Posts Tagged ‘Crystallography’

Continuous diffraction crystallographics analysis

Larry H. Bernstein, MD, FCAP, Curator



Biomolecular Structure Emerges from the Crystallographic Shadows

Here’s the caption/credit for the image: Slightly disordered crystals of complex biomolecules like that of the photosystem II molecule shown here produce a complex continous diffraction pattern (right) under X-ray light that contains far more information than the so-called Bragg peaks of a strongly ordered crystal alone (left). The degree of disorder is greatly exaggerated in the crystal on the right. [Eberhard Reimann/DESY]


In keeping with the adage, “If life gives you lemons, make lemonade,” an international team of scientists has shown that if X-crystallography relies on low-quality crystals, it can still derive high-quality structural information. In fact, resolutions can be achieved that surpass the Bragg diffraction limit.

The key, it turns out, is to make the most out of continuous diffraction data, which is ordinarily considered a nuisance in crystallographic analysis. Continuous diffraction data could be obtained from a single molecule, but would be too weak to yield any kind of analysis. But if such data could be combined from a collection of molecules, analyses would be possible. Each of the molecules in the collection, however, would have to be misaligned only in the translational sense. That is, the molecules could not be misaligned rotationally or differ intramolecularly.

With these limitations in mind, scientists based at the Center for Free-Electron Laser Science, DESY, in Germany “read” the atomic structure of complex biomolecules by crystallography without the usual need for prior knowledge and chemical insight. “This discovery has the potential to become a true revolution for the crystallography of complex matter,” said the chairman of DESY’s board of directors, Professor Helmut Dosch.

The work of the DESY-led scientific team appeared February 10 in Nature, in an article entitled “Macromolecular diffractive imaging using imperfect crystals.” The article described how the scientists took advantage of a phenomenon called continuous diffraction.

Protein crystals, particularly imperfect protein crystals, do not always “diffract,” in the traditional Bragg sense. A proper, perfect crystal scatters X-rays in many different directions, producing an intricate and characteristic pattern of numerous bright spots, called Bragg peaks (named after the British crystallography pioneers William Henry and William Lawrence Bragg). The positions and strengths of these spots contain information about the structure of the crystal and of its constituents. Using this approach, researchers have already determined the atomic structures of tens of thousands of proteins and other biomolecules.

“Continuous” scattering arises when crystals become disordered. Usually, this non-Bragg continuous diffraction is not used to derive structural information. Instead, it is used to provide insights into vibrations and dynamics of molecules. But when the disorder consists only of displacements of the individual molecules from their ideal positions in the crystal, the “background” takes on a much more complex character—and its rich structure is anything but diffuse. It then offers a much bigger prize than the analysis of the Bragg peaks: The continuously modulated “background” fully encodes the diffracted waves from individual “single” molecules.

The possibility of using continuous diffraction for structural determinations leads to a paradigm shift in crystallography—the most ordered crystals are no longer the best to analyze with the novel method. “For the first time we have access to single molecule diffraction—we have never had this in crystallography before,” explained DESY’s Professor Henry Chapman. “But we have long known how to solve single-molecule diffraction if we could measure it.” The field of coherent diffractive imaging, spurred by the availability of laser-like beams from X-ray free-electron lasers, has developed powerful algorithms to directly solve the phase problem in this case, without having to know anything at all about the molecule.

“We show for crystals of the integral membrane protein complex photosystem II that lattice disorder increases the information content and the resolution of the diffraction pattern well beyond the 4.5-ångström limit of measurable Bragg peaks, which allows us to phase the pattern directly,” wrote the authors of the Nature article. “Using the molecular envelope conventionally determined at 4.5 ångströms as a constraint, we obtain a static image of the photosystem II dimer at a resolution of 3.5 ångströms. This result shows that continuous diffraction can be used to overcome what have long been supposed to be the resolution limits of macromolecular crystallography.”


