Molecular architecture of the luminal ring of the<i>Xenopus laevis</i>nuclear pore complex

Zhang, Yanqing; Li, Sai; Zeng, Chao; Huang, Guangli; Zhu, Xinhui; Wang, Qifan; Wang, Kunpeng; Zhou, Qiang; Zhang, Wusheng; Yang, Guangwen; Liu, Minhao; Tao, Qinghua; Lei, Jianlin; Shi, Yigong

doi:10.1101/2020.03.27.009381

Cited by 11 publications

(13 citation statements)

References 55 publications

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“…Rather strikingly, we captured CA tubes threading through NUP153 NuPOD rings, which may represent the scenario of capsid passing through the NPC when the central pore is of sufficient size ( Figure 6). Although it is thought that the diameter of the NPC channel is less than the width of an HIV-1 capsid, recent evidence has shown that the NPC possesses a degree of elasticity, and the central channel may expand under certain conditions (Mahamid et al, 2016;Zhang et al, 2020). Consistently, our data provide direct evidence that assembled capsid structures can pass through a nuclear pore-like channel ( Figure 3D).…”

Section: Discussionsupporting

confidence: 85%

A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

Shen

Jang

et al. 2020

Preprint

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The capsid of human immunodeficiency virus 1 (HIV-1) plays a pivotal role in viral nuclear import, but the mechanism by which the viral core passages the nuclear pore complex (NPC) is poorly understood. Here, we use DNA-origami mimics of the NPC, termed NuPODs (NucleoPorins Organized by DNA), to reveal the mechanistic underpinnings of HIV-1 capsid nuclear entry. We found that trimeric interface formed via three capsid protein hexamers is targeted by a triple-arginine (RRR) motif but not the canonical phenylalanine-glycine (FG) motif of NUP153. As NUP153 is located on the nuclear face of the NPC, this result implies that the assembled capsid must cross the NPC in vivo. This hypothesis is corroborated by our observations of tubular capsid assemblies penetrating through NUP153 NuPODs. NUP153 prefers to bind highly curved capsid assemblies including those found at the tips of viral cores, thereby facilitating capsid insertion into the NPC. Furthermore, a balance of capsid stabilization by NUP153 and deformation by CPSF6, along with other cellular factors, may allow for the intact capsid to pass NPCs of various sizes. The NuPOD system serves as a unique tool for unraveling the previously elusive mechanisms of nuclear import of HIV-1 and other viruses.

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Section: Discussionsupporting

confidence: 85%

A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

Shen

Jang

et al. 2020

Preprint

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“…Cryo‐ET has provided insights into the structure and function of viruses [9–12,92–97], nuclear pore complexes [64,98–100], ribosomes [101–103], coated vesicles [104–109], nucleosome arrangement in chromatin [110], cytoskeletal elements [111–113], bacterial secretion systems [114–117], chemotaxis [118,119], flagellar motors [120], and S‐layers [121]. Obtaining information about the spatial arrangement of proteins by cryo‐ET has also found use in assessing protein denaturation at the air‐buffer interface for single‐particle analysis [122].…”

Section: Examplesmentioning

confidence: 99%

The promise and the challenges of cryo‐electron tomography

2020

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Structural biologists have traditionally approached cellular complexity in a reductionist manner in which the cellular molecular components are fractionated and purified before being studied individually. This ‘divide and conquer’ approach has been highly successful. However, awareness has grown in recent years that biological functions can rarely be attributed to individual macromolecules. Most cellular functions arise from their concerted action, and there is thus a need for methods enabling structural studies performed in situ, ideally in unperturbed cellular environments. Cryo‐electron tomography (Cryo‐ET) combines the power of 3D molecular‐level imaging with the best structural preservation that is physically possible to achieve. Thus, it has a unique potential to reveal the supramolecular architecture or ‘molecular sociology’ of cells and to discover the unexpected. Here, we review state‐of‐the‐art Cryo‐ET workflows, provide examples of biological applications, and discuss what is needed to realize the full potential of Cryo‐ET.

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“…This means that simply tilting the grid can have substantial benefits to improving the reconstruction. Indeed, many experimental cases have already made practical use of tilts to solve the preferred orientation problem and resolve the structure of a macromolecular object of interest [14][15][16][17][18][19][20][21], including samples as small as 40 kDa [22], and as large as the ~100 mDa nuclear pore complex [23,24]. The sampling plots, and the SCF* values, now provide quantitative insight explaining the benefits observed through experiment.…”

Section: The Effects Of Tilting the Grid On The Sampling Distribution And The Scf Valuementioning

confidence: 99%

Tools for visualizing and analyzing Fourier space sampling in Cryo-EM

Baldwin

Lyumkis

2021

Progress in Biophysics and Molecular Biology

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A complete understanding of how an orientation distribution contributes to a cryo-EM reconstruction remains lacking. It is necessary to begin critically assessing the set of views to gain an understanding of its effect on experimental reconstructions. Toward that end, we recently suggested that the type of orientation distribution may alter resolution measures in a systematic manner. We introduced the sampling compensation factor (SCF), which incorporates how the collection geometry might change the spectral signal-to-noise ratio (SSNR), irrespective of the other experimental aspects. We show here that knowledge of the sampling restricted to spherical surfaces of sufficiently large radii in Fourier space is equivalent to knowledge of the set of projection views. Moreover, the SCF geometrical factor may be calculated from one such surface. To aid cryo-EM researchers, we developed a graphical user interface (GUI) tool that evaluates experimental orientation distributions. The GUI returns plots of projection directions, sampling constrained to the surface of a sphere, the SCF value, the fraction of the empty region of Fourier space, and a histogram of the sampling values over the points on a sphere. Finally, a fixed tilt angle may be incorporated to determine how tilting the grid during collection may improve the distribution of views and Fourier space sampling. We advocate this simple conception of sampling and the use of such tools as a complement to the distribution of views to capture the different aspects of the effect of projection directions on cryo-EM reconstructions.

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Molecular architecture of the luminal ring of theXenopus laevisnuclear pore complex

Cited by 11 publications

References 55 publications

A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

A DNA-origami nuclear pore mimic reveals nuclear entry mechanisms of HIV-1 capsid

The promise and the challenges of cryo‐electron tomography

Tools for visualizing and analyzing Fourier space sampling in Cryo-EM

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