scholarly journals Raster-scanning serial protein crystallography using micro- and nano-focused synchrotron beams

2015 ◽  
Vol 71 (5) ◽  
pp. 1184-1196 ◽  
Author(s):  
Nicolas Coquelle ◽  
Aaron S. Brewster ◽  
Ulrike Kapp ◽  
Anastasya Shilova ◽  
Britta Weinhausen ◽  
...  
Keyword(s):  
Silicon Nitride ◽  
High Resolution ◽  
Room Temperature ◽  
Structural Information ◽  
Protein Crystallography ◽  
Structural Data ◽  
Analysis Software ◽  
Raster Scanning ◽  
Hit Rate ◽  
Alternative Approach

High-resolution structural information was obtained from lysozyme microcrystals (20 µm in the largest dimension) using raster-scanning serial protein crystallography on micro- and nano-focused beamlines at the ESRF. Data were collected at room temperature (RT) from crystals sandwiched between two silicon nitride wafers, thereby preventing their drying, while limiting background scattering and sample consumption. In order to identify crystal hits, new multi-processing and GUI-driven Python-based pre-analysis software was developed, namedNanoPeakCell, that was able to read data from a variety of crystallographic image formats. Further data processing was carried out usingCrystFEL, and the resultant structures were refined to 1.7 Å resolution. The data demonstrate the feasibility of RT raster-scanning serial micro- and nano-protein crystallography at synchrotrons and validate it as an alternative approach for the collection of high-resolution structural data from micro-sized crystals. Advantages of the proposed approach are its thriftiness, its handling-free nature, the reduced amount of sample required, the adjustable hit rate, the high indexing rate and the minimization of background scattering.

High-Resolution Scanning Electron Microscopy of Biological Specimens

1988 ◽  
Vol 46 ◽  
pp. 186-187
Author(s):  
M. Müller ◽  
R. Hermann
Keyword(s):  
High Resolution ◽  
Freeze Drying ◽  
Room Temperature ◽  
Structural Information ◽  
Freeze Substitution ◽  
Critical Point Drying ◽  
Sem Study ◽  
Major Factors ◽  
Structural Preservation ◽  
Preparation Techniques

Three major factors must be concomitantly assessed in order to extract relevant structural information from the surface of biological material at high resolution (2-3nm).Procedures based on chemical fixation and dehydration in graded solvent series seem inappropriate when aiming for TEM-like resolution. Cells inevitably shrink up to 30-70% of their initial volume during gehydration; important surface components e.g. glycoproteins may be lost. These problems may be circumvented by preparation techniques based on cryofixation. Freezedrying and freeze-substitution followed by critical point drying yields improved structural preservation in TEM. An appropriate preservation of dimensional integrity may be achieved by freeze-drying at - 85° C. The sample shrinks and may partially collapse as it is warmed to room temperature for subsequent SEM study. Observations at low temperatures are therefore a necessary prerequisite for high fidelity SEM. Compromises however have been unavoidable up until now. Aldehyde prefixation is frequently needed prior to freeze drying, rendering the sample resistant to treatment with distilled water.

Molecular structure and properties from high resolution Raman spectra of gases

1979 ◽  
Vol 293 (1402) ◽  
pp. 249-256
Keyword(s):  
Electronic Structure ◽  
High Resolution ◽  
Structural Information ◽  
Structural Data ◽  
Molecular Vibration ◽  
Polarizability Tensor ◽  
Spontaneous Raman Scattering ◽  
Adequate Treatment ◽  
Additional Information ◽  
Potential Availability

Despite the weakness of spontaneous Raman scattering, it can frequently provide structural information of comparable precision to the best infrared studies. In addition, because of the less restrictive selection rules which operate, more types of transitions are permitted so that rotational constants which may not be obtained from infrared spectra can be obtained via Raman studies. In addition to structural data, high-resolution Raman spectroscopy has recently been used to determine explicit values for the principal components of the polarizability tensor and it is beginning to provide information on how these components vary with the molecular vibration. The potential availability of such information is of considerable interest, since the polarizability provides an insight into the electronic structure of a molecule. It is perhaps surprising that there is at present no adequate treatment of the manner in which the molecular polarizability changes with vibration. Additional information on the components of the polarizability tensor can only serve to promote our understanding of the electronic structure of molecules.

scholarly journals Enzyme catalysis captured using multiple structures from one crystal at varying temperatures

IUCrJ ◽  
2018 ◽  
Vol 5 (3) ◽  
pp. 283-292 ◽  
Author(s):  
Sam Horrell ◽  
Demet Kekilli ◽  
Kakali Sen ◽  
Robin L. Owen ◽  
Florian S. N. Dworkowski ◽  
...  
Keyword(s):  
High Resolution ◽  
Enzyme Catalysis ◽  
Density Functional ◽  
Room Temperature ◽  
Structural Information ◽  
Density Functional Theory Calculations ◽  
State Density ◽  
X Rays ◽  
Copper Site ◽  
Multiple Structures

