Characterization of Membrane Proteins Using Cryo-Electron Microscopy

2018 ◽  
Vol 94 (1) ◽  
pp. e72
Author(s):  
Vanessa Carvalho ◽  
Joachim W. Pronk ◽  
Andreas H. Engel
Keyword(s):  
Electron Microscopy ◽  
Membrane Proteins ◽  
Cryo Electron Microscopy

scholarly journals LRRC8A homohexameric channels poorly recapitulate VRAC regulation and pharmacology

2020 ◽  
Author(s):  
Toshiki Yamada ◽  
Eric E. Figueroa ◽  
Jerod S. Denton ◽  
Kevin Strange
Keyword(s):  
Electron Microscopy ◽  
Voltage Dependence ◽  
Function Analysis ◽  
Cell Swelling ◽  
Independent Manner ◽  
Cryo Electron Microscopy ◽  
Homologous Position ◽  
Pore Properties ◽  
Hct116 Cells

Swelling-activated VRACs are heterohexameric channels comprising LRRC8A and at least one other LRRC8 paralog. Cryo-electron microscopy (EM) structures of non-native LRRC8A and LRRC8D homohexamers have been described. We demonstrate here that LRRC8A homohexamers poorly recapitulate VRAC functional properties. Unlike VRACs, LRRC8A channels heterologously expressed in Lrr8c-/- HCT116 cells are poorly activated by low intracellular ionic strength (µ) and insensitive to cell swelling with normal µ. Combining low µ with swelling modestly activates LRRC8A allowing characterization of pore properties. VRACs are strongly inhibited by 10 mM DCPIB in a voltage-independent manner. In contrast, DCPIB block of LRRC8A is weak and voltage sensitive. Cryo-EM structures indicate that DCPIB block is dependent on arginine 103. Consistent with this, LRRC8A R103F mutants are insensitive to DCPIB. However, a LRRC8 chimeric channel in which R103 is replaced by a leucine at the homologous position is inhibited ~90% by 10 mM DCPIB in a voltage-independent manner. Coexpression of LRRC8A and LRRC8C gives rise to channels with DCPIB sensitivity that is strongly µ-dependent. At normal intracellular µ, LRRC8A+LRRC8C heterohexamers exhibit strong, voltage-independent DCPIB block that is insensitive to R103F. DCPIB inhibition is greatly reduced and exhibits voltage dependence with low intracellular µ. The R103F mutation has no effect on maximal DCPIB inhibition but eliminates voltage-dependence under low µ conditions. Our findings demonstrate that the LRRC8A cryo-EM structure and the use of heterologously expressed LRRC8 heterohexameric channels pose significant limitations for VRAC mutagenesis-based structure-function analysis. Native VRAC function is most closely mimicked by chimeric LRRC8 homohexameric channels.

scholarly journals Cryo-electron Microscopy of Membrane Proteins

2013 ◽  
pp. 325-341 ◽  
Author(s):  
Kenneth N. Goldie ◽  
Priyanka Abeyrathne ◽  
Fabian Kebbel ◽  
Mohamed Chami ◽  
Philippe Ringler ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Membrane Proteins ◽  
Cryo Electron Microscopy

Uncovering the structures of modular polyketide synthases

2015 ◽  
Vol 32 (3) ◽  
pp. 436-453 ◽  
Author(s):  
Kira J. Weissman
Keyword(s):  
Electron Microscopy ◽  
Structural Biology ◽  
Structural Characterization ◽  
Small Angle ◽  
Single Particle ◽  
Polyketide Synthases ◽  
X Ray ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy

This review covers a breakthrough in the structural biology of the gigantic modular polyketide synthases (PKS): the structural characterization of intact modules by single-particle cryo-electron microscopy and small-angle X-ray scattering.

scholarly journals Characterization of Influenza Vaccine Hemagglutinin Complexes by Cryo-Electron Microscopy and Image Analyses Reveals Structural Polymorphisms

2016 ◽  
Vol 23 (6) ◽  
pp. 483-495 ◽  
Author(s):  
Dustin M. McCraw ◽  
John R. Gallagher ◽  
Audray K. Harris
Keyword(s):  
Electron Microscopy ◽  
Influenza Virus ◽  
Vaccine Efficacy ◽  
Hydrophobic Interactions ◽  
Influenza Virus Infection ◽  
Molecular Structures ◽  
Molecular Organization ◽  
Subunit Vaccines ◽  
Cryo Electron Microscopy

