Thickness measurement of glucose-embedded crotoxin complex crystals by EELS

1993 ◽  
Vol 51 ◽  
pp. 686-687
Author(s):  
R.D. Leapman ◽  
J. Brink ◽  
W. Chiu
Keyword(s):  
Energy Loss ◽  
Three Dimensional ◽  
Structural Data ◽  
Mean Free Path ◽  
Thickness Measurement ◽  
Dimensional Structure ◽  
Specimen Thickness ◽  
Electron Dose ◽  
Cell Layers ◽  
Complex Crystals

In three-dimensional structure determination of macromolccules by electron crystallography it is necessary to combine diffraction patterns and images recorded at various tilt angles from different crystals. In order to merge these data sets successfully all the crystals should have the same thickness. It has been proposed that parallel electron energy loss spectroscopy (EELS) might provide a useful means of assessing the thickness of a beam-sensitive organic crystal prior to recording the high-resolution structural data. This can be achieved by measuring the fraction of the total transmitted electrons that do not lose energy, i.e., the zero-loss intensity. If Iz is the integrated zero-loss intensity and Itot is the total integrated intensity in the energy loss spectrum, then the specimen thickness, t, is given in terms of the total inelastic mean free path, λi, by, t/λi = ln(Itot/Iz). Results recently obtained from n-paraffin crystals have shown that it is feasible to determine the number of unit cell layers under low electron dose conditions.

A New 1000kv Electron Microscope

1969 ◽  
Vol 27 ◽  
pp. 100-101
Author(s):  
J.L. Williams ◽  
K. Heathcote ◽  
E.J. Greer
Keyword(s):  
Electron Microscope ◽  
Direct Observation ◽  
High Voltage ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Specimen Thickness ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
Dynamic Experiments ◽  
Conventional Instrument

High Voltage Electron Microscope already offers exciting experimental possibilities to Biologists and Materials Scientists because the increased specimen thickness allows direct observation of three dimensional structure and dynamic experiments on effectively bulk specimens. This microscope is designed to give maximum accessibility and space in the specimen region for the special stages which are required. At the same time it provides an ease of operation similar to a conventional instrument.

The Three-dimensional structure of frozen-hydrated nudaurelia capensis β virus

1987 ◽  
Vol 45 ◽  
pp. 650-651
Author(s):  
N. H. Olson ◽  
T. S. Baker ◽  
Wu Bo Mu ◽  
J. E. Johnson ◽  
D. A. Hendry
Keyword(s):  
South African ◽  
Rna Virus ◽  
Carbon Film ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Electron Dose ◽  
Total Electron ◽  
Three Dimensional Structure ◽  
Negative Stain ◽  
Dimensional Reconstruction

Nudaurelia capensis β virus (NβV) is an RNA virus of the South African Pine Emperor moth, Nudaurelia cytherea capensis (Lepidoptera: Saturniidae). The NβV capsid is a T = 4 icosahedron that contains 60T = 240 subunits of the coat protein (Mr = 61,000). A three-dimensional reconstruction of the NβV capsid was previously computed from visions embedded in negative stain suspended over holes in a carbon film. We have re-examined the three-dimensional structure of NβV, using cryo-microscopy to examine the native, unstained structure of the virion and to provide a initial phasing model for high-resolution x-ray crystallographic studiesNβV was purified and prepared for cryo-microscopy as described. Micrographs were recorded ∼1 - 2 μm underfocus at a magnification of 49,000X with a total electron dose of about 1800 e-/nm2.

