December 4, 2010
Since GPCRs are transmembrane proteins they require to be removed from the membrane for their isolation, chromatographic purification and growth of crystals for X-ray structural analysis. This solublization process is facilitated by amphiphilic reagents that provide a microenvironment that is compatible with aqueous solutions and maintains the proper structural integrity of the membrane protein as individual particles. The list of the most useful amphile reagents, detergents, is long for membrane proteins in general, but short for GPCRs. In total, 49 unique detergents (including detergent mixtures) have been used to grown membrane protein crystals (Newstead et al., 2008). Interestingly, it turns out that with only 8 detergents, 80 % of membrane protein crystals can be grown. Below is the short table of those detergents that have been applied to the purification and crystallization of GPCRs.
Detergents used for extraction & solubilization of GPCRs for 3D crystallization:
- b1Adrenergic Receptor
- Bovine Rhodopsin
- b2Adrenergic Receptor
- Adenosine A2A Receptor
- CXCR4
- Dopamine D3 Receptor
|
- Octylthioglucoside
- Nonyl beta glucoside / heptantriol
- Dodecyl maltoside / cholesterol hemisuccinate
- Dodecyl maltoside / cholesterol hemisuccinate
- Dodecyl maltoside / cholesterol hemisuccinate
- Dodecyl maltoside / cholesterol hemisuccinate
|
Of course, for the G-protein coupled receptors that were crystallized in a lipidic cubic phase matrix, monoolein and cholesterol played a major role. Nonetheless, the extraction from the original membrane and purification requires one to select a particular detergent. Hence, for GPCRs the list of tried-and-proven detergents offers only three choices: 1. octylthioglucoside, 2. nonylbetaglucoside (with heptanetriol) or 3. dodecyl maltoside (with cholesterol hemisuccinate). Fortunately this collection has just grown dramatically. In a recent Nature Methods paper that I review in this blog post (New Detergent Class for Membrane Proteins) over at the Protein Crystallization Hits blog, a new class of detergents, called maltose-neopentyl glycol amphiphiles (MNGs) is introduced.
'Hidden' in the body of the paper is this gem of good news: one of the MNGs has produced crystals of b2AR-T4L that could not be grown by using the standard DDM (dodecylmaltoside) detergent. This bodes well for experimentalists that are stuck with the current narrow choice of detergents. And this is also good news for everybody who's been waiting for a structure of the agonist-bound beta 2 adrenergic receptor, the latter of which is advertised in the MNG methods paper (Chae et al. , 2010) to become avaliable at 3.5A resolution.
Can't wait,
Peter
References:
Newstead, S., Ferrandon, S., & Iwata, S. (2008). Rationalizing α-helical membrane protein crystallization Protein Science, 17 (3), 466-472 DOI: 10.1110/ps.073263108
Chae PS, Rasmussen SG, Rana RR, Gotfryd K, Chandra R, Goren MA, Kruse AC, Nurva S, Loland CJ, Pierre Y, Drew D, Popot JL, Picot D, Fox BG, Guan L, Gether U, Byrne B, Kobilka B, & Gellman SH (2010). Maltose-neopentyl glycol (MNG) amphiphiles for solubilization, stabilization and crystallization of membrane proteins. Nature methods, 7 (12), 1003-8 PMID: 21037590
Filed under:
Membrane Proteins, Crystallization | Posted by: Peter Nollert | Comments: 1
October 16, 2010
There's a nice write-up in GEN on the progress in the field of GPCR crystallization / structural biology and the impact this has on R&D efforts. I couldn't agree more with what Mike Hanson from Receptos says about the utility of lipidic cubic phases for the crystallization of G protein coupled receptor molecules. The List of "GPCRs of known structure" speaks for itself as it shows that the majority of the all crystal structures are based on crystals that have been grown within lipidic cubic phases.