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RNA polymerase – molecular basis for DNA transcription

Larry H. Bernstein, MD, FCAP, Curator

Leaders in Pharmaceutical Intelligence

Series E: 2; 3.1

Roger Kornberg, MD
Nobel Prize in Chemistry
Stanford University

Son of Arthur Kornberg, who received the Nobel Prize for DNA polymerase, Roger Kornberg spent decades on the problem of transcription of the genetic code in eukaryotic cells. Roger Kornberg made several contributions to the understanding of the transcription model including – recognition of the nucleosomal structure of DNA, characterization of the chromatin modifying factors, and discovering the bridging factor that mediates transcriptional activation (called Mediator). The three types of RNA are termed mRNA, tRNA, and rRNA. Kornberg recognized that chromatin consists of nucleosomes arranged along DNA in the form of beads on a string. He used electron crystallography to determine that lateral diffusion in molecules tethered to the bilayer to pack into two-dimensional crystals suitable for crystallography.   Using yeast, Kornberg identified the role of RNA polymerase II and other proteins in transcribing DNA, and he created three-dimensional images of the protein cluster using X-ray crystallography. Polymerase II is used by all organisms with nuclei, including humans, to transcribe DNA.

While a graduate student working with Harden McConnell at Stanford in the late 1960s, he discovered the “flip-flop” and lateral diffusion of phospholipids in bilayer membranes. While a postdoctoral fellow working with Aaron Klug and Francis Crick at the MRC in the 1970s, Kornberg discovered the nucleosome as the basic protein complex packaging chromosomal DNA in the nucleus of eukaryotic cells (chromosomal DNA is often termed “Chromatin” when it is bound to proteins in this manner, reflecting Walther Flemming‘s discovery that certain structures within the cell nucleus would absorb dyes and become visible under a microscope).[10] Within the nucleosome, Kornberg found that roughly 200 bp of DNA are wrapped around an octamer of histone proteins.

Kornberg’s research group at Stanford later succeeded in the development of a faithful transcription system from baker’s yeast, a simple unicellular eukaryote, which they then used to isolate in a purified form all of the several dozen proteins required for the transcription process. Through the work of Kornberg and others, it has become clear that these protein components are remarkably conserved across the full spectrum of eukaryotes, from yeast to human cells.

Using this system, Kornberg made the major discovery that transmission of gene regulatory signals to the RNA polymerase machinery is accomplished by an additional protein complex that they dubbed Mediator.[11] As noted by the Nobel Prize committee, “the great complexity of eukaryotic organisms is actually enabled by the fine interplay between tissue-specific substances, enhancers in the DNA and Mediator. The discovery of Mediator is therefore a true milestone in the understanding of the transcription process.”[12]

Kornberg took advantage of expertise with lipid membranes gained from his graduate studies to devise a technique for the formation of two-dimensional protein crystals on lipid bilayers. These 2D crystals could then be analyzed using electron microscopy to derive low-resolution images of the protein’s structure. Eventually, Kornberg was able to use X-ray crystallography to solve the 3-dimensional structure of RNA polymerase at atomic resolution.[13][14] He extended these studies to obtain structural images of RNA polymerase associated with accessory proteins.[15] Through these studies, Kornberg created an actual picture of how transcription works at a molecular level.

“I measured the molecular weight of the purified H3/H4 preparation by equilibrium ultracentrifugation, while Jean Thomas offered to analyze the material by chemical cross-linking. Both methods showed unequivocally that H3 and H4 were in the form of a double dimer, an (H3)2(H4)2 tetramer (Kornberg and Thomas, 1974). I pondered this result for days, and came to the following conclusions (Kornberg, 1974). First, the exact equivalence of H3 and H4 in the tetramer implied that the differences in relative amounts of the histones from various sources measured in the past must be due to experimental error. This and the stoichiometry of the tetramer implied a unit of structure in chromatin based on two each of the four histones, or an (H2A)2(H2B)2(H3)2(H4)2 octamer. Second, since chromatin from all sources contains roughly one of each histone for every 100 bp of DNA, a histone octamer would be associated with 200 bp of DNA. Finally, the (H3)2(H4)2 tetramer was reminiscent of hemoglobin, an a2b2 tetramer. The X-ray structures of hemoglobin and other oligomeric proteins available at the time were compact, with no holes through which a molecule the size of DNA might pass. Rather, the DNA in chromatin must be wrapped on the outside of the histone octamer.

