scholarly journals Simplified Approach for Preparing Graphene Oxide TEM Grids for Stained and Vitrified Biomolecules

Nanomaterials ◽  
2021 ◽  
Vol 11 (3) ◽  
pp. 643
Author(s):  
Anil Kumar ◽  
Nayanika Sengupta ◽  
Somnath Dutta
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Biological Macromolecules ◽  
Contrast Transfer Function ◽  
Discharge System ◽  
Simplified Approach ◽  
Heavy Equipment ◽  
Peristaltic Pumps ◽  
High Resolution Structure

In this manuscript, we report the application of graphene oxide (GO) in the preparation of cryo-electron microscopy (cryo-EM) and transmission electron microscopy (TEM) grids. We treated GO with water and organic solvents, such as, methanol, ethanol and isopropanol separately to isolate significantly large GO monolayer flake to fabricate the grids for cryo-EM and TEM study. We implemented a simplified approach to isolate flakes of GO monolayer for constructing the TEM grids, independent of expensive heavy equipment (Langmuir–Blodgett trough, glow-discharge system, carbon-evaporator or plasma-cleaner or peristaltic pumps). We employed confocal microscopy, SEM and TEM to characterize the flake size, stability and transparency of the GO monolayer and atomic force microscopy (AFM) to probe the depth of GO coated grids. Additionally, GO grids are visualized at cryogenic condition for suitability of GO monolayer for cryo-EM study. In addition, GO-Met-H2O grids reduce the effect of preferred orientation of biological macromolecules within the amorphous ice. The power-spectrum and contrast-transfer-function unequivocally suggest that GO-Met-H2O fabricated holey grids have excellent potential for application in high-resolution structural characterization of biomolecules. Furthermore, only 200 movies and ~8000 70S ribosome particles are selected on GO-coated grids for cryo-EM reconstruction to achieve high-resolution structure.

Techniques for Obtaining High Resolution Information about Periodic Arrays of Biological Macromolecules in the Electron Microscope

1982 ◽  
Vol 40 ◽  
pp. 68-71
Author(s):  
S. B. Hayward
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structure Analysis ◽  
Structural Information ◽  
Probability Distributions ◽  
High Resolution Electron Microscopy ◽  
Three Dimensions ◽  
Biological Macromolecules ◽  
Periodic Arrays ◽  
High Resolution Structure

In the past six years considerable effort has been expended in EM structure analysis of periodic arrays of biological macromolecules. Although sparked by the success of the moderately high resolution structure analysis of purple membrane J most of this work has been limited in resolution to anywhere from 30 to 15 A. Although there is much to learn out to this range of resolution, especially in three dimensions, there is a wealth of structural information awaiting us beyond it. Our ultimate goal must, of course, be information in the range of 3 A, which for proteins is enough to allow us to trace the path of the polypeptide chain. In fact, much of this information is accessible by electron microscopy, providing that the periodic arrays are ordered to this extent. However, great care must be taken to optimize all aspects of microscopy and image processing in order for structural information of a resolution close to the instrumental limitation to be obtained. Following are some of the more important factors which will determine success or failure of an attempt at high resolution imaging, in this case assumed to be of an untilted specimen. While some of these factors have been known for years by workers in the field of high resolution electron microscopy, others are problems unique to imaging biological macromolecular arrays. The recent solution of some of these problems involves the use of probability distributions to describe image phases.

High-Resolution Cryo-Electron Microscopy of Biological Macromolecules

1990 ◽  
Vol 48 (1) ◽  
pp. 126-127
Author(s):  
Yoshinori Fujiyoshi
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Diffraction Pattern ◽  
Superfluid Helium ◽  
Copper Phthalocyanine ◽  
Optical Diffraction ◽  
Irradiation Damage ◽  
Biological Macromolecules ◽  
Cross Sectional ◽  
Instrumental Resolution

