 14;6(7):eaaz4137. Epub 2020 Feb 14.

University of Geneva, Department of Cell Biology, Sciences III, Geneva, Switzerland.

The ninefold radial arrangement of microtubule triplets (MTTs) is the hallmark of the centriole, a conserved organelle crucial for the formation of centrosomes and cilia. Although strong cohesion between MTTs is critical to resist forces applied by ciliary beating and the mitotic spindle, how the centriole maintains its structural integrity is not known. Using cryo-electron tomography and subtomogram averaging of centrioles from four evolutionarily distant species, we found that MTTs are bound together by a helical inner scaffold covering ~70% of the centriole length that maintains MTTs cohesion under compressive forces. Ultrastructure Expansion Microscopy (U-ExM) indicated that POC5, POC1B, FAM161A, and Centrin-2 localize to the scaffold structure along the inner wall of the centriole MTTs. Moreover, we established that these four proteins interact with each other to form a complex that binds microtubules. Together, our results provide a structural and molecular basis for centriole cohesion and geometry.
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http://dx.doi.org/10.1126/sciadv.aaz4137DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7021493PMC
February 2020

Two new polymorphic structures of human full-length alpha-synuclein fibrils solved by cryo-electron microscopy.

Elife 2019 12 9;8. Epub 2019 Dec 9.

Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Basel, Switzerland.

Intracellular inclusions rich in alpha-synuclein are a hallmark of several neuropathological diseases including Parkinson's disease (PD). Previously, we reported the structure of alpha-synuclein fibrils (residues 1-121), composed of two protofibrils that are connected via a densely-packed interface formed by residues 50-57 (Guerrero-Ferreira, eLife 218;7:e36402). We here report two new polymorphic atomic structures of alpha-synuclein fibrils termed polymorphs 2a and 2b, at 3.0 Å and 3.4 Å resolution, respectively. These polymorphs show a radically different structure compared to previously reported polymorphs. The new structures have a 10 nm fibril diameter and are composed of two protofilaments which interact via intermolecular salt-bridges between amino acids K45, E57 (polymorph 2a) or E46 (polymorph 2b). The non-amyloid component (NAC) region of alpha-synuclein is fully buried by previously non-described interactions with the N-terminus. A hydrophobic cleft, the location of familial PD mutation sites, and the nature of the protofilament interface now invite to formulate hypotheses about fibril formation, growth and stability.
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http://dx.doi.org/10.7554/eLife.48907DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6957273PMC
December 2019

DuoMab: a novel CrossMab-based IgG-derived antibody format for enhanced antibody-dependent cell-mediated cytotoxicity.

MAbs 2019 Nov-Dec;11(8):1402-1414. Epub 2019 Sep 17.

Roche Pharma Research and Early Development (pRED), Discovery Oncology, Roche Innovation Center Zurich , Schlieren , Switzerland.

High specificity accompanied with the ability to recruit immune cells has made recombinant therapeutic antibodies an integral part of drug development. Here we present a generic approach to generate two novel IgG-derived antibody formats that are based on a modification of the CrossMab technology. MoAbs harbor two heavy chains (HCs) resulting in one binding entity and one fragment crystallizable region (Fc), whereas DuoMabs are composed of four HCs harboring two binding entities and two Fc regions linked at a disulfide-bridged hinge. The latter bivalent format is characterized by avidity-enhanced target cell binding while simultaneously increasing the 'Fc-load' on the surface. DuoMabs were shown to be producible in high yield and purity and bind to surface cells with affinities comparable to IgGs. The increased Fc load directed at the surface of target cells by DuoMabs modulates their antibody-dependent cell-mediated cytotoxicity competency toward target cells, making them attractive for applications that require or are modulated by FcR interactions.
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http://dx.doi.org/10.1080/19420862.2019.1661736DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6816436PMC
June 2020

Diverse roles of TssA-like proteins in the assembly of bacterial type VI secretion systems.

