Dear Colleagues, Thankyou, James, for a most interesting and extensive summary. There is not much more to add.
In my view microgravity has provided benchmarks for protein crystal perfection against which earth based crystal growth results can be compared. Also the general principles of crystal growth, and the concept of the depletion zone around a growing protein crystal in particular, have improved understanding for this field in my view. The resulting crystal quality also posed considerable characterisation challenges to people like me in the specialty of crystal perfection assessment. This latter was an interest I developed arising from characterising my beamline Si and Ge monochromator crystals. Gels and much smaller crystallisation drop volumes have indeed proved to be competitive in the end with microg for protein crystal growth. Also the use of magnetic and electric fields, separately or together, also provide a most interesting technology for further development of earth based protein crystal growth techniques. The reduction of entropy of amino acid side chains through molecular biology mutagenesis has also proved important and effective. Re developing of business plans, Jack, I think there are now quite a variety of methods which can be explored, as I mention above, before turning to space based methods to interest Pharma companies. Interestingly there was a distinct difference between Europe and the USA re microg protein crystal growth. In Europe I would say that we always saw the emphasis to be on fundamental understanding of microg protein crystal growth and crystal perfection. In the USA both fundamental research and sales to Pharma companies seemed to be promoted simultaneously. Best wishes, John Prof John R Helliwell On Mon, May 10, 2010 at 10:30 PM, James Holton <jmhol...@lbl.gov> wrote: > Jack Reynolds wrote: > >> My name is Jack Reynolds and I am economics major. I graduate in August >> and begin an MBA program shortly thereafter. I am exploring a business idea >> that involves protein crystal growth and x-ray diffraction in the >> microgravity environment of space. >> >> I hope the group can indulge me a few questions. If this group is not the >> proper forum for this, please feel free to let me know and I will go >> elsewhere. Thanks. >> >> > Don't worry. This is definitely not the most off-topic post I have seen on > this list! > >> 1. Have there been some new techniques developed to grow crystals in >> terrestrial labs that may produce crystals comparable in quality to those >> grown in space. Despite these advances, is there still a role for protein >> growth in an actual microg environment? >> >> > Microfluidic systems show some promise on this "front" because sufficiently > small volumes also drastically limit convection. Myself and others are > actively working on in-situ diffraction from microfluidic trays, and we > found that we could get mosaic spreads that were ... well, too small to > measure. Definitely less than 0.01 degrees, and probably much less, but our > x-ray beam divergence dominated the rocking width in those experiments. > > No, I do not get kick-backs from Fluidigm, but I did play a role in > developing the product described here: > http://www.fluidigm.com/pdf/topaz/FLDM_TOPAZ_MRKT00114.pdf > For any diffraction experiment it is really important to to reduce the > amount of background x-ray scattering (non-crystal solids and > heavier-than-carbon atoms in the x-ray beam) to less than or equal to that > of the crystal itself, and this is probably why in-situ results to date are > not usually as good a results from T-Y Teng's cryo-loop mounting technique. > Most trays are far thicker than crystals! But, the new Fluidigm trays have > the same x-ray background as a loop mount, and so I think this is actually a > major step forward. > > It is true that cryo-cooling generally ruins the advantages of > micro-gravity growth, but a more gentle cryo-cooling procedure was developed > recently (Warkentin and Thorne, J. Appl. Cryst. 2009) that demonstrated much > better preservation of mosaic spread. Also, radiation damage rates at room > temperature may not be nearly as bad as previously thought if the dose rate > is chosen properly (Southworth-Davies et al. Structure 2007 and also the > recent RD6 workshop proceedings). Combine this with other microgravity > advantages: larger crystal volumes, lower Wilson B factors, and smaller > diffracted beam spots (Holton and Frankel Acta D, 2010), and you could > potentially make room-temperature diffraction competitive with cryo-cooled > methods again. > > > > Another "alternative" technology you seem to have also found is the "Bitter > solenoid" idea. I thought this had potential ever since I first saw the > levitating frog: > http://www.ru.nl/hfml/research/levitation/diamagnetic/ > which seems to have also made it on to YouTube. > http://www.youtube.com/watch?v=A1vyB-O5i6E > > I suppose here the problem is the cost of building a 96-well > crystallization "tray" made of these solenoids, and the energy needed to run > it. Then again, this may not be too bad when compared to the energy > required to lift the tray into low-earth orbit. > > Crystallographers are notoriously penny-wise and pound-foolish. We will > balk at spending more than $5/ea for a 96-well crystallization tray, but > gladly blow $1e5 or more on a "device" that has only been shown to improve > diffraction once in a blue moon. I'm not going to name names, but I will > say that the latter becomes far far more attractive if the one case where > the device "worked" produced a Science, Nature or Cell paper. This is not > because we are mindless bandwagon-chasers, but rather because a "big splash" > is seldom made by doing an easy experiment. > > As a beamline scientist I can tell you that there is definitely a "market" > in improving diffraction. Incredible amounts of time and effort are now > typically spent trying to get a given crystal system to yield spots out > beyond the water ring (3.5 A). Typical investments are months to years for > a single structure determination. Particularly with large complexes and > membrane proteins. Nevertheless, the general approach is to exhaust all > cheap options first (such as graduate student's time), before resorting to > more exotic ideas. > > > My perception of the main problem with the "space crystals" was that there > is nothing more infuriating to a crystallographer than seeing what looks > like huge