Here at Emerald we have developed several relevant patented technologies ourselves and hold exclusive licenses to technologies that are indispensible for the practical aspects that are involved in GPCR crystallization. Some of these technologies are distributed as products via Emerald BioSystems. For instance, there is the 'Cubic LCP Cubic Kit' and the 'Cubic™ Screen', crystallization tools that are specifically designed for lipidic cubic phase - based membrane protein crystallization. These products aid researchers to set up crystallization trials with their own GPCR preparations.
Over here, at Emerald BioStructures we offer research services that apply these membrane protein crystallization technologies and tools within contract research projects. In fact, we have all the components in place to go from gene to membrane protein structure to provide our clients with unique insight into their drug targets (download a description of our membrane protein structure determination services here). At the heart of the matter, we feel that the field of membrane protein crystallography has now matured to a state where it can be applied within drug discovery projects and make a significant impact to lead optimization and to the discovery of new compounds, for instance with crystallographic fragment screening.
Soluble protein structure based drug discovery has an impressive track record of giving researchers a key advantage in their drug discvoery efforts. Demonstrating the value of crystallography for membrane proteins such as GPCRs is the logical next step.
This brings me to my shameless plug: Should you be interested in Emerald BioStructures' membrane protein structural biology services, please contact us here - or send me an email directly, I'd be happy to discuss specifics.
All the best,
Peter
Filed under:
Membrane Proteins, Crystallization, Opinion | Posted by: Peter Nollert | Comments: 0
October 8, 2010
Congratulations to Ray Stevens and team to determining and publishing the crystallographic structure of the CXCR4 Chemokine receptor in Science. The diffenent binding areas for the small molecule and peptide antagonist are nicely resolved and show extensive interactions with binding pocket residues in the 2.5 - 3.1 A crystal structures.
Wu B, Chien EY, Mol CD, Fenalti G, Liu W, Katritch V, Abagyan R, Brooun A, Wells P, Bi FC, Hamel DJ, Kuhn P, Handel TM, Cherezov V, & Stevens RC (2010). Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists. Science (New York, N.Y.), 330 (6007), 1066-71 PMID: 20929726
The corresponding structure files have not been released as of writing this. The PDB accession codes are: 3ODU (CXCR4-2–IT1t,P21), 3OE0 (CXCR4-3–CVX15, C2), 3OE8 (CXCR4-2–IT1t, P1), 3OE9 (CXCR4-3–IT1t, P1), and 3OE6 (CXCR4-1–IT1t, I222). The supplemental material lists a protocol that one could almost characterize as becoming 'the standard' for GPCR structure studies: expression of highly engineered construct (5 features) in Sf9 cells, membranes solubilized with a mix of dodecylmaltoside and cholesterol hemisuccinate (no detergent exchange) , His tag clipped off, PNGase treatment and crystallization in a matrix consisting of monoolein-based LCP with 6% cholesterol. All five crystal forms are stacked layers of membranes (as expected in LCP - grown crystals).
Lots of info to digest. What a feast!
Peter
Filed under:
Membrane Proteins, Crystallization, Structure | Posted by: Peter Nollert | Comments: 1
August 27, 2010
There's yet another great Technology Feature in NATURE, describing what it took - and what it takes - to pursue crystallographic GPCR structure determinations: The gatekeepers revealed: 10 June 2010 about gpcr xtallization.
Monya Baker has done a superb job in telling the people- and the research story behind the recent GPCR structure determination. Thankfully she does this in a language that would be suitable to share with your non-science inclined relatives, or with your colleagues that would like to get a superficial understanding on the 'state of the art'. I specifically like Monya's crystal clear, treatment of the subject matter. Not that I agree with her on all aspects of the piece, though. For example, omitting the pioneering work of Jurg Rosenbusch & Ehud Landau is more than an oversight. Indeed, the story starts in the mid 1990-ies at the Biozentrum in Basel, Switzerland when these researchers introduced for the first time lipidic cubic phases to membrane protein crystallographers. And Monya unfortunately misses to point out the ironic twist that one of the researchers mentioned in the article actually rejected an early paper for publication on precisely this subject, with the question 'who on earth would use such a complicated method for crystallization?'
