Phasing using MIRAS or MAD
Get your Electron Density Maps Here!
subtitle: It's like making sauasge; if you knew what went into it, you wouldn't eat it. 
Preparing a useful derivative

There are many things to consider:

1) Safety: When preparing heavy atom derivatives, special care must be taken to prevent poisoning/irradiating yourself or others. 
2) Selection: What heavy atoms are most likely to give you a useful derivative? Consider what side chains are available to ligate the heavy atoms. Also, consider a list of the most successfully used heavy atom reagents. 
3) Screening: Use the native gel test. A band shift is indicative of a coordination of a heavy atom by your protein. 

Requirements: Caution when handling heavy atoms, a native gel (Phast system OK), a few microliters of concentrated protein, and various heavy atom solutions, crystals not necessary at this point. (hurray!)

References:T. J. Boggon and L. Shapiro Structure 8, R143-R149, 2000.

Evaluating Derivative Quality
Isomorphous OR Anomalous
The quality of a heavy atom derivative can be first evaluated by the chi squared test performed on either isomorphous differences or anomalous differences. The chi squared test measures the significance of the signal originating from the heavy atom. If the chi squared statistics suggest that the differences are small, then the putative derivative data is not useful for phasing. If the chi squared statistics are large, then there is hope. 

A positive chi squared test based on isomorphous differences is less reliable than a positive test based on anomalous differences. The poorer reliability of the isomorphous chi squared test is due to non specific heavy atom binding which often causes non-isomorphism between native and derivative crystals ...which in turn causes large chi squared values. Anomalous differences are measured from a single crystal (i.e. perfect isomorphism) so a positive chi squared test is more indicative of a true anomalous signal. Note that the anomalous signal is enhanced by chosing the peak wavelength. To find this wavelength, see the Anomalous Scattering Web Site

Requirements: For isomorphous differences - a complete native data set in scalepack format (.sca file) and 5 degrees of integrated data (.x files) from a derivative data set.

For anomalous differences - a highly redundant derivative data set in scalepack format (.sca) and processed with "anomalous" or "scale anomalous" flags in scalepack.

Difference Patterson Maps
Isomorphous OR Anomalous

The value of a heavy atom derivative can be more accurately judged from the appearance of a difference Patterson map -- this could be an isomorphous difference Patterson map (i.e. using differences in intensities between a native crystal and a heavy atom derivative) or an anomalous difference Patterson map (i.e. using differences in intensities between Bijvoet pairs). Planning to solve your MIR structure automatically with SOLVE? Check for peaks on your isomorphous difference Patterson first. If you have significant peaks on your Harker section (greater than 4 sigma), then SOLVE is likely to work. If not, then move on to the next derivative. 

If you have identified heavy atom sites by means of SOLVE or SHELXD, You should check the difference Patterson to see whether all the sites identified are correct. Through the use of xpatpred we see site 1 has been identified correctly on the figure at left. List of Harker Sections for all space groups.

Requirements: Unit cell parameters, space group, and observed structure factors in fin format.

References: Author, Journal volume, pages, year.

Locating Heavy Atom Sites

There are several programs which will locate heavy atom positions from difference Patterson maps: HASSP (from the Heavy suite of programs and also utilized in SOLVE), Hercules (from the XtalView suite of programs), Patsol (from Liang Tong), and rsps (from the CCP4 suite of programs). However, in my opinion, use SHELXD first. 

SHELXD is easy to use and the output is the most informative and easily interpretable of all programs. It has been used successfully in locating Br in MAD experiments where other programs failed. It claims to produce "more complete and precise solutions" by integrating both Patterson and direct methods. 

Requirements: Unit cell parameters, space group, and observed intensities in Scalepack format.

References:M. G. Rossmann and D. M. Blow, Acta Cryst. 15, 24-31, 1962.

MIRAS phasing with Heavy Atoms
SIRAS phasing with Iodide

There are many steps to the phasing process. After the heavy atoms have been located 1) their positions must be refined 2) the phases are calculated using both configurations of heavy atoms related by a center of inversion 3) solvent flattening is used to improve the phases for both maps 4) visual inspection of the map to chose the correct hand 5) model building into the correct map. There are programs that can do all these steps automatically (e.g. SOLVE and ELVES). Here we describe the invidual steps used by these programs.  (1) for the general MIRAS case (multiple isomorphous replacment with anomalous scattering) and (2) for SIRAS phasing with iodide.

Requirements: Unit cell parameters, space group, and observed structure factors in Scalepack format for native and derivative, coordinates of heavy atom sites.



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