Tips and Tricks in Crystallography - Crystallization Xinhua Ji: It was recently reported that microseeding was used to produce untwinned crystals of an O-demethylase using twinned crystals as seeds (Acta Crystal. F72:897-902, 2016). Microseeding separates nucleation events from crystal growth events so that it can yield different crystal forms. Hence, it is promising that this technique can be used to produce untwinned crystals from twinned seeds. Alternative Reservoirs in Vapor Diffusion Experiments At the 45th Mid-Atlantic Protein Crystallography Meeting, Dr. Ivan Shabalin from Professor Wladek Minor's laboratory at the University of Virginia shared various tricks in crystallization with the audience, including alterntive reservoirs in vapor diffusion experiments. Wondering what it is? Follow this link for an introduction. The Cross-Linking Effect of PEG Michael Garavito: One of the unfortunate by-products of keeping PEG stock solutions in water is that they will form peroxides and aldehydes. They will slowly cross-link the surface of some crystals. However, it is dependent on the nature of your protein's composition of surface residues, so not every protein cyrstal does this. I had one case where PEG4000 grown crystals would be resistant to dissolving and would easily bend; the thinner rods would spring back staight. After placing the crystals into buffer known to dissolve them, I poked the crystals hard and the insides squeezed out like toothpaste, leaving an empty sack behind. the bottom-line is that fresh crystals diffracted better than old crystals because of this cross-linking. Suggestions: (1) Make your PEG stocks up fresh or store them in the freezer as aliquots; (2) Remove oxidized PEGs from your stocks (See Ray et al. Biochemistry, 30, 6866-6875, 1991 and Jurnak, J. Cryst. Growth, 76, 577-582, 1986); (3) Check to see if freshly grown crystals behave better. Seeded Screening with a Robot Artem Evdokimov: By
popular request here's
my favorite version of the in-screen seeding. We use a Mosquito but it
doesn't
have to be a specific robot as long as it can dispense relatively
tiny
volumes of seed stock. artem.evdokimov@GMAIL.COM Caveats: (1) If
I am desperate enough to do this, then the situation is pretty bad
indeed
and I don't mind wasting some protein; (2) my success rate is
not
hugely favorable but this does work on occasion when other things have
failed. (1) Identify
a few likely conditions. Ideally they have microcrystals but
desperation has
made me try 'lovable precipitates' in the past, with a modest degree of
success. (2) Harvest
entire drop using a few ul of mother liquor as diluent (3) Break the
existing crystals using your favorite method (sead beed, etc.) mine
involves
swirling a pipette tip in the mixture, running it along the
walls, with
rapid pipetting up and down. Dilute seed stock to useful volume (enough
for
screening). (4) I do not
normally centrifuge the resulting seed stock, but some people do (5) Dispense
your screen as always with the usual protein/reservoir ratio. Let's say
you
like drops of the 0.2ul+0.2ul variety - add 25 nanoliters of the seed
stock
*last*. Optional mixing of the condition is a fun thing to try but it
seems not
to matter very much. Note that I typically use the same tips to
dispense seed
stock, fully aware that this causes cross-contamination of conditions.
