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Definition : X-ray crystallography is a tool used for determining the atomic and
molecular structure of a crystal.
Principle :
● The crystalline atoms cause a beam of X-rays to diffract into many specific
directions .
● By measuring the angles and intensities of these diffracted beams, a 3D
picture of the density of electrons within the crystal is produced .
● From this electron density image, the mean positions of the atoms in the
crystal can be determined, as well as their chemical bonds, their disorder, and
various other information.
Application : The method revealed the structure and function of many biological
molecules, including vitamins, drugs, proteins, and nucleic acids, such as DNA.
Disadvantage :
Several factors are known to inhibit or mar crystallization. The growing crystals are generally held
at a constant temperature and protected from shocks or vibrations that might disturb their
crystallization. Impurities in the molecules or in the crystallization solutions are often inimical to
crystallization. Conformational flexibility in the molecule also tends to make crystallization less
likely, due to entropy. Molecules that tend to self-assemble into regular helices are often unwilling
[citation needed]
to assemble into crystals. Crystals can be marred by twinning, which can occur
when a unit cell can pack equally favorably in multiple orientations; although recent advances in
computational methods may allow solving the structure of some twinned crystals. Having failed to
crystallize a target molecule, a crystallographer may try again with a slightly modified version of
the molecule; even small changes in molecular properties can lead to large differences in
crystallization behavior.
Using the electron density map and the protein sequence, a computer graphics
program can insert each residue into the map and determine its overall structure.
Some challenges encountered in this step are small breaks in the continuity of the
map, which only become a problem if the path of the protein chain isn’t clear.
Another challenge is having a low resolution map, in which case a new or different
crystal would have to be analyzed.
The final output is a protein data bank (PDB) file containing atom-coordinates,
residue sequence, protein chains, and other relevant information. You can view
these PDB files on the Research Collaboratory for Structural Bioinformatics (RCSB)
website or download software programs made for viewing them, such as PyMol.
What is a crystal ?
X rays :
X-rays are a form of electromagnetic radiation with a much shorter wavelength than
visible light (0.01 to 10 nanometers)
X-ray sources :
accelerating a beam of electrons
cathode into an anode
Determines the wavelength
thin metal foil
absorbs unwanted radiation
low-order diffraction from a graphite crystal
Monochromatization
Applications Of Autoradiography
(link : https://conductscience.com/what-is-autoradiography/)
Autoradiography In Preclinical Research
A radiotracer (usually labeled with 14C and/or 3H) is administered and incubated for
specific time points.
WBA and MARG are especially suitable for the study of receptor biology, as the
radioisotopes can be coupled with ligands that are specific to cell membrane
receptors.
Thus, WBA and MARG provide data on the distribution and activity of cellular
receptors.
In microbiology, the MARG method has been combined with fluorescence in situ
hybridization (FISH), using specific oligonucleotides (DNA probes) to identify
organisms (Figure 5) (Nielsen & Nielsen, 2005).
Figure 4 WBA (A) and QWBA (B) of drug distribution in rodents. A – intravenous administration
of [14C]-ethanol (Gifford, Espaillat, & Gatley, 2008)darker areas correspond to areas of high
ethanol concentration. B – intravenous injection of [3H]-glucose (Potchoiba & Nocerini, 2004).
The region of high glucose (radioactivity) concentration is shown in green/blue.
Figure 5: MARG combined with FISH. A–C – the same microscopic view of an activated sludge
sample. A– MARG image after incubation of the sample with [14C]-propionate. B The same
microscopic field as A, but recorded for fluorescence after hybridization with a probe specific for
Meganema perioderoedes (Nielsen & Nielsen, 2005).
The radioactive decay from the radiotracer produces positrons: particles with the
same mass as electrons, but with opposite charges (i.e., +1). When a positron and
an electron collide, they annihilate each other, and two gamma rays are produced,
which are detected by a gamma camera.
Common radionuclides used in PET scans are listed in Table 1.
Proteins, lipids, sugars, and even patient cells can be used as radioactive tracers.
SPECT imaging, the radioactive tracers emit gamma rays. A list of the most common
SPECT radioisotopes is shown in Table 2.
❖ Diagnostic (link:https://pubmed.ncbi.nlm.nih.gov/2504892/)
A quantitative autoradiographic method was developed to measure
proteins in extravascular tissues with a spatial resolution sufficient to associate
these proteins with tissue morphology.
A linear relationship between measured grain density and isotope concentration was
demonstrated with uniformly-labeled standard sources of epoxy-embedded gelatin
containing [111In]albumin; half-distance of spatial resolution was 0.6 micron. The
technique was illustrated by measuring 24-hr accumulation of
diethylenetriaminepentaacetic acid-coupled 111In-labeled human polyclonal IgG and
human serum albumin (HSA) in a thigh infection model in the rat. Gamma camera
images localized the infection and showed target-to-background ratios of 2.5 +/- 0.3
for IgG and 1.4 +/- 0.02 for human serum albumin (mean +/- s.d., n = 3). Using
quantitative autoradiography, significantly higher average tissue concentrations were
found in the infected thighs at 4 to 4.5% of the initial plasma concentrations as
compared to 0.2 to 0.3% of initial plasma concentrations in the noninfected thigh (p
less than 0.05); these radiolabeled proteins were not inflammatory cell associated
and localized primarily within the edematous interstitial spaces of the infection.
Methods
The panicle and brown rice samples were collected from areas of
Minamisoma City where radiocaesium concentrations exceeding 100 Bq
kg−1 were detected in 2013. An imaging plate (BAS-SR2040, Fuji-Film,
Japan) and a reading system (Typhoon FLA 7000, GE Healthcare
Bio-Science Corp., USA) were used to obtain and analyse the images of
panicles and brown rice samples. The panicle samples were fixed onto
42 × 30-cm cardboard pieces with cellophane tape. After covering the
samples with polyvinylidene chloride resin film (Saran wrap,
AsahiKASEI, Japan), potassium chloride markers were attached to the
corners of the film to allow the autoradiogram and the photograph (or the
cardboard-mounted samples) to be superimposed. The markers were made
by packing potassium chloride (10 or 26 mg) into a frame, which was
created by cutting a silicon tube with a 6-mm inside diameter into a
1-mm-thick round slice. The filled frame was sealed inside a laminated
pouch. The cardboard-mounted panicle samples were exposed to an
imaging plate in the dark for 2 days to evaluate the contamination state,
and the imaging plates were scanned at a spatial resolution of 25 μm with
the reading system. The unhulled rice on the rachis branch was separated
into brown rice and husks. The brown rice and husks were fixed onto
cardboard with double-sided tape and exposed to an imaging plate for 3
days.
https://www.mhs.ox.ac.uk/backfromthedead/exhibition/the-structure-of-pe
nicillin/ penicillin