Artem Evdokimov: Cryoprotection of Delicate Crystals

Introduction

The procedure below summarizes one of the approaches to cryoprotection commonly employed in our lab. This approach worked for me on many occasions, but obviously I cannot promise that it works with every crystal, all the time.

I would like to mention that this is not the easiest approach, and certainly not the first that we try on a new crystal (I usually start with 20-25% ethylene glycol in crystal mother liquor; this seems to be satisfactory for at least 50% of crystals I worked with). However, if you have a few crystals to spare, and the simpler things did not pan out, this method is worth trying.

This approach has been hashed out during the late-night hours at various synchrotron beam lines, and has not been put to a rigorous statistical test, so it’s not likely that this kind of semi-anecdotal stuff will see the light of day as a real paper. Many colleagues and friends have contributed through discussion, for which I am very grateful. It is likely that someone has described similar, if not the same considerations before. If this is the case, I would appreciate the reference so that I could give proper credit for earlier work.

I am always most grateful for comments, suggestions, or anecdotes of success/failure. (Click for the FULL ARTICLE)

ARCHIVE: Introduction, Pre-crystallization, Crystallization, Post-crystallization, Derivatization, Diffraction, Symmetry, Structure Solution, Structure Refinement, Structure Analysis & Presentation.

TOPIC DISCUSSION - Test Set and R-free

Editor: Please share your practice and theory on Test Set and R-free with fellow members of the group. A list of suggested subtopics is shown below. 

(1) How many reflections do you put in Test Set for R-free calculations, a centerin percentage or a certain number?

(2) For isomorphous structures, such as a wild type and several mutant structures crystallized in the same lattice, do you use the same Test Set for all these structures?

(3) Do you use the same Test Set if you re-process your data during refinement or obtain a better data set?

(4) Do you use the same Test Set when you switch refinement programs?

(5) What do you do to the Test Set if you find out the crystal is twinned during refinement without twinning considerations?

(6) Do you include some or all data in the Test Set for the final rounds of refinement?

      
Mark Mayer: How about an addition?

(7) When you have NCS do you pick Test Set using thin shells?

Recommended Reading: CCP4 wiki: R-factors

Xinhua Ji: The more complete the data, the more accurate the stucture. It appears that R-free should be used to monitor initial refinement only. Once the model is completed and refinement is converged, all data should be included for final refinement. Besides, refinement with all data is likely to reveal additional features. However, the modern ML refinement targets require the use of test set and R-free is mandatory. Any ideas?

ARCHIVE: Twinning, Low Resolution Crystallography, PHASER, HKL2000, Parallel Expression, Structural Genomics, NCS, Missing Atoms, Trends in Crystallography,  Absorption Correction.

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"