Do note that yet another X-ray structure of a GPCR, that of sphingosine-1-phosphate receptor subtype 1 is advertised within the body of the online text right below Stephen White's # membrane protein structures / year plot.
Cheers,
Peter
Filed under:
Membrane Proteins, Crystallization, Opinion, Structure | Posted by: Peter Nollert | Comments: 0
July 23, 2010
Below is a list with recent patent filings that are relevant to X-ray crystallographic structure determinations of GPCRs.
|
Patent Application |
Assignee |
Title and Abstract |
|
US 20090148510 |
Stanford University |
GPCR crystalization method using an antibody
An antibody that specifically binds a three dimensional epitope on the IC3 loop of a GPCR is provided. The antibody may be employed in a method that comprises: contacting a GPCR with a monovalent version of the antibody binding conditions to form a complex; and crystallizing the complex |
|
US 20090271162 |
Stanford University |
Crystal structure beta2 adrenoreceptor
A computer readable medium comprising atomic coordinates for the human .beta..sub.2 adrenoreceptor is provided. The computer readable medium programming for displaying a molecular model of the human .beta..sub.2 adrenoreceptor, programming for identifying a compound that binds to said human .beta..sub.2 adrenoreceptor and/or a database of structures of known test compounds. Also provided is a method comprising computationally identifying a compound that binds to the human .beta..sub.2 adrenoreceptor using the atomic coordinates. |
|
US 20090118474 |
Stanford University |
Method and composition for crystallizing G protein-coupled receptors
Certain embodiments provide a method for crystallizing a GPCR. The method may employ a fusion protein comprising: a) a first portion of a G-protein coupled receptor (GPCR), where the first portion comprises the TM1, TM2, TM3, TM4 and TM5 regions of the GPCR; b) a stable, folded protein insertion; and c) a second portion of the GPCR, where the second portion comprises the TM6 and TM7 regions of the GPCR |
|
GB2456235 |
Medical Research Council |
Mutant beta-adrenergic receptors with improved stability |
|
WO/2009/055509 |
The Scripps Research Institute |
Cholesterol Consensus Motif of Membrane Proteins
The invention provides the structure of a human β2-adrenergic receptor, a cholesterol consensus motif, and methods of identifying modulators of G-protein coupled receptors (GPCRs). Methods of using the modulators of the receptor, GPCRs, and the cholesterol consensus motif are also provided. |
|
WO 2009055512 |
The Scripps Research Institute |
METHODS AND COMPOSITIONS FOR OBTAINING HIGH-RESOLUTION CRYSTALS OF MEMBRANE PROTEINS
The invention describes compositions and method useful for the crystallization of membrane proteins |
Cheers, Peter
Filed under:
Membrane Proteins, Crystallization, Structure | Posted by: Peter Nollert | Comments: 2
July 6, 2010
As members of the membrane protein family, GPCRs are notoriously difficult to work with in the biochemistry lab. Everything needs to be optimal for an experiment to succeed. A recent Technology Feature in Nature Methods summarizes this nicely for GPCRs: Making membrane proteins for structures: a trillion tiny tweaks.
Researchers are warned that GPCR projects are "extremely expensive" and should be "…planning for a long slog".
I personally like the analogy of scouting: you know where you want to go but at the outset have not clue about how to exactly navigate the path.
Below is a table that lists the steps that needed to be optimized for X-ray crystallographic GPCR structure determinations.
Table: Steps requiring careful optimization in X-ray crystallographic GPCR structure determination projects
|
# |
Step to optimize |
|
1 |
Design of gene with fusions/truncations/stabilizing mutations |
|
2 |
Over expression at sufficient scale in eukaryotic heterologous expression systems |
|
3 |
Stability and specific activity |
|
4 |
Sample purity and homogeneity |
|
5 |
Bound Ligand |
|
6 |
Crystallization with the appropriate amphiphile mix (such as monoglycerides, cholesterol) |
|
7 |
Crystallization in small volume and with optimized precipitation screening reagents |
|
8 |
Crystal detection, retrieval, cryoprotection and mounting |
|
9 |
X-ray micro diffraction |
The trouble really is that the optimization needs to be done FOR EVERY SINGLE TARGET. There's little what one can learn for one GPCR that can be applied to the entire target family. But a good scout find her destination quicker than a greenhorn.
