Structure of the AAVhu.37 capsid by cryoelectron microscopy

Adeno-associated viruses (AAVs) are used as in vivo gene-delivery vectors in gene-therapy products and have been heavily investigated for numerous indications. Over 100 naturally occurring AAV serotypes and variants have been isolated from primate samples. Many reports have described unique properties of these variants (for instance, differences in potency, target cell or evasion of the immune response), despite high amino-acid sequence conservation. AAVhu.37 is of interest for clinical applications owing to its proficient transduction of the liver and central nervous system. The sequence identity of the AAVhu.37 VP1 to the well characterized AAVrh.10 serotype, for which no structure is available, is greater than 98%. Here, the structure of the AAVhu.37 capsid at 2.56 Å resolution obtained via single-particle cryo-electron microscopy is presented.

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Cryo-Electron Microscopy Structure of Seneca Valley Virus Procapsid

Journal of Virology ◽

10.1128/jvi.01927-17 ◽

2017 ◽

Vol 92 (6) ◽

Cited By ~ 7

Author(s):

Mike Strauss ◽

Nadishka Jayawardena ◽

Eileen Sun ◽

Richard A. Easingwood ◽

Laura N. Burga ◽

...

Keyword(s):

Electron Microscopy ◽

Virus Infections ◽

Content Type ◽

Agricultural Industry ◽

Lung Cancers ◽

Cryo Electron Microscopy ◽

Naturally Occurring ◽

Interesting Possibility ◽

Seneca Valley Virus

ABSTRACTSeneca Valley virus (SVV), like some other members of thePicornaviridae, forms naturally occurring empty capsids, known as procapsids. Procapsids have the same antigenicity as full virions, so they present an interesting possibility for the formation of stable virus-like particles. Interestingly, although SVV is a livestock pathogen, it has also been found to preferentially infect tumor cells and is being explored for use as a therapeutic agent in the treatment of small-cell lung cancers. Here we used cryo-electron microscopy to investigate the procapsid structure and describe the transition of capsid protein VP0 to the cleaved forms of VP4 and VP2. We show that the SVV receptor binds the procapsid, as evidence of its native antigenicity. In comparing the procapsid structure to that of the full virion, we also show that a cage of RNA serves to stabilize the inside surface of the virus, thereby making it more acid stable.IMPORTANCEViruses are extensively studied to help us understand infection and disease. One of the by-products of some virus infections are the naturally occurring empty virus capsids (containing no genome), termed procapsids, whose function remains unclear. Here we investigate the structure and formation of the procapsids of Seneca Valley virus, to better understand how they form, what causes them to form, how they behave, and how we can make use of them. One potential benefit of this work is the modification of the procapsid to develop it for targetedin vivodelivery of therapeutics or to make a stable vaccine against SVV, which could be of great interest to the agricultural industry.

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Using cryo-electron microscopy maps for X-ray structure determination of homologues

Acta Crystallographica Section D Structural Biology ◽

10.1107/s2059798319015924 ◽

2020 ◽

Vol 76 (1) ◽

pp. 63-72

Author(s):

Lingxiao Zeng ◽

Wei Ding ◽

Quan Hao

Keyword(s):

Electron Microscopy ◽

Hybrid Method ◽

Model Building ◽

Test Cases ◽

X Ray ◽

X Ray Crystallography ◽

Sequence Identity ◽

Whole Process ◽

Cryo Electron Microscopy

The combination of cryo-electron microscopy (cryo-EM) and X-ray crystallography reflects an important trend in structural biology. In a previously published study, a hybrid method for the determination of X-ray structures using initial phases provided by the corresponding parts of cryo-EM maps was presented. However, if the target structure of X-ray crystallography is not identical but homologous to the corresponding molecular model of the cryo-EM map, then the decrease in the accuracy of the starting phases makes the whole process more difficult. Here, a modified hybrid method is presented to handle such cases. The whole process includes three steps: cryo-EM map replacement, phase extension by NCS averaging and dual-space iterative model building. When the resolution gap between the cryo-EM and X-ray crystallographic data is large and the sequence identity is low, an intermediate stage of model building is necessary. Six test cases have been studied with sequence identity between the corresponding molecules in the cryo-EM and X-ray structures ranging from 34 to 52% and with sequence similarity ranging from 86 to 91%. This hybrid method consistently produced models with reasonable R work and R free values which agree well with the previously determined X-ray structures for all test cases, thus indicating the general applicability of the method for X-ray structure determination of homologues using cryo-EM maps as a starting point.