As I turned these ideas over in mind, it struck me how I might explain the results of Hewish and Burgoyne. What if their sedimentation coefficient of unit length DNA fragments was measured under neutral rather than alkaline conditions? Then the DNA would have been double stranded and about 250 bp in length. Allowing for the approximate nature of the result, the correspondence with my prediction of 200 bp was electrifying. Then I recalled a reference near the end of the Hewish and Burgoyne paper to a report of a similar pattern of DNA fragments by Williamson. I rushed to the library and found that Williamson had obtained a ladder of DNA fragments from the cytoplasm of necrotic cells and measured the unit size by sedimentation under neutral conditions: the result was 205 bp! … with colleagues in Cambridge, I proved the existence of the histone octamer and the equivalence of the 200 bp unit with the particle seen in the electron microscope (Kornberg, 1977). This chapter of the chromatin story concluded with the X-ray crystal structure determination of the particle, now known as the nucleosome, showing a histone octamer surrounded by DNA, in near atomic detail (Luger et al., 1997).

I had decided to pursue the function rather than the structure of the nucleosome, and was joined in this by Yahli Lorch, who became my lifelong partner in chromatin research, and also my partner in life. We investigated the consequences of the nucleosome for transcription. It was believed that histones are generally inhibitory to transcription. We found, to the contrary, that RNA polymerases are capable of reading right through a nucleosome. Coiling of promoter DNA in a nucleosome, however, abolished initiation by RNA polymerase II (pol II) (Lorch et al., 1987). This finding, together with genetic studies of Michael Grunstein and colleagues, identified a regulatory role of the nucleosome in transcription. It has since emerged that nucleosomes play regulatory roles in a wide range of chromosomal transactions. A whole new field has emerged, one of the most active in bioscience today. It involves a bewildering variety of posttranslational modifications of the histones, and a protein machinery of great complexity for applying, recognizing, and removing these modifications.”


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Nitric Oxide Synthase Inhibitors (NOS-I)

Author: Larry H Bernstein, MD, FCAP

Curator: Stephen J. Williams, PhD


Co-Curator: Aviva Lev-Ari, PhD, RN


This recent article sheds a new light on nitric oxide and the activity of NOS in reactive oxygen species generation and the effect of NOS inhibitors in bacteria.

Structural and Biological Studies on Bacterial Nitric Oxide Synthase Inhibitors

Jeffrey K. Holdena, Huiying Lia, Qing Jingb, Soosung Kangb, Jerry Richoa, Richard B. Silvermanb,1, and Thomas L. Poulosb,1
Author contributions: J.K.H. designed research; J.K.H. and J.R. performed research; Q.J. and S.K. contributed new reagents/analytic tools; J.K.H., H.L., R.B.S., and T.L.P. analyzed data; and J.K.H., R.B.S., and T.L.P. wrote the paper.

PNAS Oct 21, 2013;
This article is a PNAS Direct Submission
Data deposition: The atomic coordinates and structure factors have been deposited in the Protein Data Bank
Edited by Douglas C. Rees, Howard Hughes Medical Institute, California Institute of Technology, Pasadena, CA, and approved September 23, 2013 (received for review July 29, 2013)
Keywords:  crystallography, antibiotics, nitric oxide, NOS inhibitors, Bacillus subtilis, gram positive bacteria


Nitric oxide (NO) produced by bacterial nitric oxide synthase has recently been shown to

Using Bacillus subtilis as a model system, we identified

  • two NOS inhibitors that work in conjunction with an antibiotic to kill B. subtilis.

Moreover, comparison of inhibitor-bound crystal structures between the bacterial NOS and mammalian NOS revealed an unprecedented

  • mode of binding to the bacterial NOS that can be further exploited for future structure-based drug design.

Overall, this work is an important advance in developing inhibitors against gram-positive pathogens.


