Easier Way to Convert Proteins into Crystals

Roland Piquepaille writes "As you might know, proteins need to be transformed into 3-D crystals before their atomic structures and their properties can be analyzed. And production of high quality crystals from proteins has been a difficult task until now. But scientists in the U.K. have successfully used a porous medium, or 'nucleant,' a material that encourages protein molecules to crystallize. Their first step towards 'holy grail' of crystallography could help speed up the development of new medicines and treatments."

Comments

Re:This is Big

[DerCed]

Yeah, you've got it mostly right.
In structural biology, the x-ray crystallographers try to find out the exact 3-dimensional structure of a single protein. Since almost everything in the biochemistry of the human body works with proteins, they are a common target for drugs!

The problem in crystallization lies in the properties of the proteins themselves. They are flexible, dynamic, fragile little machines which SHOULD NOT crystallize in your cells (exactly this happens in diseases linked to prions). So they are very soluble in water. Researchers try to find the right conditions (salts, precipitants, etc.) to get them to build little crystals. Often, this process takes months or years.

This new technique will hopefully fasten it up!

Re:This is Big but not that big

[Asam]

Sure being able to find suitable crystallization conditions for proteins is a bottleneck at the moment and this will aid in the process and add to the 20,000 plus know Protein structures, however, this is limited to certain types of proteins. A lot of really interesting proteins however are more flexible, and its this flexibility thats the key to understanding a lot of protein function. Structures derived from crystals don't give so much information about protein dynamics and molten globule like states of proteins, in fact when you crystallize proteins they tend to be locked into one conformation. Everyone in the field should keep this in mind always. A protein structure derived from a crystal structure is just a framework, its not a final representation of final function. More clues about this kind of information can be derived from NMR (Nuclear Magnetic Resonance), however the use of NMR is restricted to relatively small proteins. This is going to help, but its not huge, the real breakthrough will come when we can model any given protein sequence on a computer and have an acurate predicated 3 diminsional structure together with it molecular motions and dynamics in real time. Glad to see /. covering the field, its huge and its just started, we're at the begining of an exponential curve. Maybe /. should make a seperate section for this, I'd certainly be interested in contributing articles and news to such an effort.

Or don't crystallize at all(!)

[infolib]

It might be possible to determine protein structure with just a single molecule, no need for crystallization at all, e.g. with free electron lasers

The brilliance of x-ray sources are right now undergoing a revolution much faster than Moore's law.

Re:Ever hear of NMR structure determination?

[PDoc]

NMR is great, because it's fast, relatively easy to run, and tolerant of substrate. But it isn't absolutely accurate. Techniques such as nOesy and cosey allow for determination of stereochemistry via two-dimensionaly NMR, showing contact through bonding and through-space interactions. However the data from a nOe experiment can be inconclusive, especially in cyclic systems. Only X-ray data can actually confirm the true structure then.
A friend, also doing a Ph. D in chemistry, has just binned half his thesis because the NMR data lead to one conclusion, only to be contradicted by the X-ray data. And this wasn't a small difference either...
Oh well, back to the lab then...

Re:Roland Piquepaille knows nothing.

[cruachan]

I'm afraid not. Nothing beats having an accurate structure from Px. I spent several years as a postdoc attempting to grow large crystals of a membrane protein (at the time one of the first three or four membrane proteins to be crystallized). As we really were interested in knowing the structure rather than how we got it as light relief from purifying the protein on a near industrial scale and seeding thousands of crystallization trials we tried every other structural analysis method we could get our hands on. Many of these gave us interesting and invaluable info, but in all cases they were of most interest after the crystal structure was solved and the results could be interpreted in the light of that.

With most Protein Chemistry having the Px structure is the Gold Standard, and you can't really say you understand a protein until you have it. Trouble is, that as the article says, getting crystals is hard, slow, and extrodinarily painstaking work

crystallization is tough stuff

[!splut]

IAASB (structural biologist), and while I can't verify their findings, I can back up the premise in the article that generating diffraction-quality protein crystals is one of the two major bottlenecks to X-ray crystallography (the other being purification).

It's pretty easy to understand why. Not only do you need pure protein, but one must find conditions under which that protein forms relatively large, single crystals. The chief variables, aside from the homogeneity of the protein you're starting off with, include temperature, pH, protein concentration, choice of and concentration of precipitant (generally a chemical that drives the protein out of solution), choice of and concentration of additive compounds, in some cases detergents... The researcher must traverse this multidemensional search space by trial and error, with a limited quantity of protein, looking for the optimal conditions. On top of that, the conditions that confer the ideal level of nucleation may not be ideal for crystal growth...