amounts of money being spent on a protein that you are not > particularly interested in. Unfortunately, when you work with NASA you are > not allowed to fly experiments that have a significant probability of > "failure", and since the only protein that is pretty much guaranteed to > crystallize is lysozyme, that's what the "space crystals" had to be. The > resulting research has been much maligned because of this, but if you read > the papers carefully you will find that many of these workers managed to > learn a great deal about the basic chemistry and physics of macromolecular > crystal growth that was not known before. This is not an easy thing to > study, and there are a very small number of people who do, despite how > incredibly important it is. > > I think the current literature is convincing that microgravity can have a > positive impact on diffraction in some cases, but just like everything else > in crystallography there are plenty of cases where it won't help. The only > way to know is to try, and you have to do something to convince > crystallographers that trying out your new method is worth it. This > generally involves demonstrating a "success" with a "hard" problem (the kind > that gets into a big-name Journal), or gathering statistics from a very > large number of different proteins. You can also arrive at a fundamental > understanding of the process and do your convincing scientifically, but all > of these routes are expensive. Such is the plight of methods development. > > As a "business model", what I would recommend is some kind of "return > policy" for when the experiment "fails". As a service provider, you will be > facing a very high likelihood of failure, and a customer who will never be > entirely convinced that you "tried hard enough". So the motto: "Improved > diffraction, or your money back!" is an attractive one. Or maybe "Improved > diffraction, or (most of) your money back!". > > 2. Has the process of growing crystals been automated? Is there any step in >> the process of growing crystals that has not or cannot be automated? >> >> > Yes and no. The process itself has certainly been automated, not once but > many times. But none of these automation systems have a 100% success rate. > Not even close. There are both stochastic and systematic components to the > success rate of macromolecular crystal growth. For example, many proteins > simply don't crystallize, no matter what you do (a systematic failure). > Most that do crystallize are EXTREMELY finicky (sensitive to both > controlled and uncontrolled variables). For example, it is not uncommon for > an identical chemical mixture to produce crystals in one kind of tray, but > not in another. In fact, even identical conditions in the same tray will > not always produce crystals every time (uncontrolled variables). This is > generally regarded as being due to the stochastic (random) nature of > nucleation, and seeding can help a great deal in such cases. Seeding is > generally done by hand, but at least one group has an automation system for > it (Newman et al. Acta F 2008, Newman et al. J. Biomol. Screen. 2009). > > Nevertheless, once you ave studied a particular crystallization system > enough to understand its quirks, it can become very "routine". That is, > everything becomes easy once you know how to do it. My favorite example of > this is thaumatin. Crystals of this protein were incredibly fragile and > difficult to work with for more than ten years until the discovery of the > tartarate-dependent crystal form (Alex McPherson, personal communication). > Now thaumatin crystals are generally regarded as being "too easy" and it is > now difficult to convince people that things you learn by studying thaumatin > crystallization are relevant to "hard" experiments. > > > Possibly the weakest link in crystallization automation is at the end: > harvesting. There are a few systems out there for automated harvesting of > crystals (Viola et al. J. Struc Func. Genom. 2007), but right now almost > everyone still does this by hand. The challenges are mainly in object > recognition. > > 3. Has the process of x-ray diffraction been automated? Is there any step >> in the process of x-ray diffraction that has not or cannot be automated? >> >> > Again, everything has been "automated", but nothing is 100% effective. For > diffraction, much effort has been spent eliminating stochastic failure modes > (robots dropping crystals on the floor), but that usually comes at the > expense of introducing systematic ones (narrow range of supported > methodologies). > > I think the weakest link here is getting the crystal centered in the x-ray > beam. A tremendous amount of effort has been expended on this problem, but, > personally, I don't think it will ever be 100% effective. I formed this > opinion watching hundreds of highly intelligent human beings centering > crystals that they had grown, harvested and mounted themselves, and there > are still plenty of cases where they can't figure out where their crystals > are in the loop. Using polarized light or UV illumination to light up > tryptophan can help, but not every protein crystal is birefringent and not > every protein contains tryptophan. > > There are X-ray based centering approaches, such as "peppering" the loop > with x-ray shots (available at most beamlines), phase-contrast x-ray > tomography (Brockhauser et al. J. Appl. Cryst. 2008), or just looking at the > shadow of the sample on the x-ray detector (something I am working on). All > of these are currently time consuming (much more time consuming that > clicking on a video image of the loop), but all show promise of becoming > faster. You can also just illuminate the whole drop with a broad x-ray > beam, but then the background scattering problems I mentioned above must be > cubed (xtal volume vs illuminated volume). > > Anyway, I certainly cannot summarize the whole field in one email, but most > of the people in the field are on this BB and I'm sure they will now chime > in to correct or expand the statements I have made above. > > For shooting crystals in space, I would recommend a well-designed in-situ > growth/diffraction system (with thin walls) and as compact a light source as > you can find that can still deliver ~100 Gy/s or so. I think that an > up-and-coming gallium jet technology (Otendal et al. Rev. Sci. Inst. 2008) > could potentially have the most photons/kg. > > -James Holton > MAD Scientist > --
