@misc{14592, abstract = {Cryo-electron microscopy (cryo-EM) of cellular specimens provides insights into biological processes and structures within a native context. However, a major challenge still lies in the efficient and reproducible preparation of adherent cells for subsequent cryo-EM analysis. This is due to the sensitivity of many cellular specimens to the varying seeding and culturing conditions required for EM experiments, the often limited amount of cellular material and also the fragility of EM grids and their substrate. Here, we present low-cost and reusable 3D printed grid holders, designed to improve specimen preparation when culturing challenging cellular samples directly on grids. The described grid holders increase cell culture reproducibility and throughput, and reduce the resources required for cell culturing. We show that grid holders can be integrated into various cryo-EM workflows, including micro-patterning approaches to control cell seeding on grids, and for generating samples for cryo-focused ion beam milling and cryo-electron tomography experiments. Their adaptable design allows for the generation of specialized grid holders customized to a large variety of applications.}, author = {Schur, Florian KM}, publisher = {Institute of Science and Technology Austria}, title = {{STL-files for 3D-printed grid holders described in Fäßler F, Zens B, et al.; 3D printed cell culture grid holders for improved cellular specimen preparation in cryo-electron microscopy}}, doi = {10.15479/AT:ISTA:14592}, year = {2020}, } @inbook{6890, abstract = {Describing the protein interactions that form pleomorphic and asymmetric viruses represents a considerable challenge to most structural biology techniques, including X-ray crystallography and single particle cryo-electron microscopy. Obtaining a detailed understanding of these interactions is nevertheless important, considering the number of relevant human pathogens that do not follow strict icosahedral or helical symmetry. Cryo-electron tomography and subtomogram averaging methods provide structural insights into complex biological environments and are well suited to go beyond structures of perfectly symmetric viruses. This chapter discusses recent developments showing that cryo-ET and subtomogram averaging can provide high-resolution insights into hitherto unknown structural features of pleomorphic and asymmetric virus particles. It also describes how these methods have significantly added to our understanding of retrovirus capsid assemblies in immature and mature viruses. Additional examples of irregular viruses and their associated proteins, whose structures have been studied via cryo-ET and subtomogram averaging, further support the versatility of these methods.}, author = {Obr, Martin and Schur, Florian KM}, booktitle = {Complementary Strategies to Study Virus Structure and Function}, editor = {Rey, Félix A.}, isbn = {9780128184561}, issn = {0065-3527}, pages = {117--159}, publisher = {Elsevier}, title = {{Structural analysis of pleomorphic and asymmetric viruses using cryo-electron tomography and subtomogram averaging}}, doi = {10.1016/bs.aivir.2019.07.008}, volume = {105}, year = {2019}, } @article{5907, abstract = {Microalgae of the genus Chlorella vulgaris are candidates for the production of lipids for biofuel production. Besides that, Chlorella vulgaris is marketed as protein and vitamin rich food additive. Its potential as a novel expression system for recombinant proteins inspired us to study its asparagine-linked oligosaccharides (N-glycans) by mass spectrometry, chromatography and gas chromatography. Oligomannosidic N-glycans with up to nine mannoses were the structures found in culture collection strains as well as several commercial products. These glycans co-eluted with plant N-glycans in the highly shape selective porous graphitic carbon chromatography. Thus, Chlorella vulgaris generates oligomannosidic N-glycans of the structural type known from land plants and animals. In fact, Man5 (Man5GlcNAc2) served as substrate for GlcNAc-transferase I and a trace of an endogenous structure with terminal GlcNAc was seen. The unusual more linear Man5 structure recently found on glycoproteins of Chlamydomonas reinhardtii occurred - if at all - in traces only. Notably, a majority of the oligomannosidic glycans was multiply O-methylated with 3-O-methyl and 3,6-di-O-methyl mannoses at the non-reducing termini. This modification has so far been neither found on plant nor vertebrate N-glycans. It’s possible immunogenicity raises concerns as to the use of C. vulgaris for production of pharmaceutical glycoproteins.