We have developed shortcuts over the past 20 years, or so. Kits are available that allow one to screen through frequently successful crystallization conditions. The number of conditions one can test in one go is gradually increasing, as things miniturize somewhat.

The ease-of-crystallization varies amazingly from one protein to another, and tricks that improve one do not necessarily work for another, but anything that simplifies the process will be greatly appreciated by the field.

Re:crystallization is tough stuff

[BabyStewy]

While I agree that this is a major advance, I think calling this the "holy grail" is going too far. Currently there a several ways of doing large screens for crystallization conditions which utilize robots and nanoliter volumes of protein/condition to screen thousands of precipitant mixtures. The two real major stumbling blocks in crystallography are purification of monodisperse, nearly homogeneous protein (with respect to post-translational modifications as well as identity of the protein species) and the real "holy grail" problem, which is ab initio phase determination.
Since X-rays cannot be lensed, the Fourier transform of the diffraction pattern (which is the Fourier synthesis of the ordered electrons in the crystal) requires knowing not only the intensities, a trivial task, but also the relative phase angle of each reflection. This is a problem with possible solutions on the order of 6^n, where n is the total number of unique reflections, or about 5-10,000 for an average macromolecular structure at 3 angstrom Bragg spacing, allowing for a +/- accuracy of 30 degrees for each phase angle. Current solutions rely on searching reciprocal space with similar known structures (Molecular Replacement) or several ab initio methods that require one or more heavy element derivatives of native crystals. The first approach only works for crystals where a very similar structure is already known and available to the investigator. The second approach requires further screening for heavy metals that bind in ordered sites in the crystal without significant alteration of the native lattice, then usually a trip to a tunable X-ray source at a synchrotron. This second approach can burn through an astounding number of crystals and investigator time. There are shortcuts such as making the protein with Seleno-methionine instead of methionine (selenium has a usable X-ray edge for phasing, unlike sulfur),but this is normally done after initial purification and crystallization have been optimized. For more reading on the phase problem, I'd recommend either Alexander McPherson or Jan Drenth's excellent introductory textbooks on macromolecular crystallography.

Re:Understanding protein structure..

[Asam]

There are ways to obtain structure of proteins from DNA, just that they're not so good if a similar structure doesn't exist in the Protein Data Bank (http://pdb.org/ already, its called homology modelling. You have the protein sequence of an unknown structure then look for similiar sequences in the 20,000 known protein structures. That's why the more structures we add to the PDB makes it easier to find structures of unknown proteins.

Probably gonna start a flamewar, but...

[cr0sh]

If proteins can be crystalized (albeit with some difficulty), in order to study structure and makeup, then is the reverse true? That is, could some proteins be created from a crystal matrix?

I know this isn't a new idea. I don't have references handy to prove it isn't, I just know that I have read arguments about it. This theory is used to explain the origins of life (distinct from the theory of evolution). Basically, you have the whole "early earth molecular soup mix with electrical activity providing the spark-o-life" (Miller-Urey Experiment), forming organic compounds, which are then (in some manner) "processed" by crystal structures forming later (?).

It makes me wonder if it wouldn't be possible to study crystals in a similar manner to see whether they could (in some manner) aid the formation of the organic compounds formed by the Miller-Urey (and other similar) experiments into early proteins or protein-like structures? Does anyone know if such a study has already been undertaken? Or, is this idea nothing more than baseless speculation with no foundation in reality? I am sincerely curious...

Re:Probably gonna start a flamewar, but...

[Anonymous Coward]

There are theories of the origins of life that invoke highly ordered matricies like montmorillonite clays that have been shown to catalyze some biologically relevant reactions, as well as induce the formation of lipid bilayer micelles, which would have been important for the formation of primordial cells. But for the most part, biologists agree that RNA probably came along before proteins, so looking for a template that could create RNA would probably be a better target.

But whatever the method, the way to make proteins would almost certainly not occur on a naturally formed crystal of another protein. Protein crystals don't form easily, and it's doubtful that they'd ever get concentrated and pure enough in the wild to crystallize on their own.

Re:This is Not so Big

[poincaraux]

Crystalization is difficult and, frankly, rather unscientific
You're not kidding. My favorite example is the fact that many crystallographers add diet coke to aid in crystallization.