Stochastic chromatin packing of 3D mitotic chromosomes revealed by coherent X-rays

2021 ◽  
Vol 118 (46) ◽  
pp. e2109921118
Author(s):  
Daeho Sung ◽  
Chan Lim ◽  
Masatoshi Takagi ◽  
Chulho Jung ◽  
Heemin Lee ◽  
...  
Keyword(s):  
Information Transfer ◽  
3D Structure ◽  
Three Dimensional ◽  
Information Storage ◽  
Atomic Scale ◽  
Scaling Analysis ◽  
X Rays ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
Chromatin Packing

DNA molecules are atomic-scale information storage molecules that promote reliable information transfer via fault-free repetitions of replications and transcriptions. Remarkable accuracy of compacting a few-meters-long DNA into a micrometer-scale object, and the reverse, makes the chromosome one of the most intriguing structures from both physical and biological viewpoints. However, its three-dimensional (3D) structure remains elusive with challenges in observing native structures of specimens at tens-of-nanometers resolution. Here, using cryogenic coherent X-ray diffraction imaging, we succeeded in obtaining nanoscale 3D structures of metaphase chromosomes that exhibited a random distribution of electron density without characteristics of high-order folding structures. Scaling analysis of the chromosomes, compared with a model structure having the same density profile as the experimental results, has discovered the fractal nature of density distributions. Quantitative 3D density maps, corroborated by molecular dynamics simulations, reveal that internal structures of chromosomes conform to diffusion-limited aggregation behavior, which indicates that 3D chromatin packing occurs via stochastic processes.

scholarly journals Scanning electron microscope studies of human metaphase chromosomes

2014 ◽  
Vol 372 (2010) ◽  
pp. 20130144 ◽  
Author(s):  
L. A. Shemilt ◽  
A. K. C. Estandarte ◽  
M. Yusuf ◽  
I. K. Robinson
Keyword(s):  
Sample Preparation ◽  
Three Dimensional ◽  
X Rays ◽  
Metaphase Chromosomes ◽  
X Ray Imaging ◽  
Starting Point ◽  
Optical Fluorescence ◽  
Backscattered Electron ◽  
Staining Methods ◽  
Scanning Electron

Scanning electron microscopy (SEM) is used to evaluate potential chromosome preparations and staining methods for application in high-resolution three-dimensional X-ray imaging. Our starting point is optical fluorescence microscopy, the standard method for chromosomes, which only gives structural detail at the 200 nm scale. In principle, with suitable sample preparation protocols, including contrast enhancing staining, the surface structure of the chromosomes can be viewed at the 1 nm level by SEM. Here, we evaluate a heavy metal nucleic-acid-specific stain, which gives strong contrast in the backscattered electron signal. This study uses SEM to examine chromosomes prepared in different ways to establish a sample preparation protocol for X-rays. Secondary electron and backscattered electron signals are compared to evaluate the effectiveness of platinum-based stains used to enhance the contrast.

Detection of Single Atoms and Buried Defects in Three Dimensions by Aberration-Corrected Electron Microscope with 0.5-Å Information Limit

2008 ◽  
Vol 14 (5) ◽  
pp. 469-477 ◽  
Author(s):  
C. Kisielowski ◽  
B. Freitag ◽  
M. Bischoff ◽  
H. van Lin ◽  
S. Lazar ◽  
...  
Keyword(s):  
Electron Microscope ◽  
Information Transfer ◽  
Signal To Noise Ratio ◽  
Three Dimensional ◽  
Atomic Scale ◽  
Three Dimensions ◽  
Single Atoms ◽  
Atom Pairs ◽  
Structure Of Nanomaterials ◽  
Electron Microscopes

AbstractThe ability of electron microscopes to analyze all the atoms in individual nanostructures is limited by lens aberrations. However, recent advances in aberration-correcting electron optics have led to greatly enhanced instrument performance and new techniques of electron microscopy. The development of an ultrastable electron microscope with aberration-correcting optics and a monochromated high-brightness source has significantly improved instrument resolution and contrast. In the present work, we report information transfer beyond 50 pm and show images of single gold atoms with a signal-to-noise ratio as large as 10. The instrument's new capabilities were exploited to detect a buried Σ3 {112} grain boundary and observe the dynamic arrangements of single atoms and atom pairs with sub-angstrom resolution. These results mark an important step toward meeting the challenge of determining the three-dimensional atomic-scale structure of nanomaterials.