Comparison of Calculated and Recorded Electron Energy-Loss Spectra of Aluminium and Carbon

1990 ◽  
Vol 48 (2) ◽  
pp. 64-65
Author(s):  
L. Reimer ◽  
R. Oelgeklaus
Keyword(s):  
Fourier Transform ◽  
Energy Loss ◽  
Electron Energy ◽  
Rotational Symmetry ◽  
Mean Free Path ◽  
Single Scattering ◽  
Specimen Thickness ◽  
Two Dimensional ◽  
Image Position ◽  
Electron Energy Loss

Quantitative electron energy-loss spectroscopy (EELS) needs a correction for the limited collection aperture α and a deconvolution of recorded spectra for eliminating the influence of multiple inelastic scattering. Reversely, it is of interest to calculate the influence of multiple scattering on EELS. The distribution f(w,θ,z) of scattered electrons as a function of energy loss w, scattering angle θ and reduced specimen thickness z=t/Λ (Λ=total mean-free-path) can either be recorded by angular-resolved EELS or calculated by a convolution of a normalized single-scattering function ϕ(w,θ). For rotational symmetry in angle (amorphous or polycrystalline specimens) this can be realised by the following sequence of operations :(1)where the two-dimensional distribution in angle is reduced to a one-dimensional function by a projection P, T is a two-dimensional Fourier transform in angle θ and energy loss w and the exponent -1 indicates a deprojection and inverse Fourier transform, respectively.

Local thickness determination by Electron Energy Loss Spectroscopy

1994 ◽  
Vol 52 ◽  
pp. 944-945
Author(s):  
Suichu Luo ◽  
John R. Dunlap ◽  
Richard W. Williams ◽  
David C. Joy
Keyword(s):  
Energy Loss ◽  
Analytical Electron Microscopy ◽  
Mean Free Path ◽  
Single Scattering ◽  
Specimen Thickness ◽  
Three Dimension ◽  
Angular Correction ◽  
Electron Intensity ◽  
Image Position ◽  
Inelastic Mean Free Path

In analytical electron microscopy, it is often important to know the local thickness of a sample. The conventional method used for measuring specimen thickness by EELS is:where t is the specimen thickness, λi is the total inelastic mean free path, IT is the total intensity in an EEL spectrum, and I0 is the zero loss peak intensity. This is rigorouslycorrect only if the electrons are collected over all scattering angles and all energy losses. However, in most experiments only a fraction of the scattered electrons are collected due to a limited collection semi-angle. To overcome this problem we present a method based on three-dimension Poisson statistics, which takes into account both the inelastic and elastic mixed angular correction.The three-dimension Poisson formula is given by:where I is the unscattered electron intensity; t is the sample thickness; λi and λe are the inelastic and elastic scattering mean free paths; Si (θ) and Se(θ) are normalized single inelastic and elastic angular scattering distributions respectively ; F(E) is the single scattering normalized energy loss distribution; D(E,θ) is the plural scattering distribution,

Some problems and solutions in electron energy loss spectroscopy of cryosections

1993 ◽  
Vol 51 ◽  
pp. 566-567
Author(s):  
Zhifeng Shao ◽  
Ruoya Ho ◽  
Andrew P. Somlyo
Keyword(s):  
High Resolution ◽  
Energy Loss ◽  
Electron Energy ◽  
Electron Energy Loss Spectroscopy ◽  
Biological Research ◽  
Specimen Thickness ◽  
Elemental Distribution ◽  
Electron Dose ◽  
Simple Assumption ◽  
Electron Energy Loss

Electron energy loss spectroscopy (EELS) has been a powerful tool for high resolution studies of elemental distribution, as well as electronic structure, in thin samples. Its foundation for biological research has been laid out nearly two decades ago, and in the subsequent years it has been subjected to rigorous, but by no means extensive research. In particular, some problems unique to EELS of biological samples, have not been fully resolved. In this article we present a brief summary of recent methodological developments, related to biological applications of EELS, in our laboratory. The main purpose of this work was to maximize the signal to noise ratio (S/N) for trace elemental analysis at a minimum dose, in order to reduce the electron dose and/or time required for the acquisition of high resolution elemental maps of radiation sensitive biological materials.Based on the simple assumption of Poisson distribution of independently scattered electrons, it had been generally assumed that the optimum specimen thickness, at which the S/N is a maximum, must be the total inelastic mean free path of the beam electron in the sample.

