1. QUANTITATIVE SCREENING FOR CRYSTALLIZATION CONDITIONS Marc L. Pusey and Takahisa Minamitani MI Research, Inc., Suite 109, 515 Sparkman Drive, Huntsville, AL, 35816 FACTs Fluorescence-based Analytical Crystallization Technologies A paradigm shift in how macromolecule crystallization conditions are determined.

2. MI Research, Inc. September 2006 – MI Research, Inc. started by TM & ELM. Goals – development of biomedical instrumentation. October 2006 – MLP quits day job and joins company. Task – develop the FACTs (Fluorescence-based Analytical Crystallization Technologies*) for crystallization screening. March 2007 – Proof of concept instrument up and running Data collection from assay volumes of 25-30 µL. July 2007 – Improved ‘Phase 0’ instrument Assay volumes of 2.5 – 3.5 µL. (1.73 mg / 96 conditions) Started collecting and testing data on model proteins. (this talk). September 2007 – Additional improvements to control software Data collection time reduced to ~3-4 hrs. Now open for business… * patent pending

4.  Current methods are highly ‘digital’ – yes (crystal) / no (no crystal). Days to months to obtain results.  Data interpretation (visual image analysis) is highly subjective. machine-based methods even less subtle led to idea of trace fluorescent labeling (intensity an easier search parameter than straight lines).  Many clear or precipitated solution outcomes may be near misses, which are not evident upon visual analysis. AN IMPROVED SCREENING APPROACH  Not dependent upon appearance of crystals  Will provide feedback & let you know you are close – near misses.  Will be amenable to automation (data collection and analysis).  Will rapidly (< 2 days, purified protein to data) give screen results. Problems with the Crystallization Screening Process (my perspective) info@mi-research.com

5. Goal of the FACTs Approach A means of rapidly acquiring data about protein behavior in the presence of precipitant solutions. B6 Screen outcome Optimized results after a single 2D screen. Scale bars =200 µm. To use the FACTs data to find where this can become this

6. Builds upon B 22 work of Wilson and others, which shows that there is a range of attractive interactions which favors (but does not necessarily guarantee) protein crystallization. We propose that a quantitative approach to crystallization screening would result in more successes by indicating the proximity to likely crystallization conditions. Basis for Our Approach Crystallization Variable B 22 “Crystallization Slot” defined by B 22. Expanded slot How Far? attractive 0 info@mi-research.com

7. A NEW APPROACH TO PROTEIN CRYSTALLIZATION Current Practice Add together proteinPrecipitant Incubate Days Weeks Months Observe Crystal? YES! NO Harvest and Proceed Add together diluted protein with fluorescent label Precipitant Our Approach Measure Protein Response to Solution Conditions Pick likely Crystallization Conditions. Time to this point < 2 DAYS! Advantages – Speed and Increased Success Rate Optimize minimum feedback!! Data we can learn from Plot and Analyze Data – the quantitative component

8. Fluorescence Anisotropy F Ex Em H V SOLUBLE When exciting with vertically polarized light, if the rotation time of the fluorophore is ≥ than the fluorescent lifetime, τ, then the vertical and horizontal emission will be ~ equal and r ≈ 0 F F F F F F PRECIPITATION Aggregated protein mass rapidly builds at low concentrations, rapidly slowing down the rotational rate. Anisotropy  r 0 at low concentrations. CRYSTALLIZATION Slow self association as a function of protein concentration, as with crystallization, leads to a progressive increase in anisotropy with concentration. F F r = I VV – I VH I VV + 2*I VH r0r0 r = 1 + τ θ θ = ηVηV RT r = measured anisotropy r 0 = fundamental anisotropy a property of the fluorophore τ = probe lifetime θ = rotational correlation time η = viscosity R = gas constant T = temperature V = volume of rotator Note dependencies!! These will affect data collection. info@mi-research.com

9. Anticipated Results What we expect to see in the data and how it will be interpreted  Clear Solution outcomes – will have anisotropy vs. protein concentration curves that are flat (or with a slightly negative slope).  Precipitated Solution outcomes – will have high (and/or level) anisotropy values in anisotropy vs. concentration curves.  Crystallization conditions – will have curves showing a progressive increase in anisotropy with increasing protein concentration. info@mi-research.com

