Structural biology needs no introduction for people doing drug discovery. This wasn’t always so. Drugs were discovered back in the days when people used to argue about whether those “receptor” thingies were real objects (as opposed to useful conceptual shorthand), and before anyone had any idea of what an enzyme’s active site might look like. And even today, there are targets, and whole classes of targets, for which we can’t get enough structural information to help us out much.
But when you can get it, structure can be a wonderful thing. X-ray crystallography of proteins, and protein-ligand complexes has revealed so much useful information that it’s hard to know where to start. It’s not the magic wand – you can’t look at an empty binding site and just design something right at your desk that’ll be a potent ligand right off the bat. And you can’t look at a series of ligand-bound structures and say which one is the most potent, not in most situations, anyway. But you still learn things from X-ray structures that you could never have known otherwise.
It’s not the only game in town, either. NMR structures are very useful, although the X-ray ones can be easier to get, especially in these days of automated synchroton beamlines and powerful number-crunching. But what if your protein doesn’t crystallize? And what if there are things happening in solution that you’d never pick up on from the crystallized form? You’re not going to watch your protein rearrange into a new ligand-bound conformation with X-ray crystallography, that’s for sure. No, even though NMR structures can be a pain to get, and have to be carefully interpreted, they’ll also show you things you’d never had seen.
And there are more exotic methods. Earlier this summer, there was a startling report of a structure of the HIV surface proteins gp120 and gp41 obtained through cryogenic electron microscopy. This is a very important and very challenging field to work in. What you’ve got there is a membrane-bound protein-protein interaction, which is just the sort of thing that the other major structure-determination techniques can’t handle well. At the same time, though, the number of important proteins involved in this sort of thing is almost beyond listing. Cryo-EM, since it observes the native proteins in their natural environment, without tags or stains, has a lot of potential, but it’s been extremely hard to get the sort of resolution with it that’s needed on such targets.
Joseph Sodroski‘s group at Harvard, longtime workers in this area, published their 6-angstrom-resolution structure of the protein complex in PNAS. But according to this new article in Science, the work has been an absolute lightning rod ever since it appeared. Many other structural biologists think that the paper is so flawed that it never should have seen print. No, I’m not exaggerating:
Several respected HIV/AIDS researchers are wowed by the work. But others—structural biologists in particular—assert that the paper is too good to be true and is more likely fantasy than fantastic. “That paper is complete rubbish,” charges Richard Henderson, an electron microscopy pioneer at the MRC Laboratory of Molecular Biology in Cambridge, U.K. “It has no redeeming features whatsoever.”
. . .Most of the structural biologists and HIV/AIDS researchers Science spoke with, including several reviewers, did not want to speak on the record because of their close relations with Sodroski or fear that they’d be seen as competitors griping—and some indeed are competitors. Two main criticisms emerged. Structural biologists are convinced that Sodroski’s group, for technical reasons, could not have obtained a 6-Å resolution structure with the type of microscope they used. The second concern is even more disturbing: They solved the structure of a phantom molecule, not the trimer.
Cryo-EM is an art form. You have to freeze your samples in an aqueous system, but without making ice. The crystals of normal ice formation will do unsightly things to biological samples, on both the macro and micro levels, so you have to form “vitreous ice“, a glassy amorphous form of frozen water, which is odd enough that until the 1980s many people considered it impossible. Once you’ve got your protein particles in this matrix, though, you can’t just blast away at full power with your electron beam, because that will also tear things up. You have to take a huge number of runs at lower power, and analyze them through statistical techniques. The Sodolski HIV structure, for example, is the product of 670,000 single-particle images.
But its critics say that it’s also the product of wishful thinking.:
The essential problem, they contend, is that Sodroski and Mao “aligned” their trimers to lower-resolution images published before, aiming to refine what was known. This is a popular cryo-EM technique but requires convincing evidence that the particles are there in the first place and rigorous tests to ensure that any improvements are real and not the result of simply finding a spurious agreement with random noise. “They should have done lots of controls that they didn’t do,” (Sriram) Subramaniam asserts. In an oft-cited experiment that aligns 1000 computer-generated images of white noise to a picture of Albert Einstein sticking out his tongue, the resulting image still clearly shows the famous physicist. “You get a beautiful picture of Albert Einstein out of nothing,” Henderson says. “That’s exactly what Sodroski and Mao have done. They’ve taken a previously published structure and put atoms in and gone down into a hole.” Sodroski and Mao declined to address specific criticisms about their studies.
Well, they decline to answer them in response to a news item in Science. They’ve indicated a willingness to take on all comers in the peer-reviewed literature, but otherwise, in print, they’re doing the we-stand-by-our-results-no-comment thing. Sodroski himself, with his level of experience in the field, seems ready to defend this paper vigorously, but there seem to be plenty of others willing to attack. We’ll have to see how this plays out in the coming months – I’ll update as things develop.