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© Johan Zeelen M.P.I. of Biophysics
The crystallization of (membrane) proteins,using a flexible Sparse Matrix Screen.
Soluble proteins and membrane proteins
Screening protocol for 3 D crystallization
- flexible sparse matrix
- adjusted screen
- optimisation
Membrane proteins and detergent
Excluding regions where nucleation is not evident
© Johan Zeelen M.P.I. of Biophysics
Soluble proteins and membrane proteins
- Soluble proteins
Protein
- Membrane proteins
Protein
Detergent
(Lipid)
© Johan Zeelen M.P.I. of Biophysics
Soluble proteins and membrane proteins
The purity of the starting material is a crucial factor.
- Purity of the protein:
SDS gel and TLC
- Sample heterogeneity:
Mass spectrometry
Dynamic light scattering
- Solubility 5-10 mg/ml
- Stability Storage conditions
SDS - page-
Thin-layer chromatography-> Proteolysis and post
translational modifications-> Oligomerisation, aggregation
© Johan Zeelen M.P.I. of Biophysics
Screening protocol for 3 D crystallization
1 2 3 4 5 6 7 8 9 10ABCD
FGHIJ
3.5Å
1.5Å.
8Å
E
Triosephosphate isomerase:
P212121 1.83ÅC2 (big) 2.1 ÅC2 (small) 2.1 ÅP1 1.5 Å
The screening for suitable crystallization conditions starts with the search in a multidimensional phase diagram for conditions that favor nucleation. Experiments with Triosephospate isomerase (soluble protein) and Light-harvesting complex II (membrane protein) indicate, that the proteins can crystallize in different space groups and resolution.
Two dimensional representation of the multi dimensional crystallization space
© Johan Zeelen M.P.I. of Biophysics
Initial screenThe systematic search of the multidimensional space requires large amount of protein and time.
1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ
X XX XX XX XX XX X
To reduce the number of crystallization trails, the incomplete factorial is a powerful tool to identify the influence of different variables.
1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ
1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ
X
X
X
X
The conditions in the sparse matrix are not random but heavily biased towards published crystallization conditions. Most of the commercially available screens are sparse matrix screens.
1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ
X
XX
X
1 2 3 4 5 6 7 8 9 10ABCDEFGHIJ
In the flexible sparse matrix also information from biochemistry (pH stability, salts) is used to exclude conditions where the protein denatures / is inactive.To simplify the interpretation of the results, the crystallization conditions are sorted by precipitant. The drop size is 1 µl protein and 1 µl well solution.
X
XX
© Johan Zeelen M.P.I. of Biophysics
Interpretation of the initial screen
The crystallization experiments are examined with a stereo microscope: 1) immediately after setup, 2) each day for the first week, and 3) once a week for several weeks.
Not precipitatedPrecipitated no birefringence no edgesPrecipitated with birefringence and edges
© Johan Zeelen M.P.I. of Biophysics
No nucleation with the initial screen
Try a different screen- random sampling
- based on published conditions
0
1
2
3
4
5
3 4 5 6 7 8 9 10 11
11 Commercially Available Screens
Am
mo
niu
m S
ulf
ate
pH0
1
2
3
4
5
3 4 5 6 7 8 9 10 11
Conditions from BMCD
Am
mo
niu
m S
ulf
ate
pH
As initial screen the sparse matrix is the most popular because it is commercially available. This does not mean that every protein will crystallize under these conditions. The strategy to try different sparse matrix screens will limit the search to the most common successful crystallization conditions.
© Johan Zeelen M.P.I. of Biophysics
No nucleation with the initial screen
Adjust the initial screen
Precipitant
soluble
supersaturation
slow precipitation /nucleation
Fast precipitation
[Pro
tein
]
No precipitate -> increase/double precipitant concentrationFast precipitation -> decrease/halve precipitant concentration
Repeat until the precipitation point for all wells is determined.
A different and possibly faster strategy is to use the solubility information from the first screen to exclude areas of the phase diagram where no crystallization will occur. This knowledge is used for the design of a new adjusted screen. In the adjusted screen only the precipitant concentration is changed.
© Johan Zeelen M.P.I. of Biophysics
Optimisation screen
After identification of conditions that favour crystal growth a new set of conditions is set-up by adjusting the:
A) precipitant 1 concentration with salt
B) precipitant concentration without salt
C) buffer and pH (4.5-9.5)
D) protein concentration
Initial screen
Adjusted screen
Optimisation
25 % PEG 6000, 200 mM LiSO4 at pH=6.5, 5 mg/ml protein -> small crystals after 3 days
A) 12.5% - 25 % PEG 6000, 200 mM LiSO4 at pH=6.5 B) 12.5% - 25 % PEG 6000 at pH=6.5 C) pH = 4.5 - 9.5, 25 % PEG 6000, 200 mM LiSO4 D) Protein 1 - 6 mg/ml, 25 % PEG 6000, 200 mM LiSO4 at pH=6.5
A - B -> is salt neededA or B -> precipitant concentrationC -> pH dependenceD -> protein concentrationIdentical conditions -> reproducibility
© Johan Zeelen M.P.I. of Biophysics
Detergent
The detergent plays a crucial role in the crystallisation of membrane
proteins.
Neurospora crassa plasma membrane H+-ATPaseprecipitant
dete
rgen
t
phase separation
CMC
Detergent phase diagram At high detergent and precipitant concentrations the micelles aggregate. The solution separates in a micelle rich and a micelle poor phase. Often 3D crystals are found, close to the condition where phase separation occurs.
© Johan Zeelen M.P.I. of Biophysics
2D and 3D crystallisation of LHC II
2D EM
3D X-ray
© Johan Zeelen M.P.I. of Biophysics
Flexible sparse matrix screenInitial screen
Initial screen
Adjusted screen
Detergent/biochemistry
Optimization Crystals
Optimization
Stock solutions
Initial screen Adjusted screen
Buffer Precipitant Additive
The flexible sparse matrix is an efficient method for searching the multidimensional phase diagram for conditions that favor nucleation, by excluding regions where nucleation is not evident. Although the preparation of wells from stock solutions is more labor intensive than ready-made solutions, it has the advantage that precipitant concentration, pH, and additive can be manipulated independently.
© Johan Zeelen M.P.I. of Biophysics
Acknowledgements
E.M.B.L Heidelberg
Group R.K. Wierenga
M.P.I. of Biophysics
Department of Structural Biology
W. Kühlbrandt
H+-ATPase: M. Auer and G.A. Scarborough
LHC II : M. Lamborghini
http://www.mpibp-frankfurt.mpg.de/~johan.zeelen/xtal.html
Garavito, R.M and Picot, D (1990) The Art of Crystallizing Membrane Proteins. Methods: A Companion to Methods in Enzymology Vol. 1 No. 1 pp. 57-69
Hjelmeland, L.M. and Chrambach, A. Solubilization of Functional Membrane Proteins. Methods in enzymology Vol 104 pp. 305-318
Zeelen, J.P. (1999) Strategy 1: A Flexible Sparse Matrix Screen. In Protein Crystallization, Techniques, Strategies, and Tips, chapter 9 (ed. T.M. Bergfors) IUL Biotechnology Series.
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