Biology

Structural
Structural biology is concerned
with the arrangement of atoms
and molecules in biological
macromolecules such as
proteins, membranes and DNA.
These macromolecules are
vital to the function of most
cells and therefore, biological
systems.

Consequently, being able to understand how they achieve

their structure, the impact of biologically relevant conditions
and how alterations in their structure impact their function is
of great interest to scientists in diverse disciplines, from cell
biology to drug discovery. In this infographic, we will explore
the techniques used to solve macromolecular structures and
what information structural biology can tell us.

What do
structural
biologists
look at?

Whilst a certain amount of information can be

gained about a protein by considering its primary
structure, having the capability to look at tertiary
and quaternary structure is very important, as
it reveals how protein folding and interactions
influence its behavior and function.

ß-pleated
amino a-helices sheet
acids

ß-pleated
sheet a-helices

Primary Protein Secondary Protein Tertiary Protein Quarternary Protein

Structure Structure Structure Structure
Sequence of a chain Local folding of the Three-dimensional Protein consisting of
of amino acids polypeptide chain into folding pattern of a more than one amino
helices or sheets protein due to side acid chain
chain interactions

Considerations

The conditions in which the

structure of a macromolecule
is examined are important too.

Some sample preparations take the

macromolecule out of its natural
environment (e.g. different pH) and
consequently, observations made
may not reflect the structure in natural
settings. Consequently, information
gleaned may have limitations and
it is important to be mindful of
experimental artifacts.

For example, a protein that has been denatured

during preparation will not demonstrate the
structure seen in its native state.

A Role for Bioinformatics?

Bioinformatics can help

scientists to make predictions
about the structure of a
macromolecule or the way
it interacts.

This is achieved by taking information,

such as the position of residues and
their surrounding environment and
incorporating this with data from
known macromolecules that have
been previously analyzed.

Structural biology relies

on a host of techniques
that, used together, enable
scientists to gain insight
into multiple aspects of
structural information
without ever "seeing" them.

What
techniques
are used in
structural
biology?

Visualizing atoms is not easy. Even highly

sophisticated light microscopes cannot give the
resolution required to study macromolecular
structures. Therefore, scientists use a selection
of more targeted techniques.

The principle methods used are:

Cryogenic
X-Ray Nuclear Magnetic
Electron
Crystallography Resonance (NMR)
Microscopy
(cryo-EM)
Spectroscopy

X-Ray Crystallography

Proteins and nucleic acids are able to form crystals that can then be subjected to X-ray
diffraction experiments to reveal their atomic structure.

Sample preparation is key for obtaining good crystal structures. Conditions for crystallization are molecule-
specific and therefore require the screening of many buffers, salts and additives to get the best conditions.
The sample buffer chosen must also be compatible with buffers used during crystallization.

Nucleic acid or protein crystals must be:

◊ High purity (chromatography techniques

are often used to achieve this)

◊ Sufficiently concentrated
(typically 0.2 mM or more)

◊ Sufficiently large (crystals should be

at least 0.1 mm in all dimensions)

◊ Free from significant internal

imperfections (such as cracks or twinning)

Following crystallization, crystals are

imaged under a microscope and
checked with UV light to confirm they
are biomolecules and not salt.

Crystals are then subjected to X-ray diffraction.

CRYSTAL DIFFRACTION ELECTRON ATOMIC

PATTERN DENSITY MAP MODEL

X-Rays Phases Fitting

Refinement

1
The crystal is placed 2
The intensities 3
Each data set 4
The final refined
in an intense beam and angles of consists of many model of the atomic
of X-rays. Normally X-rays diffracted thousands of arrangement is called
a single wavelength by the crystal are reflections and a crystal structure.
is used to produce measured as the represents just
a regular reflection crystal rotates and over half a rotation
pattern. each reflection of the crystal. Using
recorded. Every informatics tools,
compound has a these datasets are
unique diffraction then combined
pattern. and, with the
addition of other
complementary
information, used
to refine a model
of the atomic
arrangement
within the crystal.

Interesting facts:

Synchrotrons can be used to improve resolution and target

the most highly defracting areas of a crystal

In 1952 Rosalind Franklin was the first person to use X-ray

crystallography to reveal the helical structure of DNA

The first protein structure ever to be solved was sperm whale

myoglobin, determined in 1958 using X-ray analysis

NMR

NMR is a powerful and non-destructive technique for solving macromolecular structures,

investigating molecular dynamics, interactions and much more.

