MemSys™
Catalogue number MD1-25
Strategy and background
MemSys offers a systematic approach to screening for initial crystallisation conditions for membrane
proteins using vapour diffusion methods. The systematic approach where hopefully membrane protein
solubility is pushed to the limit aims to provide more information than previous sparse matrix type
screens. Whilst primarily designed for alpha type transmembrane proteins this screen has also been
successfully applied to beta type outer membrane proteins (unpublished results) and is expected to
be equally applicable to all membrane protein types.
Recently there has been an important increase in the number of membrane protein structures solved
providing a much larger data base of reported conditions for successful crystallization. A previously
introduced product, MemStart™ is a sparse matix approach biased to span the 33 reported successful
crystallization conditions of membrane proteins for which atomic resolution structures have been
determined.
MemSys is a systematic screen spanning the key values of pH, precipitant type / concentration, and
salts (“Methods and Results in the Crystallization of Membrane Proteins” Ed. Iwata S. In Press,
International University Line). Molecular Dimensions acknowledges the work of Prof S Iwata, Dr M
Iwata and Dr J Abramson in designing this product.
Types and concentrations of PEGs
used for membrane protein
crystallisation. Small PEGs include
triethylene glycol, PEG400 and
PEG550 monomethylether. Medium
PEGs include PEG1500, PEG2000 and
PEG2000 monomethylether. Large
PEGs include PEG3350, PEG4000,
PEG6000 and PEG10000.
pH used for membrane
protein crystallisation.
Total concentration of
salts used for
membrane protein
crystallisation.
MemSys contains 48 conditions allowing the pH range, precipitants and salts used in membrane
protein crystallization to be screened with detergent containing protein drops. The reagents can be
easily arranged in systematic array to facilitate the interpretaion of results and the design of further
optimisation experiments.
This product is manufactured under an exclusive licence from Imperial College of Science, Technology
& Medicine, London, UK.
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Instructions for use
MemSys is intended to be used in sitting drop or hanging drop vapour diffusion crystallisation
methods. The protein drop will normally be diluted 1+1 with the screening reagent and the drop will
of course also contain detergent.
Sample preparation
Membrane proteins often form aggregates and these will not crystallize. Electron microscopy and
analytical ultracentrifugation are more appropriate than dynamic light scattering for assessing sample
homogeneity / monodispersity of membrane protein samples. Sample monodispersity can be
improved by changing the detergent, increasing salt concentration, and ultracentrifugation.
Typical protein concentrations for crystallizing membrane proteins are in the range 40-80 µM. A good
starting point would be 50 µM (10 mg/ml for a 200 kDa protein). Protein concentrations for
crystallizing membrane proteins tend to be somewhat higher than normally recommended for soluble
proteins so if 50 µM is not successful try 100 µM (or even higher, it is often easier than changing the
precipitant concentration).
Protein concentration used for membrane protein
crystallisation.
Detergents used for membrane protein
crystallisation. DDM: dodecylmaltoside, OG:
Octylglucoside, DM: decylmaltoside, UDM:
undecylmaltoside, LDAO: N,Ndimethyldodecylamine N-oxide, UDAO: N,Ndimethylundecylamine N-oxide, NG:
The most successfully used detergent is dodecylmaltoside. Then follow octylglucoside and LDAO, N,Ndimethyldodecylamine N-oxide. It is worth trying to crystallize with the detergent that was used
during purification, good first alternatives are dodecylymaltoside (0.03%), octylglucoside (1%), LDAO
(0.1%). Obviously other detergents can be tried if success is not achieved. Typically a concentration
around 2-3 times CMC (critical micelle concentration) should be used. Often the choices of detergent
or precise concentration are critical sensitive parameters for initial screening. Once a result is
obtained optimisation of detergent choice and concentration is critical to obtaining good quality
crystals and a second detergent is often used as an additive (see below).
The pH of the protein drop should not be overlooked. The kit reagents are buffered and to take full
advantage of this a low concentration (20 mM) of buffer in the protein sample is desirable. Ionic
strength can be increased with sodium chloride (50 – 100 mM) if protein solubility becomes a
problem. Crystallization conditions for soluble proteins have evolved into published screens based on
a trial and error experimental approach. This has led to it being standard practice to make up
screening reagents with stock buffer solutions to which salts and precipitants are added without any
further attention being paid to the final reagent pH. In MemSys we have tried to rationalise the
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buffers to within 0.2 pH units of the quoted pH. (See the list of reagents for details). In this way we
hope to make it easier to design optimisation experiments.
