Download The Macromolecular X-ray Crystallography (MX) ESRF Tutorial

The Macromolecular X-ray Crystallography (MX) ESRF Tutorial
Coordinators: Elspeth Gordon (ESRF, [email protected]) and Hassan Belrhali (EMBL, [email protected])
In Practice: practical will be carried out at the ID23-1 and ID23-2 ESRF beamlines where 2 X-ray
experimental stations, a sample preparation lab and graphic stations are available. The data collection
will be a mimic as the tutorial time will happen during an ESRF shutdown period.
1.
Participants will have the opportunity to make crystallization experiments of a well known
enzyme leading to the growth of protein crystals.
2.
Using the automation systems in place at the ESRF MX beamlines, a diffraction experiment,
i.e. exposing the protein crystals to the synchrotron beam, will be performed.
3.
Tutors will introduce and explain the various computational steps from data analysis to map
interpretation, looking at electron density maps and relating the shape and chemistry to the
protein structure.
Introduction:
The notes below will often refer specifically to proteins, but generally the same techniques and problems
apply to other macromolecules (DNA, RNA) or assemblies (viruses, ribosomes).
3D structure allows us to understand biological processes at the most basic level: which molecules
interact, how they interact, how enzymes catalyze reactions, how drugs act, etc…In some cases, it can
allow us to understand disease at an atomic level. We can also exploit 3D structure in developing new
drugs. There are a number of methods for studying 3D structure. But we will concentrate on X-ray
crystallography, which is one of the most effective at present. Other techniques complement effectively Xray crystallography and have a valued place in the set of tools that scientists can use.
What is X-ray crystallography?
X-ray crystallography is an experimental scientific technique that enables us to visualize protein
structures at the atomic level and therefore enhances our understanding of protein function. It can be
seen as a form of very high resolution microscopy.
Why use X-rays?
In order to see proteins in atomic detail, we need to work with electromagnetic radiation at a wavelength
of around 0.1 nm (same as 1 Å), in other words we need to use X-rays.
Why do we need a crystal?
The diffraction from a single molecule would be too weak to be measurable. So we use an ordered threedimensional array of molecules, i.e. a crystal, to magnify the signal. Even a small protein crystal (of few
tens of microns per dimension) might contain a billion molecules.
The X-rays are diffracted by the electrons in the structure and consequently the result of an X-ray
experiment is a 3-dimensional map showing the distribution of electrons in the structure.
A crystal behaves like a three-dimensional diffraction grating, which gives rise to both constructive and
destructive interference effects in such a way that they appear on the detector as discrete spots which are
known as “Bragg reflections”. This is called the diffraction pattern. As with all forms of electromagnetic
radiation, X-rays have wave properties, in other words they have both an amplitude and a phase. In order
to recombine an electronic envelop of the protein, both of these parameters are required for each
reflection. Unfortunately, only the amplitudes can be recorded experimentally – all phase information is
lost. This is known as "the phase problem". Crystallographers need to obtain phase information sufficient
to enable an interpretable electron density map to be calculated, i.e. they need to “solve the phase
problem”.
What is involved in a crystal structure determination?
Protein preparation
Firstly one needs to obtain a pure sample of the target protein. We can do this by either isolating it from
its source, or by cloning its gene into a high expression system.
Crystallization
The sample needs to be concentrated and transferred to a dilute buffer containing little or no salt if the
protein is happy under these conditions. If a similar protein has already been crystallized then it is
definitely worth trying the conditions used to grow crystals of this protein. Otherwise one would normally
submit it to one or more sparse matrix screens. To date, the total number of different conditions in our
repertoire of screens comes to about 600 using robotic platforms that handle nanoliter volumes.
Crystals (figure 1) might form overnight, but more typically they will take from several days to several
weeks to grow.
Figure 1: protein crystals
Figure 2 : a diffraction pattern
Testing crystals
Once we have crystals, then it is time to test them with X-rays. The crystal needs to be flash-cooled to
100 K in a loop to maintain its integrity during exposure to X-rays - and then attached to a device called a
goniometer which enables the sample to be accurately positioned in the X-ray beam. The focused and
monochromatic synchrotron X-rays strike the crystal to produce a diffraction pattern that will be recorded
on a special CCD X-ray detector. In ideal cases, we should see clean sharp spots, one lattice of spots
indicating we have a single crystal (figure 2).
X-ray data collection
Firstly we need to ascertain crystal parameters: symmetry, unit cell dimensions, crystal orientation and
resolution limit. Armed with this information we derive a data collection strategy which will maximize both
the resolution and completeness of the data set. The method we use is to rotate the crystal by a small
angle, typically 1 degree, and record the X-ray diffraction pattern. If the diffraction pattern is very crowded,
then the rotation angle should be reduced so that each spot can be resolved on the image. This is
repeated until the crystal has moved through at least 30 degrees and sometimes as much as 180
degrees depending on the crystal symmetry. The lower the symmetry, the more data are required. A
typical data set takes about 10 minutes to be recorded at the ESRF while would require several days
using an 'in house' X-ray source!
Structure solution
In order to visualize our structure we need to solve the phase problem, in other words we need to obtain
some phase information. For protein structure determinations we can do it in several ways:
A – If we already have the coordinates of a similar protein, we can try to solve the structure using a
process called Molecular Replacement which involves taking this model and rotating and translating it into
our new crystal system until we get a good match to our experimental data (all in silico processes). If we
are successful then we can calculate the amplitudes and phases from this solution which can then be
combined with our data to produce an electron density map.
B - If we have no starting model, we can make use of the anomalous scattering behaviour of certain
atoms at or near their X-ray absorption edges to gain phase information. The Multi-wavelength
Anomalous Dispersion (MAD) is indeed a very effective method that relies entirely on the measurement of
the anomalous differences produced by one or more anomalously scattering atoms in the crystal. In
practice three or more consecutive data sets are recorded from the same crystal at different wavelengths
around the X-ray absorption edge of the anomalous scatterer. As this method requires a tunable X-ray
source, it can only be performed at a synchrotron. The resultant phase information can often produce
very high quality electron density maps, thereby simplifying the subsequent interpretation.
Model building This is the process where the electron density map is interpreted in terms of a set of
atomic coordinates. This is more straightforward in the molecular replacement case because we already
have a coordinate set to work with. In the case of the MAD method we simply have the map. It is
essentially a three-dimensional jigsaw puzzle with the pieces being the amino acid residues. The normal
procedure is to first fit a protein backbone and then, if the resolution permits, we insert the sequence. The
amount of detail that is visible is dependent on the resolution and the quality of the phases. Shown below
is a high resolution electron density map with atomic coordinates superposed.
Figure 3: High resolution electron density map and model
Refinement
Once we have a preliminary model we can refine it against our data. This will have the effect of improving
the phases, which results in clearer maps and therefore better models. We would typically go round this
cycle several times until we get little or no further improvements.
Now is the time for biological interpretation!