Field of Science

Crystallography and chemistry: The culture issue

Image: Charles Reynolds and ACS Med Chem Letters
As the old saying goes, beware of crystallographers bearing ligands. Charles Reynolds who is a well-known structure-based drug design expert has an editorial in ACS Medicinal Chemistry Letters touching on an issue that lies at the confluence of crystallography, medicinal chemistry and modeling: flaws in protein ligand co-crystal structures. It's a problem with major ramifications for drug design, especially since it sits at the apex of the process and has the power to influence all subsequent steps. It's also an issue that has come up many times before, but like many deep-seated issues this is one that has not quite disappeared from the palette of the structure-based design scientist.

In 2003 Davis, Teague and Gerard Kleywegt (who is incidentally also one of the wittiest conference speakers I have come across) wrote an article pointing out one simple observation: in several PDB structures of proteins co-crystallized with small molecule druglike ligands, the protein seems to be well-resolved and assigned, but the small molecule is often strained, with unrealistic bond lengths, planar aromatic ring atoms, non-planar amide bonds, rings in boat or pseudo chair conformations and clashes between protein and ligand atoms. Now the protein can also be misassigned, and so can water molecules, but it turns out that the problem looms much larger for ligands.

Reynolds's editorial takes another, 2014 look at this 2003 problem. And it seems that while some people have actually become more cognizant of issues in crystal structures, things aren't exactly rosy at this point in time. He points out a 2009 study that located 75% of the structures in the data set whose geometries could be improved by using better restraints.

The first and foremost pitfall that non-specialists fall into when taking a crystal structure at face value is is to assume that whatever they see on that fancy computer screen is...real. The fact though is that, barring any structure solved to better than 1 Å (when was the last time you saw that?) every crystal structure is a model (and while we are on the topic, Morpheus's definition of "real" may also be somewhat relevant here). The raw data is those dots that you see in the x-ray diffraction; everything after that, including the pretty picture that you visualize in Pymol, comes from a series of steps undertaken by the crystallographer that involve intuition, parameter fitting, expert judgement and the divining of complete information from incomplete data. That's potentially a lot of guesswork and approximation, and so it shouldn't be surprising that it often leads to flaws in the results.

So is this problem primarily a technology issue? Not really. Reynolds points out several programs that can now fit ligands to the electron density better and get rid of strain and artifacts; Schrodinger's PrimeX and OpenEye's AFITT are only two prominent examples. Nor is it complicated to find out in the first place whether a ligand might be strained; any scientist who has access to a good molecular mechanics energy minimization program can take the ligand structure out of the protein, minimize it to the nearest local minimum, look at the energy difference (usually > 5kcal/mol for a strained ligand), visualize steric clashes between atoms and reach a reasonable conclusion regarding the feasibility of that particular ligand conformation.

The abundance of methods for both figuring out strained ligand conformations and refining them seems to point to something other than technology as the operative factor in the misinterpretation of crystal structures. I believe the problem, in significant part, is culture. Reynolds alludes to this when he says that "Crystallographers are not chemists". When you are a crystallographer and are in hot pursuit of a protein structure, you are rightly going to experience a moment of ecstasy when that huge hulking hunk of sheets and strands finally appears on your screen. But most crystallographers don't care about that little blimp in the binding site - a small molecule that's often crystallized with the purpose of stabilizing the protein as much as for aiding drug discovery - as they do about their beloved protein. In addition, many crystallographers don't have the knee-jerk, intuitive reaction to, say, rings in boat conformations that a good medicinal chemist or a medicinal chemistry-aware modeler would have.

The unfortunate consequence of all this is that the ligand often just comes along for the ride and the protein's gory structural details are exquisitely teased apart at the expense of the ligand's. Protein love often inevitably translates into ligand hate. For an organic chemist a cyclohexane boat may be a textbook violation of conformational preferences, but for a crystallographer it's a big, hydrophobic group filling up a big, fuzzy halo of electron density. Crystallographers are not chemists.

