Experimental mapping of
protein precipitation diagrams
Morten O.A. Sommer (morten@ccs.ki.ku.dk)
Centre for Crystallographic Studies
University Of Copenhagen
Look at protein crystallography
and liquid handling
Liquid handling for protein crystallization
100
10
2006
2004
2002
2000
1998
1996
1
1994
Number of chemical
experiments pr micro liter
1000
Low volume liquid
handling
technology 
more experiments
performed using
less SAMPLE
Lab automation
 more
experiments
performed using
less TIME
Low TIME and SAMPLE consumption enables new
approaches to protein crystallization
Microfluidic formulator
technology
1 mm
Experiments done by: Carl Hansen, Morten Sommer and
Stephen Quake. PNAS (2004) 101:14431-14436
Metering accurate and robust
4.5
Metering accuracy
determined by absorption
measurements
Injected volume [nL]
4
3.5
Motor oil
Raw Linseed oil (SAE 20) at
Water at
20 degrees C
20 degrees C 20 degrees C
3
2.5
2
1.5
1
Injection volume:
80 pL +/- 0.6 pL
0.5
0
0
10
20
30
40
50
Number of injection cycles
Experiments done by: Carl Hansen, Morten Sommer and
Stephen Quake. PNAS (2004) 101:14431-14436
Ideal approach to protein
crystallization
• GOAL: Further rationalization of protein
crystallization
• Using minute amounts of protein sample
to quantify:
– Protein stability, folding & activity
– Protein physical chemistry (solubility and
precipitation limits)
– Protein - protein interactions (Virial
coefficients etc.)
Why use precipitation diagrams?
Phase diagram
of: aspartyltRNA
synthetase-1
From Thermus
thermophilus
Zhu et. al. 2001
Acta Cryst. D
57:552-558
Detecting precipitation
Detection of Precipitation
25.00
STDEV
20.00
15.00
10.00
5.00
0.00
0
10
20
30
40
Titration Number
50
60
Towards a rational approach:
Tailor made screens based on
precipitation diagrams
• Characterize protein
solution and identify
potential conditions
• Map protein precipitation
diagrams
• Design and set up a tailor
made crystallization
screen based on the
precipitation diagrams of
the particular protein
Initial validation: Xylanase
Xylanase [mg/ml]
1. Make solubility
fingerprint identifying
precipitating
chemical conditions
2. Map precipitation
diagrams for
potential conditions
3. Set up crystallization
experiments near
precipitation
boundary
40
35
30
25
20
15
10
5
Experiments0 0done by: 0.5
Carl Hansen,
Morten
Sommer2 and
1
1.5
Na/K Tartrate
[M]
Stephen Quake. PNAS (2004)
101:14431-14436
Initial validation: Xylanase
Crystallization probability pr. trial
OPT (Tailor made screen): 27 hits out of 48
experiments = 56 %
Sparse matrix screens: 3 hits out of 384
experiments = 0.8 %
Experiments done by: Carl Hansen, Morten Sommer and
Stephen Quake. PNAS (2004) 101:14431-14436
Further validation
Membrane protein: SERCA
• Study the
crystallization of
membrane proteins
using the previously
crystallized calcium
pump (SERCA)
• Crystallization
conditions are know
• Reliable preparation
and purification
Sørensen et.al., (2004) Science 304, 1672-1675
Further validation
Membrane protein: SERCA
1
Relative Precipitant Strength
0.8
0.6
0.4
0.2
S
od
iu
m
Ac
C
et
al
at
ci
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id
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at
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at
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e
• Solubility fingerprint can be used to identify specific
0
protein – precipitant
interactions
• Identification of specific interaction between sodium
acetate and SERCA
• Sodium acetate is an established crystallization agent for
SERCA
Experiments done by: Morten Sommer and Sine Larsen.
Journ. of Synchrotron Rad. (2005) in press
Further validation
Membrane protein: SERCA
• Based on the characterization of specific protein
– precipitant interactions several chemical
conditions were selected for precipitation
diagram mapping
• Set up tailor made crystallization screen
• Identification of well known and new
crystallization agents
• Potentially useful for crystallizing previously
uncrystallized membrane proteins
Experiments done by: Morten Sommer and Sine Larsen.
Journ. of Synchrotron Rad. (2005) in press
Process diagram
Solubility
fingerprint
Analysis of
protein-precipitant
interaction
Protein
sample
Precipitation
diagrams
Formulator
chip
Design rational
crystallization
experiments
Crystals
Monitor
experiments
Setup
crystallization
experiments
Perspectives
Rational approach to protein crystallization
using minute sample volumes
Task
Volume consumption (μL)
Solubility characterization
10
Setup of 300 crystallization exp.
25
TOTAL
35
Rational approaches are possible for
many targets that are available in low
amounts (Membrane proteins, protein
complexes, and proteins purified from
native tissue).
Testing previously uncrystallized
membrane proteins
The ultimate test of the rational approach:
3 previously uncrystallized membrane
proteins are tested.
1. Voltage gated channel
2. DsbB: disulfide bond forming
membrane protein.
3. AIDA: adhesin autotransporter
protein
Voltage-gated channel:
Solubility mapping
Voltage-gated channel
in 0.1 M Linear Buffer pH 6.5
Based on the
solubility fingerprint
40 precipitation
diagrams are mapped
out.
