Hi James – thx for starting a riveting thread. (Of course) I agree with Dom, Janet, Artem and the cosmic cats that crystallization is key.
I also agree with Artem a relatively modest investment in the fundamentals of crystallization could make a big difference – even a 10% improvement in productivity would save the community $ millions. A high proportion of Nobel prizes (and highly-cited papers) are essentially about method development. So why is it so hard to get grants for new scientific methods? It’s as though current funding is only about charging up the scientific motorways (interstates/autobahns for US/German citizens) while ignoring the side-roads. You have to claim that you will cure cancer, diabetes, Ebola *and *ageing in less than six months for your grant application to be considered. But the side-roads have often yielded the most important scientific breakthroughs. The current approach to crystallization is (I’m told) a type of "martingale" – that is, a betting scheme/stochastic process where your next bet is based on analysis of previous rounds of betting. The problem is that if your starting assumptions are flawed it may take many decades to arrive at a good solution. What would fundamental research into crystallization look like? I used to think it was a matter of “boiling down” the dimensions in a typical crystallization screen to say 10 “underlying variables” using multivariate analysis. Now I think this view is wrong. There are a few variables that are common to all crystallization experiments (temperature, pH and “saturation” – whatever that is) but we also have to explore the space of all the possible small molecules that can interact with - and probably bind to - our target protein and help it to crystallize. So crystallization space is similar to chemical space – very big indeed. I (now) think the targets of crystallization experiments are like this picture: *https://www.douglas.co.uk/f_ftp1/How%20desperate%20are%20you.jpg <https://www.douglas.co.uk/f_ftp1/How%20desperate%20are%20you.jpg>* There are some proteins that just need to be pushed out of solution to crystallize – like lysozyme. Then there are others that need to combine somehow with one particular small molecule to make crystals. For example, thaumatin crystallizes very easily if there’s tartrate in the drop. Others need two small molecule additives. Presumably still others could crystallize if only we could find the right combination of 3, 4 or 5 small molecules. (Tartrate in the example above is in a sense a “silver bullet”. Hampton Research called their screen that because they had the idea the additives would self-select, so you could put lots of them in each condition. Strangely, their Silver Bullet screen never worked as well as some of us expected - I don’t know why. But there isn’t one silver bullet – there are, or could be, thousands.) Random microseeding including cross-seeding works really well and is one of Artem's silver bullets of crystallization methods – but we still have to get our first crystals somehow. Practical questions that could (and should!) be answered include: 1. Which are the best precipitants and how many do we need? Maybe 4 or 5 would be enough. 2. How can we identify the best set of several hundred small molecules to use as additives? 3. How many small molecules should we put in each crystallization trial? 4. What should we do about pH and temperature? I think these questions could be answered by one lab, with good experimental design and automation, using say 25 target proteins – not including lysozyme, see above - that can be bought from Sigma etc (Artem, making them yourself is too much like hard work!) This can make both screening and optimization more efficient. Yes it would be a lot of work, but the current approach is a lot of work for hundreds (thousands?) of labs all over the world - work that is largely wasted. But it needs proper funding – and I don’t know how to get that. Best wishes to all, Patrick Ps Of course I agree with Janet, Tom and others that good record-keeping is essential. But there is a limit to what we will learn if we insist on solving structures at the same time. I say just buy in the model proteins by the gram and focus what is *really *going on in crystallization. On Sun, Jul 21, 2019 at 4:29 PM Artem Evdokimov <artem.evdoki...@gmail.com> wrote: > Excellent question :) > > First of all, thank you for putting this out to the community! > > Secondly, I agree with several of us who've written that a single > conference is not enough to discuss all the possible topics. > > Thirdly, in my opinion all the other problems are secondary to the main > (and only remaining!) problem in crystallography: getting > diffraction-quality protein crystals reproducibly and quickly > > The amount of funding for serious crystallization research seems to be > close to non-existent. In general methodology funding is hard to get, but > crystallization seems to me like the absolute underdog of the method pool - > the true 'red headed stepchild' of the methods development funders. > > At risk of repeating myself - the other problems (worthy, significant, and > urgent as they are!) are subservient to the main issue at hand - namely > that crystallization remains an unpredictable and artful phenomenon while > literally all other aspects of structure determination process (the gene to > structure pipeline, whatever you might call it)have made astronomic leaps > forward. > > Artem > - Cosmic Cats approve of this message > > > On Mon, Jul 15, 2019 at 3:44 PM Holton, James M < > 0000270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote: > >> Hello folks, >> >> I have the distinct honor of chairing the next Gordon Research >> Conference on Diffraction Methods in Structural Biology (July 26-31 >> 2020). This meeting will focus on the biggest challenges currently >> faced by structural biologists, and I mean actual real-world >> challenges. As much as possible, these challenges will take the form of >> friendly competitions with defined parameters, data, a scoring system, >> and "winners", to be established along with other unpublished results >> only at the meeting, as is tradition at GRCs. >> >> But what are the principle challenges in biological structure >> determination today? I of course have my own ideas, but I feel like I'm >> forgetting something. Obvious choices are: >> 1) getting crystals to diffract better >> 2) building models into low-resolution maps (after failing at #1) >> 3) telling if a ligand is really there or not >> 4) the phase problem (dealing with weak signal, twinning and >> pseudotranslation) >> 5) what does "resolution" really mean? >> 6) why are macromolecular R factors so much higher than small-molecule >> ones? >> 7) what is the best way to process serial crystallography data? >> 8) how should one deal with non-isomorphism in multi-crystal methods? >> 9) what is the "structure" of something that won't sit still? >> >> What am I missing? Is industry facing different problems than >> academics? Are there specific challenges facing electron-based >> techniques? If so, could the combined strength of all the world's >> methods developers solve them? I'm interested in hearing the voice of >> this community. On or off-list is fine. >> >> -James Holton >> MAD Scientist >> >> >> ######################################################################## >> >> To unsubscribe from the CCP4BB list, click the following link: >> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 >> > > ------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1 > -- patr...@douglas.co.uk Douglas Instruments Ltd. Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK Directors: Peter Baldock, Patrick Shaw Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090 US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB&A=1
