Protein Crystallization Hits

Many X-ray crystallographers have told me that images of protein structure have contributed in their decision to become structural biologists. For me, it was a David Goodsells 1992 book "The Machinery of Life"  that showed protein structures in the cellular context (here's some of Goodsell's current work). Such images give a tangible impression that the inner workings of cells and show that protein molecules are akin to engineered machines with precisely developed mechanics. 

I was happy to see that there are scientists/artists following this tradition of creating such fascinating imagery.  But rather than drawing cartoons of protein particles (based on experimentally derived structures, mind you) Sean R. McGuffee and Adrian Elcock  have set up brownian dynamics simulations of a cytoplasm consisting of 50 proteins and, and this is the crucial control, reproduce experimental parameters.

This is a snapshot of their model of macromolecules in the cytoplasm:

As real as it gets in a computer: a fascinating image of a computational simulation of macromolecules in the cytoplasm. Taken from: http://www.flickr.com/photos/dullhunk/4464549449/sizes/o/in/photostream/

McGuffee, S. R. and A. H. Elcock 

Diffusion, crowding & protein stability in a dynamic molecular model of the bacterial cytoplasm.

PLoS Computational Biology 6 (3), e1000694+ (2010)

dx.doi.org/10.1371/journal.pcbi.1000694

Enjoy,

Peter

It's getting time again to do some spring-cleaning in the protein crystallization lab.

A well-organized lab is more productive, is fun to work in, produces better results and costs less money. That's why I'd recommend spring cleaning to everyone running a protein crystallization lab. Depending on how your particular lab is set up you may want to do some or all of the following:

1. Toss out all old crystallization plates for those crystallization projects that have been completed to make room for new crystallization trials.
2. Replace all opened crystallization reagent containers ('crystallization screens'). Using evaporated formulations can produce more annoying salt crystals and fewer crystallization hits due to lower organic solvent concentration; contaminated formulations proteolyze your protein solution and light sensitive reagents such as MES or PEG can react with protein molecules, rendering them uncrystallizable. And of course: replace these crystallization screens with new Emerald Bio Wizard kits! and stock solutions. Buying in bundles makes sense! Ask us for your reagent kit combination or stock solution libraries. 
3. Clean working surfaces to minimize contamination of crystallization experiments with dust.
4. Organize: Put tools, such as pipettors, tip boxes, pins, wands, tongs, razor blades and fresh crystallization screens in labeled boxes or on shelves. You may be surprised by the amount of work space you can gain.
5. Check your tools, and if necessary earmark them for cleaning or calibration : microscopes, robotic instrumentation, liquid dispensing instruments, dewars.
6. Computer: Archive old files, check backup routine and clean up desktops (Disk Cleanup)
7. Safety-check the protein crystallization work area: do you have appropriate gloves, lab goggles, coats, clean-up kits, containers and cabinets available? Lab safety matters!  
8. Pick up the trash. Empty out disposables containers for sharps, various liquids (heavy metal, organics, halides, hazardous liquids) and plastics. 

So, show some of your inner Martha Stewart and spring-clean your protein crystallization lab!

Peter

P.S. Now that I think of it- maybe you want to keep some of these old formulations for use as a 'last resort'. These moldy, evaporated and reactive formulations may be of use in some special cases. More about that at another time.

Darren Begley recently put up an interesting post about "Grooming your fragment library: Checking the quality of commercially-sourced fragments". 

The point he's making is that tight QC measures are key to the success of fragment screening: 

"These results highlight the importance of implementing tight quality control measures for preliminary vetting of commercially-sourced materials, as well as maintaining and curating a fragment screening library. It also puts forth a statistical likelihood of around 10-15% failure, regardless of vendor. Most importantly, we have seen our methods reduce risks while accelerating drug discovery. "

This is certainly true for NMR-based experiments and of course also applies to crystallography-based fragment screening.