Temperature
Temperature is a critical parameter for crystallization due to the temperature dependence of
solubility. Membrane protein crystals are often temperature sensitive and so experiments should be
observed at the temperature at which they have been performed. The most successful reported
temperature is 4 ºC followed by room temperature (18 – 20 ºC).
Additives & optimisation
The use of additives in the protein drop has often been found useful, or even essential, for optimising
the crystal quality of membrane proteins. Whilst additives are normally added to the protein drop,
volatile additives must also be included in the well (reservoir) solution. 1,2,3-heptantriol (1-6%) has
been the most successfully used additive. Other additives often used are: benzamidine (2-4%),
glycerol (10-20%), ethanol (5-10%) and DMSO ((5-10%). As mentioned above second detergents are
also often used as additives to optimise crystal quality.
The strategy of MemSys is also designed to provide clues for optimisation experiments. The layout of
the results page below allows comparisons to be easily made between precipitant, pH, and salt. Like
all protein crystal growth screening, backing off a concentration of a precipitating reagent, changing
the buffer and pH, using an alternative ion (eg Ca2+, Mg2+, Zn2+), modifying the precipitant can all be
tried.
Observation of results
Under optimised conditions crystals can grow quite quickly. A useful regime is to check for crystal
growth at 1, 3, 7, 14, and 30 days. MemSys reagents are numbered according to precipitant and pH
to facilitate layout of the crystallisation tray as illustrated on the results page below as an aid to the
analysis of screening results, and to plan optimisation experiments.
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MemSys™
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0.1M
0.1M
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0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
Sodium citrate/citric acid pH 5.5
Sodium citrate/citric acid pH 3.5
Sodium acetate/acetic acid pH 4.5
Sodium citrate/citric acid pH 5.5
Sodium citrate/citric acid pH 5.5
Sodium citrate/citric acid pH 5.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MOPS/Sodium hydroxide pH 7.0
Sodium Hepes/HCl pH 7.5
MOPS/Sodium hydroxide pH 7.0
Sodium Hepes/HCl pH 7.5
Sodium Hepes/HCl pH 7.5
Sodium Hepes/HCl pH 7.5
Sodium Hepes/HCl pH 7.5
Tris/HCl pH 8.5
Tris/HCl pH 8.5
Tris/HCl pH 8.5
Tris/HCl pH 8.5
CAPSO/Sodium hydroxide pH 9.5
CAPSO/Sodium hydroxide pH 9.5
Sodium citrate/citric acid pH 5.5
Sodium citrate/citric acid pH 3.5
Sodium acetate/acetic acid pH 4.5
Sodium citrate/citric acid pH 5.5
Sodium citrate/citric acid pH 5.5
Sodium citrate/citric acid pH 5.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MES/Sodium hydroxide pH 6.5
MOPS/Sodium hydroxide pH 7.0
Sodium Hepes/HCl pH 7.5
MOPS/Sodium hydroxide pH 7.0
Sodium Hepes/HCl pH 7.5
Sodium Hepes/HCl pH 7.5
Sodium Hepes/HCl pH 7.5
Sodium Hepes/HCl pH 7.5
Tris/HCl pH 8.5
Tris/HCl pH 8.5
Tris/HCl pH 8.5
Tris/HCl pH 8.5
CAPSO/Sodium hydroxide pH 9.5
CAPSO/Sodium hydroxide pH 9.5
Contents
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
Sodium
Sodium
Sodium
Sodium
Sodium
chloride
chloride
chloride
chloride
chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium
Sodium
Sodium
Sodium
Sodium
chloride
chloride
chloride
chloride
chloride
Sodium
Sodium
Sodium
Sodium
Sodium
chloride
chloride
chloride
chloride
chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium chloride
