On protein crystallisation…

February 23, 2012
Synthetic insulin crystals synthesized using r...

Image via Wikipedia

A bit of a departure from the usual quack-bashing…

Crystallography is great. I love it. The reason that I love it is that it is to my mind one of the most powerful techniques we have at our disposal for getting mechanistic detail about how life works at a molecular level. Biological crystallography allows us to determine the precise three dimensional structure of biologically relevant macromolecules – like proteins and DNA, but also small molecules such as ligands and metabolites necessary for life to thrive and survive.

As the name suggests, crystallography requires the growth of crystals. I think that one of the more interesting facets of crystallography is the dichotomy between the very rigorous and precise methods of data processing and model refinement, and the act of crystallising a protein, which is often described as ‘black magic’.

Crystallisation is essentially controlled precipitation – the protein comes out of solution in an ordered manner, and forms a crystal lattice. In order to get the protein to fall out of solution in this way, we have to alter the chemical environment of protein by mixing it with different concentrations of buffers, salt and precipitating agents.

The complex nature of both the process of crystallisation and the chemical nature of proteins means that we cannot currently predict the chemical conditions in which protein might crystalize. So, we just try stuff that has worked previously. We take our hard won protein and mix it with random solutions, and let it concentrate slowly using a process called vapour diffusion*

Crystallisation is now routinely carried out using crystallisation robots – liquid handling machines that make the whole process easier, faster and more repeatable. They also can dispense nanolitre volumes of protein, making the meager amounts of  proteins that we have struggled to produce and purify go much further. Initial screening is carried out using screens that can be brought in deep well block format from companies such as Molecular Dimensions and Hampton Research.

Labs will have their preferred range of screens and preferred suppliers. FWIW, my first line of attack is to use the JCSG+, Pact Premier, Morpheus and Clear Strategy I & II screens from molecular dimensions. It is the follow-up optimisation of any crystallization ‘hits’ that I want to discuss here.

In days of old (~10 years ago) crystallisation was setup on a micro/millilitre scale. We would setup 24-well trays with 0.5-1 ml of well solution, and drop sizes of 1-10µl. Now, with the robots, we setup 96 well trays with ~80µl of well solution and drop sizes of 200-600 nanolitres. When optimising crystallisation hits, one might suppose that bigger drops might be more likely to yield bigger crystals – which is probably true, but the crystallisation conditions do not necessarily scale up in a simple fashion – this is due to changes in the crystallisation setup, such as the ratio of well solution volume to total volume within the experiment and the drop surface area to volume ratio .

I will admit that I had previously struggled to make the transition from robot-setup nanolitre-scale screens to hand made microlitre-scale optimisation screens. However, I now would argue that hand-setup drops are no-longer required in routine cases of structure solution. Cases where ligand or heavy atoms soaks are required may still need 24-well plate style setups and whathaveyou.

Rather than screen around potential crystallisation hits, I now setup bespoke deep well blocks to screen around them as follows.

I setup 16 15ml falcon tubes. A-H (low) and A-H (high).  Each set of falcon tubes will contain crystallisation conditions with different extremes of one variable, for a given condition.

Let us suppose that I get a hit in 100mM MES pH 6, 20% PEG 3350, 0.2M CaCl2. The first set of variables that we might want to screen are PEG concentration, pH and salt concentration. I would setup 3 sets of falcon tubes as follows.

A (low) 100mM MES pH 6, 10% PEG 3350, 0.2M CaCl2

A (high) 100mM MES pH 6, 30% PEG 3350, 0.2M CaCl2

B (low) 100mM MES pH 5.5, 20% PEG3350, 0.2M CaCl2

B (high) 100mM MES pH 6.5, 20% PEG3350, 0.2M CaCl2

C (low) 100mM MES pH 6, 20% PEG3350,

C (high) 100mM MES pH 6, 20% PEG3350, 0.4M CaCl2

I would then setup a gradient within rows running from positions 1-11 as follows:

  1. 1ml of ‘low’
  2. 0.9ml of ‘low’, 0.1ml of ‘high’
  3. 0.8ml of ‘low’, 0.2ml of ‘high’
  4. and so on up to 11, which contains 1ml of ‘high’
This is a relatively straight forward setup for fine screening of crystallisation hits for those of us without really big liquid handling robots that make up your own screens from stock solutions**. You can also incorporate increasing amounts of additives such as cryoprotective agents or perhaps ligands into screens as well, although I tend to use column 12 for screening a couple of low concentrations of cryoprotective additives – e.g. 1% and 5% (v/v) of Ethylene Glycol, Glycerol, MPD and PEG400 – in the base conditions.
I find that this setup is extremely useful as the deep well block can be stored in the fridge for several months (if properly sealed) and used to screen successive batches of material or perhaps to screen point mutants, (that may or may not crystalize in the same conditions, depending upon how lucky you are 😉 ). You get the benefit of bespoke optimisation screens with fine gradients AND the reproducibility, ease and low protein use of a robot. This obviously works best for a system like the Art Robbins Pheonix robot (my weapon of choice) where the crystallisation screen is dispensed into the crystallisation tray by the robot.
Another benefit of this system is the ease at which one can obtain rudimentary phase diagrams, if you use a crystallisation plate with 3 drops per well (I use Art Robbins 3-well intelliplates) and set up screens with 3 different protein concentrations, you will generate data which allows you to create such a phase diagram.
More often than not, this optimisation can yield crystals of the sizes required for routine synchrotron data collection (i.e. >100µm along at least one edge), thus rendering the stock of 24-well plate gathering dust in the corner of the lab pretty much obsolete. Clearly, there will always be occasions when a larger crystallisation drop is necessary, but on the whole, I believe that simple fine screening done like this is sufficient for most structure determination efforts.