Beyond X-rays: an overview of emerging structural biology methods

2021 ◽  
Author(s):  
Jason E. Schaffer ◽  
Vandna Kukshal ◽  
Justin J. Miller ◽  
Vivian Kitainda ◽  
Joseph M. Jez
Keyword(s):  
Structure Determination ◽  
De Novo ◽  
Three Dimensional ◽  
Atomic Scale ◽  
X Rays ◽  
Time Resolved ◽  
X Ray ◽  
X Ray Crystallography ◽  
New Methods ◽  
Main Technique

Structural biologists rely on X-ray crystallography as the main technique for determining the three-dimensional structures of macromolecules; however, in recent years, new methods that go beyond X-ray-based technologies are broadening the selection of tools to understand molecular structure and function. Simultaneously, national facilities are developing programming tools and maintaining personnel to aid novice structural biologists in de novo structure determination. The combination of X-ray free electron lasers (XFELs) and serial femtosecond crystallography (SFX) now enable time-resolved structure determination that allows for capture of dynamic processes, such as reaction mechanism and conformational flexibility. XFEL and SFX, along with microcrystal electron diffraction (MicroED), help side-step the need for large crystals for structural studies. Moreover, advances in cryogenic electron microscopy (cryo-EM) as a tool for structure determination is revolutionizing how difficult to crystallize macromolecules and/or complexes can be visualized at the atomic scale. This review aims to provide a broad overview of these new methods and to guide readers to more in-depth literature of these methods.

scholarly journals 3D atomic-scale imaging of mixed Co-Fe spinel oxide nanoparticles during oxygen evolution reaction

2022 ◽  
Vol 13 (1) ◽  
Author(s):  
Weikai Xiang ◽  
Nating Yang ◽  
Xiaopeng Li ◽  
Julia Linnemann ◽  
Ulrich Hagemann ◽  
...  
Keyword(s):  
Oxygen Evolution ◽  
Oxygen Evolution Reaction ◽  
Active Sites ◽  
3D Structure ◽  
Three Dimensional ◽  
Atom Probe ◽  
Vital Role ◽  
Atomic Scale ◽  
Hydroxyl Groups ◽  
Compositional Distribution

AbstractThe three-dimensional (3D) distribution of individual atoms on the surface of catalyst nanoparticles plays a vital role in their activity and stability. Optimising the performance of electrocatalysts requires atomic-scale information, but it is difficult to obtain. Here, we use atom probe tomography to elucidate the 3D structure of 10 nm sized Co2FeO4 and CoFe2O4 nanoparticles during oxygen evolution reaction (OER). We reveal nanoscale spinodal decomposition in pristine Co2FeO4. The interfaces of Co-rich and Fe-rich nanodomains of Co2FeO4 become trapping sites for hydroxyl groups, contributing to a higher OER activity compared to that of CoFe2O4. However, the activity of Co2FeO4 drops considerably due to concurrent irreversible transformation towards CoIVO2 and pronounced Fe dissolution. In contrast, there is negligible elemental redistribution for CoFe2O4 after OER, except for surface structural transformation towards (FeIII, CoIII)2O3. Overall, our study provides a unique 3D compositional distribution of mixed Co-Fe spinel oxides, which gives atomic-scale insights into active sites and the deactivation of electrocatalysts during OER.

scholarly journals Proteome Analysis of Condensed Barley Mitotic Chromosomes

2021 ◽  
Vol 12 ◽  
Author(s):  
Zdeněk Perutka ◽  
Kateřina Kaduchová ◽  
Ivo Chamrád ◽  
Jana Beinhauer ◽  
René Lenobel ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Functional Characterization ◽  
Three Dimensional ◽  
Nuclear Genome ◽  
Proteome Analysis ◽  
Reversed Phase ◽  
Mitotic Chromosomes ◽  
Metaphase Chromosomes ◽  
Chromosomal Proteins ◽  
Nucleolar Proteins