The resolution of direct images of biological macromolecules is normally restricted to far less than 0.3 nm. This is not due instrumental resolution, but irradiation damage. The damage to biological macromolecules may expect to be reduced when they are cooled to a very low temperature. We started to develop a new cryo-stage for a high resolution electron microscopy in 1983, and successfully constructed a superfluid helium stage for a 400 kV microscope by 1986, whereby chlorinated copper-phthalocyanine could be photographed to a resolution of 0.26 nm at a stage temperature of 1.5 K. We are continuing to develop the cryo-microscope and have developed a cryo-microscope equipped with a superfluid helium stage and new cryo-transfer device.The New cryo-microscope achieves not only improved resolution but also increased operational ease. The construction of the new super-fluid helium stage is shown in Fig. 1, where the cross sectional structure is shown parallel to an electron beam path. The capacities of LN2 tank, LHe tank and the pot are 1400 ml, 1200 ml and 3 ml, respectively. Their surfaces are placed with gold to minimize thermal radiation. Consumption rates of liquid nitrogen and liquid helium are 170 ml/hour and 140 ml/hour, respectively. The working time of this stage is more than 7 hours starting from full LN2 and LHe tanks. Instrumental resolution of our cryo-stage cooled to 4.2 K was confirmed to be 0.20 nm by an optical diffraction pattern from the image of a chlorinated copper-phthalocyanine crystal. The image and the optical diffraction pattern are shown in Fig. 2 a, b, respectively.

scholarly journals Rapid high-resolution structure analysis of small, biotechnologically relevant enzymes by cryo-electron microscopy

2021 ◽  
Author(s):  
Nicole Dimos ◽  
Carl P.O. Helmer ◽  
Andrea M. Chanique ◽  
Markus C. Wahl ◽  
Robert Kourist ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Biology ◽  
Structure Analysis ◽  
Cryo Electron Microscopy ◽  
High Resolution Structure ◽  
Fast Development ◽  
Pharmaceutical Industries ◽  
Exquisite Specificity ◽  
Resolution Structure

Enzyme catalysis has emerged as a key technology for developing efficient, sustainable processes in the chemical, biotechnological and pharmaceutical industries. Plants provide large and diverse pools of biosynthetic enzymes that facilitate complex reactions, such as the formation of intricate terpene carbon skeletons, with exquisite specificity. High-resolution structural analysis of these enzymes is crucial to understand their mechanisms and modulate their properties by targeted engineering. Although cryo-electron microscopy (cryo-EM) has revolutionized structural biology, its applicability to high-resolution structure analysis of comparatively small enzymes is so far largely unexplored. Here, we show that cryo-EM can reveal the structures of ~120 kDa plant borneol dehydrogenases at or below 2 Å resolution, paving the way for the fast development of new biocatalysts that provide access to bioactive terpenes and terpenoids.

scholarly journals Graphene Oxide Decorated Nanometal-Poly(Anilino-Dodecylbenzene Sulfonic Acid) for Application in High Performance Supercapacitors

Micromachines ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 115 ◽  
Author(s):  
Nomxolisi R. Dywili ◽  
Afroditi Ntziouni ◽  
Chinwe Ikpo ◽  
Miranda Ndipingwi ◽  
Ntuthuko W. Hlongwa ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Specific Capacitance ◽  
Sulfonic Acid ◽  
High Performance ◽  
Pt Nanoparticles ◽  
Metal Nanoparticle ◽  
Vibration Band ◽  
Dodecylbenzene Sulfonic Acid

Graphene oxide (GO) decorated with silver (Ag), copper (Cu) or platinum (Pt) nanoparticles that are anchored on dodecylbenzene sulfonic acid (DBSA)-doped polyaniline (PANI) were prepared by a simple one-step method and applied as novel materials for high performance supercapacitors. High-resolution transmission electron microscopy (HRTEM) and high-resolution scanning electron microscopy (HRSEM) analyses revealed that a metal-decorated polymer matrix is embedded within the GO sheet. This caused the M/DBSA–PANI (M = Ag, Cu or Pt) particles to adsorb on the surface of the GO sheets, appearing as aggregated dark regions in the HRSEM images. The Fourier transform infrared (FTIR) spectroscopy studies revealed that GO was successfully produced and decorated with Ag, Cu or Pt nanoparticles anchored on DBSA–PANI. This was confirmed by the appearance of the GO signature epoxy C–O vibration band at 1040 cm−1 (which decreased upon the introduction of metal nanoparticle) and the PANI characteristic N–H stretching vibration band at 3144 cm−1 present only in the GO/M/DBSA–PANI systems. The composites were tested for their suitability as supercapacitor materials; and specific capacitance values of 206.4, 192.8 and 227.2 F·g−1 were determined for GO/Ag/DBSA–PANI, GO/Cu/DBSA–PANI and GO/Pt/DBSA–PANI, respectively. The GO/Pt/DBSA–PANI electrode exhibited the best specific capacitance value of the three electrodes and also had twice the specific capacitance value reported for Graphene/MnO2//ACN (113.5 F·g−1). This makes GO/Pt/DBSA–PANI a very promising organic supercapacitor material.