Some atomic nuclei have magnetic properties called “spin” that respond when placed in a weak magnetic field.
The surrounding environment of that nucleus impacts this response which can then be interpreted to determine
atomic arrangements, map binding sights and measure affinities.

a b
solid-
bacteria state NMR collection 3D structure of the
+ plasmid
of solid- protein assembly
state NMR
labeled structural
protein subunit
magnetic
data
expression purification
field

c d 54.7° modeling
from solid-
state NMR type 1 pilus of
data uropathogenic
in vitro protein assembly E.coli
assembly control (eg. EM)

1
Biomolecules are 2
Samples (which 3
Sequential 4
A structure is
labeled with nuclei can be solid or assignment is used calculated and
that have spin liquid and therefore on the spectra refined; other data
(e.g. 1H, 13C and 15N). have dynamic to assign 1H, 15N types, such as crystal
movement) are and 13C etc. to the structure information,
bombarded with peaks observed. can be incorporated
radio waves and in this step.
spectral data
collected.

Cryo-EM

Cryo-EM is a form of transmission electron microscopy (TEM) which allows samples to be

studied at cryogenic temperatures.

There are two principal types of cryo-EM, single particle analysis cryo-EM and cryo-electron tomography.

sample

Cryo-EM Image Identification Combination

Acquisition and Alignment

1
Thin films of samples 2
Samples are then bombarded with electrons, 3
With single particle
are placed in ethane creating a magnified image on the detector and cryo-EM, multiple 2D
baths at -180 °C, enabling the structure to be elucidated. images of the same
flash freezing them. kind of biological
object within the
frozen structure are
captured and then
processed using
algorithms into 3D
structures.

With cryo-electron tomography, multiple images of a single biological object are captured from different angles
from which a 3D structure can be created.

e- beam tilt alignment &

series extractions
+70º

thin
sample

inverse
0% problem

forward
problem -70º

3D reconstruction 3D reconstruction,
detector of individual regularization, and
molecules refinement

Cryo-EM has gained popularity in structural biology in the last 20 years over NMR and X-ray crystallography.
Whilst resolution has been the technique's stumbling block, two recent preprints now indicate that atomic
resolution protein structural determination is possible. This is however yet to become standard for cryo-EM.

Here is a comparison of the techniques discussed:

ADVANTAGES DISADVANTAGES SAMPLES SUITED TO

◊ X-Ray ◊
◊
High resolution
Well established
◊  rystallization can be
C
problematic
◊ C rystallizable samples
such as soluble proteins,
Crystall- ◊  uitable for macromolecules
S ◊ Diffraction can be difficult membrane proteins, DNA/
RNA, ribosomes, and protein
◊
ography of broad molecular weight
range
 tatic structure and
S
typically solid
complexes

◊ Easy to build models from

◊ NMR ◊
◊
High resolution
 an look at dynamic structure
C
◊  ample preparation can
S
be challenging
◊
◊
Water soluble samples
Macromolecules under 50 kDa
and structures in solution ◊  amples must have high
S
purity
◊ C omputational simulation
difficult

◊ Cryo-EM ◊
◊
Requires smaller samples
 o need to crystallize the
N
◊ C omparatively low resolution
(although this is improving)
◊  acromolecules over 150 kDa,
M
such as membrane proteins,
target (can be difficult and ◊  nly suited to high molecular
O large proteins, ribosomes,
changes environment of the target weight targets complex compounds and
virions
macromolecule)
◊  ependent on use of EM for
D
◊  o chemical fixing or staining,
N which equipment can be
retaining the native environment expensive
◊  an flash freeze macromolecule
C
in many different conformations

Other techniques used in structural biology include mass spectrometry, neutron diffraction, proteolysis, electron
paramagnetic resonance (EPR), electron crystallography and microcrystal electron diffraction, multiangle light
scattering, small angle scattering ultrafast laser spectroscopy and dual-polarization interferometry.

Additional molecular information can also be gained using biophysical techniques such as absorption spectrophotometry, circular dichroism,
electrospray mass spectrometry, fluorescence spectroscopy and isothermal titration calorimetry.

What can
structural
biology
tell us?

Structure-Based Drug Design

Many biological molecules are targeted by drugs. Understanding their structure and
interactions with candidate drugs is vital to optimize target binding and avoid unwanted
off-target effects.

+
+

Docking Docking

Anopheles dirus Species B

The Asian mosquito Anopheles dirus species B, an important malarial

vector, has developed resistance to some insecticides. Solving the structure
of the detoxifying enzyme glutathione S-transferase has helped scientists
to understand the mechanism behind resistance and to inform the design
of new insecticides.

Discovery of Molecular Function

Biomolecule structural changes

underly the basis of many
diseases. Understanding what
these changes are aids
scientists in deciphering disease
pathology.

By comparing data from healthy,

functional systems to those in a diseased
state, understanding of the disease
mechanism can be obtained, helping to
inform therapeutic development.

By comparing the structure of hemoglobin in red

blood cells from patients with sickle cell anemia
with those of healthy individuals, the structural
consequence of the genetic mutation that leads
to the condition is now understood.