scholarly journals SphereCon—a method for precise estimation of residue relative solvent accessible area from limited structural information

2020 ◽  
Vol 36 (11) ◽  
pp. 3372-3378
Author(s):  
Alexander Gress ◽  
Olga V Kalinina
Keyword(s):  
Protein Function ◽  
Structural Information ◽  
Solvent Accessibility ◽  
Three Dimensional ◽  
Structural Data ◽  
Supplementary Information ◽  
Dimensional Structure ◽  
Relative Solvent Accessibility ◽  
Precise Measure ◽  
The Impact

Abstract Motivation In proteins, solvent accessibility of individual residues is a factor contributing to their importance for protein function and stability. Hence one might wish to calculate solvent accessibility in order to predict the impact of mutations, their pathogenicity and for other biomedical applications. A direct computation of solvent accessibility is only possible if all atoms of a protein three-dimensional structure are reliably resolved. Results We present SphereCon, a new precise measure that can estimate residue relative solvent accessibility (RSA) from limited data. The measure is based on calculating the volume of intersection of a sphere with a cone cut out in the direction opposite of the residue with surrounding atoms. We propose a method for estimating the position and volume of residue atoms in cases when they are not known from the structure, or when the structural data are unreliable or missing. We show that in cases of reliable input structures, SphereCon correlates almost perfectly with the directly computed RSA, and outperforms other previously suggested indirect methods. Moreover, SphereCon is the only measure that yields accurate results when the identities of amino acids are unknown. A significant novel feature of SphereCon is that it can estimate RSA from inter-residue distance and contact matrices, without any information about the actual atom coordinates. Availability and implementation https://github.com/kalininalab/spherecon. Contact [email protected] Supplementary information Supplementary data are available at Bioinformatics online.

Thickness Measurement by EELS

1985 ◽  
Vol 43 ◽  
pp. 398-399
Author(s):  
R. F. Egerton ◽  
S. C. Cheng
Keyword(s):  
Energy Loss ◽  
Electron Energy Loss Spectroscopy ◽  
Incident Energy ◽  
Mean Free Path ◽  
Thickness Measurement ◽  
Loss Peak ◽  
Wide Range ◽  
Integrated Intensity ◽  
The Mean ◽  
Electron Energy Loss

Electron energy-loss spectroscopy offers a rapid method of estimating the local thickness of a TEM specimen. The best-known procedure requires only measurement of the integrated intensity IO under the zero-loss peak and of the integral It under the whole spectrum (up to some suitable energy loss Δ). The thickness t is obtained from the formula:where λ(β) is the mean free path for inelastic scattering up to some angle β which is determined by the collection aperture (e.g. objective aperture in CTEM). In agreement with previous work we find that Eq. (1) is applicable over a wide range of thickness, typically 10-500 nm for EO = 100keV incident energy; see Fig. 1. Some deviation at large thickness might be expected as a result of the angular broadening produced by plural scattering, and because of contributions from electrons elastically scattered through angles greater than β.

An Improved Double-Tilt Stage for the AEI EM7 High-Voltage Electron Microscope

1980 ◽  
Vol 38 ◽  
pp. 54-55
Author(s):  
J. N. Turner ◽  
A. J. Ratkowski
Keyword(s):  
Degrees Of Freedom ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Specimen Thickness ◽  
Large Specimen ◽  
Voltage Electron ◽  
High Voltage Electron Microscope ◽  
Sampling Statistics ◽  
Image Detail ◽  
Image Details

Specimens approximately a micron thick are routinely examined in the HVEM as a result of its increased penetrating power. This large specimen thickness is advantageous for the study of three-dimensional structure, and it increases the sampling statistics significantly for observing a particular image detail. However, each image in the HVEM represents a volume of the specimen and cannot be assumed to be a plane as is common practice for the CTEM. Therefore, image details in the HVEM often overlap or are not optimally oriented with respect to the beam. Thus, a specimen stage with sufficient degrees of freedom to position the specimen in any orientation relative to beam is required (Turner and Chang, these proceedings). Our double-tilt stage shown in fig. 1 and built by P. R. Swann (1972) has this capability.