10. Clear Solution Outcomes - Xylanase - Clear solution conditions typically have a flat or slightly negative slope in a plot of concentration vs. anisotropy. info@mi-research.com

11. Precipitated Solution Outcomes - Xylanase - More variable, but generally higher anisotropy values and/or no positive slope in the anisotropy vs. concentration data. info@mi-research.com

12. Crystallization Conditions Glucose Isomerase Initial testing is being carried out using model proteins, for which we know the crystallization outcomes for the solutions being tested and which are available in high purity and relatively large amounts. These tests are also used to develop the methodology for making the measurements. info@mi-research.com

13. Glucose Isomerase - continued These gave crystals with higher protein concentration In a ‘standard’ crytallization screen, a number of wells resulted in clear solutions. Some of these gave anisotropy data suggesting that they could be crystallization conditions. A total of 8 conditions were tested, with protein concentrations of 50, 100, and 150 mg/mL, of which 6 produced crystals. info@mi-research.com

14. UNCUT CANAVALIN Starting with this protein we decided to aggressively pursue all leads found using anisotropy measurements. “Standard” crystallization conditions are 0.1-0.2 M MgCl 2, 15-20% MPD, pH 6.5 to 8.0 (cacodylate, bis-tris, hepes, tris). P2 1 2 1 2 1 a=80.32, b=85.2, c=211.93 Screen results, condition B6 Anisotropy data Divalent cations facilitate UCAN crystallization 0.2 M MgAcetate, 20% PEG 8K,.1 M NaCacodylate, pH 6.5 5% PEG 8K 0.05 M MgAc 0.1 M NaCac pH 6.5 scale bar = 500 µm. info@mi-research.com 1:12:14:1

15. UNCUT CANAVALIN 4:12:11:1 In some cases it only took a simple 2D screen to convert precipitation conditions to crystallization conditions. HSHT Condition H3 0.2 M MgCl2 0.1 M Tris, pH 8.5 3.4M 1,6 Hexanediol MgAc hex-diol 0.05 M Mg Acetate 2.55 M Hex diol 0.1 M Hepes, pH 7.5 Scale bar = 200 µm.05.2.85 3.4 info@mi-research.com

16. UNCUT CANAVALIN For other conditions it took several screens to convert precipitation conditions to crystallization conditions. PEG 8K 1:12:14:1 HSHT condition C4 0.2 M Na Acetate 0.1 M Na Cacodylate, pH 6.5 30% PEG 8K Second screen – all wells made 0.1 M Mg Acetate Na Acetate 0.05 M NaAcetate 13.3% PEG 8K 0.1 M NaCac, pH 6.5 0.1 M MgAc Scale bar = 100 µm 5 30.05.2 info@mi-research.com

17. UNCUT CANAVALIN A number of anisotropy-derived leads had citrate as a component. Condition A9 0.2 M Ammonium acetate 30% PEG 4K 0.1 M Na Citrate, pH 5.6 Screen outcomes 10 % PEG 4K 0.2 M Ammonium Ac. 0.1 M NaCitrate, pH 5.6 Scale bar = 500 µm 15% PEG 4K 0.2 M Ammonium Ac 0.1 M NaCacodylate, pH 6.5 0.1 M Mg Acetate Scale bar = 100 µm Replace the Citrate & added Mg 2+ PEG 4K Am Acetate 1:12:14:1 info@mi-research.com

18. UNCUT CANAVALIN Some anisotropy leads were at pH values < UCAN pI (~5.2) HSHT condition D1 0.1 M Na Acetate, pH 4.6 8% PEG 4K BmimCl PEG 4K 8 2 0.40.1 1:12:14:1 0.1 M BmimCl 1 6% PEG 4K 0.1M Na Acetate, pH 6.5 0.1 M Mg Acetate scale bar = 200 µm 1 Pusey et al., Crystal Growth & Design (2007) 7;787-792 info@mi-research.com