Nitric oxide (NO) produced by bacterial NOS functions as

  • a cytoprotective agent against oxidative stress in Staphylococcus aureusBacillus anthracis, and Bacillus subtilis.

The screening of several NOS-selective inhibitors uncovered two inhibitors with potential antimicrobial properties. These two compounds

  • impede the growth of B. subtilis under oxidative stress, and
  • crystal structures show that each compound exhibits a unique binding mode.

Both compounds serve as excellent leads for the future development of antimicrobials against bacterial NOS-containing bacteria.

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Quantum Biology And Computational Medicine

Curator: Larry H. Bernstein, MD, FCAP Biology And Computational Medicine

There has been a huge impetus given to bioinformatics that has provided an underpinning to the accelerated discovery and insights in molecular structure with an impact on genomics in the last 15 years.
The most notable is an Israeli metallurgical physicist and crystallographer, Daniel Schechtman, who did his most important work at the National Bureau of Standards, recipient of the 2011 Nobel Prize in chemistry. He is a Professor of Chemistry at Technion University and the fourth Israeli scientist to receive the recognition since 2002.  The achievement was so remarkable that the work was initially discredited as violation of established laws of crystal structure that were proved to be incomplete.
In 1619 the great German mathematician and astronomer Johannes Kepler paid his attention on the sextuple symmetry of snowflakes. He attempted to explain of its nature by the fact that the crystals are constructed from the smallest identical marbles, which are connected closely one to another. Subsequently many great minds made many efforts to uncover the secret of crystals. According to the main law of the crystallographic symmetry that came to be accepted it is possible for the crystals only the symmetry axis’s of the first, second, third, fourth and sixth orders. The main crystallography law rejects a possibility of the symmetry axis of the fifth order in the crystallographic lattices.
The essence of the discovery is that crystals have repeating patterns, but Schechtman’s examination of the crystallography of a synthetic alloy did not fit that requirement.  He identified an Icosohedral Phase, which is referred to as quasicrystals.  The alloy of the aluminum and the manganese discovered by Shechtman is formed at the super-fast cooling of the melt with the speed 106 K per second. Thus there is formed the alloy ordered in the pattern, which is characteristic for the symmetry of the regular icosahedron having alongside with the dodecahedron the symmetry axes of the 5th order.
The tiling of a plane in a non-periodic fashion was first noted in 1963 (Wang tiles), and then in 1976, Roger Penrose proposed a set of non-periodic tiles referred to as Penrose tiles.  Penrose was engaged in the “parquet’s problem” consisting of the dense filling of the plane with the help of polygons. In 1972 he found the method to cover flatness only with two simple polygons arranged non-periodically. In their simplest form “Penrose’s tiles” represent a nonrandom set of the diamond-shaped figures of two types, one of them is with the interior angle of 36°, the other one with the angle of 72°. Just as simple curves in a plane can be obtained as sections from a three dimensional double cone, aperiodic arrangements were obtained from hyperlattices with four or more dimensions.
The pentagram has a number characteristic of isosceles triangles. Take the triangle of the kind of ADC, where the acute angle at the vertex of A is equal to 36° and the ratio of the side AC = AD to the base DC is equal to the golden proportion, that is, the given triangle is the “golden” one. If we combine two such triangles so that their bases coincide, we will get “Penrose’s rhombus”. Another type of the isosceles triangle is available in the pentagram, for example, EBK. It has the acute angles at the vertex of E and B equal to 36°, and the obtuse angle at the vertex of K is equal to 108°. The ratio of the base EB of the triangle EBK to its side EK is again equal to the golden proportion, so this triangle also is the “golden” one. If we connect such two triangles together so that their bases coincided we will get another “Penrose rhombus”. “Penrose’s tile” can be formed by using the “golden” rhombuses. We can cover the plane using only two “golden” rhombuses of the kinds of formed from the “golden triangles”.  At the end, there is some non-periodic structure called “Penrose’s tile”. It was proved, that the ratio of the number of the “thick” rhombuses to the number of the “thin” rhombuses in such structure strives for in the limit to the golden proportion!
