100 Years of X-ray Crystallography (IYCr2014)
AND TRICKS -
Effect of PEG
(Click for PDF reader to view articles)
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.
ARCHIVE: Introduction, Pre-crystallization, Crystallization, Post-crystallization, Derivatization, Cryoprotection, Diffraction, Symmetry, Structure Solution, Structure Refinement, Structure Analysis & Presentation.
- Data for
Refinement and Deposition/Publication
Xinhua Ji (NCI): High-resolution data, even not complete, always helps improve electron density that reveals additional structure features. Therefore, it is beneficial to include more data in the refinement. Claiming resolution for structure deposition/publication can be done at the final stage of the refiment. A guide line I have been using in my lab is shown below. Please comment and/or advise.
* Files for PDB deposition.
Mark Mayer (NICHD): I understand the benefit of using weak and incomplete data in high resolution shells for calculating maps and improving model building, especially with the routine use of rpim, cc and cc* at the stage of scaling supporting use of reflections in shells with with I/sigma < 2, but I don't understand how to proceed to the deposition/publication stage. After completing model building and refinement using all the data, why would we drop weak and incomplete data in the last round of refinement to achieve > 70% completeness and I/sigma > 2 or some other arbitrary cut off that will satisfy reviewers/PDB annotaters? If maps improve with weak and incomplete data in high resolution shells, then there is useful structural information, so why throw it away?
Mariusz Jaskolski (Polish Acadamy of Sciences): Thanks very much for initiating a discussion about the use of high-resolution reflections for refinement and at other stages of structure determination/publication. I have a lot of comments and practical remarks in this area, and I have summarized some of them in a one-page document.
A number of similar questions are discussed in my chapter in one of the recent Erice books: M.Jaskolski (2013), High resolution macromolecular crystallography. In: Advancing Methods for Biomolecular Crystallography. R.Read, A.G.Urzhumtsev, V.Y.Lunin eds. Springer, 259-275.
ARCHIVE: Test-set-and-R-free, Twinning, Low Resolution Crystallography, PHASER, HKL2000, Parallel Expression, Structural Genomics, NCS, Missing Atoms, Trends in Crystallography, Absorption Correction.
AND TUTORIALS NEW ADDITION - Structure Factor
RCAKU WEBINAR SERIES (2009 - PRESENT)
LOW RESOLUTION PHASING AND REFINEMENT (2011)
CRYSTALLOGRAPHY: SEEING THINGS IN A DIFFERENT LIGHT (2013)
CRYSTALLOGRAPHY: FOR ASPIRING CRYSTALLOGRAPHERS (2013)
STRUCTURE FACTOR TUTORIAL BY KEVIN COWTAN (2014)
- NEW ADDITION - Protein Geometry
Databases: BMCD, CryoD, DisProt, ExPASy, HAD, HIC-Up, Metal Sites, NDB, PDB, PDBe,
Protein Geometry Database, Scattering
Programs: CCP4, COOT, DSSR, HKL, PHENIX, PyMOL, SOLVE, USF, XDS
Servers: Anisotropy, CheckMyMetal, Crystal, C6, Dali, DSSR, ESPript, Grade,
PDBePISA, Phyre, Probity, Protein, Robetta Fragment & HHpred,
Facilities: APS SER-CAT, APS SAXS Capabilities
|Copyright © NIH X-Ray Diffraction Group Maintained by Dr. Xinhua Ji|
|on the NIH-NCI-CCR-MCL server (http://mcl1.ncifcrf.gov)|