Three finger salute,
Peter
Filed under:
Membrane Proteins, Crystallization, Opinion | Posted by: Peter Nollert | Comments: 0
June 29, 2010
So far eight different types of GPCR structures have been determined by X-ray crystallography: β2 Adrenergic Receptor, β1 Adrenergic Receptor, Adenosine Receptor, Rhodopsin, CXCR4 Chemokine receptor, Dopamine D3 receptor, the Histamine Receptor and the S1P1 Sphingosine 1-phosphate receptor.
Updates:
-
7 Oct 2010: added CXCR4 Chemokine Receptor
-
18 Nov 2010: added Dopamine D3 Receptor
-
The tables below makes it easier to find them either as a PDB code or the original reference.
-
21 Jan 2011: β1 and β2 Adrenergic Receptor structures
stabilized active state of β2 Adrenergic Receptor: 3P0G with irreversible agonist: 3PDS partial agonist and agonist structures of β1 2y00, 2Y01, 2Y02, 2Y03, 2Y04
-
27 March 2011: A2A Adenonsine Receptor with bound agonist: 3QAK
-
30 March 2011: Dobutamin bound beta 1 adrenergic receptor (turkey): 2Y01
-
22 June 2011: Histamine H1 Recepotor bound to doxepin: 3RZE ; A2A Adenosine Receptor with bound agonists Adenosine 2YDO & NECA 2YDV
-
20 July 2011: agonist-occupied β2 adrenergic receptor (active) in complex with the (nucleotide free) Gs heterotrimer: 3SN6
-
17 Feb 2012: sphingosine 1-phosphate receptor 1 (S1P1-T4L) with a bound sphingolipid mimic (antagonist): 3v2W
If there's a new GPCR structure out there and it's not on this list: drop me a line so I can included it here.
Thanks!
Peter
Histamine H1 Receptor
| Structure Notes | PDB | Resolution [A] | Reference |
| H1R with bound drug molecule doxepin | 3RZE | 3.1 | T.Shimamura, M. Shiroishi, S. Weyand, H.Tsujimoto, G. Winter, V. Katritch, R. Abagyan, V. Cherezov, W. Liu, G.W. Han, T. Kobayashi, R.C. Stevens & So Iwata
Structure of the human histamine H1 receptor complex with doxepin
Nature (2011) doi:10.1038/nature10236 |
Sphingosine 1-phosphate Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| sphingosine 1-phosphate receptor 1 (S1P1-T4L) with a bound sphingolipid mimic (antagonist) |
3V2W |
3.35 |
Crystal Structure of a Lipid G Protein–Coupled Receptor
Michael A. Hanson, Christopher B. Roth, Euijung Jo,Mark T. Griffith, Fiona L. Scott, Greg Reinhart, Hans Desale, Bryan Clemons, Stuart M. Cahalan, Stephan C. Schuerer, M. Germana Sanna, Gye Won Han, Peter Kuhn, Hugh Rosen, Raymond C. Stevens
Science Vol. 335 no. 6070 pp. 851-855; 2012 |
Dopamine D3 Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| D(3) dopamine receptor, T4 lysozyme insertion in 3rd intracellular loop, in complex with Eticlopride, crystallized in LCP (Cholesterol additive) |
3PBL |
2.9 A |
No publication yet. PDB authors: Chien, E.Y.T., Liu, W., Han, G.W., Katritch, V., Zhao, Q., Cherezov, V., Stevens, R.C., Accelerated Technologies Center for Gene to 3D Structure (ATCG3D) |
Rhodopsin
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| first experimental GPCR structures |
1F88, 1HZX |
2.8 |
Palczewski K, Kumasaka T, Hori T, Behnke CA, Motoshima H, Fox BA, Le Trong I, Teller DC, Okada T, Stenkamp RE, Yamamoto M, Miyano M. Science. 2000 Aug Crystal structure of rhodopsin: A G protein-coupled receptor. 4;289(5480):739-45.
