Dear Haytham, may I address your points (although some nice hints have already come from previous replies)
1- if i have anomalous peak of unknown heavy atom, How can i identify this heavy atom in general. (different methods) To see anomalous peak I guess you have done the experiment at a synchrotron (Cu and Zn do not show much anomalous if measured at home sources). It is always good practice to run some fluorescence scan on a new sample (say, a novel protein). This can be done at all tunable beamlines (you might be able to have a broad scan of the entire range of wavelength, such as an MCA scan first. Once you have found a region of intrerest you can do a proper energy scan, exactly as if you were looking for peak remote and inflection wavelengths for a MAD experiment). XANES (the proper absorption peak scan) and EXAFS are complementary techniques. They can be performed in solution at dedicated beamlines and they can provide precious information on the first and second coordination spheres of the metal, but for the elemental recognition it might be sufficient what you measure from your crystal. Additional information can come from the coordination geometry of your ion, as mentioned above. 2- in my case, i see anomalous peak in heavy atom binding site (without any soaking). preliminary i did mass spec. i got Zn++ and Cu, How can i know which one give the anomalous peak in my protein. They might both give you anomalous signal... if you are working around 12.5 keV (near the Se edge), both metals have anomalous signal of 2-3 electrons... if you have only 1 heavy atom binding site you might consider collecting anomalous at both the peak of Zn and Cu (9.7 and 9.0 keV approximately)... however this experiment would not be necessary if you first find from an energy scan that only one is the metal binding your protein. If you have a mixture of the two, your final structure should take this into account (i.e. the appropriate relative occupancies for the two ions) 3- there is way to know if i have Cu+ or Cu++. Apart from the coordination geometry for the two oxidation states of Copper, I know of people doing UV-Vis spectroscopy on the crystals alongside their data collection. Micro-spectrophotometers are available on demand at most (if not all) of the synchrotron sources. If your species is radiation sensitive, a dose-dependent oxidation could be monitored by collecting multiple spectra. HTH Best regards, Marco