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Structural insight into nucleosome transcription by RNA polymerase II with elongation factors

Science ◽

10.1126/science.aav8912 ◽

2019 ◽

Vol 363 (6428) ◽

pp. 744-747 ◽

Cited By ~ 38

Author(s):

Haruhiko Ehara ◽

Tomoya Kujirai ◽

Yuka Fujino ◽

Mikako Shirouzu ◽

Hitoshi Kurumizaka ◽

...

Keyword(s):

Electron Microscopy ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Transcription Elongation ◽

Chromosomal Dna ◽

Elongation Factors ◽

Cryo Electron Microscopy ◽

Structural Insight ◽

Insight Into

RNA polymerase II (RNAPII) transcribes chromosomal DNA that contains multiple nucleosomes. The nucleosome forms transcriptional barriers, and nucleosomal transcription requires several additional factors in vivo. We demonstrate that the transcription elongation factors Elf1 and Spt4/5 cooperatively lower the barriers and increase the RNAPII processivity in the nucleosome. The cryo–electron microscopy structures of the nucleosome-transcribing RNAPII elongation complexes (ECs) reveal that Elf1 and Spt4/5 reshape the EC downstream edge and intervene between RNAPII and the nucleosome. They facilitate RNAPII progression through superhelical location SHL(–1) by adjusting the nucleosome in favor of the forward progression. They suppress pausing at SHL(–5) by preventing the stable RNAPII-nucleosome interaction. Thus, the EC overcomes the nucleosomal barriers while providing a platform for various chromatin functions.

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Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.

The Journal of Cell Biology ◽

10.1083/jcb.109.3.955 ◽

1989 ◽

Vol 109 (3) ◽

pp. 955-970 ◽

Cited By ~ 126

Author(s):

C W Akey

Keyword(s):

Electron Microscopy ◽

Mirror Symmetry ◽

Nuclear Lamina ◽

Nucleocytoplasmic Transport ◽

Nuclear Pore ◽

Effective Diameter ◽

Nuclear Pore Complexes ◽

Cryo Electron Microscopy ◽

Symmetrical Configuration

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.

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Template-free 13-protofilament microtubule–MAP assembly visualized at 8 Å resolution

The Journal of Cell Biology ◽

10.1083/jcb.201007081 ◽

2010 ◽

Vol 191 (3) ◽

pp. 463-470 ◽

Cited By ~ 91

Author(s):

Franck J. Fourniol ◽

Charles V. Sindelar ◽

Béatrice Amigues ◽

Daniel K. Clare ◽

Geraint Thomas ◽

...

Keyword(s):

Electron Microscopy ◽

Structural Information ◽

Binding Mode ◽

Microtubule Associated Proteins ◽

Cryo Electron Microscopy ◽

Associated Proteins ◽

Template Free ◽

Map Function ◽

Insight Into

Microtubule-associated proteins (MAPs) are essential for regulating and organizing cellular microtubules (MTs). However, our mechanistic understanding of MAP function is limited by a lack of detailed structural information. Using cryo-electron microscopy and single particle algorithms, we solved the 8 Å structure of doublecortin (DCX)-stabilized MTs. Because of DCX’s unusual ability to specifically nucleate and stabilize 13-protofilament MTs, our reconstruction provides unprecedented insight into the structure of MTs with an in vivo architecture, and in the absence of a stabilizing drug. DCX specifically recognizes the corner of four tubulin dimers, a binding mode ideally suited to stabilizing both lateral and longitudinal lattice contacts. A striking consequence of this is that DCX does not bind the MT seam. DCX binding on the MT surface indirectly stabilizes conserved tubulin–tubulin lateral contacts in the MT lumen, operating independently of the nucleotide bound to tubulin. DCX’s exquisite binding selectivity uncovers important insights into regulation of cellular MTs.

