THE C6 WEBTOOL
Mapping crystallization space to see where to go
Janet Newman
Uppsala, May 2011
COLLABORATIVE CRYSTALLISATION CENTRE (C3)
• History:
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•
•
•
•
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Grant application in 2004 (Bio21 NPCF)
First equipment in 2005 (Fluidigm Topaz system)
CSIRO first equipment in 2006
First plates 9-03-2006
On-line booking July 2006
CSIRO C3 July 2010
• Numbers
•
•
•
•
Users – 158
Blocks created – 855
Crystal plates – 10239
(distinct reservoir_designs in xtal plates) – 1055
CSIRO. Core Facilities
WHAT IS A SCREEN?
•A screen is a collection of crystallants
•There are often 24, 48 or 96 conditions in a screen
•The crystallant
•“reservoir” “well” “conditions” “cocktail” “precipitant” “precipitating
solution”
•consists of 2-5 different chemicals, from a set of 300-500 chemicals
•buffer
(0.1M)
•salt
(0.2-2M)
•polymer
(5-30%)
•Screens
•Available commercially
•Made locally
QUESTIONS THAT ARE OFTEN ASKED OF US:
•What screen(s) should I use?
• Which ones do you have in-house?
•What screens do you have which contain/don‟t
contain chemical x?
•I‟ve set up screen Y, what should I try now?
•Literature conditions for protein Z are
available…
IS THIS A PROBLEM?
•Currently there are at least 230 commercially
available screens
• (195 in September, 2008)
•≈14000 conditions
WHICH SCREENS/CONDITIONS TO USE?
•Use them all:
•Assume setups of 100 nl protein +100 nl
crystallant
•~145 x 96 well plates need to be set up
• over 3 ml protein
• Around €30K
• 5-10K for plastic consumables etc
• 7 days to set up (in C3)
• 200000 images to review
CAN WE STREAMLINE THIS?
• We could set up only the unique crystallants.
• Still a big problem
• And how do you work out which ones are unique?
• Set up only the distinct crystallants
QUESTIONS ABOUT „DISTINCTNESS‟
•Can we define “likeness” for crystallisation
conditions?
•When are two crystallants similar?
• How different are they?
•When are two crystallisation screens similar?
• How different are they?
DO THIS EXPERIMENTALLY (“OUTPUT”)
Take a number of proteins
Set up in each (unique) condition
Observe…
Conditions that produce the same crystals are
similar
PROBLEMS
•How many proteins?
•Which proteins?
•What about other conditions?
•How do we extrapolate to similarity between
different screens?
AND this assumes:
If a crystal can happen, it will happen.
Or to put it another way
That crystallisation is a reliable process.
REPLICATION EXPERIMENT
•Lysozyme (same solution)
•Hampton HT (4 different blocks-same batch)
•Same pipetting robot (Phoenix)
•Same imagers (Rigaku)
•Same plate type (Greiner low profile)
•Same seals/phase of moon/blah blah blah
•
•
•
•
50+50nl or 100+100nl drops
4C or 20C
Imaged four days after setup
Crystals detected by inspection of images
NOT JUST LYSOZYME
• Catalase
• Set up 6 times in the HWI 1536 well screen
• <85% correlation between the replicants
AN “INPUT” WAY OF DETERMINING SIMILARITY
•Look at the make-up of the crystallisation
condition and compare that to the make-up of
other crystallisation conditions
What groundwork need to be in place?
•Consider two conditions:
• 100mM citric acid pH5
• 25% PEG MME 5K
• 200mM MgSO4•7H2O
• 0.1M sodium citrate pH 5.0
• 0.2M magnesium sulfate
• 25w/v MPEG 5000
•Data standards
•
•
•
•
Naming
Spelling
Units
Ordering
MEASURING DIVERSITY
•A metric is a distance between two points in a
space.
•In normal Euclidian space, distance is given
by
•What about chemical space?
METRICS
•We define an initial dissimilarity metric for
crystallisation space
• T is the number of distinct chemical species in conditions i and j
• [sti] is the concentration of chemical t in condition i
• max[st] is the maximum concentration found for that chemical
within chemical space (solubility)
CRYSTALLISATION DISSIMILARITY METRIC
•This returns a value between 0 and 1, where
•0 means the two conditions are identical
•1 means the two conditions are maximally dissimilar
(nothing in common)
Worked examples
•Condition i
• 100mM Hepes pH 7.5
• 200mM sodium acetate
• 2M Ammonium sulfate
•Condition j
• 100mM MES pH 5.5
• 0.5M Ammonium sulfate
• 20w/v PEG 4K
1/5 (1 + 1 + 1 + 1 + (2M-0.5M)/3.5M) = 0.88
• 100mM Acetate pH 4.6
• 200mM Li2SO4
• 20w/v PEG 3350
• 100mM Acetate pH 4.6
• 200mM MgCl2
• 25w/v PEG 3350
1/4 (0 + 1 + 1 + (25-20)/35) = 0.53
REFINEMENT OF THE INITIAL METRIC
• Include a pH term, which uses information that is
available about pH (to normalise this value, use a
denominator which spans the pH range found over all
commercial conditions 2.4-11.6)
• Include a similarity term (based on name) into the metric,
so that lithium sulfate will be more similar to lithium
chloride than it will be to sodium acetate. (include a
penalty for guessing)
• Look at conditions which both have a single PEG
species, and if 1/2  [PEGi]/[PEGj]  2 then term
becomes ([PEGi]-[PEGj])/25 (includes the denominator,
which is essentially a penalty for guessing)
CURRENT METRIC
+
+
COMPARING SCREENS
•Two screens (or sets of conditions) may be
compared, given a metric using
•Not so good for comparing a set of conditions
to itself
• average distance better in this case
DO WE HAVE TO GET THE METRIC “RIGHT”?
