Understanding ribosome assembly: the structure of in vivo assembled immature 30S subunits revealed by cryo-electron microscopy

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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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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Cryo-electron microscopy structure of the H3-H4 octasome without histones H2A and H2B

10.1101/2021.10.27.466091 ◽

2021 ◽

Author(s):

Kayo Nozawa ◽

Yoshimasa Takizawa ◽

Leonidas Pierrakeas ◽

Kazumi Saikusa ◽

Satoko Akashi ◽

...

Keyword(s):

Electron Microscopy ◽

Base Pair ◽

Genome Architecture ◽

Histone H2a ◽

Cryo Electron Microscopy ◽

Eukaryotic Chromatin ◽

And Function ◽

In Cells ◽

Dna Wrapping

The canonical nucleosome, which represents the predominant packaging unit in eukaryotic chromatin, has an octameric core made up of two histone H2A-H2B and H3-H4 dimers with ~147 base-pair (bp) DNA wrapping around it. Non-nucleosome particles with alterative histone stoichiometries and DNA wrapping configurations have been found, and they could profoundly influence genome architecture and function. Here we solved the structure of the H3-H4 octasome, which is a nucleosome-like particle with a core made up of four H3-H4 dimers. Two conformations, open and closed, are determined at 3.9 angstrom and 3.6 angstrom resolutions by cryo-electron microscopy, respectively. The H3-H4 octasome, made up of a di-tetrameric core, is wrapped by ~120 bp DNA in 1.5 negative superhelical turns. The symmetrical halves are connected by a unique H4-H4' interface along the dyad axis. In vivo crosslinking of cysteine probes placed at another unique H3-H3' interface demonstrated the existence of the H3-H4 octasome in cells.

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Time Resolved Cryo-Electron Microscopy Of Ribosome Assembly using Microfluidic Mixing

Microscopy and Microanalysis ◽

10.1017/s1431927609096925 ◽

2009 ◽

Vol 15 (S2) ◽

pp. 942-943 ◽

Cited By ~ 1

Author(s):

D Barnard ◽

Z Lu ◽

TR Shaikh ◽

A Yassin ◽

H Mohamed ◽

...

Keyword(s):

Electron Microscopy ◽

Ribosome Assembly ◽

Microfluidic Mixing ◽

Time Resolved ◽

Cryo Electron Microscopy

Extended abstract of a paper presented at Microscopy and Microanalysis 2009 in Richmond, Virginia, USA, July 26 – July 30, 2009

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Cryo-EM structure of the mammalian ATP synthase tetramer bound with inhibitory protein IF1

Science ◽

10.1126/science.aaw4852 ◽

2019 ◽

Vol 364 (6445) ◽

pp. 1068-1075 ◽

Cited By ~ 46

Author(s):

Jinke Gu ◽

Laixing Zhang ◽

Shuai Zong ◽

Runyu Guo ◽

Tianya Liu ◽

...

Keyword(s):

Electron Microscopy ◽

Atp Synthase ◽

Mammalian Cells ◽

Low Ph ◽

Inhibitory Protein ◽

Cryo Electron Microscopy ◽

The Matrix ◽

Microscopy Method ◽

Electron Microscopy Method

The mitochondrial adenosine triphosphate (ATP) synthase produces most of the ATP required by mammalian cells. We isolated porcine tetrameric ATP synthase and solved its structure at 6.2-angstrom resolution using a single-particle cryo–electron microscopy method. Two classical V-shaped ATP synthase dimers lie antiparallel to each other to form an H-shaped ATP synthase tetramer, as viewed from the matrix. ATP synthase inhibitory factor subunit 1 (IF1) is a well-known in vivo inhibitor of mammalian ATP synthase at low pH. Two IF1 dimers link two ATP synthase dimers, which is consistent with the ATP synthase tetramer adopting an inhibited state. Within the tetramer, we refined structures of intact ATP synthase in two different rotational conformations at 3.34- and 3.45-Å resolution.

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