Quasicrystals don’t have a repeating pattern. They have an Arabic mosaic pattern that relies on nonrepeating patterns. The quasicrystal is ordered but not periodic. It lacks translational symmetry, where crystals, according to the classical crystallographic restriction theorem, can possess only two, three, four, and six-fold rotational symmetries.  Now quasicrystals is a three dimensional analogy to Penrose’s tiles.  It has been stated by Gratia (1988) that the concept of the quasi-crystal presents a fundamental interest because it extends and completes the definition of the crystal. The theory based on this concept replaces the traditional idea about the “structural unit repeated in the space by the strictly periodic mode” by the key concept of the distant order.  Its significance in the mineral world can be put in one row with attachment of the irrational numbers concept to the rational ones in mathematics”. Gratia notes “the mechanical strength of the quasi-crystal alloys increases sharply; the absence of periodicity results in slowing down of the dislocations distribution in comparison to the traditional metals… ”  The quasi-crystals shattered the conventional presentation about the insuperable watershed between the mineral world where the “pentagonal” symmetry was prohibited, and the living world, where the “pentagonal” symmetry is one of most widespread.
This discovery came 20 years after the discovery of aperiodic crystals by mathematicians in the 1960s, but it was only later, in 2009, that finding of a naturally occurring mineral icosohedrite in the Khatyra River in eastern Russia, provided the proof for existence of naturally occurring quasicrystals and produced a paradigm shift in crystallography. The extended concept of crystal structure is  that an ordering can be non-periodic because it lacks translational symmetry so that a shifted copy does not match the original pattern.  Mathematically, it states that there is never translational symmetry in more than n-1 linearly independent directions.  The ability to diffract is due to the existence of an indefinite number of regularly spaced elements.The asymmetry is displayed in orders other than two, three, four or six.Dan Schechtman observed the unusual diffraction patterns in Aluminium-Manganese alloys in1982.  He did not publish the results until two years after his discovery.
When Schechtman first made the observation he showed it to Ilan Blech, who noted that such diffraction patterns had been seen before.  When Blech looked at the pattern again two years later, it was immediately clear that the common explanation was ruled out ny the experiments.  Blech decided to use a computer simulation to calculate the diffraction intensity from a cluster of such a material without long-range translational order but still not random. He termed this new structure multiple polyhedral. the computer simulation showed sharp ten-fold diffraction patterns, similar to the observed ones, emanating from the three-dimensional structure devoid of periodicity. The multiple polyhedral structure was termed later by many researchers as icosahedral glass but in effect it embraces any arrangement of polyhedra connected with definite angles and distances.  At he request of John Cahn, a final paper, “The Microstructure of Rapidly Solidified Al6Mn” was submitted and accepted in the Physical Review Letters, which caused considerable interest.
This natural quasicrystal exhibits high crystalline quality, equalling the best artificial examples. The natural quasicrystal phase, with a composition of Al63Cu24Fe13, was named icosahedrite and it was approved by the International Mineralogical Association in 2010. On further analysis it was thought to meteoritic in origin, possibly from a carbonaceous chondrite asteroid.  In 1992 the International Union of Crystallography altered its definition of a crystal, broadening it as a result of Shechtman’s findings, reducing it to the ability to produce a clear-cut diffraction pattern and acknowledging the possibility of the ordering to be either periodic or aperiodic.

