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QUANTITATIVE SCREENING FOR
CRYSTALLIZATION CONDITIONS
Marc L. Pusey and Takahisa Minamitani MI Research, Inc., Suite 109, 515 Sparkman Drive, Huntsville, AL, 35816
FACTsFluorescence-based Analytical Crystallization Technologies
A paradigm shift in how macromolecule crystallization conditions are determined.
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 AnalyticalCrystallization Technologies*) for crystallization screening.
March 2007 – Proof of concept instrument up and runningData collection from assay volumes of 25-30 µL.
July 2007 – Improved ‘Phase 0’ instrumentAssay 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 softwareData collection time reduced to ~3-4 hrs.
Now open for business…* patent pending
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 subtleled 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)
Goal of the FACTs Approach
0
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concentration, mg/mL
anis
otr
op
y, r
A6
0
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A means of rapidly acquiring data about protein behavior in the presence of precipitant solutions.
A6-a A6-b
B6-a B6-b
A6-a A6-bA6-a A6-b
B6-a B6-bB6-a B6-b
B6
Screen outcomeOptimized results aftera single 2D screen. Scalebars =200 µm.
To use the FACTs data to find where this can become this
Builds upon B22 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
CrystallizationVariable
B2
2
“Crystallization Slot”defined by B22.
Expanded slotHow Far?
attr
acti
ve
0
A NEW APPROACH TO PROTEIN CRYSTALLIZATION
Current Practice
Add together
protein Precipitant
Incubate
DaysWeeksMonths
Observe
Crystal?YES!
NO
Harvest and
Proceed
Add together
dilutedproteinwith fluorescentlabel
Precipitant
Our ApproachMeasureProteinResponseto SolutionConditions Pick likely
CrystallizationConditions.Time to this point < 2 DAYS!
Advantages – Speed and Increased Success Rate
Optim
ize
minimumfeedback!!
Data we canlearn from
Plot and AnalyzeData – the quantitativecomponent
Fluorescence Anisotropy
FEx
EmH
V
SOLUBLEWhen 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
FF
F
F F
F
PRECIPITATIONAggregated protein mass rapidly builds at low concentrations, rapidly slowing down the rotationalrate. Anisotropy r0 at low concentrations.
CRYSTALLIZATIONSlow self association as a function of protein concentration, as with crystallization, leads to a progressive increase in anisotropy with concentration.
FF
r =IVV – IVH
IVV + 2*IVH
r0
r= 1 +
τ
θθ =
ηV
RTr = measured anisotropyr0 = fundamental anisotropy a property of the fluorophoreτ = probe lifetimeθ = rotational correlation timeη = viscosityR = gas constantT = temperature V = volume of rotator
Note dependencies!!These will affect data collection.
Anticipated ResultsWhat 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.
-0.05
0.00
0.05
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0.15
0.20
0.25
0.0 1.0 2.0 3.0 4.0
concentration, mg/mL
an
iso
tro
py
, r
B1
B9
E3
G9
E8
C9
Clear Solution Outcomes- Xylanase -
Clear solution conditions typically have a flat or slightly negativeslope in a plot of concentration vs. anisotropy.
Precipitated Solution Outcomes- Xylanase -
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concentration, mg/mL
an
iso
tro
py
, r
C8
D5
D8
F3
F11
F12
G8
More variable, but generally higher anisotropy values and/or no positive slope in the anisotropy vs. concentration data.
Crystallization ConditionsGlucose 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.
Known Crystallization Conditions
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0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0
concentration, mg/mL
anis
otr
op
y, r
A1B2B3B5B9B11D2
Glucose Isomerase - continued
Clear Solution Outcomes
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concentration, mg/mL
anis
otro
py, r
A5A7B4C1C5
These gavecrystals withhigher proteinconcentration
In a ‘standard’ crytallization screen, a number of wells resulted inclear 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.
UNCUT CANAVALIN
Starting with this protein we decided to aggressively pursue all leads foundusing anisotropy measurements. “Standard” crystallization conditions are 0.1-0.2 M MgCl2, 15-20% MPD, pH 6.5to 8.0 (cacodylate, bis-tris, hepes, tris). P212121 a=80.32, b=85.2, c=211.93
0
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0.0 1.0 2.0 3.0 4.0
concentration, mg/mL
Screen results, condition B6
Anisotropy data
Divalent cations facilitateUCAN crystallization
0.2 M MgAcetate, 20% PEG 8K, .1 M NaCacodylate, pH 6.5
5% PEG 8K0.05 M MgAc0.1 M NaCac pH 6.5scale bar = 500 µm.