Teller DC, Okada T, Behnke CA, Palczewski K, Stenkamp RE.
Advances in determination of a high-resolution three-dimensional structure of rhodopsin, a model of G-protein-coupled receptors (GPCRs). Biochemistry. 2001 Jul 3;40(26):7761-72. |
| shows functional water molecules |
1L9H |
2.6 |
Okada T, Fujiyoshi Y, Silow M, Navarro J, Landau EM, Shichida Y.
Functional role of internal water molecules in rhodopsin revealed by X-ray crystallography.
Proc Natl Acad Sci U S A. 2002 Apr 30;99(9):5982-7. Epub 2002 Apr 23. |
| focus on retinal conformation |
1U19 |
2.2 |
Okada T, Sugihara M, Bondar AN, Elstner M, Entel P, Buss V. The retinal conformation and its environment in rhodopsin in light of a new 2.2 A crystal structure. J Mol Biol. 2004 Sep 10;342(2):571-83. |
| Thermostable N2C/D282C mutant heterologously expressed in COS cells |
2J4Y |
3.4 |
Standfuss J, Xie G, Edwards PC, Burghammer M, Oprian DD, Schertler GF. Crystal structure of a thermally stable rhodopsin mutant. J Mol Biol. 2007 Oct 5;372(5):1179-88. Epub 2007 Mar 12. |
| photoactivated and ground state |
2I35, 2I36, 2I37 |
3.8, 4.1, 4.15 |
Salom D, Lodowski DT, Stenkamp RE, Le Trong I, Golczak M, Jastrzebska B, Harris T, Ballesteros JA, Palczewski K. Crystal structure of a photoactivated deprotonated intermediate of rhodopsin. Proc Natl Acad Sci U S A. 2006 Oct 31;103(44):16123-8. Epub 2006 Oct 23 |
| Retinal removed: Opsin |
3CAP |
2.9 |
Park JH, Scheerer P, Hofmann KP, Choe HW, Ernst OP. Nature. 2008 Jul 10;454(7201):183-7. Epub 2008 Jun 18. Crystal structure of the ligand-free G-protein-coupled receptor opsin. |
| activated form of Ops*-GalphaCT peptide complex |
3DQB |
3.2 |
Scheerer P, Park JH, Hildebrand PW, Kim YJ, Krauss N, Choe HW, Hofmann KP, Ernst OP. Crystal structure of opsin in its G-protein-interacting conformation. Nature. 2008 Sep 25;455(7212):497-502. |
| Squid Rhodopsin |
2Z73 |
2.5 |
Murakami M, Kouyama T. Crystal structure of squid rhodopsin. Nature. 2008 May 15;453(7193):363-7. |
CXCR4 Chemokine Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| complex with small molecule antagonist IT1t and cyclic peptide antagonist CVX15, T4 lysozyme insertion in 3rd intracellular loop, stabilizing mutations, crystallized in LCP (Cholesterol additive) |
3ODU, 3OE0, 3OE8, 3OE9, 3OE6 |
2.5 A, 2.9 A, 3.1 A, 3.1 A, 3.2 A |
Wu, B. et al., Structures of the CXCR4 Chemokine GPCR with Small-Molecule and Cyclic Peptide Antagonists. Science 7 Oct 2010 |
β1 Adrenergic Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| Dobutamin bound beta 1 adrenergic receptor (turkey) |
2Y01 |
2.6 |
Warne, A. et al. (2011) TURKEY BETA1 ADRENERGIC RECEPTOR WITH STABILISING MUTATIONS AND BOUND PARTIAL AGONIST DOBUTAMINE (CRYSTAL DOB102) Nature 469: 241 |
| Thermostabilized turkey receptor |
2VT4 |
2.7 |
Warne T. et al., (2008) Structure of a beta1-adrenergic G-protein-coupled receptor
Nature 454, 486-491 |
Adenosine Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| Bound antagonist ZM241385 |
3EML |
2.6 |
Jaakola VP, Griffith MT, Hanson MA, Cherezov V, Chien EY, Lane JR, Ijzerman AP, Stevens RC. The 2.6 angstrom crystal structure of a human A2A adenosine receptor bound to an antagonist. Science. 2008 Nov 21;322(5905):1211-7. Epub 2008 Oct 2. |
| Bound agonist UK-432097 |
3QAK |
2.7 |
Structure of an Agonist-Bound Human A2A Adenosine Receptor
Xu, F., Wu, H., Katritch, V., Han, G.W., Jacobson, K.A., Gao, Z-D., Cherezov, V., Stevens, R.C.