Proteins play a major role in the three-dimensional organization of nuclear genome and its function. While histones arrange DNA into a nucleosome fiber, other proteins contribute to higher-order chromatin structures in interphase nuclei, and mitotic/meiotic chromosomes. Despite the key role of proteins in maintaining genome integrity and transferring hereditary information to daughter cells and progenies, the knowledge about their function remains fragmentary. This is particularly true for the proteins of condensed chromosomes and, in particular, chromosomes of plants. Here, we purified barley mitotic metaphase chromosomes by a flow cytometric sorting and characterized their proteins. Peptides from tryptic protein digests were fractionated either on a cation exchanger or reversed-phase microgradient system before liquid chromatography coupled to tandem mass spectrometry. Chromosomal proteins comprising almost 900 identifications were classified based on a combination of software prediction, available database localization information, sequence homology, and domain representation. A biological context evaluation indicated the presence of several groups of abundant proteins including histones, topoisomerase 2, POLYMERASE 2, condensin subunits, and many proteins with chromatin-related functions. Proteins involved in processes related to DNA replication, transcription, and repair as well as nucleolar proteins were found. We have experimentally validated the presence of FIBRILLARIN 1, one of the nucleolar proteins, on metaphase chromosomes, suggesting that plant chromosomes are coated with proteins during mitosis, similar to those of human and animals. These results improve significantly the knowledge of plant chromosomal proteins and provide a basis for their functional characterization and comparative phylogenetic analyses.

Measurements in 3D images

1991 ◽  
Vol 49 ◽  
pp. 408-409
Author(s):  
John C. Russ
Keyword(s):  
Three Dimensional ◽  
Image Plane ◽  
X Rays ◽  
Traditional Use ◽  
3D Images ◽  
Confocal Scanning Laser Microscope ◽  
Hard Materials ◽  
Depth Direction ◽  
Confocal Scanning ◽  
Confocal Scanning Laser

Three-dimensional (3D) images consisting of arrays of voxels can now be routinely obtained from several different types of microscopes. These include both the transmission and emission modes of the confocal scanning laser microscope (but not its most common reflection mode), the secondary ion mass spectrometer, and computed tomography using electrons, X-rays or other signals. Compared to the traditional use of serial sectioning (which includes sequential polishing of hard materials), these newer techniques eliminate difficulties of alignment of slices, and maintain uniform resolution in the depth direction. However, the resolution in the z-direction may be different from that within each image plane, which makes the voxels non-cubic and creates some difficulties for subsequent analysis.

Studies of metaphase chromosomes in the scanning transmission x-ray microscope: Image fidelity, radiation damage and specimen preparation

1992 ◽  
Vol 50 (2) ◽  
pp. 1038-1039
Author(s):  
Shawn Williams ◽  
Xiaodong Zhang ◽  
Susan Lamm ◽  
Jack Van’t Hof
Keyword(s):  
Visible Light ◽  
Radiation Damage ◽  
Cross Section ◽  
Imaging Techniques ◽  
Dose Range ◽  
Specimen Preparation ◽  
X Rays ◽  
Metaphase Chromosomes ◽  
X Ray ◽  
Scanning Transmission

The Scanning Transmission X-ray Microscope (STXM) is well suited for investigating metaphase chromosome structure. The absorption cross-section of soft x-rays having energies between the carbon and oxygen K edges (284 - 531 eV) is 6 - 9.5 times greater for organic specimens than for water, which permits one to examine unstained, wet biological specimens with resolution superior to that attainable using visible light. The attenuation length of the x-rays is suitable for imaging micron thick specimens without sectioning. This large difference in cross-section yields good specimen contrast, so that fewer soft x-rays than electrons are required to image wet biological specimens at a given resolution. But most imaging techniques delivering better resolution than visible light produce radiation damage. Soft x-rays are known to be very effective in damaging biological specimens. The STXM is constructed to minimize specimen dose, but it is important to measure the actual damage induced as a function of dose in order to determine the dose range within which radiation damage does not compromise image quality.

Methods for three-dimensional reconstruction of cellular components within a thick section

1986 ◽  
Vol 44 ◽  
pp. 18-21
Author(s):  
J. Frank ◽  
B. F. McEwen ◽  
M. Radermacher ◽  
C. L. Rieder
Keyword(s):  
Tilt Angle ◽  
Tomographic Reconstruction ◽  
3D Structure ◽  
Three Dimensional ◽  
Thick Section ◽  
Three Dimensional Reconstruction ◽  
Axis Ratio ◽  
Dimensional Reconstruction ◽  
Cellular Components

The tomographic reconstruction from multiple projections of cellular components, within a thick section, offers a way of visualizing and quantifying their three-dimensional (3D) structure. However, asymmetric objects require as many views from the widest tilt range as possible; otherwise the reconstruction may be uninterpretable. Even if not for geometric obstructions, the increasing pathway of electrons, as the tilt angle is increased, poses the ultimate upper limitation to the projection range. With the maximum tilt angle being fixed, the only way to improve the faithfulness of the reconstruction is by changing the mode of the tilting from single-axis to conical; a point within the object projected with a tilt angle of 60° and a full 360° azimuthal range is then reconstructed as a slightly elliptic (axis ratio 1.2 : 1) sphere.