EMBO J 2019 09 12;38(18):e100825. Epub 2019 Aug 12.

Biozentrum, University of Basel, Basel, Switzerland.

Protein translocation by the bacterial type VI secretion system (T6SS) is driven by a rapid contraction of a sheath assembled around a tube with associated effectors. Here, we show that TssA-like or TagA-like proteins with a conserved N-terminal domain and varying C-terminal domains can be grouped into at least three distinct classes based on their role in sheath assembly. The proteins of the first class increase speed and frequency of sheath assembly and form a stable dodecamer at the distal end of a polymerizing sheath. The proteins of the second class localize to the cell membrane and block sheath polymerization upon extension across the cell. This prevents excessive sheath polymerization and bending, which may result in sheath destabilization and detachment from its membrane anchor and thus result in failed secretion. The third class of these proteins localizes to the baseplate and is required for initiation of sheath assembly. Our work shows that while various proteins share a conserved N-terminal domain, their roles in T6SS biogenesis are fundamentally different.
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http://dx.doi.org/10.15252/embj.2018100825DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6745524PMC
September 2019

"Differential Visual Proteomics": Enabling the Proteome-Wide Comparison of Protein Structures of Single-Cells.

J Proteome Res 2019 09 12;18(9):3521-3531. Epub 2019 Aug 12.

Center for Cellular Imaging and NanoAnalytics, Biozentrum , University of Basel , Mattenstrasse 26 , 4058 Basel , Switzerland.

Proteins are involved in all tasks of life, and their characterization is essential to understand the underlying mechanisms of biological processes. We present a method called "differential visual proteomics" geared to study proteome-wide structural changes of proteins and protein-complexes between a disturbed and an undisturbed cell or between two cell populations. To implement this method, the cells are lysed and the lysate is prepared in a lossless manner for single-particle electron microscopy (EM). The samples are subsequently imaged in the EM. Individual particles are computationally extracted from the images and pooled together, while keeping track of which particle originated from which specimen. The extracted particles are then aligned and classified. A final quantitative analysis of the particle classes found identifies the particle structures that differ between positive and negative control samples. The algorithm and a graphical user interface developed to perform the analysis and to visualize the results were tested with simulated and experimental data. The results are presented, and the potential and limitations of the current implementation are discussed. We envisage the method as a tool for the untargeted profiling of the structural changes in the proteome of single-cells as a response to a disturbing force.
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http://dx.doi.org/10.1021/acs.jproteome.9b00447DOI Listing
September 2019

Imaging of post-mortem human brain tissue using electron and X-ray microscopy.

Curr Opin Struct Biol 2019 10 23;58:138-148. Epub 2019 Jul 23.

Center for Cellular Imaging and NanoAnalytics (C-CINA), Biozentrum, University of Basel, Switzerland.

Electron microscopy imaging of post-mortem human brain (PMHB) comes with a unique set of challenges due to numerous parameters beyond the researcher's control. Nevertheless, the wealth of information provided by the ultrastructural analysis of PMHB is proving crucial in our understanding of neurodegenerative diseases. This review highlights the importance of such studies and covers challenges, limitations and recent developments in the application of current EM imaging, including cryo-ET and correlative hybrid techniques, on PMHB.
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http://dx.doi.org/10.1016/j.sbi.2019.06.003DOI Listing
October 2019

Microfluidic protein isolation and sample preparation for high-resolution cryo-EM.

Proc Natl Acad Sci U S A 2019 07 10;116(30):15007-15012. Epub 2019 Jul 10.

