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No hits, what next
No hits from a screen, what next? You can always set up another screen! Or, you can make a list of all drops that have precipitate and use the precipitate as a potential seed stock. Streak seed from the precipitate into new drops that have been set up at 50% and 75% of the original screening solution. There may be nucleation sites or microscopic seeds in the precipitates that may grow at lower precipitate saturation. Better still, streak seed from the precipitate to a clear zone into the same drop to recycle the drop.
Anil Mistry
Reproducible seeding
For reproducible micro-seeding by hand use a cryoloop to fish out your seed from the seed stock and transfer them to the drop. Use a 0.3-0.4 mm cryoloop.
Anna Aagaard
Reduce to enlarge
For bigger crystals try to add 0.5-1 microliter of 14 M beta-mercaptoethanol to the reservoir after the protein drop was set up.
Zhanna Druzina
Difficult ligands
Problem: Can grow crystal but no protein-ligand crystals. Tip: Take the conditions from the apo crystals and develop a focused optimization screen (24 well maximum). Screen complexes using cross seeding and the focused screen and three drop ratios (1:1, 2:1 and 2:3).
Annie Hassell
Time for a change
Problem: Poor crystal quality. Tip: Change tray type or crystallization method. For example, initial screens done in sitting drop tray and crystal quality improved in 96 well hanging drop tray.
Annie Hassell
Avoid the rut
Every project, every protein, every construct is unique. Be careful of knowing too much. Just because things did or did not work in the past does not mean things will work that way for the next project.
Brandon Collins
Get heavy to stabilize
Problem: Poor diffraction. Tip: Heavy atom soaks to stabilize floppy regions of the protein.
Lose focus
Don't focus all of your optimization efforts on a single crystallization condition. If you have several different crystallization conditions identified for a sample go after them. Crystals of the same protein produced from different chemical conditions and/or temperatures will have unique physical properties. These properties will determine how easy the crystal can be looped (physical stability), cryoprotected and ultimately how well the crystal diffracts X-rays. Avoid single points of failure, go after several hits.
Joseph Luft
Ligand seeding
If ligands can't be soaked into crystal or co-crystallization is ligand specific try seeding into drops that contain ligand of interest. When soaking crystals with insoluble ligands try adding the cryoprotectant to the soaking solution. This can help solubilize the ligand and also cryoprotect (so less handling). Doesn't work so well when salt is the cryoprotectant, but may well when it's glycerol, DMSO or ethylene glycol.
Doug Marcotte
Mostly clear
Problem: Drops are all/mostly clear. Tip: Remove stabilizing agents (salt, glycerol, etc) from the protein buffer. Then do crystallization screens.
Annie Hassell
Hydrate or die
At suboptimal protein concentrations the interface between protein solution and crystallization screen solution may exhibit excessive precipitation. To avoid this, before adding screen solution add 1 microliter of water. Downside of this is equilibration will take slightly longer. Upside of this is decreased osmotic shock for protein and less precipitation.
Vaheh Oganesyan
What failure looks like
Put one conditions of 40% TCA into your standard screen. This should precipitate out all of your protein, so that you have an idea of what heavy precipitate should look like.
Janet Newman
Think global act local
Don't consider a crystallization result in isolation. Look for neighbors in chemical space and use those results to provide chemical directions for optimization. If a cryocooled crystal does not diffract well. You cannot tell if it is the crystal, cryoprotectant or cooling that is causing the problem. Look at room temperature data before moving on.
Edward Snell
X marks the spot
Consider the case of poor nucleation and seeding did not work. Tip: Mix protein and well solution then use pipette tip to cross the drop into branched shape. Crystal may grow in the branches of the drop.
Mei Xu
Robotic seeding
To increase you choices of producing more optimal crystal condition or conditions using seeds, try the following. Program a small volume liquid handler to dispense your protein and seed solution directly into 96 well commercial screens and/or an additive screen.
Paris Ward
Delete to succeed
Recent success with loop deletions. Sequence alignments reveal either charged loops and/or loop insertions relative to homologues. I have removed 3 to 34 amino acids and retained high expression soluble protein and novel crystals.
Heidi Schubert
PEG to the rescue
Problem: Unstable protein. Tip: Add 1-5% low molecular weight (200 to 1,000) PEG directly to the protein and then screen.
Annie Hassell
Doh!
Start with big crystals! Add at least 5% glycerol to everything.
Jim Pflugrath
Cryo stabilization
Stabilize crystals in cryo by adding protein buffer components into the cryo. For example, most commonly I add 100 to 150 mM NaCl plus reservoir components plus cryoprotectant(s).
Laura Pelletier
Play with the ratio
Play with protein-mother liquor ratio, especially with low solubility proteins. Try different concentration of protein coupled with streak seeding.
Ayse Sinem Ozyurt
Repeat after me
After looking at the results of initial screening or of additive screen, pick several of the best "hits" and screen in 96 well format, 6 or 8 identical drops of each favorite before scaling up. Helps to eliminate "one offs" and save time.
Nancy Bump
Complex it
Apo protein is monidisperse but won't crystallize. Complex it! Complex it! Complex it!
Paul Reichert
Matrix seeding
If your crystal seeds withstand large serial dilutions, try matrix seeding via the reservoir by doing the following. 1) Create crystal seeds as described by Allan D'Arcy. 2) Dispense seed into reservoirs containing reservoir. 3) Aispirate/dispense to mix seed in reservoir. 4) Dispense mother liquor droplet containing seed onto protein drop.
Armando Villasenor
Salt that sucker out
If you do not see any crystal growth in several days after set-up (more than 1 to 2 weeks) and the drop are not all clear, add salt (such as ammonium sulfate) to 0.5 M to the drops. Even though ammonium sulfate salt crystals might form, you might actually get protein crystals. This worked for me recently.