Cryomicroscopy of crotoxin complex crystals at 400 KV

1989 ◽  
Vol 47 ◽  
pp. 826-827
Author(s):  
Jaap Brink ◽  
Wah Chiu
Keyword(s):  
Signal To Noise Ratio ◽  
3D Structure ◽  
Three Dimensional ◽  
Carbon Films ◽  
Depth Of Field ◽  
Basic Subunit ◽  
Ewald Sphere ◽  
Diffraction Patterns ◽  
Complex Crystals ◽  
Acidic Subunit

The crotoxin complex is a potent neurotoxin composed of a basic subunit (Mr = 12,000) and an acidic subunit (M = 10,000). The basic subunit possesses phospholipase activity whereas the acidic subunit shows no enzymatic activity at all. The complex's toxocity is expressed both pre- and post-synaptically. The crotoxin complex forms thin crystals suitable for electron crystallography. The crystals diffract up to 0.16 nm in the microscope, whereas images show reflections out to 0.39 nm2. Ultimate goal in this study is to obtain a three-dimensional (3D-) structure map of the protein around 0.3 nm resolution. Use of 100 keV electrons in this is limited; the unit cell's height c of 25.6 nm causes problems associated with multiple scattering, radiation damage, limited depth of field and a more pronounced Ewald sphere curvature. In general, they lead to projections of the unit cell, which at the desired resolution, cannot be interpreted following the weak-phase approximation. Circumventing this problem is possible through the use of 400 keV electrons. Although the overall contrast is lowered due to a smaller scattering cross-section, the signal-to-noise ratio of especially higher order reflections will improve due to a smaller contribution of inelastic scattering. We report here our preliminary results demonstrating the feasability of the data collection procedure at 400 kV.Crystals of crotoxin complex were prepared on carbon-covered holey-carbon films, quench frozen in liquid ethane, inserted into a Gatan 626 holder, transferred into a JEOL 4000EX electron microscope equipped with a pair of anticontaminators operating at −184°C and examined under low-dose conditions. Selected area electron diffraction patterns (EDP's) and images of the crystals were recorded at 400 kV and −167°C with dose levels of 5 and 9.5 electrons/Å, respectively.

Detection, Averaging, and 3D Reconstruction of Biological Specimens on Hypercubes (Transputer-based) Computers

1990 ◽  
Vol 48 (1) ◽  
pp. 454-455
Author(s):  
Jose-Maria Carazo ◽  
I. Benavides ◽  
S. Marco ◽  
J.L. Carrascosa ◽  
E.L. Zapata
Keyword(s):  
3D Reconstruction ◽  
3D Structure ◽  
Three Dimensional ◽  
Software Tools ◽  
Mapping Method ◽  
Computer Architectures ◽  
Parallel Computer ◽  
Reconstruction Process ◽  
Computing Power ◽  
Order Of Magnitude

Obtaining the three-dimensional (3D) structure of negatively stained biological specimens at a resolution of, typically, 2 - 4 nm is becoming a relatively common practice in an increasing number of laboratories. A combination of new conceptual approaches, new software tools, and faster computers have made this situation possible. However, all these 3D reconstruction processes are quite computer intensive, and the middle term future is full of suggestions entailing an even greater need of computing power. Up to now all published 3D reconstructions in this field have been performed on conventional (sequential) computers, but it is a fact that new parallel computer architectures represent the potential of order-of-magnitude increases in computing power and should, therefore, be considered for their possible application in the most computing intensive tasks.We have studied both shared-memory-based computer architectures, like the BBN Butterfly, and local-memory-based architectures, mainly hypercubes implemented on transputers, where we have used the algorithmic mapping method proposed by Zapata el at. In this work we have developed the basic software tools needed to obtain a 3D reconstruction from non-crystalline specimens (“single particles”) using the so-called Random Conical Tilt Series Method. We start from a pair of images presenting the same field, first tilted (by ≃55°) and then untilted. It is then assumed that we can supply the system with the image of the particle we are looking for (ideally, a 2D average from a previous study) and with a matrix describing the geometrical relationships between the tilted and untilted fields (this step is now accomplished by interactively marking a few pairs of corresponding features in the two fields). From here on the 3D reconstruction process may be run automatically.

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