I don't
mind :) (5a) Variation
- Add seed stock to protein, then dispense ASAP. Surprisingly not a bad
option,
practically speaking. (5b) Variation
- Crosslink seed stock very gently in solution (with trace of
glutaraldehyde)
before use. Buffers/additives with primary or secondary amine groups do
interfere, of course. (5c) Variation
- Mix seeds from SEVERAL initial hit conditions, then use as one seed
stock. Be
ready for fireworks as they may not be compatible! (6) Endure
nail-biting wait for results :) As noted
earlier, it's not a sure-fire way to get new hit conditions but it does
seem to
work and it's a fun way to put to use a remainder of
otherwise
useless protein (when you've tried all other tricks you like to try). Ligand Concentration Herman
Schreuder: If you
do the
calculations, you will find that you need a FREE ligand concentration
of >10
* Kd to get >90% occupancy of the binding site. Seaver: Crystallization Resources - P212121 Hubbard: Crystal Screen Optimization Server It is a GUI written by P Hubbard to calculate 2D gradients for crystal screen optimization (much like a web 2.0 version of Hampton’s “Make Tray” utility). It gives a handy print out of recipes and records for people who make their optimization trays by hand. CCP4
wiki: Crystallization of Protein-DNA Complexes
Annie Hassell: Hapton Research Crystal Screens 1
& 2 as Addititives They generally use 5% of the sparse matrix screen added to the well. When they get a hit, they further optimize the concentration of the reagent to add to the well. Drs. Jichun Ma
and Di Xia
(NCI): The Use of Blue-Native PAGE in the Evaluation of Membrane
Protein Aggregation States for Crystallization Dr.
Susan Buchanan (NIDDK): Crystallization of Integral Membrane
Proteins Editor:
Tantalum
Cluster [(Ta6Br12)2+] Derivatization
Kit - Available Wei Yang
(NIDDK): Crystallization
of Protein-DNA Complexes (updated 2007) Editorial: The Silver Bullets: At the ACA 2006, Bob Cudney (Hampton Research) and Alexander McPherson (University of California Irvine) presented an alternative stretage for crystallizing macromolecules, as they put it, by searching the silver bullets. Examples of the silver bullets incude hexammine cobalt (III) chloride, 1,3-propanediol, sebacic acid, 4-aminobezonic acid, terephthalic acid, arginine, pentaglycine, glycerol 2-phosphate, trans-aconitic acid, trimesic acid, and putrescine. As you may realize, they are in fact additives. They tested 120 additives in the crystallization experiment of 81 proteins using two fundamental conditions: (1) 30% w/v PEG 3350, 0.1 M HEPES pH 7.0; and (2) 50% TacsimateTM pH 7.0. The succesful rate was very impressive: 65 out of 81 (85%) proteins crystallized. Most significant was that 35 of the 65 (54%) crystallized only in the presence of one or more reagent mixes, but not in control samples lacking any additives! As crystallographers zest for
larger and
larger molecular machinery (spoiled public is part to blame), what used
to be a novelty: crystallization of protein-protein complexes, has
indeed become a way of life for most of us. Like everyone else, our lab
often struggles to obtain that elusive crystal of so-and-so complex.
Over the years, it became clear to us that protein complexes often
favored certain conditions of crystallizations. Although they were
not as well defined as DNA-protein complex crystallizations, these
conditions appeared more narrowly distributed than those for general
soluble proteins. This lead us to conduct a survey on
protein-protein complex crystallizations a few years ago, which
resulted in a 48-condition sparse matrix screening kit. More recently,
we revisited the survey using a much larger database of published
structures and expanded the initial 48-condition to a 96-condition
sparse matrix kit (Radaev,
Li, and Sun, Acta Cryst. D62:605-612).