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A microtubule RELION-based pipeline for cryo-EM image processing

10.1101/673566 ◽

2019 ◽

Author(s):

Alexander D. Cook ◽

Szymon W. Manka ◽

Su Wang ◽

Carolyn A. Moores ◽

Joseph Atherton

Keyword(s):

Electron Microscopy ◽

Image Processing ◽

Structural Similarity ◽

Geometric Lattice ◽

Contrast Transfer Function ◽

Biologically Relevant ◽

Cryo Electron Microscopy

AbstractMicrotubules are polar filaments built from αβ-tubulin heterodimers that exhibit a range of architectures in vitro and in vivo. Tubulin heterodimers are arranged helically in the microtubule wall but many physiologically relevant architectures exhibit a break in helical symmetry known as the seam. Noisy 2D cryo-electron microscopy projection images of pseudo-helical microtubules therefore depict distinct but highly similar views owing to the high structural similarity of α- and β-tubulin. The determination of the αβ-tubulin register and seam location during image processing is essential for alignment accuracy that enables determination of biologically relevant structures. Here we present a pipeline designed for image processing and high-resolution reconstruction of cryo-electron microscopy microtubule datasets, based in the popular and user-friendly RELION image-processing package, Microtubule RELION-based Pipeline (MiRP). The pipeline uses a combination of supervised classification and prior knowledge about geometric lattice constraints in microtubules to accurately determine microtubule architecture and seam location. The presented method is fast and semi-automated, producing near-atomic resolution reconstructions with test datasets that contain a range of microtubule architectures and binding proteins.AbbreviationsMiRP, Microtubule RELION-based Pipeline; cryo-EM, cryo-electron microscopy; MT, microtubule; CTF, contrast transfer function; PF, protofilament.

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Structural basis of prokaryotic ubiquitin-like protein engagement and translocation by the mycobacterial Mpa-proteasome complex

Nature Communications ◽

10.1038/s41467-021-27787-3 ◽

2022 ◽

Vol 13 (1) ◽

Author(s):

Mikhail Kavalchuk ◽

Ahmad Jomaa ◽

Andreas U. Müller ◽

Eilika Weber-Ban

Keyword(s):

Electron Microscopy ◽

Mycobacterium Tuberculosis ◽

Early Stage ◽

Structural Basis ◽

Human Pathogen ◽

Core Particle ◽

Cryo Electron Microscopy ◽

Complementary Interactions

AbstractProteasomes are present in eukaryotes, archaea and Actinobacteria, including the human pathogen Mycobacterium tuberculosis, where proteasomal degradation supports persistence inside the host. In mycobacteria and other members of Actinobacteria, prokaryotic ubiquitin-like protein (Pup) serves as a degradation tag post-translationally conjugated to target proteins for their recruitment to the mycobacterial proteasome ATPase (Mpa). Here, we use single-particle cryo-electron microscopy to determine the structure of Mpa in complex with the 20S core particle at an early stage of pupylated substrate recruitment, shedding light on the mechanism of substrate translocation. Two conformational states of Mpa show how substrate is translocated stepwise towards the degradation chamber of the proteasome core particle. We also demonstrate, in vitro and in vivo, the importance of a structural feature in Mpa that allows formation of alternating charge-complementary interactions with the proteasome resulting in radial, rail-guided movements during the ATPase conformational cycle.

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Comparative Analysis of the Capsid Structures of AAVrh.10, AAVrh.39, and AAV8

Journal of Virology ◽

10.1128/jvi.01769-19 ◽

2019 ◽

Vol 94 (6) ◽

Cited By ~ 3

Author(s):

Mario Mietzsch ◽

Candace Barnes ◽

Joshua A. Hull ◽

Paul Chipman ◽

Jun Xie ◽

...

