The Role of High-Density and Low-Density Amorphous Ice on Biomolecules at Cryogenic Temperatures: A Case Study with Polyalanine

2021 ◽  
Author(s):  
Ali Hussein Eltareb ◽  
Gustavo E. Lopez ◽  
Nicolas Giovambattista
Keyword(s):  
Electron Microscopy ◽  
Biological Samples ◽  
Bulk Water ◽  
Experimental Techniques ◽  
Low Density ◽  
Cryogenic Temperatures ◽  
Amorphous Ice ◽  
Cryo Electron Microscopy

Experimental techniques, such as cryo-electron microscopy, require biological samples to be recovered at cryogenic temperatures (T ≈ 100 K) with water being in an amorphous ice state. However, (bulk) water...

scholarly journals Protocol for preparation of heterogeneous biological samples for 3D electron microscopy: a case study for insects

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Alexey A. Polilov ◽  
Anastasia A. Makarova ◽  
Song Pang ◽  
C. Shan Xu ◽  
Harald Hess
Keyword(s):  
Electron Microscopy ◽  
Biological Samples ◽  
Focused Ion Beam ◽  
Ion Beam ◽  
Three Dimensional ◽  
Entire Volume ◽  
Simple Protocol ◽  
3D Electron Microscopy ◽  
3D Em

AbstractModern morphological and structural studies are coming to a new level by incorporating the latest methods of three-dimensional electron microscopy (3D-EM). One of the key problems for the wide usage of these methods is posed by difficulties with sample preparation, since the methods work poorly with heterogeneous (consisting of tissues different in structure and in chemical composition) samples and require expensive equipment and usually much time. We have developed a simple protocol allows preparing heterogeneous biological samples suitable for 3D-EM in a laboratory that has a standard supply of equipment and reagents for electron microscopy. This protocol, combined with focused ion-beam scanning electron microscopy, makes it possible to study 3D ultrastructure of complex biological samples, e.g., whole insect heads, over their entire volume at the cellular and subcellular levels. The protocol provides new opportunities for many areas of study, including connectomics.

Cryo-Electron Microscopy of Trichocyte (Hard α-Keratin) Intermediate Filaments Reveals a Low-Density Core

2002 ◽  
Vol 137 (1-2) ◽  
pp. 109-118 ◽  
Author(s):  
Norman R. Watts ◽  
Leslie N. Jones ◽  
Naiqian Cheng ◽  
Joseph S. Wall ◽  
David A.D. Parry ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Intermediate Filaments ◽  
Low Density ◽  
Cryo Electron Microscopy ◽  
Keratin Intermediate Filaments

Domain topology of human Rasal

2017 ◽  
Vol 399 (1) ◽  
pp. 63-72 ◽  
Author(s):  
Jorge Cuellar ◽  
José María Valpuesta ◽  
Alfred Wittinghofer ◽  
Begoña Sot
Keyword(s):  
Electron Microscopy ◽  
Small Gtpase ◽  
Full Length ◽  
Gtpase Activating Protein ◽  
Ras Gtpase ◽  
Cryo Electron Microscopy ◽  
Domain Protein ◽  
Negative Staining Electron Microscopy ◽  
Resolution Structure

AbstractRasal is a modular multi-domain protein of the GTPase-activating protein 1 (GAP1) family; its four known members, GAP1m, Rasal, GAP1IP4BPand Capri, have a Ras GTPase-activating domain (RasGAP). This domain supports the intrinsically slow GTPase activity of Ras by actively participating in the catalytic reaction. In the case of Rasal, GAP1IP4BPand Capri, their remaining domains are responsible for converting the RasGAP domains into dual Ras- and Rap-GAPs, via an incompletely understood mechanism. Although Rap proteins are small GTPase homologues of Ras, their catalytic residues are distinct, which reinforces the importance of determining the structure of full-length GAP1 family proteins. To date, these proteins have not been crystallized, and their size is not adequate for nuclear magnetic resonance (NMR) or for high-resolution cryo-electron microscopy (cryoEM). Here we present the low resolution structure of full-length Rasal, obtained by negative staining electron microscopy, which allows us to propose a model of its domain topology. These results help to understand the role of the different domains in controlling the dual GAP activity of GAP1 family proteins.

scholarly journals The divergent mitotic kinesin MKLP2 exhibits atypical structure and mechanochemistry

eLife ◽  
2017 ◽  
Vol 6 ◽  
Author(s):  
Joseph Atherton ◽  
I-Mei Yu ◽  
Alexander Cook ◽  
Joseph M Muretta ◽  
Agnel Joseph ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Motor Function ◽  
Nucleotide Binding ◽  
Motor Domain ◽  
Kinesin Motor ◽  
Binding Motifs ◽  
Cryo Electron Microscopy ◽  
Specific Sequences