Neil Grodsky
Helpful urea
Non denaturing (less than 3 M) concentrations of urea can be helpful to solubilize your protein.
Gloria Borgstahl
Precise dosing
Try stoichiometric levels of multivalents, cations as additives. They may be necessary for crystallization but sometimes the levels found in commercial screens are too high and toxic.
Simon Low
Cook to crystallize
Heat treatment of protein complex to obtain diffraction quality crystals. Original complex has no initial crystal hits. Heat treat protein complex (25 to 80 degrees Celsius) for various times (5 to 30 minutes). Centrifuge to get rid of aggregated protein and screen again.
Liping Wang
Gap it
When using pre greased trays, take a toothpick and remove a bit of grease from each well. Now as you push down on your cover slip, you turn it a few degrees. This will allow the air to escape and the turn will form an airtight seal over each well.
Paul Reinfelds
Thermostability
Thermostability. Monitor the effect of additives, buffers, ligands, etc. on melt temp of your protein. We have seen in multiple cases that the most thermostable construct, buffers, additive yields the best or only crystals. How? We use Bio-Rad's iQ5 iCycler (a PCR instrument) as it has 5 sets of filters for excitational emission and hydrophobic dyes that fluoresce upon binding (protein unfolding).
Jackie Day
Sticky situation
Problem: Crystals adhering to plastic of sitting drop plate, and mechanical dislodging (by cryoloop, tool, etc) does not work. Solution: Stan a fine gauge syringe needle into the plastic, near but not into the crystal. This often distorts/disrupts the plastic near the crystal and breaks the seal.
Michael Wiener
Big low tech
Getting bigger crystal by low tech / low cost counter diffusion. If you are faced with either no nucleation or showers of crystals and the usual tricks including seeding do not work, try this: On a cover slide, set the protein and precipitant drop (example 1 microliter plus 1 microliter) separate, but very close to each other. Then, with a whisker or pipette tip streak through the drops to form a connecting bridge between the protein and precipitant solution. Invert cover slide and place over well. Crystals will form along the gradient and "self screen" for best conditions.
Margarete Neu
Insoluble compounds
When working with compounds for co-crystallization, if the compounds are highly insoluble in protein buffer (50 micromolar or less) we often employ low concentration complexing. We dilute the protein and then add in diluted compound, so tha the compound is added close, or at least closer to a concentration where it is soluble and the content of DMSO in the protein sample remains less than 2%. The protein-compound complex is then concentrated for crystallization trials. This has helped us with several projects with highly insoluble compounds.
Elizabeth Fry
Ionic liquids
In experiments using model proteins we found that Ionic Liquids (IL's) specifically 1-Butyl-2-methyl imidazolium chloride gave increased numbers of crystallization outcomes compared to the IL controls. A large number of the crystals obtained had precipitated outcomes in the IL controls. In many other cases the IL and crystal had an improved morphology (needles to plates, plates to 3D crystals) over the IL controls. Tip: Using an IL such as 1-Butyl-2-methyl imidazolium chloride as an additive to improve chances of getting a crystal from conditions which otherwise would give precipitate. Marc Pusey, MI Research, Inc. Our favorite cryoprotectant. 1x UCP (Ultimate Cryo Protectant) 8% Glycerol, 8% Ethylene glycol, 9% Sucrose, 2% Glucose. We make a 2x solution. Generally add this 1:1 with reservoir. The ratio can be modified, for example 1.2 microliter 2x UCP : 0.8 microliter reservoir, or 1.4 microliter 2x UCP : 0.6 microliter reservoir, or 0.6 microliter 2x UCP : 1.4 microliter reservoir, etc. I believe this has been successful in cryoprotecting some 70% of all of our systems, resulting in more than 50 solutions of these targets regardless of previous cryogenic treatments. Author in unknown to me, but the credit is published in a singled Hencrickson paper. I was tipped off 6 years ago.
Christopher Bonagura
Se-met stuff
If you can't get your Se-met protein to crystallize try leaving ou the DTT/TCEP during purification and crystallization.Sometimes there are disulfide bridges near crystallization contacts that need preserving for crystallization to take place. Collected the Se edge is still possible.
Shirley Robert
DMSO cryo
Try 20 to 30% DMSO as cryo. This has worked well in a number of cases for me and I've added this to a very short list of cryos that I personally use. Note: If your mother solution contains a high salt concentration the DMSO will cause it to precipitate out of solution. So beware!
Rich Romero
Stack em up
4 degrees Celsius crystallization plates prepared at room temperature always have a condensation problem on the plate seal. To avoid condensation cover the finished plates with two lids and plate it in the cold room for 20 minutes or on top of a cold metal block, The will reduce the temperature of the reservoir solution while the 2 lids delay the temperature change from the top long enough to avoid condensation.
Beat Blattmann
Anti skid
Ever been manually sealing a plate only have it slip out from under you? The result is usually death for hanging drop plates, and with sitting drop plates your best bet is hoping the drop is not splashed onto the seal above. To ensure the plate stays put when applying pressure, we use a "grip pad" in our lab. Simply place the grip pad onto the bench, set plate on top and seal as usual. The grip pad prevents the plate from slipping out from under the compression tool, usually a brayer, used for ensuring the please seal is applied correctly. The grip pad can be cut from the material commercially available for lining tool shop drawers. In addition, we created a fixed plate holder that encloses the entire plate to guarantee the brayer does not slip off the plate when sealing, a common occurrence when manually sealing many plates. Our plate holder is custom cut from a hard rubber to fit both thee 24 well and 96 well plates. The plates sit slightly above the platform to ensure both ends are sealed and for easy removal. The sides of the platform are rounded to ensure the brayer has a smooth path of travel.
Barbra Pagarigan
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