Chemical bonding effects in the determination of protein structures by electron crystallography

1999 ◽  
Vol 55 (2) ◽  
pp. 305-313 ◽  
Author(s):  
Steven Chang ◽  
Teresa Head-Gordon ◽  
Robert M. Glaeser ◽  
Kenneth H. Downing
Keyword(s):  
Chemical Bonding ◽  
Protein Structures ◽  
Electron Crystallography ◽  
Small Unit ◽  
Protein Fragments ◽  
Structure Factors ◽  
Angle Scattering ◽  
Sensitive Determination ◽  
Nucleotide Binding Proteins

Scattering of electrons is affected by the distribution of valence electrons that participate in chemical bonding and thus change the electrostatic shielding of the nucleus. This effect is particularly significant for low-angle scattering. Thus, while chemical bonding effects are difficult to measure with small-unit cell materials, they can be substantial in the study of proteins by electron crystallography. This work investigates the magnitude of chemical bonding effects for a representative collection of protein fragments and a model ligand for nucleotide-binding proteins within the resolution range generally used in determining protein structures by electron crystallography. Electrostatic potentials were calculated by ab initio methods for both the test molecules and for superpositions of their free atoms. Differences in scattering amplitudes can be well over 10% in the resolution range below 5 Å and are especially large in the case of ionized side chains and ligands. We conclude that the use of molecule-based scattering factors can provide a much more accurate representation of the low-resolution data obtained in electron crystallographic studies. The comparison of neutral and ionic structure factors at resolutions below 5 Å can also provide a sensitive determination of charge states, important for biological function, that is not accessible from X-ray crystallographic measurements.

Assessment of structure factors for analysis of small-angle scattering data from desired or undesired aggregates

2020 ◽  
Vol 53 (4) ◽  
pp. 991-1005
Author(s):  
Andreas Haahr Larsen ◽  
Jan Skov Pedersen ◽  
Lise Arleth
Keyword(s):  
Small Angle ◽  
Structure Factor ◽  
Protein Structures ◽  
Simulated Data ◽  
Small Angle Scattering ◽  
Scattering Data ◽  
Spherical Cluster ◽  
Structure Factors ◽  
Angle Scattering ◽  
Aggregation Processes

Aggregation processes are central features of many systems ranging from colloids and polymers to inorganic nanoparticles and biological systems. Some aggregated structures are controlled and desirable, e.g. in the design of size-controlled clustered nanoparticles or some protein-based drugs. In other cases, the aggregates are undesirable, e.g. protein aggregation involved in neurodegenerative diseases or in vitro studies of single protein structures. In either case, experimental and analytical tools are needed to cast light on the aggregation processes. Aggregation processes can be studied with small-angle scattering, but analytical descriptions of the aggregates are needed for detailed structural analysis. This paper presents a list of useful small-angle scattering structure factors, including a novel structure factor for a spherical cluster with local correlations between the constituent particles. Several of the structure factors were renormalized to get correct limit values in both the high-q and low-q limit, where q is the modulus of the scattering vector. The structure factors were critically evaluated against simulated data. Structure factors describing fractal aggregates provided approximate descriptions of the simulated data for all tested structures, from linear to globular aggregates. The addition of a correlation hole for the constituent particles in the fractal structure factors significantly improved the fits in all cases. Linear aggregates were best described by a linear structure factor and globular aggregates by the newly derived spherical cluster structure factor. As a central point, it is shown that the structure factors could be used to take aggregation contributions into account for samples of monomeric protein containing a minor fraction of aggregated protein. After applying structure factors in the analysis, the correct structure and oligomeric state of the protein were determined. Thus, by careful use of the presented structure factors, important structural information can be retrieved from small-angle scattering data, both when aggregates are desired and when they are undesired.

The Pseudo-Atom Approximation in Direct Phase Determination of Protein Structures.

1997 ◽  
Vol 3 (S2) ◽  
pp. 1025-1026
Author(s):  
Douglas L. Dorset
Keyword(s):  
Fourier Transform ◽  
High Resolution ◽  
Protein Structures ◽  
Direct Methods ◽  
Direct Solution ◽  
Phase Problem ◽  
Electron Crystallography ◽  
Phase Determination ◽  
The Fourier Transform

In principle, the availability of high-resolution micrographs in electron crystallography is a direct solution of the phase problem that has been used to great advantage for the study of proteins. However, as the resolution of the determination increases, the Fourier transform of the micrograph becomes a less accurate phase source. Hence, alternative direct methods for phase determination have been evaluated, if only to extend the resolution of most reliable lower resolution phases to the limit of the electron diffraction pattern. The first demonstration of its feasibility was published in a study of bacteriorhodopsin extending 15 Å image phases to beyond 3 Å by maximum entropy and likelihood procedures i. Later studies demonstrated that convolutional methods also can be effective.In protein crystallography, there is always an interest in carrying out a true ab initio determinations, if only because of the challenge to traditional direct methods that become statistically less reliable as the number of atoms in the unit cell increases.

scholarly journals Three-dimensional electron crystallography of protein microcrystals

eLife ◽  
2013 ◽  
Vol 2 ◽  
Author(s):  
Dan Shi ◽  
Brent L Nannenga ◽  
Matthew G Iadanza ◽  
Tamir Gonen
Keyword(s):  
Electron Diffraction ◽  
Protein Structures ◽  
Three Dimensional ◽  
Electron Diffraction Data ◽  
Electron Crystallography ◽  
Electron Dose ◽  
Tilt Series ◽  
Diffraction Patterns ◽  
A New Technique