High-resolution crystal structures of enzymes in relevant redox states have transformed our understanding of enzyme catalysis. Recent developments have demonstrated that X-rays can be used, via the generation of solvated electrons, to drive reactions in crystals at cryogenic temperatures (100 K) to generate `structural movies' of enzyme reactions. However, a serious limitation at these temperatures is that protein conformational motion can be significantly supressed. Here, the recently developed MSOX (multiple serial structures from one crystal) approach has been applied to nitrite-bound copper nitrite reductase at room temperature and at 190 K, close to the glass transition. During both series of multiple structures, nitrite was initially observed in a `top-hat' geometry, which was rapidly transformed to a `side-on' configuration before conversion to side-on NO, followed by dissociation of NO and substitution by water to reform the resting state. Density functional theory calculations indicate that the top-hat orientation corresponds to the oxidized type 2 copper site, while the side-on orientation is consistent with the reduced state. It is demonstrated that substrate-to-product conversion within the crystal occurs at a lower radiation dose at 190 K, allowing more of the enzyme catalytic cycle to be captured at high resolution than in the previous 100 K experiment. At room temperature the reaction was very rapid, but it remained possible to generate and characterize several structural states. These experiments open up the possibility of obtaining MSOX structural movies at multiple temperatures (MSOX-VT), providing an unparallelled level of structural information during catalysis for redox enzymes.

Tissue Engineered and Native Ovine Pulmonary Valves: A High Resolution Three-Dimensional Structural Characterization

2009 ◽  
Author(s):  
Chad E. Eckert ◽  
Brandon T. Mikulis ◽  
Dane Gerneke ◽  
Ian LeGrice ◽  
Danielle Gottlieb ◽  
...  
Keyword(s):  
High Resolution ◽  
Heart Valves ◽  
Structural Information ◽  
Three Dimensional ◽  
Structural Data ◽  
Native Valve ◽  
Tissue Engineered Heart Valves

Tissue engineered heart valves (TEHV) have received much attention as a potential pediatric valve replacement therapy, offering a variety of prospective long-term functional improvements over current options. Early in vivo and in vitro efforts have produced TEHV showing increasingly equivalent mechanical and structural properties compared to native valves [1]. Despite these advances, a significant gap in the literature exists regarding detailed 3D structural information of TEHV prior to implantation (in vitro) and after implantation (explants) as well as that of the native valve. The present work was performed to provide high resolution 3D structural data of implanted TEHV, the native pulmonary valve (PV), and pre-implant scaffold to develop an accurate understanding of developing tissue.

A Liquid Nitrogen Cooled Stage for STEM

1974 ◽  
Vol 32 ◽  
pp. 444-445
Author(s):  
Louis T. Germinario
Keyword(s):  
Electron Microscope ◽  
High Resolution ◽  
Liquid Nitrogen ◽  
Room Temperature ◽  
Objective Lens ◽  
Thermal Drift ◽  
Specimen Holder ◽  
Resolution Capability ◽  
Focal Position

A liquid nitrogen stage has been developed for the JEOL JEM-100B electron microscope equipped with a scanning attachment. The design is a modification of the standard JEM-100B SEM specimen holder with specimen cooling to any temperatures In the range ~ 55°K to room temperature. Since the specimen plane is maintained at the ‘high resolution’ focal position of the objective lens and ‘bumping’ and thermal drift la minimized by supercooling the liquid nitrogen, the high resolution capability of the microscope is maintained (Fig.4).

A TEM investigation of silicon nitride whiskers

1987 ◽  
Vol 45 ◽  
pp. 160-161
Author(s):  
Tapan Roy
Keyword(s):  
Silicon Nitride ◽  
High Resolution ◽  
Ceramic Matrix Composites ◽  
High Resolution Electron Microscopy ◽  
Molten Silicon ◽  
Matrix Composites ◽  
Ceramic Fibers ◽  
Resolution Electron ◽  
Point To Point ◽  
Technological University

Ceramic fibers are being used to improve the mechanical properties of metal matrix and ceramic matrix composites. This paper reports a study of the structural and other microstructural characteristics of silicon nitride whiskers using both conventional TEM and high resolution electron microscopy.The whiskers were grown by T. E. Scott of Michigan Technological University, by passing nitrogen over molten silicon in the presence of a catalyst. The whiskers were ultrasonically dispersed in chloroform and picked up on holey carbon grids. The diameter of some whiskers (<70nm) was small enough to allow direct observation without thinning. Conventional TEM was performed on a Philips EM400T while high resolution imaging was done on a JEOL 200CX microscope with a point to point resolution of 0.23nm.