*other crystallisation methods are available.

** pH might not necessarily scale linearly with different buffer mixes, especially if buffer types change – but this can be checked with a pH meter if a particularly successful mix of buffers is found

This is the first time I have posted about work and technical aspects of it on my blog – I didn’t think that this method would merit a write up as a technical note in a journal, and as far as I know, most labs might be doing this already. However, I have found this technique to be very successful and easy, and so thought it might be beneficial for some to post it online. If you either already use this setup, or you have used this setup after reading it here and it was successful or not, please post below and give me feedback!

My favourite… Homeopathic remedy

April 17, 2010

Celebrating the end of WHAW.
(world Homeopathy awareness week, of course)

You may not be aware of it, but we have just survived world homeopathy awareness week (WHAW), the amazing non-event that was marked by ever-so-slightly elevated levels of pro-homeo spam on twitter, and a plethora of pithy science blogs that carefully pointed out that there was no evidence that homeopathy works, or indeed, how it might conceivably work. This was topped off by a Dilbert cartoon that handily lampooned both homeopathy and astrology.


Homeopaths might want take note of the current plight of UK chiropractors – who this week, finally folded in their attempts to sue Simon Singh for libel. In suing Simon Singh, the British Chiropractic Association (BCA) stuck their collective heads above the parapet of public ignorance/apathy, and were struck down by a combination of artillery fire from organisations like Index on Censorship, English Pen and the “Keep libel laws out of science” campaign, and some precision snipping from science/skeptical bloggers such as Zeno and Simon Perry. As a result of all this:

  1. the libel laws look set to change,
  2. the BCA lost,
  3. the BCA will be paying a proportion Simon Singh’s costs.
  4. ~25% of all chiropractors are currently under investigation for making unsubstantiated claims on their websites

Given this, one would hope that other “unreality enthusiasts” (© Dr_Aust_PhD) might keep their heads down for a while – but oh no. Not the homeopaths. Despite the 1023 campaign drawing attention to homeopathy, and STFC report into homeopathy concluding that homeopathy is not deserving of NHS funding, the homeopaths continue to draw attention to themselves – by having an “awareness week”.

Of all the “remedies” that homeopaths use1, from the light of Venus, to fragments of the Berlin wall, Natrum Mur has to be one of the most ridiculous, from a scientific point of view. According to this webpage, Natrum Mur is used for:

Homeopaths give this remedy for emotional problems, such as anxiety and depression, that are caused by suppressed grief and other emotions.

Natrum mur. is also used to treat conditions with a watery discharge, such as colds, and phlegm or profuse, clear mucus. Complaints that are generally worse with heat and that are often brought on by stuffy heat or exposure to hot sun are helped by this remedy. These include: migraines with zigzag lines in front of the eyes; eyestrain with aching eyes; headaches that come on after menstruation; and cold sores.

It is also good for mouth problems, for example, gingivitis (inflamed gums), dry, cracked lips, mouth ulcers, and bad breath (halitosis).

Skin complaints, for example, warts, dry cuticles, hangnails, boils, and painful acne are helped by Natrum mur. It is also effective for goiter; anemia; indigestion; constipation with dry, hard stools; bleeding anal fissures; backache; and delayed urine flow.

In women, Natrum mur. is given for absent menstruation induced by shock or grief; irregular menstruation; and a general feeling of being unwell both before and after menstruation. It is also good for a dry or sore vagina, vaginal discharge, and vaginismus (vaginal pain during sexual intercourse).

When ill, people who need this remedy are chilly but dislike heat.

Clearly a highly potent remedy with a wide range of clinical applications. *cough*

But what, I hear you ask, is was in Natrum Mur? It is some exotic herb, a distillation of fermented sheep eyes? No.

It’s table salt. Sodium chloride. The same stuff you put on your chips. The main flavouring in “ready salted” crisps. Salt.

Incidentally, the first crystal structure ever determined was NaCl, which earned Sir W.H. Bragg and his son Sir W.L. Bragg the Nobel prize for physics in 1915.

Salt is an important part of our diet, but problems generally stem from having too much of the stuff. The food standard agency recommend that an adult eat no more that 6g of salt per day. Aficionados of Avogadro’s constant will be able to tell you that this is 6/(22.99+35.453) X 6×1023 = 6.15×1022 molecules of salt – which is “some” salt.