19. UNCUT CANAVALIN HSHT condition F1 0.2 M Ammonium Sulfate 0.1 M Na Acetate, pH 4.6 30 % PEG MME 2K Crystals at pH < UCAN pI, continued HSHT screen condition F1 – screen outcome was a clear solution. Increasing the protein concentration (1x40 mg/mL, 2x40 mg/mL) gave precipitate. 1 st screen around pcpt. conditions, w/BmimCl 1 to reduce pcptn, no xtls. 2 nd screen, replacing BmimCl w/ 0.1 M MgAc  crystals. (protein @ 40 mg/mL) 1 Pusey et al., Crystal Growth & Design (2007) 7;787-792 PEG MME2K AmmSulf 0.2 0.05 30 15 15 % PEG MME2K 0.15, 0.2 M AmSulf 0.1 M NaAc, pH 4.6 0.1 M MgAcetate scale bar = 200 µm info@mi-research.com

20. UCAN vs. CCAN Hits CCANUCAN pp p p p p p pp p p ppp p p p ppp A A AA A AA A A AAA A AA A AA A A A A A A A A A A A A A A A A A p = by crystallization plate A = by anisotropy assay ABCDEFGHABCDEFGH 1 2 3 4 5 6 7 8 9 10 11 12 A info@mi-research.com

21. Data Summary for Model * Proteins * model = any protein for which we have plate screening data. GI (33 20) 11 / 10 8 / 6 6 / 3 75 / 50 27.3 XLN (12 3)0 1 / 28 0 1 / 24 0 1 / 17 0 / 70.8 141.67 CCAN (15 8) 27 / 12 27 / 8 8 / 7 29.6 / 87.5 100 UCAN (6 2) 22 / 18 22 / 14 10 / 11 45.5 / 78.6 350 Overall (66 33) 60 / 68 57 / 52 24 / 38 42.1 / 73.1 93.9 New leads found by FACTs 1 / 2-5 # of FACTs Leads tested 1 / 2-5 Crystals obtained % Success 1 / 2-5 Plate score Protein (plate xtls 1:1 only) % Increase over plate Values based on outcomes at 1:1, 2:1, & 4:1 conditions 1 All clear solution FACTs leads @ 1:1 had xtls at 2:1 &/or 4:1 1 = clear solution 2-5 = precipitate

22. Current Requirements Protein ~2 mg protein required per 96 condition screen (using 2.5 µL assay solution volumes). Goals – reduce this to ~0.8 mg (1.0 µL assay volume) within 6 months and ~8 µg (10 nL assay volume) within 2 years.  4 mg protein for screen + 5-10 mg for subsequent optimization. Time To prepare derivatized protein ~ 2 hrs. To set up Assay ~1 hr. To carry out 96 condition screen ~3-4 hrs. Less than 1 day, protein to screen results. NOTE: Labeled protein is only used for the assay, not subsequent optimizations. info@mi-research.com

23. FUTURE GOALS  Assay volume reduction – to 1.0 µL solution/assay – manual solution dispensing methods - within 6 months (691 µg protein/96 condition screen), to ≤ 10 nL – robotic solution dispensing methods - within 2 years.  Faster measurements – reduce the time to collect a 96 condition screen data set to < 1 - 2 hrs.  Temperature control – temperature sensitive crystallizations, membrane proteins.  Automated data analysis – a rational (vs. artsy) approach to protein crystallization.  Rational screen design – Incomplete factorial approach with two (or more) levels of screening.

24. SUMMARY What this approach WILL NOT do  Anisotropy leads are not automatically crystallization conditions. However – Knowing where to look - analysis of the data - may indicate those factors that are most important to obtaining crystals.  No indication or guarantee of crystal quality. However – An expanded set of crystallization conditions may help in finding better conditions. Not a method for getting crystals from bad protein.  Not a method for getting crystals from bad protein. Bad protein may give good looking data, but see above.

25. SUMMARY  Faster – The fluorescence anisotropy screen approach can be used to carry out the initial crystallization screen in ~8 hrs.  More Successes – The anisotropy screen finds more lead conditions – outcomes that would be clear or precipitated solutions in ‘normal’ crystallization screens.  Quantitative – Anisotropy screen data is highly amenable to machine analysis.

26. finis Contact Information: MI Research, Inc. Suite 109 515 Sparkman Drive Huntsville, AL 35816 info@mi-research.com