QUESTIONS YOU SHOULD ASK
ABOUT CRYSTALLOGRAPHIC MODELS

Gale Rhodes
Chemistry Department
University of Southern Maine

INTRODUCTION

Molecular modelling programs and fine graphics computers are becoming common, making it possible for many researchers and students to explore the wealth of structural information that comes from x-ray crystallography. Many students, teachers, and researchers in biochemistry and molecular biology use crystallographic models to help them understand structure-function relationships. Despite the best educational efforts of crystallographers, many users still treat molecular structures as objects that have been seen directly, rather than as models resulting from a demanding interpretative process. Such users are often unaware of the strengths and weakness of crystallgraphic models.

REVEALING THE ESOTERICA OF CRYSTALLOGRAPHY

According to the American Heritage Dictionary, esoterica are mysteries of a special type: "What is esoteric is mysterious because it is known and understood by only a small, select group, as by a circle of initiates or the members of a profession." Following is an attempt to disseminate widely some of the esoterica of x-ray crystallography, and thus to enlarge the circle of those who might discern more clearly the elusive truths that lie behind each crystallographic model.

I find in conversations with noncrystallographers that a significant number of them are surprised to learn the following facts about crystallographic models (some are annotated with the gist of their response to dawning awareness):

1. that the structure obtained is not of one molecule, but of the average of many molecules ("Oh yes, that's a basic point of all molecular science, I guess, but sometimes I don't stop to think about it.");
2. that the model is obtained from molecules in the solid state, rather than in solution ("Oh -- I guess that's what crystallography means...but it never sank in that those pictures are not of the molecule in solution"); but
3. that many macromolecules are demonstrably functional in the crystalline state ("Do you mean that enzyme molecules can sit in a crystal and still be active???"); and in fact,
4. that crystallographers go to great lengths to demonstrate that the crystalline substance is still functional, and that it is consistent with what is known about the molecule in solution ("Well, that's a comfort...");
5. that macromolecular crystals contain a large amount of water, some ordered and thus detectable, and some disordered ("Oh, so in a sense, the molecules are still in solution...? That would help to explain how they might still be active.").
6. that in some published models, the crystallographer has been unable to locate all of the amino acid residues ("What? They can't even find parts of the molecule???");
7. that in some published models, there is unexplained electron density, to which no known parts of the protein or associated cofactors can be assigned ("You mean like when I reassembled my carburetor and had parts left over?");
8. that some macromolecules in the crystalline state contain distortions due to crystal packing ("Well, I'm not surprised -- but why isn't it more common, and how can you detect it??");
9. that, despite being in the solid state, macromolecules are still in motion ("Now wait a minute, I thought you said they were sitting pretty in the crystal..."), and that crystallographic study provides some suggestions about the relative mobility of various parts of the molecule ("Hey, that might be useful! Can I view that information in the form of different colors on a graphics model??");
10. that the resolution of the model is not constant throughout, because i) different portions of macromolecules in crystals possess different ranges and types of motion, and ii) some portions adopt different ordered conformations in different unit cells ("You mean that all unit cells are not identical???"); and for this and other reasons,
11. that there is some tolerance or uncertainty in the atom positions, usually expressed in a statistical way for the molecule as a whole, and that this tolerance, in part, reflects the quality of the model ("Whattaya mean, quality -- are some models better than others?"); and finally,
12. that you do not have to be a crystallographer to assess, at least roughly, the quality of a model from data in the original publication of a crystallographic structure ("Oh, I can never make heads or tails of the experimental section in a new structure paper -- but I love the stereo pictures!").

(Now, that's a long sentence!)

TOWARDS BETTER-INFORMED USE OF MODELS

The responses (parodied in parentheses above) of noncrystallographers to these bits of crystallographic common knowledge were part of the motivation that led me to write a little book on macromolecular crystallography for biochemists, molecular biologists, and other users of crystallographic models.* In writing the book, I hoped to help turn the statements listed above into common knowledge in much wider circles, as well as to equip noncrystallographers to read structure publications with greater understanding. I review the major book's major concepts in a chapter on how to read a crystallographic paper. The chapter includes annotated excerpts from a recently published structure determination. If you or your colleagues want to understand crystallography better, you might find this book useful.

When we study a striking computer display of an enzyme's active site or a protein/DNA complex, we are able to make discerning use of what we see only if we are fully aware of the strengths and limitations of crystallographic models. The facts listed above suggest a series of questions that protein scientists should ask of all models before using them in attempts to explain their own observations. Crystallographers, in turn, should not assume that other researchers are aware of these points of common crystallographic knowledge, and should make a special effort to enlarge the proportion of users who can extract the most from the fruits of structure determination.

And by the way, what questions should we ask about macromolecular structures derived from NMR data?

* Crystallography Made Crystal Clear: A Guide for Users of Macromolecular Models, 2nd Edition, Gale Rhodes, San Diego: Academic Press, 2000, (ISBN 0-12-587072-8).

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