Science DOI: 10.1126/science.1202793 |
| Bound to agonists adenosine and NECA |
2YDO 2YDV |
3.0 & 2.6 |
G. Lebon, T. Warne, P. C. Edwards, K. Bennett, C. J. Langmead, A. G. W. Leslie & C. G. Tate
Agonist-bound adenosine A(2A) receptor structures reveal common features of GPCR activation
Nature 474, 521–525 (23 June 2011) |
| A2A adrenergic receptor bound to Fab2839 |
3VG9
3VGA |
3.1 & 2.7 |
Hino, T., Arakawa, T., Iwanari, H., Yurugi-Kobayashi, T., Ikeda-Suno, C., Nakada-Nakura, Y., Kusano-Arai, O., Weyand, S., Shimamura, T., Nomura, N., Cameron, A., Kobayashi, T., Hamakubo, T., Iwata, S., & Murata, T. (2012). G-protein-coupled receptor inactivation by an allosteric inverse-agonist antibody |
β2 Adrenergic Receptor
| Structure Notes |
PDB |
Resolution [A] |
Reference |
| b2AR365-Fab5 complex |
2R4S, 2R4R |
3.4 / 3.4 |
Rasmussen SG, Choi HJ, Rosenbaum DM, Kobilka TS, Thian FS, Edwards PC, Burghammer M, Ratnala VR, Sanishvili R, Fischetti RF, Schertler GF, Weis WI, & Kobilka BK (2007). Crystal structure of the human β2 adrenergic G-protein-coupled receptor. Nature 450:383-387. |
| Complex with Carazolol ligand and bound Cholesterol; T4 lysozyme fusion in 3rd intracellular loop |
2RH1 |
2.4 |
Cherezov et al. (2007). High-resolution crystal structure of an engineered human β2-adrenergic G protein-coupled receptor. Science 318:1258-1265 |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol |
3D4S |
2.8 |
Hanson et al., (2008)
A specific cholesterol binding site is established by the 2.8 A structure of the human beta2-adrenergic receptor. Structure 16: 897-905 |
| methylated receptor |
3KJ6 |
3.4 |
Bokoch et al., (2010) Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor.
(2010) Nature 463: 108-112 |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol, mutations: E122W, N187E, C1054T, C1097A; inverse agonist ICI 118,551 |
3NY8 |
2.84 |
To be Published: Crystal structure of the human beta2 adrenergic receptor in complex with the inverse agonist ICI 118,551 |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol, mutations: E122W, N187E, C1054T, C1097A; Timolol |
3NY9 |
2.48 |
To be published: Crystal structure of the human beta2 adrenergic receptor in complex with a novel inverse agonist |
| T4 lysozyme fusion in 3rd intracellular loop, bound cholesterol, mutations: E122W, N187E, C1054T, C1097A; antagonist alprenolol |
3NYA |
3.16 |
Conserved binding mode of human beta2 adrenergic receptor inverse agonists and antagonist revealed by X-ray crystallography |
| agonist-occupied β2 adrenergic receptor (active) in complex with the (nucleotide free) Gs heterotrimer |
3SN6 |
3.2 |
Rasmussen et al.