1:1 2:1 4:1
00.050.1
0.150.2
0.250.3
0.0 1.0 2.0 3.0 4.0
concentration, mg/mL
anis
otro
py, r
UNCUT CANAVALIN
4:12:11:1
In some cases it only took a simple 2D screen to convertprecipitation conditions to crystallization conditions.
HSHT Condition H30.2 M MgCl20.1 M Tris, pH 8.53.4M 1,6 Hexanediol
MgAc
hex-d
iol
0.05 M Mg Acetate2.55 M Hex diol0.1 M Hepes, pH 7.5Scale bar = 200 µm
.05 .2.85
3.4
UNCUT CANAVALIN
For other conditions it took several screens to convertprecipitation conditions to crystallization conditions.
0
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concentration, mg/mL
anis
otr
op
y, r
PE
G 8
K
1:1 2:1 4:1 HSHT condition C40.2 M Na Acetate0.1 M Na Cacodylate, pH 6.530% PEG 8K
Second screen – all wells made0.1 M Mg Acetate
Na Acetate
0.05 M NaAcetate13.3% PEG 8K0.1 M NaCac, pH 6.50.1 M MgAcScale bar = 100 µm
5
30
.05 .2
0
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concentration, mg/ mL
UNCUT CANAVALIN
A number of anisotropy-derived leads had citrate as a component.Condition A90.2 M Ammonium acetate30% PEG 4K0.1 M Na Citrate, pH 5.6
Screen outcomes
10 % PEG 4K0.2 M Ammonium Ac.0.1 M NaCitrate, pH 5.6Scale bar = 500 µm
15% PEG 4K0.2 M Ammonium Ac0.1 M NaCacodylate, pH 6.50.1 M Mg AcetateScale bar = 100 µm
Replace the Citrate & added Mg2+
PEG 4K
Am
Ace
tate
1:1 2:1 4:1
UNCUT CANAVALIN
Some anisotropy leads were at pH values < UCAN pI (~5.2)
00.050.1
0.150.2
0.250.3
0.0 1.0 2.0 3.0 4.0
concentration, mg/mL
anis
otro
py, r
HSHT condition D10.1 M Na Acetate, pH 4.68% PEG 4K
BmimCl
PE
G 4
K
8
2
0.40.1
1:1 2:1 4:1
0.1 M BmimCl1
6% PEG 4K0.1M Na Acetate, pH 6.50.1 M Mg Acetatescale bar = 200 µm
1 Pusey et al., Crystal Growth & Design (2007) 7;[email protected]
UNCUT CANAVALIN
00.050.1
0.150.2
0.250.3
0.0 1.0 2.0 3.0 4.0
concentration, mg/mL
anis
otro
py, r
HSHT condition F10.2 M Ammonium Sulfate0.1 M Na Acetate, pH 4.630 % 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.1st screen around pcpt. conditions, w/BmimCl1 to reduce pcptn, no xtls.2nd 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
Am
mS
ulf
0.2
0.0530
15
15 % PEG MME2K0.15, 0.2 M AmSulf0.1 M NaAc, pH 4.60.1 M MgAcetatescale bar = 200 µm
UCAN vs. CCAN Hits
CCAN UCAN
ppp
p
pp
p
pp
p p
p p p
p
p
p
pp p
AA
AA
A
AAAA
AA A
A
AA
AAA
AA
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
p = by crystallization plate A = by anisotropy assay
A
B
C
D
E
F
G
H 1 2 3 4 5 6 7 8 9 10 11 12
A
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 27.3XLN (12 3) 01 / 28 01 / 24 01 / 17 0 / 70.8 141.67CCAN (15 8) 27 / 12 27 / 8 8 / 7 29.6 / 87.5 100UCAN (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
lead
s
foun
d by
FA
CTs
1 / 2-5
# of
FA
CTs
Lead
s te
sted
1 / 2-5 1 / 2-5
Cryst
als
obta
ined
% S
ucce
ss
1 / 2-5Plate score
Protein (plate xtls 1:1 only)
% In
crea
se
over
pla
te
Values based on outcomes at 1:1, 2:1, & 4:1 conditions1 All clear solution FACTs leads @ 1:1 had xtls at 2:1 &/or 4:1
1 = clear solution2-5 = precipitate
Current Requirements
Protein~2 mg protein required per 96 condition screen (using 2.5 µL assaysolution volumes). Goals – reduce this to ~0.8 mg (1.0 µL assayvolume) within 6 months and ~8 µg (10 nL assay volume) within 2 years. 4 mg protein for screen + 5-10 mg for subsequent optimization.
TimeTo 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.
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.
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.
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.
finis
Contact Information:MI Research, Inc.Suite 109515 Sparkman DriveHuntsville, AL [email protected]