}, author = {Mócsai, Réka and Figl, Rudolf and Troschl, Clemens and Strasser, Richard and Svehla, Elisabeth and Windwarder, Markus and Thader, Andreas and Altmann, Friedrich}, journal = {Scientific Reports}, number = {1}, publisher = {Nature Publishing Group}, title = {{N-glycans of the microalga Chlorella vulgaris are of the oligomannosidic type but highly methylated}}, doi = {10.1038/s41598-018-36884-1}, volume = {9}, year = {2019}, } @article{6343, abstract = {Cryo-electron tomography (cryo-ET) provides unprecedented insights into the molecular constituents of biological environments. In combination with an image processing method called subtomogram averaging (STA), detailed 3D structures of biological molecules can be obtained in large, irregular macromolecular assemblies or in situ, without the need for purification. The contextual meta-information these methods also provide, such as a protein’s location within its native environment, can then be combined with functional data. This allows the derivation of a detailed view on the physiological or pathological roles of proteins from the molecular to cellular level. Despite their tremendous potential in in situ structural biology, cryo-ET and STA have been restricted by methodological limitations, such as the low obtainable resolution. Exciting progress now allows one to reach unprecedented resolutions in situ, ranging in optimal cases beyond the nanometer barrier. Here, I review current frontiers and future challenges in routinely determining high-resolution structures in in situ environments using cryo-ET and STA.}, author = {Schur, Florian KM}, issn = {0959-440X}, journal = {Current Opinion in Structural Biology}, number = {10}, pages = {1--9}, publisher = {Elsevier}, title = {{Toward high-resolution in situ structural biology with cryo-electron tomography and subtomogram averaging}}, doi = {10.1016/j.sbi.2019.03.018}, volume = {58}, year = {2019}, } @article{5770, abstract = {Retroviruses assemble and bud from infected cells in an immature form and require proteolytic maturation for infectivity. The CA (capsid) domains of the Gag polyproteins assemble a protein lattice as a truncated sphere in the immature virion. Proteolytic cleavage of Gag induces dramatic structural rearrangements; a subset of cleaved CA subsequently assembles into the mature core, whose architecture varies among retroviruses. Murine leukemia virus (MLV) is the prototypical γ-retrovirus and serves as the basis of retroviral vectors, but the structure of the MLV CA layer is unknown. Here we have combined X-ray crystallography with cryoelectron tomography to determine the structures of immature and mature MLV CA layers within authentic viral particles. This reveals the structural changes associated with maturation, and, by comparison with HIV-1, uncovers conserved and variable features. In contrast to HIV-1, most MLV CA is used for assembly of the mature core, which adopts variable, multilayered morphologies and does not form a closed structure. Unlike in HIV-1, there is similarity between protein–protein interfaces in the immature MLV CA layer and those in the mature CA layer, and structural maturation of MLV could be achieved through domain rotations that largely maintain hexameric interactions. Nevertheless, the dramatic architectural change on maturation indicates that extensive disassembly and reassembly are required for mature core growth. The core morphology suggests that wrapping of the genome in CA sheets may be sufficient to protect the MLV ribonucleoprotein during cell entry.}, author = {Qu, Kun and Glass, Bärbel and Doležal, Michal and Schur, Florian and Murciano, Brice and Rein, Alan and Rumlová, Michaela and Ruml, Tomáš and Kräusslich, Hans-Georg and Briggs, John A. G.}, issn = {00278424}, journal = {Proceedings of the National Academy of Sciences}, number = {50}, pages = {E11751--E11760}, publisher = {Proceedings of the National Academy of Sciences}, title = {{Structure and architecture of immature and mature murine leukemia virus capsids}}, doi = {10.1073/pnas.1811580115}, volume = {115}, year = {2018}, } @article{150, abstract = {A short, 14-amino-acid segment called SP1, located in the Gag structural protein1, has a critical role during the formation of the HIV-1 virus particle. During virus assembly, the SP1 peptide and seven preceding residues fold into a six-helix bundle, which holds together the Gag hexamer and facilitates the formation of a curved immature hexagonal lattice underneath the viral membrane2,3. Upon completion of assembly and budding, proteolytic cleavage of Gag leads to virus maturation, in which the immature lattice is broken down; the liberated CA domain of Gag then re-assembles into the mature conical capsid that encloses the viral genome and associated enzymes. Folding and proteolysis of the six-helix bundle are crucial rate-limiting steps of both Gag assembly and disassembly, and the six-helix bundle is an established target of HIV-1 inhibitors4,5. Here, using a combination of structural and functional analyses, we show that inositol hexakisphosphate (InsP6, also known as IP6) facilitates the formation of the six-helix bundle and assembly of the immature HIV-1 Gag lattice. IP6 makes ionic contacts with two rings of lysine residues at the centre of the Gag hexamer. Proteolytic cleavage then unmasks an alternative binding site, where IP6 interaction promotes the assembly of the mature capsid lattice. These studies identify IP6 as a naturally occurring small molecule that promotes both assembly and maturation of HIV-1.}, author = {Dick, Robert and Zadrozny, Kaneil K and Xu, Chaoyi and Schur, Florian and Lyddon, Terri D and Ricana, Clifton L and Wagner, Jonathan M and Perilla, Juan R and Ganser, Pornillos Barbie K and Johnson, Marc C and Pornillos, Owen and Vogt, Volker}, issn = {1476-4687}, journal = {Nature}, number = {7719}, pages = {509–512}, publisher = {Nature Publishing Group}, title = {{Inositol phosphates are assembly co-factors for HIV-1}}, doi = {10.1038/s41586-018-0396-4}, volume = {560}, year = {2018}, }