Designing successful membrane protein crystallisation screens: how we designed MemGold. Simon Newstead

Transcription

1 Designing successful membrane protein crystallisation screens: how we designed MemGold Simon Newstead

2 Barriers to structure X-ray diffraction is currently the most successful method Requires good crystals Sufficient amount of protein In a form that is stable and compatible with forming well ordered 3D crystal lattices Bottlenecks Recombinant protein production Purification Crystal screening Data collection, phasing, building and refinement

3 What are we dealing with? Lipid 2D crystal Solubilisation & purification Detergent Type I 3D crystal protein Detergent Lipid cubic phase Protein-detergent complex

4 Improving our MP crystal screen Our strategy involved mining the PDB to investigate which conditions were being reported for MPs. Use this information to design a more rational sparse matrix screen. MemGold was released in 2007 currently the most popular screen purchased from Molecular Dimensions. 96 condition screen based on the crystallisation conditions reported for alpha helical membrane proteins up to What else is out there?

5 Current commercially available screens Molecular Dimensions MemStart MemSys MemGold MemBeta Hampton Research MemFac Emerald Biosystems BetaMem Jena Bioscience JBScreen-Membrane Qiagen Mbclass screens

6 What did our database tell us? Breakdown of families Trends in the successful detergents Crystallisation precipitants Buffers, ph Salts Additives Respiratory complexes Transporters Channels Photosynthetic & Light Harvesting complexes GPCR ATPases Others Bacterial Rhodopsins

7 Detergent selection Critical to obtaining well ordered crystals Many tools/methods now available to test experimentally which detergents are suitable for your target Most likely maltoside based detergents with longer alkyl chains and provide greater stability (C12M; C11M; C10M) although these will give on average lower resolution diffraction. How many proteins were crystallised with multiple detergents and are any trends visible, i.e. favourable couplings of different detergent classes?

8 Detergent and Crystal quality

9 Detergents (2007) 60 Respiratory complexes Transporters Channels Photosynthetic & Light Harvesting complexes GPCR ATPases Others Bacterial Rhodopsins No. of successful crystallisations Maltoside s Glucoside s LDAO 10 0 Maltosides Glucosides Polyoxyethylene Glycols HEGAS MEGAS Dodecanoyl Sucrose Lipid-Like (Foscholines) Zwittergents Amine Oxides Short Chain Lipids

10 Detergent summary The most commonly used detergent is DDM, followed by OG and DM. Although no clear rules exist, use the tools described in the literature to make an informed decision. Keep it simple initially, try to crystallise in the detergent used for prufication. Common detergents used initially in screening DDM (0.03 %) DM (0.2 %) LDAO (0.1%) C12E9 (0.03 %)

11 New amphiphile detergents.. Improved detergents for crystallisation/handling Neopentyl glycol s (Anatrace) Seok Chae et al., Nat Meth. 7, (2010) Façade-EM (Avanti Polar Lipids) Zhang, Q. X. et al., Angew Chem Int Ed 46, (2007) Both represent departures from the traditional detergent molecule (single head and tail construction). Neopentyl glycol s Façade-EM

12 Trends in precipitants (2007) Within the 96 conditions used in MemGold, 89 % were obtained using PEG, 9% using salts and 1 using Jeffamine M600. Organic solvents are generally much less successful in crystallising alpha helical membrane proteins. If your protein behaves best in zwitterion detergents then investigate different salts for crystallisation Effects of PEG on detergent micelles: implications for the crystallisation of integral membtrane proteins. Hitscherich et al. Acta Cryst. (2001). D57,

13 Precipitant trends (2007) No. of successful crystallisations Organic Molecules Salts Large MW PEGs ( , 000 Da) Medium MW PEGs ( Da) Small MW PEGs ( Da) No. of successful crystallisations Large MW PEGs Medium MW PEGs Small MW PEGs Bacterial Rhodopsins GPCRs Channels Transporters Photosynthetic & Light Harvesting Complexes ATPases Respiratory Complexes Others 0 < > Concentration of PEG (%) Figure 3 b Figure 3 a

14 Precipitant trends (2007) Respiratory complexes Transporters Channels Photosynthetic & Light Harvesting complexes GPCR ATPases Others Bacterial Rhodopsins TEG PEG 200 PEG 300 PEG 350 MME PEG 400 PEG 550 PEG 550 MME PEG 600 Jeffamine M600 PEG 1000 PEG 1450 PEG 1500 PEG 2000 PEG 2000 MME Jeffamine ED2001 PEG 3000 PEG 3350 PEG 4000 PEG 5000 PEG 5000 MME PEG 6000 PEG 8000 Tri-Sodium Citrate NaCl Ammonium Sulphate Lithium Sulphate Sodium Phosphate 5/4 PO/OH MPD No. of successful crystallisations