Recommended Readings: Rational Protein Crystallization by Mutational Surface Engineering; Anomalous-scatterer-mediated crystal-packing interactions; Strategies in making cross-linked enzyme crystals. Dr. David Waugh (NCI): When an otherwise well-behaved protein fails to crystallize, what do you (suggest to) do? It is not uncommon for proteins to have disordered termini, which may impede the formation of crystals. Therefore, when an otherwise well-behaved protein fails to crystallize, the first thing we do is subject it to limited proteolysis with thermolysin. We prefer thermolysin because its major specificity determinant is a hydrophobic residue in the P1’ position. Hydrophobic residues occur much less frequently than arginine and lysine in solvent-exposed loops of proteins. Consequently, thermolysin is less likely than trypsin or chymotrypsin to yield misleading results. We have also found that in general, digestion patterns generated by thermolysin are cleaner, which simplifies the identification of the digestion products. Metastable digestion products can be identified by mass spectrometry and N-terminal amino acid sequencing, and then new vectors can be constructed to overproduce the truncated polypeptides. Secondary structure prediction and sequence alignments can sometimes used to make educated guesses about the locations of domain boundaries when the proteolysis approach fails to yield definitive results. We also recommend trying reductive alkylation with formaldehyde and dimethylamine-borane complex (Rayment, I. Methods Enzymol. 276, 171-179 [1997]). The net result of this reaction is the dimethylation of all accessible lysine side chains (and the N-terminal amino group). Although this does not change the intrinsic charge of a protein, it may alter its isoelectric point slightly. One rational behind this strategy is that dimethylation of lysine side chains will reduce their interaction with solvent, thereby causing them to adopt more “ordered” conformations that may facilitate crystallization. Reductive methylation also frequently reduces the solubility of proteins, and so it may be a good approach to try when mostly clear drops are obtained from crystallization screens even with very concentrated solutions of protein. The nice thing about this approach is that it can be performed on the existing sample of protein (i.e., no new constructs need to be made). Surface entropy reduction mutagenesis, a strategy pioneered by Zygmunt Derewenda and coworkers, is another option. In this method, linear clusters of amino acid side chains with high conformational entropy (e.g., Lys and Glu), which are presumed to lie on the surface of the protein, are replaced by methyl groups (Ala) in an effort to create new epitopes that will facilitate crystallization. A growing number of proteins have been crystallized in this manner, suggesting that the method may be of general utility. Yet, because is impossible to predict which cluster mutant(s) will crystallize, the probability of a successful outcome is proportional to the number of mutants that are screened. Consequently, surface entropy reduction mutagenesis can be a very labor intensive undertaking. Finally, if the opportunity exists, working with multiple orthologs of a target protein generally improves the odds of obtaining crystals. Tutorial: The pictorial library of crystallization drop phenomena Dr. Wei Yang
(NIDDK):
Crystallization of protein-DNA complexes Macromolecular
interactions within a living organism achieve many remarkable feats
from formation of complex multi-molecular machines to coordination of
cascades of signaling or metabolic events. To
fully understand biology one must understand the structure and
interplay between components of macromolecular complexes and reaction
pathways. Some macromolecular complexes
are stable, such as tetrameric hemoglobin, chromatin, and the ribosome. Most macromolecular complexes are formed
only transiently however, notable examples being an enzyme and its
substrate, a growth factor and its receptor, and a transcription factor
and its DNA recognition site. To determine
structures of macromolecular complexes, whether stable or transient,
has become the goal of structural biologists in the 21st
century. Protein-DNA complexes were among the first macromolecular complexes characterized by X-ray crystallography. Since the isolation of the lac repressor with the lac operator by Gilbert and Müller-Hill, understanding the nature of specific protein-DNA interactions has captivated and occupied many scientists. How are protein-DNA interactions turned on and off? How do such interactions alter the involved macromolecules, protein or DNA, so that they initiate the next reaction step? The crystallographic studies of repressor-DNA and CAP-DNA complexes in the 1980s and early 1990s (Anderson et al., 1987; Jordan and Pabo, 1988; Otwinowski et al., 1988; Schultz et al., 1991) paved the road to pursuit of more complex and intricate protein-DNA assemblies in the last 10 years. In the following sections, I will describe strategies and current approaches employed to obtain cocrystals of protein-DNA complexes. [061] Mr. Jerry Alexandratos (NCI): Conditions 49 and 50 of
HRCS 1. Dr. Peter Sun (NIAID):
Crystallization of protein-protein complexes. Reference: Dr. Traci Hall
(NIEHS): Crystallization of Protein-RNA complexes |
This site is maintained by Dr. Xinhua Ji (jix@ncifcrf.gov) on the NCI-CCR-MCL server (http://mcl1.ncifcrf.gov). |