Center for Cellular Imaging and Nanoanalytics, Biozentrum, 4058 Basel, Switzerland;

High-resolution structural information is essential to understand protein function. Protein-structure determination needs a considerable amount of protein, which can be challenging to produce, often involving harsh and lengthy procedures. In contrast, the several thousand to a few million protein particles required for structure determination by cryogenic electron microscopy (cryo-EM) can be provided by miniaturized systems. Here, we present a microfluidic method for the rapid isolation of a target protein and its direct preparation for cryo-EM. Less than 1 μL of cell lysate is required as starting material to solve the atomic structure of the untagged, endogenous human 20S proteasome. Our work paves the way for high-throughput structure determination of proteins from minimal amounts of cell lysate and opens more opportunities for the isolation of sensitive, endogenous protein complexes.
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http://dx.doi.org/10.1073/pnas.1907214116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6660723PMC
July 2019

Cryo-EM structure of the rhodopsin-Gαi-βγ complex reveals binding of the rhodopsin C-terminal tail to the gβ subunit.

Elife 2019 06 28;8. Epub 2019 Jun 28.

Division of Biology and Chemistry / Laboratory of Biomolecular Research, Paul Scherrer Institute, Villigen, Switzerland.

One of the largest membrane protein families in eukaryotes are G protein-coupled receptors (GPCRs). GPCRs modulate cell physiology by activating diverse intracellular transducers, prominently heterotrimeric G proteins. The recent surge in structural data has expanded our understanding of GPCR-mediated signal transduction. However, many aspects, including the existence of transient interactions, remain elusive. We present the cryo-EM structure of the light-sensitive GPCR rhodopsin in complex with heterotrimeric Gi. Our density map reveals the receptor C-terminal tail bound to the Gβ subunit of the G protein, providing a structural foundation for the role of the C-terminal tail in GPCR signaling, and of Gβ as scaffold for recruiting Gα subunits and G protein-receptor kinases. By comparing available complexes, we found a small set of common anchoring points that are G protein-subtype specific. Taken together, our structure and analysis provide new structural basis for the molecular events of the GPCR signaling pathway.
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http://dx.doi.org/10.7554/eLife.46041DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6629373PMC
June 2019

Lewy pathology in Parkinson's disease consists of crowded organelles and lipid membranes.

Nat Neurosci 2019 07 24;22(7):1099-1109. Epub 2019 Jun 24.

Roche Pharma Research and Early Development, Lead Discovery, Roche Innovation Center Basel, Basel, Switzerland.

Parkinson's disease, the most common age-related movement disorder, is a progressive neurodegenerative disease with unclear etiology. Key neuropathological hallmarks are Lewy bodies and Lewy neurites: neuronal inclusions immunopositive for the protein α-synuclein. In-depth ultrastructural analysis of Lewy pathology is crucial to understanding pathogenesis of this disease. Using correlative light and electron microscopy and tomography on postmortem human brain tissue from Parkinson's disease brain donors, we identified α-synuclein immunopositive Lewy pathology and show a crowded environment of membranes therein, including vesicular structures and dysmorphic organelles. Filaments interspersed between the membranes and organelles were identifiable in many but not all α-synuclein inclusions. Crowding of organellar components was confirmed by stimulated emission depletion (STED)-based super-resolution microscopy, and high lipid content within α-synuclein immunopositive inclusions was corroborated by confocal imaging, Fourier-transform coherent anti-Stokes Raman scattering infrared imaging and lipidomics. Applying such correlative high-resolution imaging and biophysical approaches, we discovered an aggregated protein-lipid compartmentalization not previously described in the Parkinsons' disease brain.
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http://dx.doi.org/10.1038/s41593-019-0423-2DOI Listing
July 2019

Tumor-targeted 4-1BB agonists for combination with T cell bispecific antibodies as off-the-shelf therapy.

Sci Transl Med 2019 06;11(496)

Roche Innovation Center Zurich, Roche Pharmaceutical Research and Early Development (pRED), Wagistrasse 10, 8952 Schlieren, Switzerland.