Protein-ligand Docking Simulations with AutoDock4 Focused on the Main Protease of SARS-CoV-2

2021 ◽  
Vol 28 ◽  
Author(s):  
Walter Filgueira de Azevedo Junior ◽  
Gabriela Bitencourt-Ferreira ◽  
Joana Retzke Godoy ◽  
Hilda Mayela Aran Adriano ◽  
Wallyson André dos Santos Bezerra ◽  
...  
Keyword(s):  
Scientific Evidence ◽  
Three Dimensional ◽  
Structural Data ◽  
Ligand Docking ◽  
Dimensional Structure ◽  
Computational Approaches ◽  
Docking Simulations ◽  
Protein Target ◽  
Main Protease ◽  
Docking Protocol

Background: The main protease of SARS-CoV-2 (Mpro) is one of the targets identified in SARS-CoV-2, the causative agent of COVID-19. The application of X-ray diffraction crystallography made available the three-dimensional structure of this protein target in complex with ligands, which paved the way for docking studies. Objective: Our goal here is to review recent efforts in the application of docking simulations to identify inhibitors of the Mpro using the program AutoDock4. Method: We searched PubMed to identify studies that applied AutoDock4 for docking against this protein target. We used the structures available for Mpro to analyze intermolecular interactions and reviewed the methods used to search for inhibitors. Results: The application of docking against the structures available for the Mpro found ligands with an estimated inhibition in the nanomolar range. Such computational approaches focused on the crystal structures revealed potential inhibitors of Mpro that might exhibit pharmacological activity against SARS-CoV-2. Nevertheless, most of these studies lack the proper validation of the docking protocol. Also, they all ignored the potential use of machine learning to predict affinity. Conclusion: The combination of structural data with computational approaches opened the possibility to accelerate the search for drugs to treat COVID-19. Several studies used AutoDock4 to search for inhibitors of Mpro. Most of them did not employ a validated docking protocol, which lends support to critics of their computational methodology. Furthermore, one of these studies reported the binding of chloroquine and hydroxychloroquine to Mpro. This study ignores the scientific evidence against the use of these antimalarial drugs to treat COVID-19.

Mapping of monoclonal antibodies specific to P64k: A common antigen of several isolates of Neisseria meningitidis

2001 ◽  
Vol 47 (2) ◽  
pp. 158-164 ◽  
Author(s):  
C Nazábal ◽  
T Carmenate ◽  
S Cruz ◽  
S González ◽  
R Silva ◽  
...  
Keyword(s):  
Monoclonal Antibodies ◽  
Neisseria Meningitidis ◽  
Three Dimensional ◽  
Structural Data ◽  
Dimensional Structure ◽  
Bacterial Surface ◽  
A Minor ◽  
The Relationship ◽  
Cell Elisa ◽  
Competition Assays

P64k is a minor outer membrane protein from Neisseria meningitidis. This protein has been produced at high levels in Escherichia coli. We generated a group of monoclonal antibodies (mAbs) against recombinant P64k, which recognise four non-overlapping epitopes, as shown using competition assays with biotinylated mAbs. The P64k sequences involved in mAbs binding were mapped with synthetic overlapping peptides derived from the P64k protein, and located in the previously determined three-dimensional structure of the protein. These antibodies were also characterised by whole-cell ELISA and bactericidal tests against N. meningitidis. Only two of the recognised epitopes were exposed on the bacterial surface, and none of the mAbs showed bactericidal activity. The relationship between these results and the structural data on the epitopes bound by the mAbs is discussed.Key words: Neisseria meningitidis, P64k, monoclonal antibodies, epitope mapping.

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