MKLP2, a kinesin-6, has critical roles during the metaphase-anaphase transition and cytokinesis. Its motor domain contains conserved nucleotide binding motifs, but is divergent in sequence (~35% identity) and size (~40% larger) compared to other kinesins. Using cryo-electron microscopy and biophysical assays, we have undertaken a mechanochemical dissection of the microtubule-bound MKLP2 motor domain during its ATPase cycle, and show that many facets of its mechanism are distinct from other kinesins. While the MKLP2 neck-linker is directed towards the microtubule plus-end in an ATP-like state, it does not fully dock along the motor domain. Furthermore, the footprint of the MKLP2 motor domain on the MT surface is altered compared to motile kinesins, and enhanced by kinesin-6-specific sequences. The conformation of the highly extended loop6 insertion characteristic of kinesin-6s is nucleotide-independent and does not contact the MT surface. Our results emphasize the role of family-specific insertions in modulating kinesin motor function.

scholarly journals Phase behaviors of deeply supercooled bilayer water unseen in bulk water

2018 ◽  
Vol 115 (19) ◽  
pp. 4839-4844 ◽  
Author(s):  
Toshihiro Kaneko ◽  
Jaeil Bai ◽  
Takuma Akimoto ◽  
Joseph S. Francisco ◽  
Kenji Yasuoka ◽  
...  
Keyword(s):  
Solid Phase ◽  
Bulk Water ◽  
Order Transition ◽  
Heterogeneous Structure ◽  
Low Density ◽  
First Order ◽  
Amorphous Ice ◽  
First Order Transition ◽  
Theoretical Evidence ◽  
Phase Behaviors

Akin to bulk water, water confined to an isolated nanoslit can show a wealth of new 2D phases of ice and amorphous ice, as well as unusual phase behavior. Indeed, 2D water phases, such as bilayer hexagonal ice and monolayer square ice, have been detected in the laboratory, confirming earlier computational predictions. Herein, we report theoretical evidence of a hitherto unreported state, namely, bilayer very low density amorphous ice (BL-VLDA), as well as evidence of a strong first-order transition between BL-VLDA and the BL amorphous ice (BL-A), and a weak first-order transition between BL-VLDA and the BL very low density liquid (BL-VLDL) water. The diffusivity of BL-VLDA is typically in the range of 10−9 cm2/s to 10−10 cm2/s. Similar to bulk (3D) water, 2D water can exhibit two forms of liquid in the deeply supercooled state. However, unlike supercooled bulk water, for which the two forms of liquid can coexist and merge into one at a critical point, the 2D BL-VLDL and BL high-density liquid (BL-HDL) phases are separated by the highly stable solid phase of BL-A whose melting line exhibits the isochore end point (IEP) near 220 K in the temperature−pressure diagram. Above the IEP temperature, BL-VLDL and BL-HDL are indistinguishable. At negative pressures, the metastable BL-VLDL exhibits a spatially and temporally heterogeneous structure induced by dynamic changes in the nanodomains, a feature much less pronounced in the BL-HDL.

scholarly journals Why POSS-Type Compounds Should Be Considered Nanomodifiers, Not Nanofillers—A Polypropylene Blends Case Study

Polymers ◽  
2021 ◽  
Vol 13 (13) ◽  
pp. 2124
Author(s):  
Dariusz Brząkalski ◽  
Robert Przekop ◽  
Bogna Sztorch ◽  
Miłosz Frydrych ◽  
Daria Pakuła ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
Differential Scanning Calorimetry ◽  
Scanning Calorimetry ◽  
Functional Additives ◽  
Plastic Processing ◽  
Polypropylene Blends ◽  
Scanning Electron

In this work, a seriTables of silsesquioxanes (SSQ) and spherosilicates (SS), comprising a group of cage siloxane (CS) compounds, was tested as functional additives for preparation of isotactic polypropylene (iPP)-based nanocomposites and discussed in the aspect of their rationale of applicability as such additives. For this purpose, the compounds were prepared by condensation and olefin hydrosilylation reactions. The effect of these cage siloxane products on properties of obtained CS/iPP nanocomposites was analyzed by means of mechanical, microscopic (scanning electron microscopy-energy dispersive spectroscopy), thermal (differential scanning calorimetry, thermogravimetry), thermomechanical (Vicat softening point) analyses. The results were compared with the previous findings on CS/polyolefin composites. The role of CS compounds was discussed in terms of plastic processing additives.

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