Tubulin structure at intermediate resolution

1995 ◽  
Vol 53 ◽  
pp. 852-853
Author(s):  
K. H. Downing ◽  
S. G. Wolf ◽  
E. Nogales
Keyword(s):  
High Resolution ◽  
Structural Data ◽  
Therapeutic Drug ◽  
Zinc Ions ◽  
Two Dimensional ◽  
X Ray Diffraction ◽  
X Ray ◽  
Negative Stain ◽  
Functional Mechanisms ◽  
Tubulin Structure

Microtubules are involved in a host of critical cell activities, many of which involve transport of organelles through the cell. Different sets of microtubules appear to form during the cell cycle for different functions. Knowledge of the structure of tubulin will be necessary in order to understand the various functional mechanisms of microtubule assemble, disassembly, and interaction with other molecules, but tubulin has so far resisted crystallization for x-ray diffraction studies. Fortuitously, in the presence of zinc ions, tubulin also forms two-dimensional, crystalline sheets that are ideally suited for study by electron microscopy. We have refined procedures for forming the sheets and preparing them for EM, and have been able to obtain high-resolution structural data that sheds light on the formation and stabilization of microtubules, and even the interaction with a therapeutic drug.Tubulin sheets had been extensively studied in negative stain, demonstrating that the same protofilament structure was formed in the sheets and microtubules. For high resolution studies, we have found that the sheets embedded in either glucose or tannin diffract to around 3 Å.

Fine structural morphology of immunocytes of some molluscs and insects

1990 ◽  
Vol 48 (3) ◽  
pp. 386-387
Author(s):  
S.G. Pal ◽  
G. Baur ◽  
B. Ghosh ◽  
S. Palit ◽  
S. Modak ◽  
...  
Keyword(s):  
Blood Cells ◽  
Phosphate Buffer ◽  
Room Temperature ◽  
Structural Information ◽  
Uranyl Acetate ◽  
Meretrix Meretrix ◽  
Lead Citrate ◽  
Bivalve Molluscs ◽  
Structural Morphology ◽  
Ultrathin Sections

In recent years some of the blood cells of several molluscs and insects are characterised as immunocytes. Similar cells from a few invertebrates from India have been looked into under conventional TEM to register the ultrastructural features. This type of study is first of its kind in the subcontinent. Immunocytes from bivalve molluscs Meretrix meretrix, Laroellidens marqinalis and two insect species, apterygote Ctenolepism a longicaudata and pterygote Gesonula punctifrons provide a new set of fine structural information which forms a basis of comparison with those studied earlier.Immunocytes have been collected from the fresh live species of bivalve molluscs and insects obtained locally at Calcutta. These were fixed in icecold 2% glutaraldehyde in 0.1M phosphate buffer (pH 7.2-7.4) for 1-2 hours at 4-5°C. Subseguently pellets were post-osmicated in 1% OsO4 at room temperature for 1-2 hours. Following dehydration these were embedded in Araldite mixture in plastic capsules and polymerization was effected for 2 days at 60°C. Ultrathin sections were cut in a ultrotome and sections were double stained with Uranyl acetate and lead citrate. These were viewed in a TEM.

Progress in high-resolution CRYO-SEM imaging of viral particles

1996 ◽  
Vol 54 ◽  
pp. 818-819
Author(s):  
Ya Chen ◽  
Geoffrey Letchworth ◽  
John White
Keyword(s):  
Electron Microscopy ◽  
Scanning Electron Microscopy ◽  
High Resolution ◽  
Structural Information ◽  
Signal To Noise Ratio ◽  
Flexible Structures ◽  
Simian Virus ◽  
Topographic Features ◽  
Viral Particles ◽  
Scanning Electron

Low-temperature high-resolution scanning electron microscopy (cryo-HRSEM) has been successfully utilized to image biological macromolecular complexes at nanometer resolution. Recently, imaging of individual viral particles such as reovirus using cryo-HRSEM or simian virus (SIV) using HRSEM, HV-STEM and AFM have been reported. Although conventional electron microscopy (e.g., negative staining, replica, embedding and section), or cryo-TEM technique are widely used in studying of the architectures of viral particles, scanning electron microscopy presents two major advantages. First, secondary electron signal of SEM represents mostly surface topographic features. The topographic details of a biological assembly can be viewed directly and will not be obscured by signals from the opposite surface or from internal structures. Second, SEM may produce high contrast and signal-to-noise ratio images. As a result of this important feature, it is capable of visualizing not only individual virus particles, but also asymmetric or flexible structures. The 2-3 nm resolution obtained using high resolution cryo-SEM made it possible to provide useful surface structural information of macromolecule complexes within cells and tissues. In this study, cryo-HRSEM is utilized to visualize the distribution of glycoproteins of a herpesvirus.

Atomic chemistry by HREM and image simulations?

1986 ◽  
Vol 44 ◽  
pp. 828-829
Author(s):  
J.M. Howe ◽  
R. Gronsky
Keyword(s):  
Electron Microscopy ◽  
High Resolution ◽  
Structural Information ◽  
High Resolution Electron Microscopy ◽  
Resolution Electron ◽  
Image Simulations

The technique of high-resolution electron microscopy (HREM) is invaluable to the materials scientist because it allows examination of microstructural features at levels of resolution that are unobtainable by most other methods. Although the structural information which can be determined by HREM and accompanying image simulations has been well documented in the literature, there have only been a few cases where this technique has been used to reveal the chemistry of individual columns or planes of atoms, as occur in segregated and ordered materials.

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