Properties of proteins
Ideal metal coordination and bond distances[edit | edit source]
- classic papers: Harding, M.M. (1999) The geometry of metal-ligand interactions relevant to proteins; (2004) The architecture of metal coordination groups in proteins. The latter has supporting information: zip archive of the website on architecture of metal coordination groups in proteins, and PDFs containing additional tables.
- 2016 archive of original website set up by M. Harding; MESPEUS database (successor website but also now offline:) 2016 archive of website
- Check my metal
Distinguishing ions from waters[edit | edit source]
A paper exists about the bond valence method (Acta Cryst. 2003 D59 32-37). Subsequent experience has shown that although this method works well for identifying ions such as Mg2+ and Ca2+ with good resolution data, it is not reliable in other cases such as Na+.
Whereas Cl- can often be seen in anom maps, Na+ has a lower f".
There are however some tentative indicators for Na+. The bond valence sum (the sum of the 'bond orders' to the surrounding atoms estimated from the distances) tends to be higher than for Cl- or H2O. Tetrahedral coordination is more likely to be water or Cl-, Na+ prefers 5 or 6 neighbors. And of course two cations (or two anions) that are close to each other should not have an occupancy sum greater than unity.
- From the Phenix package, try: mmtbx.water_screen model.pdb data.mtz elements=NA,K
- WASP analyse water molecules in high-resolution protein structure to check if some of those could be metal ions. WASP could be run as a part of STAN server. STAN - the STructure ANalysis server from USF (http://xray.bmc.uu.se/cgi-bin/gerard/rama_server.pl )
Melting point[edit | edit source]
Thermofluor is a fluorescence-based thermal stability assay. From : The experiment is easy to perform and requires no prior knowledge of the protein properties. Every standard real-time PCR machine available in many research labs is suitable to perform this high‐throughput experiment in a short time (1‐2 hours). Only relatively little protein (500 µl of protein at 1‐2 mg/ml for a 96‐well experiment) and a dye (e.g. SYPRO® Orange, Invitrogen) are required. Proteins containing prosthetic groups (e.g. FAD) may not even require an external fluorophore if the intrinsic signal can be used during the Thermofluor experiment. For FAD‐containing proteins this method has been termed ThermoFAD.
Ku T, Lu P, Chan C, Wang T, Lai S, Lyu P, Hsiao N. (2009) Predicting melting temperature directly from protein sequences. Comput Biol Chem. 33, 445-450 at . They have a list of 35 different proteins with their Tms with the references from where they obtained their data, and their Tm Index program is available at .
A specific case which shows that Tm does not need to be high: the protein with PDB-ID 1ofc had a melting temperature of 37°C (from CD), which was supported by the fact that it did not express in E.coli at that temperature. At 20°C it expressed to about 60mg / (liter LB), could be concentrated to more than 100mg/ml, crystallised at room temperature and diffracted to 1.9A. The initial purification steps were done at 4°C.
Modifications of amino acids[edit | edit source]
- instead of CYS you might encounter S-hydroxycysteine (CSO) or S-oxycysteine (CSX). The only difference, crystallographically, is that the CSO S-O bond is about 1.78A and the CSX S-O bond is about 1.50A. You may not be able to differentiate this difference in your electron density maps. A SH - HOH contact should be considerably longer than 1.8 A between heavy atoms, certainly more like 3.0A+/-. You can substitute either CSO or CSX for CYS in Coot by deleting the CYS residue and then using File...Get Monomer and typing in either CSO or CSX. A "surprise CSO" residue was encountered when solving the structure of 3UAO. The protein had been stored in a non-reducing medium, resulting in the oxidation of the active site Cys residue. The electron density near Cys was too close to be a water molecule, but CSO modeled nicely.