Keyword(s):

Electron Microscopy ◽

Central Nervous System ◽

Nervous System ◽

Comparative Analysis ◽

Image Reconstruction ◽

Gene Delivery ◽

Variable Regions ◽

Structure Information ◽

Cryo Electron Microscopy ◽

Therapeutic Genes

ABSTRACT Adeno-associated viruses (AAVs) from clade E are often used as vectors in gene delivery applications. This clade includes rhesus isolate 10 (AAVrh.10) and 39 (AAVrh.39) which, unlike representative AAV8, are capable of crossing the blood-brain barrier (BBB), thereby enabling the delivery of therapeutic genes to the central nervous system. Here, the capsid structures of AAV8, AAVrh.10 and AAVrh.39 have been determined by cryo-electron microscopy and three-dimensional image reconstruction to 3.08-, 2.75-, and 3.39-Å resolution, respectively, to enable a direct structural comparison. AAVrh.10 and AAVrh.39 are 98% identical in amino acid sequence but only ∼93.5% identical to AAV8. However, the capsid structures of all three viruses are similar, with only minor differences observed in the previously described surface variable regions, suggesting that specific residues S269 and N472, absent in AAV8, may confer the ability to cross the BBB in AAVrh.10 and AAVrh.39. Head-to-head comparison of empty and genome-containing particles showed DNA ordered in the previously described nucleotide-binding pocket, supporting the suggested role of this pocket in DNA packaging for the Dependoparvovirus. The structural characterization of these viruses provides a platform for future vector engineering efforts toward improved gene delivery success with respect to specific tissue targeting, transduction efficiency, antigenicity, or receptor retargeting. IMPORTANCE Recombinant adeno-associated virus vectors (rAAVs), based on AAV8 and AAVrh.10, have been utilized in multiple clinical trials to treat different monogenetic diseases. The closely related AAVrh.39 has also shown promise in vivo. As recently attained for other AAV biologics, e.g., Luxturna and Zolgensma, based on AAV2 and AAV9, respectively, the vectors in this study will likely gain U.S. Food and Drug Administration approval for commercialization in the near future. This study characterized the capsid structures of these clinical vectors at atomic resolution using cryo-electron microscopy and image reconstruction for comparative analysis. The analysis suggested two key residues, S269 and N472, as determinants of BBB crossing for AAVrh.10 and AAVrh.39, a feature utilized for central nervous system delivery of therapeutic genes. The structure information thus provides a platform for engineering to improve receptor retargeting or tissue specificity. These are important challenges in the field that need attention. Capsid structure information also provides knowledge potentially applicable for regulatory product approval.

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Catalytic synthesis of PEGylated EGCG conjugates that disaggregate Alzheimer’s tau

Synthesis ◽

10.1055/a-1509-5904 ◽

2021 ◽

Author(s):

Anton El Khoury ◽

Paul M. Seidler ◽

David S. Eisenberg ◽

Patrick G. Harran

Keyword(s):

Electron Microscopy ◽

Epigallocatechin Gallate ◽

Catalytic Synthesis ◽

Protecting Group ◽

Linker Length ◽

Cryo Electron Microscopy ◽

Naturally Occurring ◽

Brain Extracts ◽

Tau Fibrils

The naturally occurring flavonoid (–)-epigallocatechin gallate (EGCG) is a potent disaggregant of tau fibrils. Guided by the recent cryo-electron microscopy (cryoEM) structure of EGCG bound to fibrils of tau derived from an Alzheimer’s brain donor, we report methods to site-specifically modify the EGCG D-ring with aminoPEGylated linkers. The resultant molecules inhibit tau fibril seeding in Alzheimer’s brain extracts. Formulations of aminoPEGylated EGCG conjugated to the (quasi)-brain-penetrant nanoparticle Ferumoxytol inhibit seeding by AD-tau with linker length affecting activity. The protecting group free catalytic cycloaddition of amino azides to mono propargylated EGCG described here provides a blueprint for access to stable nanoparticulate forms of EGCG potentially useful as therapeutics to eliminate Alzheimer’s-related tau tangles.

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