Sample preparation

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 :

● Difficult to obtain a crystal of virus particles, which is a prerequisite for X-ray

crystallography.
● It requires placing the samples in nonphysiological environments, which can
occasionally lead to functionally irrelevant conformational changes.
Proteins themselves are not crystals, and many do not crystallize readily. Therefore,
protein crystallography is a very involved process. There are many steps necessary
to produce a “protein crystal” and determine its structure. First, the protein must be
isolated through a series of purification processes that may vary depending on its
unique properties. The goal is to generate a homogeneous solution of soluble,
properly folded, and stable protein. It may take years to perfect this process for every
new protein being crystallized.Under the right conditions, a supersaturated protein
solution will form crystals.A common and much more gentle way to achieve protein
supersaturation is through vapor diffusion. In this process, the protein sample is
placed in a buffered precipitant solution. A drop of this is placed inside a sealed
container that houses a reservoir of a more concentrated solution. As water moves
to equalize the concentration between the drop and the reservoir, the precipitant and
protein concentrations increase in the drop. If this process is sufficiently gradual,
well-ordered crystals will develop in the drop.

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 ?

● It is a periodic (or repetitive) arrangement of molecules in three-dimensional

space.
● Molecules precipitating from a solution tend to reach the lowest free energy
state.
● It is accomplished by packing in a regular way. This regular packing is formed
by the repetition in the three space dimensions of the unit cell .

X rays :
X-rays are a form of electromagnetic radiation with a much shorter wavelength than
visible light (0.01 to 10 nanometers)

Diffraction is a phenomenon that occurs when a wave (such as an X-ray) hits an

obstacle. Diffraction works through elastic scattering. This is a type of scattering
where the wave, interacting with a particle, bounces off in some direction without its
wavelength changing.
Crystallographers use X-ray diffraction to produce a pattern on a detector or film.
The diffraction pattern is then analysed to determine the arrangement of the atoms.

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

To obtain a brighter source, the anode can be made to

revolve (rotating anode generator) and is water-cooled to prevent it from melting. An
alternative source of X- ray is obtained when a beam of electrons is bent by a
magnet. This is the principle behind the synchrotron radiation sources that are
capable of producing X-ray beams some thousand times more intense than a
rotating anode generator. A consequence of this high-intensity radiation source is
that the data collection times have been drastically reduced. A further advantage is
that the X-ray spectrum is continuous from around 0.05-0.3 nm .
INTRODUCTION
(link:https://pubmed.ncbi.nlm.nih.gov/25604842/#:~:text=Autoradiography%20is%20
a%20technique%20using,tissues%20and%20cells%20for%20decades.)
auto radiographic studies, both in vitro and in vivo, have been used to characterize
the properties of radiolabeled drugs used in nuclear medicine.
results from autoradiographic studies can be used to adequately define the
characteristics of a radiotracer system before embarking upon the more costly, and
technically challenging non-invasive imaging study.
For example, the imaging of glucose metabolism in man using
2-[18F]fluoro-2-deoxy-Dglucose has relied heavily on ex vivo autoradiographic
studies conducted in animals.
One of the major uses of autoradiography in the pharmaceutical industry is to
determine drug disposition (usually Carbon-14 labeled) by sectioning rats (whole
body studies) at various times after injection.

Applications Of Autoradiography

(link : https://conductscience.com/what-is-autoradiography/)
Autoradiography In Preclinical Research

Whole-body autoradiography (WBA) and Microautoradiography (MARG) – In

vivo autoradiography techniques used to determine the tissue distribution of
radiolabeled compounds in laboratory animals (Figure 4)

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).

WBA can be quantified (quantitative WBA) using dosimetry techniques, i.e.,

comparing the different radioactivity intensities with radioactive standards of known
concentrations. An alternative method to QWBA is autoradioluminography, in which
whole-body sections are exposed to a phosphor imaging plate, and scanned by a
phosphor image scanner (Figure 4) (Solon & Kraus, 2001)

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).

Autoradiography In The Clinical Context

Positron-emission tomography (PET) – Diagnostic imaging method used to

observe biological processes in the body.