Endogenous costimulatory molecules on T cells such as 4-1BB (CD137) can be leveraged for cancer immunotherapy. Systemic administration of agonistic anti-4-1BB antibodies, although effective preclinically, has not advanced to phase 3 trials because they have been hampered by both dependency on Fcγ receptor-mediated hyperclustering and hepatotoxicity. To overcome these issues, we engineered proteins simultaneously targeting 4-1BB and a tumor stroma or tumor antigen: FAP-4-1BBL (RG7826) and CD19-4-1BBL. In the presence of a T cell receptor signal, they provide potent T cell costimulation strictly dependent on tumor antigen-mediated hyperclustering without systemic activation by FcγR binding. We could show targeting of FAP-4-1BBL to FAP-expressing tumor stroma and lymph nodes in a colorectal cancer-bearing rhesus monkey. Combination of FAP-4-1BBL with tumor antigen-targeted T cell bispecific (TCB) molecules in human tumor samples led to increased IFN-γ and granzyme B secretion. Further, combination of FAP- or CD19-4-1BBL with CEA-TCB (RG7802) or CD20-TCB (RG6026), respectively, resulted in tumor remission in mouse models, accompanied by intratumoral accumulation of activated effector CD8 T cells. FAP- and CD19-4-1BBL thus represent an off-the-shelf combination immunotherapy without requiring genetic modification of effector cells for the treatment of solid and hematological malignancies.
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http://dx.doi.org/10.1126/scitranslmed.aav5989DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7181714PMC
June 2019

Cryo-EM structures of the pore-forming A subunit from the Yersinia entomophaga ABC toxin.

Nat Commun 2019 04 26;10(1):1952. Epub 2019 Apr 26.

School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia Queensland, 4072, Australia.

ABC toxins are pore-forming virulence factors produced by pathogenic bacteria. YenTcA is the pore-forming and membrane binding A subunit of the ABC toxin YenTc, produced by the insect pathogen Yersinia entomophaga. Here we present cryo-EM structures of YenTcA, purified from the native source. The soluble pre-pore structure, determined at an average resolution of 4.4 Å, reveals a pentameric assembly that in contrast to other characterised ABC toxins is formed by two TcA-like proteins (YenA1 and YenA2) and decorated by two endochitinases (Chi1 and Chi2). We also identify conformational changes that accompany membrane pore formation by visualising YenTcA inserted into liposomes. A clear outward rotation of the Chi1 subunits allows for access of the protruding translocation pore to the membrane. Our results highlight structural and functional diversity within the ABC toxin subfamily, explaining how different ABC toxins are capable of recognising diverse hosts.
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http://dx.doi.org/10.1038/s41467-019-09890-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6486591PMC
April 2019

Retrieving high-resolution information from disordered 2D crystals by single-particle cryo-EM.

Nat Commun 2019 04 12;10(1):1722. Epub 2019 Apr 12.

Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, CH-4058, Basel, Switzerland.

Electron crystallography can reveal the structure of membrane proteins within 2D crystals under close-to-native conditions. High-resolution structural information can only be reached if crystals are perfectly flat and highly ordered. In practice, such crystals are difficult to obtain. Available image unbending algorithms correct for disorder, but only perform well on images of non-tilted, flat crystals, while out-of-plane distortions are not addressed. Here, we present an approach that employs single-particle refinement procedures to locally unbend crystals in 3D. With this method, density maps of the MloK1 potassium channel with a resolution of 4 Å were obtained from images of 2D crystals that do not diffract beyond 10 Å. Furthermore, 3D classification allowed multiple structures to be resolved, revealing a series of MloK1 conformations within a single 2D crystal. This conformational heterogeneity explains the poor diffraction observed and is related to channel function. The approach is implemented in the FOCUS package.
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http://dx.doi.org/10.1038/s41467-019-09661-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6461647PMC
April 2019

Cryo-EM structure of phosphodiesterase 6 reveals insights into the allosteric regulation of type I phosphodiesterases.

Sci Adv 2019 02 27;5(2):eaav4322. Epub 2019 Feb 27.