Crystal structure of the β2 adrenergic receptor–Gs protein complex
Nature (2011) |
Filed under:
Membrane Proteins, Crystallization, Lists, Structure | Posted by: Peter Nollert | Comments: 10
June 28, 2010
With the first medically relevant GPCR structures published about 2 years ago, we can now see, for a few select examples, the 'inner workings' of GPCRs in great detail. The key to all of this was of course sample preparation, and that is: production of GPCR samples in sufficient quantity and quality and then their crystallization into well-ordered protein crystals. The fact that we can visualize GPCR molecules on an atom scale is due to identifying appropriate crystallization conditions for the respective target. Below is a compilation with the described crystallization conditions.
More to come ;)
Peter
|
GPCR Target and Crystallization Technique |
Protein Sample |
Precipitation Reagent |
TimeTemp. |
PDB, Resolution, Ref. |
|
human β2 adrenergicreceptor, b2AR365-Fab5 complex crystallized in DMPC/CHAPSO bicelles; hanging drop vapour diffusion |
8-12 mg/ml of b2AR-Fab5 complex; concentrated to 60 mg/ml in 10 mM HEPES pH 7.5, 100 mM NaCl, 0.1 % Dodecylmaltoside, 10 uM carazolol |
Ammonium Sulphate, sodium acetate and EDTA, pH 6.5-7.5 |
7-10 days |
2R4S 3.4A REF |
|
human β2 adrenergicreceptor complex with Carazolol ligand and bound Cholesterol crystallized within Lipidic Cubic Phase; receptor with T4 lysozyme fusion in 3rd intracellular loop |
In Monoolein-based lipidic cubic phase, doped with ca. 8-10% Cholesterol; portions of 25-50 nL of protein-laden LCP were overlaid with 0.8uL of precipitant solution |
30-35%(v/v) PEG400, 100-200 mM NaSulfate, 100mM Bis-tris propane pH 6.5-7.0, 5-7%(v/v) 1,4-butanediol |
21-23 C |
2RH1 2.4A REF |
|
human β2 adrenergicreceptor receptor with T4 lysozyme fusion in 3rd intracellular loop; Lipidic cubic phase based crystallization; receptor bound to Cholesterol and Timolol |
Crystals grown in25 nL or 50 nL portions of a 10% w/w cholesterol spiked monoolein-based lipidic cubic phase. Receptor concentrated to 30 mg/mL. |
0.8 uL overlaying solution: 28% w/v PEG 400, 300 mM K Formate, 100 mM Bis-tris propane pH 7.0 and 2 mM timolol |
|
3D4S 2.8A REF |
|
Turkey β1 Adrenergic Receptor containing stabilizing mutations and bound Cyanopindolol crystallized by traditional vapor diffusion from micellar solution |
Protein concentration 6 mg/ml |
10 mM Tris-HCL, pH 7.7; 50 mM NaCl, 0.1 mM EDTA, 0.35% Octylthiglucoside, 0.5 mM Cyanopindolol against reservoir filled with 100 mM ADA pH 6.9-7.3; 29-32% PEG600 |
|
2VT4 2.7A REF |
|
Human A2a Adenonsine Receptor with T4lysozyme in 3rd intracellular loop, Lipidic cubic phase based crystallization |
Lipidic matrix consisting of Monoolein and Cholesterol; receptor concentrated to 70 mg/ml; In Monoolein-based lipidic cubic phase, doped with ca. 12% Cholesterol; portions of 50 nL of protein-laden LCP were overlaid with 0.8uL of precipitant solution |
30% (v/v) PEG 400 (range of 28-32%), 186 mM Lithium sulfate (range of 180 to 220 mM), 100 mM Sodium citrate (pH 6.5) (Range of 5.5 to 6.5) and 200 μM ZM241385 |
21-23 C |
3EML 2.6A Science, Science supp. |
|
Methylated beta 2 Adrenergic Receptor-Fab complex |
methylated receptor |
|
|
3KJ6 3.4A REF |
Filed under:
Membrane Proteins, Crystallization | Posted by: Peter Nollert | Comments: 0