15 How does this compare to non MPs? Core 67 JCSG (84 %) MemGold Core 24 Toronto (94 %) Small MW PEGs ( Da) Medium MW PEGs ( Da) Large MW PEGs ( , 000 Da) Salts Organic Molecules R.Page, R.C. Stevens / Methods 34 (2004)

16 Concentration ranges Small: Medium: Large: , Small: Medium: Large: ,000 Counts MemGold Counts Core 67 JCSG < > % Concentration of PEG 0 < > % Concentration PEG 8 7 Small: Medium: Large: , Core 24 Toronto Counts < > % Concentration PEG

17 Buffers Buffers can have profound effects on the success of crystallisation trials Act as bridging ligands due to useful polar groups Tris and HEPES are the most commonly used buffers, in the ph ranges 7-8. Try to screen a wide range of ph values ADA HEPES Bis-Tris MES

18 Buffers ph values Ammonium Acetate Bicine Bis-Tris Citric Acid HEPES Sodium Cacodylate Sodium Acetate No. of successful crystallisations Sodium Citrate No. of successful crystallisations Sodium Phosphate Tris Tricine MES Glycine Potassium Citrate Potassium Phosphate < > ph

19 Salts Often critical for PEG conditions Polyvalent cations and anions can be critical for crystallisation and often stabilise crystal contacts Important parameter for optimisation Screen divalent ions Mn Mg Cd Fe Co Ni You get the picture

20 Salt trends (2007) No. of successful crystallisations Sodium Acetate Sodium Citrate Sodium Chloride Sodium Formate Sodium Sulphate Sodium Phosphate Lithium Sulphate Lithium Citrate Potassium Citrate Potassium Chloride Potassium Nitrate Ammonium Sulphate Barium Chloride Magnesium Chloride Magnesium Nitrate Magnesium Acetate Magnesium sulphate Zinc Acetate Calcium Chloride Cadmium Chloride Nickel Sulphate

21 Additives, a useful resource Well established small molecule screens for non MPs 25 Hampton additive screen Silver bullets Detergent screening No good data yet on whether any trends are emerging as to successful combinations of secondary detergents.. However, you should always screen secondary detergents No clear trends yet No. of successful crystallisations Multivalent Salts Monovalent Salts Linkers Lipids Amphiphiles Reducing Agents Chelating Agents Carbohydrates Polyalcohols Detergents Organic, Non-Volatile Organic, Volatile Heavy Water

22 Other useful parameters Protein concentration Start at 50 um, then try 100 to 200 um. Complexes often require higher protein concentrations. Sample buffer Keep the concentration in the protein sample as low as possible to extract the most out the screens. 20mM is usually sufficient. Temperature 4 and 18ºC usually tried Observation Should usually see something happen within a week, sooner for smaller volume experiments.

23 Updating the database for 2011/ we had 121 structures in the database 2011 we now have 264

24 What has changed 2007 vs Respiratory complexes Transporters Channels Photosynthetic & Light Harvesting complexes GPCR ATPases Others Bacterial Rhodopsins

25 Crystallisation method Recent success in LCP methods for GPCRs have changed the options for crystallisation Helped by advances in technology at the beamlines microfocus optics and low noise detector systems. 9%

26 Precipitant trends Organic Molecules Salts Large MW PEGs ( , 000 Da) Medium MW PEGs ( Da) Small MW PEGs ( Da) No. of successful crystallisations Bacterial Rhodopsins GPCRs Channels Transporters Photosynthetic & Light Harvesting Complexes ATPases Respiratory Complexes Others 5 0 Figure 3 a 2007

27 Detergent trends 2011 Respiratory complexes Transporters Channels Photosynthetic & Light Harvesting complexes GPCR ATPases Others Bacterial Rhodopsins No. of successful crystallisations Maltosides Glucosides Polyoxyethylene Glycols HEGAS MEGAS Dodecanoyl Sucrose Lipid-Like (Foscholines) Zwittergents Amine Oxides Short Chain Lipids 2007

28 Further reading Methods and Results in Crystallisation of Membrane Proteins IUL Biotechnology Series Membrane Protein Purification and Crystallisation, A practical guide Academic Press Tricks of the trade used to accelerate high-resolution structure determination of membrane proteins Sonoda et al., FEBS Lett 584, (2010) Rationalising alpha helical membrane protein crystallisation Newstead et al., Protein Science 17, (2008)

29 Acknowledgements So Iwata David Drew Alex Cameron Liz Carpenter Bernedette Berne Sebestian Farrendon Past lab member s