Center for Cellular Imaging and NanoAnalytics, Biozentrum, University of Basel, Mattenstrasse 26, 4058 Basel, Switzerland.

Cyclic nucleotide phosphodiesterases (PDEs) work in conjunction with adenylate/guanylate cyclases to regulate the key second messengers of G protein-coupled receptor signaling. Previous attempts to determine the full-length structure of PDE family members at high-resolution have been hindered by structural flexibility, especially in their linker regions and N- and C-terminal ends. Therefore, most structure-activity relationship studies have so far focused on truncated and conserved catalytic domains rather than the regulatory domains that allosterically govern the activity of most PDEs. Here, we used single-particle cryo-electron microscopy to determine the structure of the full-length PDE6αβ2γ complex. The final density map resolved at 3.4 Å reveals several previously unseen structural features, including a coiled N-terminal domain and the interface of PDE6γ subunits with the PDE6αβ heterodimer. Comparison of the PDE6αβ2γ complex with the closed state of PDE2A sheds light on the conformational changes associated with the allosteric activation of type I PDEs.
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http://dx.doi.org/10.1126/sciadv.aav4322DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6392808PMC
February 2019

Supramolecular architectures of molecularly thin yet robust free-standing layers.

Sci Adv 2019 Feb 22;5(2):eaav4489. Epub 2019 Feb 22.

Institute of Chemistry and Bioanalytics, School of Life Sciences, University of Applied Sciences and Arts Northwestern Switzerland, Hofackerstrasse 35, CH-4132 Muttenz, Switzerland.

Stable, single-nanometer thin, and free-standing two-dimensional layers with controlled molecular architectures are desired for several applications ranging from (opto-)electronic devices to nanoparticle and single-biomolecule characterization. It is, however, challenging to construct these stable single molecular layers via self-assembly, as the cohesion of those systems is ensured only by in-plane bonds. We herein demonstrate that relatively weak noncovalent bonds of limited directionality such as dipole-dipole (-CN⋅⋅⋅NC-) interactions act in a synergistic fashion to stabilize crystalline monomolecular layers of tetrafunctional calixarenes. The monolayers produced, demonstrated to be free-standing, display a well-defined atomic structure on the single-nanometer scale and are robust under a wide range of conditions including photon and electron radiation. This work opens up new avenues for the fabrication of robust, single-component, and free-standing layers via bottom-up self-assembly.
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http://dx.doi.org/10.1126/sciadv.aav4489DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6386556PMC
February 2019

Molecular structure and function of myelin protein P0 in membrane stacking.

Sci Rep 2019 01 24;9(1):642. Epub 2019 Jan 24.

Department of Biomedicine, University of Bergen, Bergen, Norway.

Compact myelin forms the basis of nerve insulation essential for higher vertebrates. Dozens of myelin membrane bilayers undergo tight stacking, and in the peripheral nervous system, this is partially enabled by myelin protein zero (P0). Consisting of an immunoglobulin (Ig)-like extracellular domain, a single transmembrane helix, and a cytoplasmic extension (P0ct), P0 harbours an important task in ensuring the integrity of compact myelin in the extracellular compartment, referred to as the intraperiod line. Several disease mutations resulting in peripheral neuropathies have been identified for P0, reflecting its physiological importance, but the arrangement of P0 within the myelin ultrastructure remains obscure. We performed a biophysical characterization of recombinant P0ct. P0ct contributes to the binding affinity between apposed cytoplasmic myelin membrane leaflets, which not only results in changes of the bilayer properties, but also potentially involves the arrangement of the Ig-like domains in a manner that stabilizes the intraperiod line. Transmission electron cryomicroscopy of native full-length P0 showed that P0 stacks lipid membranes by forming antiparallel dimers between the extracellular Ig-like domains. The zipper-like arrangement of the P0 extracellular domains between two membranes explains the double structure of the myelin intraperiod line. Our results contribute to the understanding of PNS myelin, the role of P0 therein, and the underlying molecular foundation of compact myelin stability in health and disease.
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http://dx.doi.org/10.1038/s41598-018-37009-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6345808PMC
January 2019

Cerebral Corpora amylacea are dense membranous labyrinths containing structurally preserved cell organelles.