However, an honest assessment of the problem would not unfairly pin the blame for bad ligand structures on crystallographers alone. The fact is that structure-based drug design is an intimate covenant between crystallographers, medicinal chemists and modelers and true appreciation and progress can only come from each side speaking or at least understanding the other's language. To this end, chemists and modelers need to be aware of crystallographic parameters and need to ask the right questions to the crystallographer, beginning with a simple question about the resolution (even this question is rarer than you may think). A medicinal chemist or modeler who simply plucks the provided structure out of the PDB file and starts using it to design drugs is as guilty as a chemistry-challenged crystallographer.

A typical set of questions a modeler or medicinal chemist might ask the crystallographer is: 

- What's the resolution?
- What are the R-factors and the B-factors
- Do you have equal confidence in all parts of the structure? Which parts are more uncertain?
- Are the amides non-planar? 
- Where are the water molecules located? How much confidence do you have in their placement?
- Are atoms supposed to be planar non-planar? 
- Are there any gauche or eclipsed interactions? 
- Are there boats in rings? 
- Have you looked at the strain energy of the ligand?
- How did you refine the ligand?

These questions are not meant to be posed to the crystallographer by men in dark suits in a dimly lit room with bars on the windows, but rather are supposed to provide a reality check on the fidelity of the structure and its potential utility in drug design for all three arms of the SBDD process. The questions are part of a process that allows all three departments to confer and reach an agreement; anyone can and should ask them. They are meant to bring hands together, not to point fingers.

One of the cultural problems in drug discovery is still the reluctance of one group of scientists to adopt at least parts of the cultural behavior of other groups. Organic chemists are quick to look at stereochemistry or unstable functional groups, modelers are not. Modelers are much more prone to look at conformation, organic chemists are not. Crystallographers are far more likely to bear multiple conformations of loops and flexible protein side chains in their minds, the other two parties are not.

The best way to fill these gaps is for each group to speak the language of the other, but until then the optimal solution is to have all of them look at the evidence and emphasize what they think is the most important part. But for that to happen each party has to make as many details of its own domain accessible to the others, and that is partly what is being said here.

Update: As usual, the Yoda of chemistry blogging got there first.


  1. Sometimes the problem with the ligand geometry can manifest itself at remote location. We used a crystal structure of an amide of 4-hydroxy piperidine and the nitrogen was pyramidal. It appeared that an equatorial geometry had been assumed for the hydroxyl and it was shown subsequently (by the same group) that an axial geometry was more correct and the amide was more planar. I should stress that having access to the original structure was still extremely valuable and I didn't consider the axial alternative at the time so I am in no position to criticize the crystallographers. Disorder needs to be kept in mind because it is possible that no single conformation is sufficient to explain the measured electron density. Strain energy is not always easy to estimate because some relaxation of the crystallographic ligand conformation is usually (always?) necessary for energy conformations to be meaningful. Also the ligand may actually bind in a strained conformation which usually represents a molecular design opportunity ( see 10.1016/j.bmcl.2005.03.068 ).

  2. Very interesting - and potentially project-shattering!
    I have a slightly tangential question to the topic: How big of a problem is uncertainty of the structural assignment for small molecule x-ray structures - without any of those pesky proteins that is?

  3. @Sven, small molecule x-ray structures are measured at much better resolution: <0.85 Angstrom barring exceptions. There are sometimes difficulties, but they are mostly in the assignment of atom types (it may be hard to see the difference between atoms with almost the same number of electrons) rather than geometry. Geometry can be difficult for small molecule structures if hydrogen atoms are involved (but most of the positions of hydrogen atoms can be easily calculated unambiguously) or if the structure is disordered (there are multiple positions for (part of) the molecule in the unit cell.

  4. Very interesting article. This study shows how far we can go in the field of medicinal chemistry. Everyone should ready and share it.


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