How do you measure “rightness”?
• The metric correctly finds all similar conditions for
every protein?
• The metric predicts as well as any
crystallographer?
OR
• The metric enables one to answer questions that
couldn‟t be answered before?
THE C6 WEBTOOL
• http://c6.csiro.au
WHICH SCREENS ARE SIMILAR?
•How similar are they?
HOW SIMILAR ARE THEY?
• Easier to see graphically
CSIRO. Insert presentation title, do not remove CSIRO from start of footer
HOW SIMILAR ARE THEY?
• Similarity comes in different ways
CSIRO. Insert presentation title, do not remove CSIRO from start of footer
THERE ARE DIFFERENT TYPES OF SCREENS
What type of screen is it?
1
0.9
0.8
Screen diversity
0.7
0.6
Minimum distance
0.5
Average distance
0.4
0.3
0.2
0.1
0
1
17 33 49 65 81 97 113 129 145 161 177 193 209 225
screen number
SPY0210 conditions?
SPY0210 conditions?
What is in that screen?
WHAT SHOULD REALLY BE IN THE METRIC?
• Physico-chemical properties
• The protein doesn‟t „see‟ ammonium sulfate
• What might be important?
• Specific activity
• Dielectric constant
• Viscosity
• Surface tension
• pH
• conductivity
•Acknowledgments
•State Government of Victoria
•Vincent Fazio
•CSIRO bosses (Tom, Tim, Tim, Paul)
•Shane, Tam, Christine
•CSIRO IT
•C3 Users
Thank you
Web: www.csiro.au/c3
Email: c3@csiro.au
Twitter: @C3CSIRO
PH
ASSAY - BACKGROUND
• The pH of the crystallisation experiment affects the charges of
ionisable sidechains (asp, glu, his, arg, lys)
• 30% of the commercially available crystallisation conditions are
provided with no associated pH information.
• The pH value is usually the pH of a minor “buffer” component
• Currently there are 10 (ok, 9) vendors of crystallisation screens,
231 screens available, or 13,868 individual conditions
• In c3
• 855 total deep well blocks
• (475 are unique)
• Any assay will be repeated A LOT.
Should be cheap, fast and easy
MEASURING PH
• Litmus used since 1200s
• extracted from one of several lichens – often Roccella tinctoria
• Robert Boyle (1727-91) showed that the indicator could be
adsorbed onto white paper
• 1871
• phenolphthalein first synthesized – introduced in 1877 as an
acid/base indicator (clear→fuchsia > pH 8.2)
• 1935
• Beckman (California) and Radiometer (Denmark) produced electric
pH meters (sensitivity 0.02 pH units)
UNIVERSAL INDICATORS
• Use combinations of different indicators to
span a large pH range
PH MEASUREMENTS IN HIGH-THROUGHPUT
• Needs to be plate based
• Needs to use small volumes of
reagents
• Accuracy not as important as
precision
• Yamada Universal Indicator (Bromothymol blue,
thymol blue, Methyl red, Phenolphthalein)
HOW BEST TO DO THIS?
• Add 1:10 diluted dye to unknown solutions
(dye:solution:water)
• 50:50:0
• 50:25:25
• 50:10:40
• pH is concentration independent!
• Dilution of solutions removes problem with intrinsic colour.
HOW TO QUANTIFY THE COLOUR?
• Create a standard curve with pH from 4 to 10
• (Use a plate reader with
• Monochromator (not filters)
• Measure from 300nm to 800nm)
CHC curve (1:5 dye)
1
0.9
0.8
Absorbance
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
Wavelength
WHY DOESN‟T THIS WORK?
• Colours are a continuum –
• different colours are formed by changes in relative
absorbance over a wide spectrum of wavelengths,
rather than by summing discrete peaks
• cf. gel filtration curves – colours aren‟t well
separated
• Metamerism
• two light sources, made up of different mixtures of
various wavelengths, may appear to be the same
colour
• Colour detectors
• Our colour detector (eye) not being uniformly
sensitive to light of different wavelengths
RGB COLOUR MATCH
• Take an image of the dyed solution
• Compare that to a standard curve using an
interpolation of the Red Green Blue (RGB) hue
values
• Colours can be collected on the crystal imager
• The images are passed through a program (pH
Hue-ristic) to convert to pH values, and can be
uploaded directed into our database
HOW WELL DOES IT WORK?
QUESTIONS
• What chemicals should be used to make the
standard curve?
• What concentration should the dye be?
• Does the camera that is used to take the
pictures matter?
• What could be done to get around the nonlinearity at around pH 7?
Different cameras (temperatures)
dye dilutions at 2 temps
600
500
400
Hue
1:10
1:20
300
1:10b
1:20b
200
100
0
3
3.5
4
4.5
5
5.5
6
6.5
7
pH
7.5
8
8.5
9
9.5
10
10.5
11
Comparison to pH meter values
pH (pH meter) vs. pH (heuristic)
12
y = 0.9312x + 0.538
R² = 0.9309
pH measured with Hue-ristic
10
8
6
4
2
0
0
2
4
6
pH measured with pH meter
8
10
12
Comparison of the same screen over time
Old
New
3
2.5
2
1.5
1
0.5
0
-4
16
36
56
76
96
PEGs over time
pH estimation for very small samples
THE PERFECT WORLD OF MACROMOLECULAR
CRYSTALLISATION
• 100% accuracy with
crystallisability prediction from
sequence
• “Rational” crystallisation:
understand link between the
crystallisation condition and the
resultant packing in a crystal
• Tools to get all salient
information from an drop image
• Crystal quality – tools for
optimising diffraction from a
crystal rather than visual
perfection of the crystal