by Peter Nollert
March 17, 2012 06:50
In a protein crystallization laboratory you typically see a lot of stock solutions on the shelf. These are used to create optimization screens to improve the quality of protein crystals. Grid-screening is a tried-and-proven way to identify better crystal growth conditions. How many do you really need?
Depends - of course. Generally, the number and type of stock solutions that you should maintain in the wet lab is directly correlated to the type of primary protein crystallization screens that are typically applied used. For instance if all your first pass crystallizations are carried out with JCSG+, it would make sense to have the 84 stock solutions on the shelf, ready to be dispensed into a protein crystallization tray. From my own experience I can tell that if these stock solutions are not handy, researchers tend to use shortcut. No Tricine buffer on the shelf? - what the heck, let's go with Tris. This may work for some crystallizations, but you're out of luck if the buffer molecule is required for providing crystal contacts. The issue is that taking such shortcuts has the potential to derail your entire structure determination project.
Clearly, having these stock solutions on the shelf improves the speed and success rate of crystallographic protein structure determination. Have you ever counted and made a list with the stock solutions that you should have handy? If not, the list below may be a good starting point for you. I'm listing number of different stock solutions that go into the production of protein crystallization screens from Hampton Research, Jena BioScience, Fluidigm, Molecular Dimensions, Qiagen, and of course from Emerald Bio.
Supplier, name and the associated number of stock solutions that are required for the production and optimization of protein crystallization hits. How this data was generated: Here at Emerald Bio we produce a lot of sparse matrix screens and we accomplish this with our fleet of Matrix Maker instruments that are instructed from a database of screen definitions. Since we keep track of many crystallization screens we can identify the number of stock solutions that are used in a number of commercial protein crystallization screens.
In average there are 40 different stocks (+- 22) that are required for these protein crystallization screens.
That's a lot of stock solutions.
by Peter Nollert
May 17, 2011 04:44
Once you've got a hit with your membrane protein crystallization trial, your life may get really exciting. After confirming that the hit is actually a protein crystal and obtaining even the weakest X-ray diffraction patterns, you're getting into the optimization game. One of the obvious parameter to optimize is the crystallization cocktail. How can that be done without wasting your precious protein sample on unproductive crystallization conditions?
Designing and making grid-screens around a particular hit condition is complicated due to the presence of detergent in the crystallization experiment. An established approach is to be close to the detergent phase separation boundary while avoiding the 'heartland' of phase separation. How can this be done practically? You could check out the various papers on this topic and spend a lot of time searching and possibly finding hints on formulations that are relevant to your hit. You could also search the phase boundary conditions in a pre-screen type of experiment (a la Song & Gouaux: Membrane protein crystallization: application of sparse-matrices to the alpha-hemolysin heptamer Methods in Enzymology(1997) (60-73)). The quickest way though to obtain guidance on optimization screen design is through mining of existing detergent phase boundary data. Fortunately Mary Koszelak-Rosenblum et al. from HWI have made such data mining a quick and simple experience:
Koszelak-Rosenblum M, Krol A, Mozumdar N, Wunsch K, Ferin A, Cook E, Veatch CK, Nagel R, Luft JR, Detitta GT, & Malkowski MG (2009). Determination and application of empirically derived detergent phase boundaries to effectively crystallize membrane proteins. Protein science : a publication of the Protein Society, 18 (9), 1828-39 PMID: 19554626
The data comes in an Excel spreadsheet called SLICKSPOT. This tool is available for download here.
The input parameters for this nifty tool are type and concentrations of:
- Detergent (data is available for C10M, C12M, b-HG, b-OG, b-NG, CHAPS, LDAO, C12E8, C8E4, C8E5, FC-12),
- crowding agent PEG (data is available for: PEG400, PEG1000, PEG2000, PEG2000MME, PEG3350, PEG 4000, PEG5000ME, PEG6000, PEG8000, PEG20000)
- Salt (data is available for CaCl2, KCl, LiCl, Li2So4, MgCl2, Na2C3H2O4, NaCl, NaH2PO4, (NH4)2SO4, NH4H2PO4, (NH4)2HPO4)
So, let's say the membrane protein crystal hit was produced at room temperature with a formulation that contained 1% Octylglucoside, Ammonium sulfate and PEG4K. For these conditions Slickspot produces the following output as a chart:
Example of a Slickspot output. How to read this customized chart: Salt and PEG concentrations below the blue curve form a single phase, those above are phase separated. Sticking to conditions around the blue line is preferred.
So, what's to do from here?
Design your customized formulation screen with Ammonium sulfate and PEG4000 conditions near to the blue line.
Pretty slick, hm?