Sodium
Sodium
Sodium
Sodium
Sodium
chloride
chloride
chloride
chloride
chloride
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Catalogue No. MD1-25
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
0.1M
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Magnesium chloride
Lithium sulphate
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
Lithium sulphate
Magnesium chloride
2.5M Ammonium sulphate
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
2.5M Ammonium sulphate
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
2.5M Ammonium sulphate
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
1.5M Lithium sulphate
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
30% v/v PEG 400
1.5M Sodium phosphate
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
1.5M Sodium phosphate
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
1.5M Potassium phosphate
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
1.5M Potassium phosphate
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
12% w/v PEG 4000
MemSys™– Results Table
1
0.1M Na citrate pH5.5
2.5M (NH4)2SO4
2
0.1M Na citrate pH3.5
0.1M NaCl
0.1M Li2SO4
30% v/v PEG 400
3
0.1M Na acetate
pH4.5
0.1M NaCl
0.1M MgCl2
30% v/v PEG 400
4
0.1M Na citrate pH5.5
0.1M NaCl
30% v/v PEG 400
5
0.1M Na citrate pH5.5
0.1M NaCl
0.1M Li2SO4
30% v/v PEG 400
6
0.1M Na citrate p 5.5
0.1M NaCl
0.1M MgCl2
30% v/v PEG 400
7
0.1M MES pH6.5
2.5M (NH4)2SO4
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0.1M MES pH6.5
30% v/v PEG 400
9
0.1M MES pH6.5
0.1M NaCl
30% v/v PEG 400
10
0.1M MES pH6.5
0.1M NaCl
0.1M Li2SO4
30% v/v PEG 400
11
0.1M MES pH6.5
0.1M NaCl
0.1M MgCl2
30% v/v PEG 400
12
0.1M MOPS pH7.0
30% v/v PEG 400
13
0.1M HEPES pH7.5
2.5M (NH4)2SO4
14
0.1M MOPS pH7.0
0.1M NaCl
30% v/v PEG 400
15
0.1M HEPES pH7.5
30% v/v PEG 400
16
0.1M HEPES pH7.5
0.1M NaCl
30% v/v PEG 400
17
0.1M HEPES pH7.5
0.1M NaCl
0.1M Li2SO4
30% v/v PEG 400
18
0.1M HEPES pH7.5
0.1M NaCl
0.1M MgCl2
30% v/v PEG 400
19
0.1M Tris pH8.5
1.5M Li2SO4
20
0.1M Tris pH8.5
0.1M NaCl
30% v/v PEG 400
21
0.1M Tris pH8.5
0.1M NaCl
0.1M Li2SO4
30% v/v PEG 400
22
0.1M Tris pH8.5
0.1M NaCl
0.1M MgCl2
30% v/v PEG 400
23
0.1M CAPSO pH9.5
0.1M NaCl
0.1M Li2SO4
30% v/v PEG 400
24
0.1M CAPSO pH9.5
0.1M NaCl
0.1M MgCl2
30% v/v PEG 400
25
0.1M Na citrate pH5.5
1.5M NaH2PO4
26
0.1M Na citrate pH3.5
0.1M NaCl
0.1M MgCl2
12% w/v PEG 4000
27
0.1M Na acetate
pH4.5
0.1M NaCl
0.1M Li2SO4
12% w/v PEG 4000
28
0.1M Na citrate pH5.5
0.1M NaCl
12% w/v PEG 4000
29
0.1M Na citrate pH5.5
0.1M NaCl
0.1M Li2SO4
12% w/v PEG 4000
30
0.1M Na citrate pH5.5
0.1M NaCl
0.1M MgCl2
12% w/v PEG 4000
31
0.1M MES pH6.5
1.5M NaH2PO4
32
0.1M MES pH6.5
12% w/v PEG 4000
33
0.1M MES pH6.5
0.1M NaCl
12% w/v PEG 4000
34
0.1M MES pH6.5
0.1M NaCl
0.1M Li2SO4
12% w/v PEG 4000
35
0.1M MES pH6.5
0.1M NaCl
0.1M MgCl2
12% w/v PEG 4000
36
0.1M MOPS pH7.0
12% w/v PEG 4000
37
0.1M HEPES pH7.5
1.5M K2HPO4
38
0.1M MOPS pH7.0
0.1M NaCl
12% w/v PEG 4000
39
0.1M HEPES pH7.5
12% w/v PEG 4000
40
0.1M HEPES pH7.5
0.1M NaCl
12% w/v PEG 4000
41
0.1M HEPES pH7.5
0.1M NaCl
0.1M Li2SO4
12% w/v PEG 4000
42
0.1M HEPES pH7.5
0.1M NaCl
0.1M MgCl2
12% w/v PEG 4000
43
0.1M Tris pH8.5
1.5M K2HPO4
44
0.1M Tris pH8.5
0.1M NaCl
12% w/v PEG 4000
45
0.1M Tris pH8.5
0.1M NaCl
0.1M Li2SO4
12% w/v PEG 4000
46
0.1M Tris pH8.5
0.1M NaCl
0.1M MgCl2
12% w/v PEG 4000
47
0.1M CAPSO pH9.5
0.1M NaCl
0.1M Li2SO4
12% w/v PEG 4000
48
0.1M CAPSO pH9.5
0.1M NaCl
0.1M MgCl2
12% w/v PEG 4000
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