In a PET scan, a radioisotope is coupled to a radioactive tracer and then introduced

into the body.

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.

Gamma cameras record several 2D images that correspond to the radioactivity in

the capillary vessels near the target organ or in the whole body. Using multiple
projections of the acquired images, a computed tomogram, i.e, a 3D reconstruction,
is generated. The 3D tomogram can then be digitally manipulated to analyze the
radioactivity distribution in the sample (Figure 6).

Proteins, lipids, sugars, and even patient cells can be used as radioactive tracers.

For example, to determine the source of internal bleeding in a patient, it is possible

to radiolabel the patient’s red blood cells, inject them back into the body and then
follow the accumulation of radioactivity in a specific part of the body using a PET
scan (NIH, 2016).

Table 1 Common positron-emitting radioisotopes used in PET

Figure 6 PET / SPECT scan. A – PET scan machine (www.itonline.com). B – Left side: 3D
reconstruction (tomogram) of a brain; Right side: 2D sectional images obtained after computed
tomography (HBOT, 2019)

Single-photon emission computerized tomography (SPECT) –

Similar to PET scan but uses different radiotracers.

SPECT imaging, the radioactive tracers emit gamma rays. A list of the most common
SPECT radioisotopes is shown in Table 2.

Table 2 Common gamma-ray emitting radioisotopes used in SPECT

(link:
https://www.hilarispublisher.com/open-access/autoradiography-detection-and-analysi
s-of-radioactive-entities-2155-6180-1000361.pdf)
Conclusion Today, autoradiography is employed as an important detection tool for
the identification of different target receptors in various tissues to provide us with a
better understanding of molecular pharmacological pathways.

❖ 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.

Case study for food contamination

(link:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4740882/)

Evaluation of the cause of unexplained radiocaesium

contamination of brown rice in Fukushima in 2013 using
autoradiography

Methods

We identified radiocaesium contamination in rice by autoradiography and

estimated the 134Cs/137Cs radioactivity ratio in brown rice and soil samples
by gamma-ray spectrometry.
Autoradiography

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.

To evaluate the effect of the direct contamination of brown rice by

radiocaesium, the brown rice grains in each sample from the southern area
of Minamisoma City with high levels of radiocaesium contamination were
divided into highly contaminated grains and less contaminated grains.
Brown rice (80 g, approximately 4000 grains) was affixed to a piece of
cardboard with spray adhesive (3 M Super 77 Multipurpose Spray
Adhesive, 3 M Japan Limited, Japan) and then exposed to an imaging plate
for 2 weeks to isolate the highly contaminated grains as much as possible.
Visible spots with the radioactivity in the samples and the markers on the
autoradiogram of brown rice were traced onto the clear plastic sheet, and
the sheets were superimposed accurately on the cardboard-mounted brown
rice samples at marked positions. The spots on the autoradiogram were
several times larger than the actual grains due to the sample thickness and
because the radiation was emitted in all directions. Therefore, it was
difficult to distinguish the highly contaminated grains from neighbouring
less contaminated grains. Accordingly, the grains of interest were selected,
set at appropriate intervals on another piece of cardboard with
double-sided tape, and exposed to an imaging plate for a further 2 weeks
to confirm their high radioactivity. In total, 320–400 g of each of the
brown rice samples was used to identify highly contaminated grains. Each
group was weighed, pulverized, and then placed in a cylindrical
polypropylene container (U-8 container, 65 mm in height and 50 mm in
diameter).

Map showing the radiocaesium contamination in brown rice in Minamisoma City in

2013.
The grey areas represent areas subject to the evacuation order. The × symbols mark places
where radioactivity concentrations in brown rice were over 100 Bq kg−1. The map was
generated based on the data from the Ministry of Agriculture, Forestry and Fisheries of Japan
(MAFF)4. The map was created using Adobe Photoshop Elements 13 and Microsoft
PowerPoint 2013 software.

https://www.mhs.ox.ac.uk/backfromthedead/exhibition/the-structure-of-pe
nicillin/ penicillin