3
Protein [mg/ml]
2.5
2
1.5
Volume consumption pr.
precipitation diagram:
100 nL
1
0.5
0
0
5
10
PEG 400 [% w/v]
15
20
Total consumption for
solubility screen and
precipitation diagrams:
8 μL (44.8 μg)
Experiments done by: Morten Sommer, Jens-Christian Navarro
Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
Voltage-gated channel:
Crystallization experiments
A tailor made screen
of 288 conditions is
designed.
The screen is set up
as sitting drop exp.
using an ORYX 6 at
Douglas Instruments
using 17 μL sample
(95 μg of protein)
Voltage-gated channel
in 0.1 M Linear Buffer pH 6.5
3
Protein [mg/ml]
2.5
2
1.5
1
0.5
0
0
5
10
PEG 400 [% w/v]
15
20
An additional screen
is set up testing
different additives
Experiments done by: Morten Sommer, Jens-Christian Navarro
Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
Voltage-gated channel:
Crystallization experiments
Scalebars =
100 microns
Crystals tested at
ESRF beamline ID 29.
Not protein crystals
Experiments done by: Morten Sommer, Jens-Christian Navarro
Poulsen, Sine Larsen, Jose Santos and Mauricio Montal
DsbB:
Solubility mapping
Using a total of 4 μL (40
μg of protein).
A tailor made screen
consisting of 288
conditions was
designed and set up
using 18 uL (180 μg)
DsbB in 0.1 M Linear Buffer pH 9 and
80 mM Calcium Acetate
7
Protein Conc. [mg/ml]
40 chemical conditions
are chosen for
determination of their
precipitation diagram.
6
5
4
3
2
1
0
0
10
20
30
40
PEG 4000 [% w/v]
Experiments done by: Morten Sommer, Jens-Christian Navarro
Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
DsbB:
Crystallization experiments
Crystals tested at
ESRF ID 29
Some were
not protein.
Scalebars =
100 microns
Some did not
diffract  cryo
optimization
Experiments done by: Morten Sommer, Jens-Christian Navarro
Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
AIDA:
Solubility characterization
40 precipitation diagrams
are selected for mapping
based on solubility
fingerprint
AIDA with
with 0.1
0.1 M
M Linear
Linear Buffer
Buffer pH
pH 44 and
and
AIDA
8%
% v/v
v/v Glycerol
Glycerol
8
5
5
4.5
4.5
Protein [mg/ml]
[mg/ml]
Protein
4
4
Based on the diagrams a
576 experiment screen is
designed and set up
3.5
3.5
3
3
2.5
2
2
1.5
1.5
1
Volume consumption:
0.5
0.5
0
0
0
0
5
5
10
10
15
15
PEG
ether [%
[% w/v]
w/v]
PEG 1500
1500 monomethyl
monomethyl ether
20
20
Solubility mapping: 8 μL
Crystallization exp.: 22 μL
Experiments done by: Morten Sommer, Jens-Christian Navarro
Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
AIDA:
Crystallization experiments
Crystals tested
at ESRF ID 29
 Some did not diffract
optimize cryo conditions
Scalebars =
100 microns
Experiments done by: Morten Sommer, Jens-Christian Navarro
Poulsen, Sine Larsen, Brian Vad and Daniel Otzen
Protein consumption
Solubility
char.
Cryst. Exp
Crystal Hits
Total protein
consumption
VGC
DsbB
AIDA
>5000 exp.
8 μL (45μg)
~576 exp.
34 μL(190μg)
No
>5000 exp.
8 μL (80μg)
~ 288 exp.
18 μL(180μg)
Yes
>5000 exp.
8 μL (80μg)
~ 576 exp.
22 μL(220μg)
Yes
235 μg
260 μg
300 μg
Summarizing remarks
• As liquid handling technologies have achieved
~1 nL experimental volumes.
A rationalization of protein crystallization in terms of
precipitation diagrams is possible
• Rational approaches to protein crystallization are
performed using < 300 μg of protein sample.
• Hope: This method and technology will allow for
a better understanding of the crystallization
process - and that complementary low volume
technology will be developed to address other
aspects of protein crystallization
Acknowledgements
• Univ. Of Copenhagen
–
–
–
–
Jens-Christian Poulsen
Prof. Sine Larsen
Flemming Hansen
Centre for Crystallographic
Studies
• Univ. Of Aalborg
– Prof. Daniel Otzen
– Brian Vad
• Univ. Of Aarhus
– Ass. Prof. Poul Nissen
– Prof. Jesper Vuust Møller
• Tech. Univ. Of Denmark
– Ass. Prof. Jörg Kutter
– Detlef Snakenborg
• Stanford
– Prof. Stephen R. Quake
• Univ. Of British Columbia
– Ass. Prof. Carl L. Hansen
• Univ. of California – San Diego
– Prof. Mauricio Montal
– Dr. Jose Santos
• Douglas Instruments
– James Smith
– Peter Baldock
– Patrick Shaw Stewart
• ESRF – ID29
– Gordon Leonard
Experimental mapping of
protein precipitation diagrams
Morten O.A. Sommer (morten@ccs.ki.ku.dk)
Centre for Crystallographic Studies
Univ. Of Copenhagen