Peter
PS: I just realized that Slickspot fails to update the legend when additional conditions are queried; a cosmetics-only bug I hope.
by Peter Nollert
May 6, 2011 09:39
This week I'll prepare my first Iodide soak ever. While we've determined many protein structures at Emerald BioStructures using anomalous data from iodide soaked crystals (see a previous blog post on this topic), I've never prepared iodide-soaked crystals myself. Being a novice I asked Tom Edwards who is an expert in this methodology. Turns out that he's preparing a webinar on this topic for next week: SAD phasing at rotating anode wavelengths using iodide ions. The goal of course is to obtain phases from anomalous X-ray diffraction data, the key to de-novo crystallographic structure determination.
Tom Edwards on "SAD phasing at rotating anode wavelengths using iodide ions"
I asked Tom for his 'standard iodide crystal soaking recipe'. Here is it:
1. prepare a 5 M Sodium Iodide stock and a formulation at 2 x of the crystallization cocktail.
2. Mix iodide to a final concentration of 1 M with the 2 x crystallization cocktail and include the cryo reagent.
3. Transfer a single crystal into 1 uL of 1 M soak and check for crystal damage. If there's no visible damage, test X-ray diffraction. Back down with the iodide concentration (0.75 M, 0.5 M etc.) if the quality of X-ray diffraction pattern suffers (mosaicity, resolution, split spots etc.).
4. Harvest, cool, mount, diffract, collect...
The fun part - such as data treatment - will be covered in Tom's webinar
I'm off to the lab.
Peter
by Peter Nollert
April 9, 2011 04:13
The proteins residing in the outer membrane of gram-negative bacteria all have beta barrel structure. The crystallization of these porins and other beta transmembrane barrel structured proteins is aided by the fact that the protein chain can often be over expressed in substantial quantities in E.coli (as inclusion bodies) and the protein can be readily extracted in urea, purified and refolded into detergent micelles. The crystallization cocktails that have produced beta barrel membrane protein crystals are sufficiently different from those of those that have worked for alpha helical transmembrane proteins that beta barrel membrane proteins warrant their own crystallization screen. Two of such crystallization screens have been recently described:
Beta-Mem™, designed by Mikiko Tanabe and Tina Iverson from Vanderbilt University is composed of 96 formulations that are based on the analysis of previously described crystallizations of beta barrel membrane proteins:
Tanabe, M., & Iverson, T. M. (2009) A Practical Guide to X-Ray Crystallography of beta-barrel Membrane Proteins: Expression, Purification, Detergent Selection, and Crystallization. Membrane Protein Crystallization, 63, 229-267.
By the way, no need to prepare this screen yourself by hand - Beta Mem is available through Emerald Biosystems.
More than boring holes: beta barrel membrane proteins
And there's a second screen, designed primarily by So Iwata's group. it is named MemPlus™ and has 48 formulations that are based on a similar rationale: 'what has worked for many similar proteins should work for my own target as well'.
Crystallization of beta barrel membrane proteins:
Newstead, S., Hobbs, J., Jordan, D., Carpenter, E., & Iwata, S. (2008). Insights into outer membrane protein crystallization Molecular Membrane Biology, 25 (8), 631-638 DOI: 10.1080/09687680802526574
Unfortunately you can't do your own sorting with the published data since the advertised supplemental file 'beta-MP-database.xls' is not available (was not available at the time of writing this post). This file gives a nice overview, though.
All the best,
Peter
by Peter Nollert
March 28, 2011 09:49
Earlier this month I received an email from John Wiley & Sons, confirming that 'The supplement file is now available at http://onlinelibrary.wiley.com/doi/10.1110/ps.073263108/suppinfo'. More than half a year ago I had pointed out that the 'supplementary material' to a paper was not available online. Now it is. Took a while, but THANKS A LOT for following through, John Wiley & Sons!
When you go to this page, you can download Simon Newstead's alpha-MP-database.xls.
Although somewhat outdated, this spreadsheet contains a concise summary of crystallization conditions from 121 alpha helical membrane protein structures that are listed in the PDB. The extensive analysis of crystallization conditions is described in the accompanying paper:
Newstead S, Ferrandon S, & Iwata S (2008).
Rationalizing alpha-helical membrane protein crystallization.
Protein science, 17 (3), 466-72
PMID: 18218713
While this accounting exercise is not the sexiest of all science, it is of great interest to all of us membrane protein crystallizers . The database is a handy tool that can be consulted when only small quantities of membrane protein sample is available and one is forced to focus on 'what's worked in the past'. And of course, this data summary is incredibly for the design of new (although highly biased) crystallization matrices. This is exactly what Simon has done: based on his own analysis, a new sparse matrix crystallization screen, called MemGold is described.
Great work!
Peter