Lipidic Cubic Phases: Frequently Asked Questions

        Questions:

1. I have a membrane protein sample that I'd like to crystallize in lipidic cubic phases. What's a simple way to get started?
2. What's a good protein concentration to start crystallization in lipidic cubic phases?
3. Which detergents work and what detergent concentration should I use?
4. Is there a size limit of proteins that can be crystallizaed in lipidic cubic phases?
5. Which papers should I read to get started on this topic of membrane protein crystallization in lipidic cubic phases?
6. Which membrane proteins have yielded crystals using the Lipidic Cubic Phase methodology?

7. I'd like to crystallize bacteriorhodopsin - how do I do it? 
   
   Answers: 

1. I have a membrane protein sample that I'd like to crystallize in lipidic cubic phases. What's a simple way to get started?

There are several papers that have been written on this subject. These are a good start. Emerald BioSystems offers a Cubic^TM LCP Kit that has all components (lipid, syringes, mixer, dispenser, Cubic screen and a manual) to set up ca. 1,000 microcrystallization experiments. Order here, and download  LCP Kit Instructions.

2. What's a good protein concentration to start crystallization in LCP?

As with any crystallization, the goal is reaching supersaturation, therefore, the higher the better. As a rule of thump I'd say 10 mg/ml is a good start. Some crystallizations work at higher, some at lower concentration. If you start out at a high concentration and you get a lot of small crystals, reducing the membrane protein crystallization may be a good way to optimize. If you're not getting any crystals at all, increasing the protein crystallization may be the key.

3. Which detergents work and what detergent concentration should I use?

There's no real good answer to this question. I'd recommend to just go for it and not worry too much about this questions. The lipidic cubic phase may not form if the detergent concentration is too high. See for instance this paper about Dodecylmaltoside and monoolein. I'd say: so what? There are a lot of membraneous structures around to aid the crystallization process.

4. Is there a size limit of proteins that can be crystallized in lipidic cubic phases?

Size does not seem to matter - see this paper. Small or large hydrophilic domains, seven or 13 transmembrane helices, helices or beta barrels - I don't see a pattern. However, based on theoretical considerations (see here) membrane proteins with fewer than four transmembrane helices may not crystallize in lipidic cubic phases.

5. Which papers should I read to get started on this topic of membrane protein crystallization in lipidic cubic phases?

List of relevant papers:

The landmark paper with the first description of the crystallization of a membrane protein in lipidic cubic phases, ever:

Landau E., M. & Rosenbusch, J. P. (1996)
Lipidic cubic phases: A novel concept for the crystallization of membrane proteins
PNAS, 93(25), 14532-14535

The title says it all. A method is described that allows the preparation of ca. 200 nanoliter LCP-based membrane protein crystallization experiments.

Nollert, P. (2002)
From test tube to plate: a simple procedure for the rapid preparation of microcrystallization experiments using the cubic phase method.
J.Appl.Cryst., 35, 637-640

A proposal for a mechanism of membrane protein crystallization is described in:

Nollert, P., Qiu, H., Caffrey, M., Rosenbusch, J., Landau, E. (2001)
Molecular mechanism for the crystallization of bacteriorhodopsin in lipidic cubic phases
FEBS Letters, 504(3), 179-186

A quantitative biophysical model for the crystallization mechanism is described in:

Grabe, M., Neu, J., Oster, G., Nollert, P. (2003)
Protein Interactions and Membrane Geometry
Biophys. J., 84, 854-868

Lyotropic and thermotropic phase diagram of Monoolein:

Qiu, H. and Caffrey, M. (2000)
The phase diagram of the monoolein/water system: metastability and equilibrium aspects
Biomaterials 21, 223-234

Compatibility of lipidic cubic phases with detergents:

Misquitta, Y., Caffrey, M. (2003)
Detergents Destabilize the Cubic Phase of Monoolein: Implications for Membrane Protein Crystallization
Biophys.J., 85, 3084-3096

6. Which membrane proteins have yielded crystals using the Lipidic Cubic Phase methodology?

Here's a list of X-ray crystallographic structures obtained for a variety of different membrane proteins, the crystals of which were grown within monoolein containing lipid phases.

Representative samples of the high(est) resolution structures deposited in the PDB are listed below for such crystal structures:

Bacteriorhodopsin: 1C3W 1.55Å

Sensory Rhodopsin II: 1H68 2.1Å

Halorhodopsin: 2JAF 1.8Å

SRII transducer complex: 1H2S 1.93Å

Photosynthetic reaction centre RC sph: 1OGV 2.35 Å

beta-2-Adrenergic Receptor-T4L: 2RH1 2.2 Å

OpcA Adhesin from N. meningitidis (beta-barrel): 2VDF 1 95 Å

BtuB cobalamin transporter beta-barrel: 2GUF 1.95 Å 

LH2 from Rps. acidophila: 2FKW 2.45 Å

7. I'd like to crystallize bacteriorhodopsin - how do I do it? 

It is possible to crystallize bacteriorhodopsin in LCP starting from Purple Membrane. At first the membrane protein is solubilized in Octylglucoside then passed over a sizing column and concentrated. The recipe for solubilization and crystallization can be found here:

Nollert, P.
Lipidic cubic phases as matrices for membrane protein crystallization.
Methods. 2004; 34(3):348-353

Send an email if you know of a new PDB deposited membrane protein structure that was determined using crystals grown within lipidic cubic phases. Thanks!