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7 Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1–1° and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9 Å resolution. This proof of principle paves the way for the implementation of a new technique, which we name ‘MicroED’, that may have wide applicability in structural biology.

scholarly journals MicroED: Three Dimensional Electron Crystallography of Protein Micro-Crystals

2014 ◽  
Vol 70 (a1) ◽  
pp. C1063-C1063
Author(s):  
Tamir Gonen
Keyword(s):  
Electron Diffraction ◽  
Protein Structures ◽  
Three Dimensional ◽  
Electron Diffraction Data ◽  
Molecular Replacement ◽  
Electron Crystallography ◽  
Electron Dose ◽  
Tilt Series ◽  
Diffraction Patterns

We demonstrate that it is feasible to determine high-resolution protein structures by electron crystallography of three-dimensional crystals in an electron cryo-microscope (CryoEM). Lysozyme microcrystals were frozen on an electron microscopy grid, and electron diffraction data collected to 1.7Å resolution. We developed a data collection protocol to collect a full-tilt series in electron diffraction to atomic resolution. A single tilt series contains up to 90 individual diffraction patterns collected from a single crystal with tilt angle increment of 0.1 - 10and a total accumulated electron dose less than 10 electrons per angstrom squared. We indexed the data from three crystals and used them for structure determination of lysozyme by molecular replacement followed by crystallographic refinement to 2.9Å resolution. In this seminar I will present our initial proof of principle study and highlight the major advances since the first publication.

Enhanced information from biological materials through technological improvements in cryo-electron microscopy

1992 ◽  
Vol 50 (1) ◽  
pp. 788-789
Author(s):  
Marc J.C. de Jong ◽  
Wim M. Busing ◽  
Max T. Otten
Keyword(s):  
Electron Microscopy ◽  
Electron Beam ◽  
Biological Materials ◽  
Electron Crystallography ◽  
Cryo Electron Microscopy ◽  
Transmission Electron ◽  
The Many ◽  
Technological Improvements ◽  
Beam Damage

Biological materials damage rapidly in the electron beam, limiting the amount of information that can be obtained in the transmission electron microscope. The discovery that observation at cryo temperatures strongly reduces beam damage (in addition to making it unnecessaiy to use chemical fixatives, dehydration agents and stains, which introduce artefacts) has given an important step forward to preserving the ‘live’ situation and makes it possible to study the relation between function, chemical composition and morphology.Among the many cryo-applications, the most challenging is perhaps the determination of the atomic structure. Henderson and co-workers were able to determine the structure of the purple membrane by electron crystallography, providing an understanding of the membrane's working as a proton pump. As far as understood at present, the main stumbling block in achieving high resolution appears to be a random movement of atoms or molecules in the specimen within a fraction of a second after exposure to the electron beam, which destroys the highest-resolution detail sought.

Direct phase determination in electron crystallography: small organic molecules

1992 ◽  
Vol 50 (2) ◽  
pp. 1166-1167
Author(s):  
Douglas L. Dorset
Keyword(s):  
Organic Molecules ◽  
Data Sets ◽  
Temperature Structure ◽  
3D Analysis ◽  
Intensity Data ◽  
Electron Crystallography ◽  
Phase Determination ◽  
Measured Intensity ◽  
3D Data

The quantitative use of electron diffraction intensity data for the determination of crystal structures represents the pioneering achievement in the electron crystallography of organic molecules, an effort largely begun by B. K. Vainshtein and his co-workers. However, despite numerous representative structure analyses yielding results consistent with X-ray determination, this entire effort was viewed with considerable mistrust by many crystallographers. This was no doubt due to the rather high crystallographic R-factors reported for some structures and, more importantly, the failure to convince many skeptics that the measured intensity data were adequate for ab initio structure determinations.We have recently demonstrated the utility of these data sets for structure analyses by direct phase determination based on the probabilistic estimate of three- and four-phase structure invariant sums. Examples include the structure of diketopiperazine using Vainshtein's 3D data, a similar 3D analysis of the room temperature structure of thiourea, and a zonal determination of the urea structure, the latter also based on data collected by the Moscow group.

Development of a colorimetric protein phosphorylation assay for detecting cyanobacterial toxins

1995 ◽  
Vol 31 (5-6) ◽  
pp. 47-49 ◽  
Author(s):  
C. Ash ◽  
C. MacKintosh ◽  
R. MacKintosh ◽  
C. R. Fricker
Keyword(s):  
Protein Phosphorylation ◽  
Colorimetric Assay ◽  
Cyanobacterial Toxins ◽  
Sensitive Determination

A new colorimetric assay is described, based on inhibition of protein phosphotases, that enables the rapid, simple and sensitive determination of the concentration of toxins from cyanobacteria.

Non-enzymatic determination of purine nucleotides using a carbon dot modified glassy carbon electrode

2019 ◽  
Vol 11 (30) ◽  
pp. 3866-3873 ◽  
Author(s):  
R. Karthikeyan ◽  
D. James Nelson ◽  
S. Abraham John
Keyword(s):  
Glassy Carbon ◽  
Low Cost ◽  
Phosphate Buffer Solution ◽  
Purine Nucleotides ◽  
Buffer Solution ◽  
Solution Ph ◽  
Carbon Dot ◽  
Enzymatic Determination ◽  
Sensitive Determination

Selective and sensitive determination of one of the purine nucleotides, inosine (INO) using a low cost carbon dot (CD) modified glassy carbon (GC) electrode in 0.2 M phosphate buffer solution (pH 7.2) was demonstrated in this paper.

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