Sci Rep 2018 12 21;8(1):18046. Epub 2018 Dec 21.

Department of Biology and Chemistry, Paul Scherrer Institute, Villigen, Switzerland.

Corpora amylacea are cell-derived structures that appear physiologically in the aged human brain. While their histological identification is straightforward, their ultrastructural composition and microenvironment at the nanoscale have remained unclear so far, as has their relevance to aging and certain disease states that involve the sequestration of toxic cellular metabolites. Here, we apply correlative serial block-face scanning electron microscopy and transmission electron tomography to gain three-dimensional insight into the ultrastructure and surrounding microenvironment of cerebral Corpora amylacea in the human brainstem and hippocampal region. We find that cerebral Corpora amylacea are composed of dense labyrinth-like sheets of lipid membranes, contain vesicles as well as morphologically preserved mitochondria, and are in close proximity to blood vessels and the glymphatic system, primarily within the cytoplasm of perivascular glial cells. Our results clarify the nature of cerebral Corpora amylacea and provide first hints on how they may arise and develop in the aging brain.
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http://dx.doi.org/10.1038/s41598-018-36223-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6303404PMC
December 2018

TDP-43 extracted from frontotemporal lobar degeneration subject brains displays distinct aggregate assemblies and neurotoxic effects reflecting disease progression rates.

Nat Neurosci 2019 01 17;22(1):65-77. Epub 2018 Dec 17.

Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.

Accumulation of abnormally phosphorylated TDP-43 (pTDP-43) is the main pathology in affected neurons of people with amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Morphological diversity and neuroanatomical distribution of pTDP-43 accumulations allowed classification of FTLD cases into at least four subtypes, which are correlated with clinical presentations and genetic causes. To understand the molecular basis of this heterogeneity, we developed SarkoSpin, a new method for biochemical isolation of pathological TDP-43. By combining SarkoSpin with mass spectrometry, we revealed proteins beyond TDP-43 that become abnormally insoluble in a disease subtype-specific manner. We show that pTDP-43 extracted from brain forms stable assemblies of distinct densities and morphologies that are associated with disease subtypes. Importantly, biochemically extracted pTDP-43 assemblies showed differential neurotoxicity and seeding that were correlated with disease duration of FTLD subjects. Our data are consistent with the notion that disease heterogeneity could originate from alternate pathological TDP-43 conformations, which are reminiscent of prion strains.
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http://dx.doi.org/10.1038/s41593-018-0294-yDOI Listing
January 2019

Self-Assembly of a Designed Nucleoprotein Architecture through Multimodal Interactions.

ACS Cent Sci 2018 Nov 15;4(11):1578-1586. Epub 2018 Nov 15.

Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093, United States.

The co-self-assembly of proteins and nucleic acids (NAs) produces complex biomolecular machines (e.g., ribosomes and telomerases) that represent some of the most daunting targets for biomolecular design. Despite significant advances in protein and DNA or RNA nanotechnology, the construction of artificial nucleoprotein complexes has largely been limited to cases that rely on the NA-mediated spatial organization of protein units, rather than a cooperative interplay between protein- and NA-mediated interactions that typify natural nucleoprotein assemblies. We report here a structurally well-defined synthetic nucleoprotein assembly that forms through the synergy of three types of intermolecular interactions: Watson-Crick base pairing, NA-protein interactions, and protein-metal coordination. The fine thermodynamic balance between these interactions enables the formation of a crystalline architecture under highly specific conditions.
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http://dx.doi.org/10.1021/acscentsci.8b00745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6276041PMC
November 2018