I was glad to see Derek Lowe pick up the discussion in this blog post: Not What It Says On the Label, Though

Several of the comments to this post support Darren's findings and drive this message home:  Buyer beware!

Peter

Genetic Engineering & Biotechnology News has a separate Expert Tips section. If you are looking for start-up advice for growing membrane protein crystals, you may want to have a look at this recent post:

8 Often-Overlooked Tips for Membrane Protein Crystallization

Enjoy,

Peter

One of our scientists - Tim Craig - has turned his attention to a fundamental barrier that needs to be overcome in any membrane protein research project: functional extraction of membrane proteins from their biological membrane.  Every membrane protein target needs to be matched with a particular detergent reagent or a set of detergents. Since Tim was working with fluorescently labeled membrane proteins, he applied an experimental approach that has gained popularity in recent years: FSEC. That is: Fluorescence-Detection Size-Exclusion Chromatography; this is primarily a pre-crystallization method for membrane proteins that has been developed in Eric Gouaux' lab (see references below). 

This is how it works: solubilize a membrane with a particular detergent and apply the resulting sample to size exclusion chromatography with a benign detergent (i.e. dodecyl maltoside) in the mobile phase. Repeat with as many detergents as possible. Inspect the traces for symmetry of the (largest) peak and absence of signal in the void volume.  Pick the best detergent(s) to establish a purification and crystallization procedure. The neat trick here is that such crystallization-relevant information can be obtained at a very early stage in your membrane protein project, for instance once you can prepare membranes. This methodology solves the hen-and-egg problem (need to have a detergent in order to be able to purify a particular membrane protein, need to have a pure membrane protein sample in order to test which detergent is compatible) and is simple to automate with standard HPLC instrumentation.   

Since preparing many different detergents is a laborious process we thought we'd pack them into a kit. And this is it, the Wizard: TIME screen Use the Wizard: TIME screen to conveniently determine the detergent that successfully extracts your target membrane protein from a membrane preparation.

In order to maximize both, extraction efficiency and membrane protein stabilization, each of the 84 different detergent reagent is formulated at 2% (w/v) together with the co-detergent cholesterolhemisuccinate. Positive and negative controls specifically for FSEC make this screen a very convenient tool for fast, simple and information-rich membrane protein extraction assessment. 

The complete listing of detergents in the Wizard: TIME screen are available here.

Our recommended Wizard: TIME screen sample treatment protocol:

1. Mix 125μL of your membrane preparation with the contents of each well in a fresh 96-well plate (membranes should be prepared at a concentration that allows for good signal:noise in the analytical assay of your choice). 

2. Incubate the mixture to allow for protein extraction (different incubation times and temperatures will influence extraction efficiency, however typically 1 hour at 4°C can serve as a good starting point for extraction optimization). 

3. Separate extracted material from debris by ultracentrifugation at >100,000 x g for 20 minutes using a small volume rotor or by filtration using a filter. Keep either the ultracentrifugation supernatant or the flitration flow through and

4. Analyze samples with an analytical fluorescence detection SEC-HPLC.  

Identification of suitable detergent for membrane protein detergent extraction with the Wizard: TIME screen using FSEC. A) LDAO produces large, in-homogenous membrane protein particles, a sizeable fraction of which running in the ‘void’ volume. B) CYGLU-4  extracted membrane protein runs mainly as a symmetric single peak. 

 Here is a link to order info for the Wizard: TIME screen. More details on the FSEC protocols are described here: 

Hattori M, Hibbs RE, & Gouaux E (2012). A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening. Structure (London, England : 1993), 20 (8), 1293-9 PMID: 22884106

 KAWATE, T., & GOUAUX, E. (2006). Fluorescence-Detection Size-Exclusion Chromatography for Precrystallization Screening of Integral Membrane Proteins Structure, 14 (4), 673-681 DOI: 10.1016/j.str.2006.01.013 

Final tip for dealing with detergent-solubilized membrane protein samples: no bubbles!

Peter