Two-dimensional aperiodic tiling

A Fibonacci crystal or quasicrystal is a model used to study systems with aperiodic structure. Fibonacci ‘chains’ or ‘lattices’ are synonyms used with regard to the dimensionality of the model. The elements of a Fibonacci crystal structure are arranged in one or more spatial dimensions. The Fourier transform of such arrangements consists of discrete values, which is the defining property for crystals. This feature guarantees that its Fourier transform is discrete.The Fibonacci-based constructions  have a diffraction pattern with the intensities arranged in a fractal pattern.
This discussion is not complete without mention of another giant of modern physics who received the Nobel Prize in Chemistry 1977. He was born in Russia in 1917, and his family moved to Germany, moving again to Belgium in 1929.  His notable work was in Statistical Mechanics and Thermodynamics.  He established the Center for Statistical Mechanics and Thermodynamics, which later became the Center for Complex Quantum Systems in Austin, Texas in 1967 after leaving the Enrico Fermi Institute at the University of Chicago, where he was from 1961-1966.
He is well known for “Order Out of Chaos”, and “End of Certainty” (1997), written with Isabella Stengers.  He developed his “dissipative structure theory” to account for self-organizing systems, which might well be considered complexity theory.
Prigogine viewed Newtonian physics as limited, then extended by the wave function in quantum mechanics, and then again with the introduction of spacetime in general relativity.  He concluded that the recognition of indeterminism is essential in the study of unstable systems, particulary because the initial state is not known, and the problem of “time’s arrow”.  He studies determinism in nonlinear systems, defining Liouville space, a vector space formed by linear operators mapping into Liouville space, but not every adjoint operator maps into a Hilbert space.  He was strongly influenced by Boltzmann and by Turing.  He distinguishes between the behavior of gases in terms of populations of particles and the concept of behavior of individual particles.  Accordingly, in deterministic physics, all processes are time-reversible, meaning that they can proceed backward as well as forward through time.  But in statistical mechanics gases undergo irreversible processes.
Prigogine declared that determinism is fundamentally a denial of the arrow of time. With no arrow of time, there is no longer a privileged moment known as the “present,” which follows a determined “past” and precedes an undetermined “future.”  With irreversibility, the arrow of time is reintroduced to physics. He notes that irreversibility includes diffusion, and the emergence and evolution of life. He concludes that organisms are unstable systems existing far from thermodynamic equilibrium. Instability resists standard deterministic explanation.  This view appears to have much relevance to an understanding of disease, mutation,  adaptation, and aging as we learn more about the interactions between cells, between organelles within cells, and in genomic regulation.
Radoslav Bozov
Date: 3/26/2013
Subject: RE: comment
The process of genomic evolution cannot be revealed throughout comparative genomics as structural data representation does not illuminate either the integral path of particles-light interference, as Richard Feynman suggests, in stable forms of matter such as interference/entanglement of the nature of particles/strings/waves to first approximation as I have claimed. Towards the compressibility principle realization, I have claimed that DNA would be entropic- favorable stable state going towards absolute ZERO temp in the space defined itself. In other words themodynamics measurement in subnano discrete space would go negative towards negativity. DNA is sort of like a cold melting/growing crystal, quite stable as it appears not due to hydrogen bonding , but due to interference of C-N-O. That force is contradicted via proteins onto which we now know large amount of negative quantum redox state carbon attaches. Chemistry is just a language as it is math following certain rules based on observation. Most stable states are most observed ones. The more locally one attempts to observe, the more hidden variables would emerge as a consequence of discrete energy spaces opposing continuity of matter/time. Still, stability emerges out of non stability states. And if life was in absolute stability, there will be neither feelings nor freedom. What is feelings and freedom is a far reaching philosophical question with sets of implications, to one may be a driving car, to another riding a horse or a bicycle etc cetera or simply seeing the unobservable …No wonder genome size differs among organisms and even tissue types as an outcome of carbon capacity.
Penrose triangle

Penrose triangle (Photo credit: Wikipedia)

English: A Penrose tiling (P3) using thick and...

English: A Penrose tiling (P3) using thick and thin rhombi. Note the aperiodic structure, shared by all Penrose tilings. This particular Penrose tiling is special in that it exhibits exact five-fold symmetry. (Photo credit: Wikipedia)

An isosceles triangle with equal sides and ang...

An isosceles triangle with equal sides and angles marked (Photo credit: Wikipedia)

Penrose Tiling

Penrose Tiling (Photo credit: CORE-Materials)

Penrose tiles

Penrose tiles (Photo credit: Peter Hilton)


Quasicrystals (Photo credit: Aranda\Lasch)

Penrose star tiling: 25"

Penrose star tiling: 25″ (Photo credit: domesticat)

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