scholarly journals High-resolution structure determination of sub-100 kilodalton complexes using conventional cryo-EM

2018 ◽  
Author(s):  
Mark A. Herzik ◽  
Mengyu Wu ◽  
Gabriel C. Lander
Keyword(s):  
High Resolution ◽  
Structure Determination ◽  
Drug Target ◽  
Phase Plate ◽  
Biological Macromolecules ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
High Resolution Structure ◽  
Resolution Structure

Determining high-resolution structures of biological macromolecules with masses of less than 100 kilodaltons (kDa) has long been a goal of the cryo-electron microscopy (cryo-EM) community. While the Volta Phase Plate has enabled cryo-EM structure determination of biological specimens of this size range, use of this instrumentation is not yet fully automated and can present technical challenges. Here, we show that conventional defocus-based cryo-EM methodologies can be used to determine the high-resolution structures of specimens amassing less than 100 kDa using a transmission electron microscope operating at 200 keV coupled with a direct electron detector. Our ~2.9 Å structure of alcohol dehydrogenase (82 kDa) proves that bound ligands can be resolved with high fidelity, indicating that these methodologies can be used to investigate the molecular details of drug-target interactions. Our ~2.8 Å and ~3.2 Å resolution structures of methemoglobin demonstrate that distinct conformational states can be identified within a dataset for proteins as small as 64 kDa. Furthermore, we provide the first sub-nanometer cryo-EM structure of a protein smaller than 50 kDa.

scholarly journals Long shelf-life streptavidin support-films suitable for electron microscopy of biological macromolecules

2016 ◽  
Author(s):  
Bong-Gyoon Han ◽  
Zoe Watson ◽  
Hannah Kang ◽  
Arto Pulk ◽  
Kenneth H. Downing ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Shelf Life ◽  
Single Crystals ◽  
Convenient Method ◽  
Test Specimen ◽  
Biological Macromolecules ◽  
Crystalline Order ◽  
Binding Function ◽  
Biotin Binding

ABSTRACTWe describe a rapid and convenient method of growing streptavidin (SA) monolayer crystals directly on holey-carbon EM grids. As expected, these SA monolayer crystals retain their biotin-binding function and crystalline order through a cycle of embedding in trehalose and, later, its removal. This fact allows one to prepare, and store for later use, EM grids on which SA monolayer crystals serve as an affinity substrate for preparing specimens of biological macromolecules. In addition, we report that coating the lipid-tail side of trehalose-embedded monolayer crystals with evaporated carbon appears to improve the consistency with which well-ordered, single crystals are observed to span over entire, 2 μm holes of the support films. Randomly biotinylated 70 S ribosomes are used as a test specimen to show that these support films can be used to obtain a high-resolution cryo-EM structure.

scholarly journals Amino and PEG-amino graphene oxide grids enrich and protect samples for high-resolution single particle cryo-electron microscopy

2020 ◽  
Vol 209 (2) ◽  
pp. 107437 ◽  
Author(s):  
Feng Wang ◽  
Zanlin Yu ◽  
Miguel Betegon ◽  
Melody G. Campbell ◽  
Tural Aksel ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Graphene Oxide ◽  
High Resolution ◽  
Single Particle ◽  
Cryo Electron Microscopy ◽  
Resolution Single
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