More than “some” salt may cause you problems – hypertension, cardiovascular disease, renal stones, osteoporosis, stomach cancer to name but a few. Now, given that homeopaths follow the law of similars, which states that “like cures like,” one might expect these diseases/pathologies would be the same that homeopaths might claim to cure with Natrum Mur. Curiously, these are all absent from the list.

Salt is also used to balance electrolytes in saline solutions for fluid replacement, and washing wounds with salt water has an antiseptic effect (killing bacteria by “osmotic shock“).

But then we all contain “some” salt  – according to Wikipedia (yes, I know!) the human body has about 0.15% by mass of both chlorine and sodium – this will all be present as sodium and chloride ions. However, if you could extract all this as sodium chloride from an average 70kg person, you’d end up with about 150g of salt.

So, given that we contain ~150g of salt, and we consume/excrete 6g of salt per day, how is adding the memory of salt going to make a blind bit of difference to “anxiety and depression”, “migraines with zigzag lines in front of the eyes” etc?

My favourite homeopathic remedy is Natrum Mur – because for me, it really drives home the inane, reality-deprived nature of homeopathy.

1 – of course, homeopathic remedies above a potency of 12C don’t actually contain anything.


My favourite… protein structure.

April 15, 2010

A bit of deadly but beautiful biology…

Crystal structures are intricate models of great complexity and beauty. Some of them may simply just resemble 3 dimensional squiggles that happen to bring the right combination of molecular groups together in space to allow some funky chemistry to happen, or some highly specific binding event. Some other them, however, look like pieces of art…

Bacteria are scavengers – tiny little single celled organisms whose raison d’etre is to proliferate, thrive and survive. (This is also your raison d’etre, but humans have developed all sorts of clever distractions to make us think that life is something more that just an advanced way of passing genetic information from generation to generation.) In order for bacteria to do this, they need lots of  chemicals – both to metabolise for energy and to serve as building blocks for components they need to fulfil their task.

It just so happens that nature has provided bountiful supplies of those chemicals almost everywhere you look – Us.

Human (and indeed all animal) cells are essentially bags of chemicals – exactly the chemicals that are so highly prized by bacteria. Which is why the bacteria have devised many cunning ways of extracting those chemicals from the bags in which they reside – one of the more formidable weapons in the bacterial arsenal is that of the “pore forming toxin” or PFT.

Pore forming toxins do exactly what they say on the tin – they form pores in cell membrane. In the short term, these pores will lead to nutrients flowing out of the cell – where the bacteria can get hold of them. In the long term, loss of membrane integrity often leads to the death of the cell – important cellular processes stop due to lack of important chemicals, and the concomitant influx of water into the cell makes it swell uncontrollably and burst – even more goodies for the bug to get hold of!

PFTs are interesting proteins to work with – partly because they are schizophrenic. They have a happy and often easy to handle soluble form, where then generally exist as single molecules (monomers) and act as normal soluble proteins. Then they have a hydrophobic (water “hating”) pore form, where they exist as large complexes (multiple proteins join together) to form the active pore. The pore mode is generally a pain in the backside to work with, as they aggregate and precipitate very easily. As a result of this, many crystal structures of PFT tend to capture them in the soluble state. However, there is one very notable exception to this rule.

In 1996, Song et al crystallised α-hemolysin (grr, US spelling) and managed to isolate it in the pore conformation. And it’s a beaut.

Sometimes crystal structures need months of evaluation and further experiments to determine the implications of the structure and develop a full picture of how the protein accomplishes it’s biological role.

Fig1: alpha-hemolysin's stalk is perfectly designed to span the plasma mebmrane of eukaryotes

In the case of α-hemolysin, one look is all you need – it is (hopefully) very obvious how structure relates to function.

To get a better sense of the 3D-nature of this beasty, I’ve knocked up a quick animated gif – view here (give it a while – it’s a bit jerky to start with)

7 monomers (each a different colour in the figures) come together in a ring – each monomer donates 2 -beta- hairpins – these form the ‘stalk’ that protrudes from the bottom of the structure. This stalk is hydrophobic/lipophilic, and is 2.8nm long. The cell membrane it’s designed to punch though? Made of lipids and ~2.5nm deep. So the stalk of the protein has just the right dimensions  and chemical composition to span the membrane of your cells.

Fig 2: Looking down alpha-hemolysin's pore...

In figure 1, what you can’t see is the pore – running from top to bottom of the stucture – figure 2 shows this better. It’s identical to figure 1 but rotated 90º top-towards you.

You can see that right though the middle of the structure is a hole – the pore through which the nutrients flow out, and water flows in.

The 7-fold rotational symmetry (a bit of a rarity) also adds to the curious attraction of this molecule.

Anyway – ever since I first stumbled across this structure in 2000, it’s been my favourite, even though my own attempts to replicate it failed. ( or did they? – work still in progress 😉 )

In my humble opinion, this structure is one of the best example of how the structure of a protein is related to its function, and also how well adapted bacteria are to explioting host biology.

EDIT – Since I did this, the wonderful guys at proteopedia have been in touch via the medium of twitter (@proteopedia) about using this as a basis for a page on α-hemolysin. If you’re interested, follow them on twitter and have a look around the proteopedia wiki. Good page to start with is ‘the ribosome