ABSTRACTInfluenza virus afflicts millions of people worldwide on an annual basis. There is an ever-present risk that animal viruses will cross the species barrier to cause epidemics and pandemics resulting in great morbidity and mortality. Zoonosis outbreaks, such as the H7N9 outbreak, underscore the need to better understand the molecular organization of viral immunogens, such as recombinant influenza virus hemagglutinin (HA) proteins, used in influenza virus subunit vaccines in order to optimize vaccine efficacy. Here, using cryo-electron microscopy and image analysis, we show that recombinant H7 HA in vaccines formed macromolecular complexes consisting of variable numbers of HA subunits (range, 6 to 8). In addition, HA complexes were distributed across at least four distinct structural classes (polymorphisms). Three-dimensional (3D) reconstruction and molecular modeling indicated that HA was in the prefusion state and suggested that the oligomerization and the structural polymorphisms observed were due to hydrophobic interactions involving the transmembrane regions. These experiments suggest that characterization of the molecular structures of influenza virus HA complexes used in subunit vaccines will lead to better understanding of the differences in vaccine efficacy and to the optimization of subunit vaccines to prevent influenza virus infection.

scholarly journals Structure of the Ebola virus nucleoprotein – RNA complex

2019 ◽  
Author(s):  
Robert N. Kirchdoerfer ◽  
Erica Ollmann Saphire ◽  
Andrew B. Ward
Keyword(s):  
Electron Microscopy ◽  
Single Particle ◽  
Ebola Virus ◽  
Viral Proteins ◽  
Cryo Electron Microscopy ◽  
Emerging Virus ◽  
Deadly Disease ◽  
Rna Complex ◽  
Viral Nucleocapsid

AbstractEbola virus is an emerging virus capable of causing a deadly disease in humans. Replication, transcription and packaging of the viral genome is carried out by the viral nucleocapsid. The nucleocapsid is a complex of the viral nucleoprotein, RNA and several other viral proteins. The nucleoprotein NP forms large, RNA-bound, helical filaments and acts as a scaffold for additional viral proteins. The 3.1 Å single-particle cryo-electron microscopy structure of the nucleoprotein-RNA helical filament presented here resembles previous structures determined at lower resolution while providing improved molecular details of protein-protein and protein-RNA interactions. The higher resolution of the structure presented here will facilitate the design and characterization of novel and specific Ebola virus therapeutics targeting the nucleocapsid.SynopsisThe 3.1 Å single-particle cryo-electron microscopy structure of the RNA-bound, Ebola virus nucleoprotein helical filament provides molecular details of protein-protein and protein-RNA interactions.

Direct Imaging of Sodium Stearate Crystals Dispersed in Waterpropylene Glycol Mixtures by Cryo-Electron Microscopy

2001 ◽  
Vol 7 (S2) ◽  
pp. 734-735
Author(s):  
Yue Ma ◽  
J. Liang ◽  
Y. Zheng ◽  
S. L. Erlandsen ◽  
L. E. Scriven ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Practical Importance ◽  
Sodium Stearate ◽  
Original Structure ◽  
Replica Technique ◽  
X Ray Scattering ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
Surface Active

Cryo-scanning electron microscopy (cryo-SEM) and cryo-transmission electron microscopy (cryo- TEM), in conjunctions with rheological measurements, light and confocal microscopy, x-ray scattering, and solid state NMR, are used to characterize sodium stearate (NaSt) crystals dispersed in waterpropylene glycol (PG) mixtures at macroscopic, microscopic, molecular, and atomic levels. NaSt is a surface-active, structural agent in household and personal cleaning products, including deodorant sticks and soap bars. A better structural characterization of NaSt/PG/water systems has practical importance in personal care and cosmetic industries. NaSt crystals and other soap crystal morphologies have been studied by the TEM/replica technique. However, the replicas were made of the residue after the original sample or its aqueous dilution were dried, and the original structure may have been lost during drying. Cryo-SEM was not used to study NaSt crystals because of its lower resolution and because the crystals are highly susceptible to radiation damage by electron beam.

scholarly journals Structure of the posttranslational Sec protein-translocation channel complex from yeast

Science ◽  
2018 ◽  
Vol 363 (6422) ◽  
pp. 84-87 ◽  
Author(s):  
Samuel Itskanov ◽  
Eunyong Park
Keyword(s):  
Electron Microscopy ◽  
Saccharomyces Cerevisiae ◽  
Endoplasmic Reticulum ◽  
Membrane Proteins ◽  
Binding Sites ◽  
Protein Translocation ◽  
Secretory Proteins ◽  
Conducting Channel ◽  
Cryo Electron Microscopy ◽  
Lateral Gate

The Sec61 protein-conducting channel mediates transport of many proteins, such as secretory proteins, across the endoplasmic reticulum (ER) membrane during or after translation. Posttranslational transport is enabled by two additional membrane proteins associated with the channel, Sec63 and Sec62, but its mechanism is poorly understood. We determined a structure of the Sec complex (Sec61-Sec63-Sec71-Sec72) from Saccharomyces cerevisiae by cryo–electron microscopy (cryo-EM). The structure shows that Sec63 tightly associates with Sec61 through interactions in cytosolic, transmembrane, and ER-luminal domains, prying open Sec61’s lateral gate and translocation pore and thus activating the channel for substrate engagement. Furthermore, Sec63 optimally positions binding sites for cytosolic and luminal chaperones in the complex to enable efficient polypeptide translocation. Our study provides mechanistic insights into eukaryotic posttranslational protein translocation.

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