| Newsletter No. 253
|| March 5, 2012
| TIPS AND TRICKS - 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.
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).
Comments and suggestions are always welcome!
ARCHIVE: Introduction, Pre-crystallization, Crystallization, Post-crystallization, Derivatization, Cryoprotection, Diffraction, Symmetry, Structure Solution, Structure Refinement, Structure Analysis & Presentation.
DISCUSSION - Low Resolution Phasing and Refinement
NE-CAT hosted a workshop on Advances in Low to Moderate Resolution Phasing and Refinement in September and compiled abstracts, PowerPoint presentations, video, and audio of the event at their website. If you are interested in this topic, please visit their website at:
ARCHIVE: Test-set-and-R-free, Twinning, Low Resolution Crystallography, PHASER, HKL2000, Parallel Expression, Structural Genomics, NCS, Missing Atoms, Trends in Crystallography, Absorption Correction.
| DR. ZBIGNIEW DAUTER'S LECTURE AT
THE NIH (2005)
Part 1: "How to read international tables?"
Part 2: "Data collection strategy" and "Twinning"
"Phasing methods - a general introduction to all methods"
Part 3: "SAD phasing, Quick halide soaking, and Radiation damage
with possible use of it for phasing"
New Addition: RIGAKU WEBINAR SERIES (2009 - PRESENT)
Databases: BMCD, CryoD, DisProt, ExPASy, HAD, HIC-Up, Metal Sites, NDB, PDB, PDBe,
Programs: CCP4, COOT, HKL, PHENIX, PyMOL, SOLVE, USF, XDS
Servers: CheckMyMetal, Crystal, C6, Dali, ESPript, Phyre, Probity, Protein
|Copyright © NIH X-Ray Diffraction Group Maintained by Dr. Xinhua Ji|
|on the NIH-NCI-CCR-MCL server (http://mcl1.ncifcrf.gov)|