In situ structure determination by subtomogram averaging

Electron microscopy at 0.2nm point-to-point resolution, 10-10 torr specimei region vacuum and facilities for in-situ specimen cleaning presents intere; ing possibilities for surface structure determination. Three methods for examining the surfaces are available: reflection (REM), transmission (TEM) and profile imaging. Profile imaging is particularly useful because it giv good resolution perpendicular as well as parallel to the surface, and can therefore be used to determine the relationship between the surface and the bulk structure.

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In situ structure and organization of the influenza C virus surface glycoprotein

Nature Communications ◽

10.1038/s41467-021-21818-9 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Steinar Halldorsson ◽

Kasim Sader ◽

Jack Turner ◽

Lesley J. Calder ◽

Peter B. Rosenthal

Keyword(s):

Membrane Fusion ◽

Hexagonal Lattice ◽

Surface Glycoprotein ◽

Structural Basis ◽

Virus Surface ◽

The Matrix ◽

Influenza C Virus ◽

Subtomogram Averaging ◽

Electron Cryotomography

AbstractThe lipid-enveloped influenza C virus contains a single surface glycoprotein, the haemagglutinin-esterase-fusion (HEF) protein, that mediates receptor binding, receptor destruction, and membrane fusion at the low pH of the endosome. Here we apply electron cryotomography and subtomogram averaging to describe the structural basis for hexagonal lattice formation by HEF on the viral surface. The conformation of the glycoprotein in situ is distinct from the structure of the isolated trimeric ectodomain, showing that a splaying of the membrane distal domains is required to mediate contacts that form the lattice. The splaying of these domains is also coupled to changes in the structure of the stem region which is involved in membrane fusion, thereby linking HEF’s membrane fusion conformation with its assembly on the virus surface. The glycoprotein lattice can form independent of other virion components but we show a major role for the matrix layer in particle formation.

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ChemInform Abstract: EXAFS Investigations of Metal Organic Molecules - Structure Determination and in situ Observation of Chemical Reactions

ChemInform ◽

10.1002/chin.199638320 ◽

2010 ◽

Vol 27 (38) ◽

pp. no-no

Author(s):

T. S. ERTEL ◽

W. HOERNER ◽

U. KOLB ◽

H. BERTAGNOLLI

Keyword(s):

Chemical Reactions ◽

Structure Determination ◽

Organic Molecules ◽

In Situ Observation ◽

Metal Organic

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A guided approach for subtomogram averaging of challenging macromolecular assemblies

10.1101/2020.02.01.930297 ◽

2020 ◽

Author(s):

Danielle Grotjahn ◽

Saikat Chowdhury ◽

Gabriel C. Lander

Keyword(s):

Electron Tomography ◽

A Priori ◽

Three Dimensional ◽

Structural Heterogeneity ◽

Dimensional Structure ◽

Diverse Range ◽

Macromolecular Assemblies ◽

Cryo Electron Tomography ◽

Subtomogram Averaging

AbstractCryo-electron tomography is a powerful biophysical technique enabling three-dimensional visualization of complex biological systems. Macromolecular targets of interest identified within cryo-tomograms can be computationally extracted, aligned, and averaged to produce a better-resolved structure through a process called subtomogram averaging (STA). However, accurate alignment of macromolecular machines that exhibit extreme structural heterogeneity and conformational flexibility remains a significant challenge with conventional STA approaches. To expand the applicability of STA to a broader range of pleomorphic complexes, we developed a user-guided, focused refinement approach that can be incorporated into the standard STA workflow to facilitate the robust alignment of particularly challenging samples. We demonstrate that it is possible to align visually recognizable portions of multi-subunit complexes by providing a priori information regarding their relative orientations within cryo-tomograms, and describe how this strategy was applied to successfully elucidate the first three-dimensional structure of the dynein-dynactin motor protein complex bound to microtubules. Our approach expands the application of STA for solving a more diverse range of heterogeneous biological structures, and establishes a conceptual framework for the development of automated strategies to deconvolve the complexity of crowded cellular environments and improve in situ structure determination technologies.

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In situ structure determination using single particle cryo-electron microscopy images

10.1101/2020.09.04.282509 ◽

2020 ◽

Author(s):

Jing Cheng ◽

Bufan Li ◽

Long Si ◽

Xinzheng Zhang

Keyword(s):

Electron Microscopy ◽

Structure Determination ◽

Single Particle ◽

Structure Refinement ◽

Target Function ◽

Target Protein ◽

Particle Analysis ◽

Cryo Electron Microscopy ◽

Tilt Series

AbstractCryo-electron microscopy (cryo-EM) tomography is a powerful tool for in situ structure determination. However, this method requires the acquisition of tilt series, and its time consuming throughput of acquiring tilt series severely slows determination of in situ structures. By treating the electron densities of non-target protein as non-Gaussian distributed noise, we developed a new target function that greatly improves the efficiency of the recognition of the target protein in a single cryo-EM image without acquiring tilt series. Moreover, we developed a sorting function that effectively eliminates the false positive detection, which not only improves the resolution during the subsequent structure refinement procedure but also allows using homolog proteins as models to recognize the target protein. Together, we developed an in situ single particle analysis (isSPA) method. Our isSPA method was successfully applied to solve structures of glycoproteins on the surface of a non-icosahedral virus and Rubisco inside the carboxysome. The cryo-EM data from both samples were collected within 24 hours, thus allowing fast and simple structural determination in situ.

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