Locating Heavy Atom Sites
Using SHELXD
Heavy Atom Sites in Protein Crystal. Three mercury ions are bound to each protein molecule. Displayed with Setor.
Using SHELXD:A sample script for running SHELXD is given below. The program runs on Linux machines (e.g. lorentz.mbi.ucla.edu) not on DEC alphas. Isomorphous or Anomalous differences are input in HKL format. To make the proper HKL format file, you may first use xprep. Xprep also runs only on linux machines (e.g. lorentz.mbi.ucla.edu). Xprep will accept Scalepack output and also produce some useful statistics which will help you chose the optimum resolution range to use in SHELXD. A guide for using xprep is also given below. Example output from SHELXD is also given below.

Manual for SHELXD:
/joule2/programs/shelx/shelxd/shelxd.htm A bare essentials manual. 

Manual for SHELX:
/joule2/programs/shelx/shelx97_2/ps/ch_12.ps More bare essentials you may need. 

SHELXD Workshop:
file:/joule2/programs/shelx/shelxd/aca2000.htm More informative documentation about how SHELXD works. 

xprep help file:
http://bluechex.cchem.berkeley.edu/chexray/XPREP.html A somewhat helpful help page. 

reference for SHELXD (pdf file):
Uson, I. & Sheldrick, G. M.  Advances in direct methods for protein crystallography, Curr. Op. Struct. Biol., 1999, 9:643-648.


Guide for Preparing an HKL file with XPREP.
An example, using anomalous differences.
EXAMPLES

Log into a linux machine (e.g. telnet lorentz).
change directories to where your scalepack files are.
cd ~sawaya/data/sawaya/fe 

To execute the program, type:

/joule2/programs/xprep/xprepx 

type in the name of the scalepack file:

hg.sca

select option "D for DENZO/SCALEPACK format input.

Yes. Use the cell from the Scalepack file. 
 
 
 
 
 
 
 
 

In this example, the space group is P61, so select "P" for primitive. 

Select option "S" to input space group information.

Select option "I" to input known space group. In this example, type, "P6(1)".

Select "Y" to accept your choice.
 
 
 
 
 
 
 
 
 
 
 
 

Select option "D" to read, modify or merge DATASETS.

Select option "A" for MAD, SAS, SIR or SIRAS.

Select option "A" for SAS. We are using anomalous differences in this case.

At this point, accept the defaults for the next three questions by hitting the enter three times.

The anomalous signal/noise ratio is given in resolution shells. The signal is significant to about 2.6 Angstroms, where signal/noise drops to just under 2.0. So at the next prompt, we cut the data to 2.6.

Hit the enter key for no renormalization, then type a filename for the output hkl file. (e.g. hg.hkl)


 


 
 


Script for Running SHELXD
An example, using anomalous differences.
COMMENTS

Log into a linux machine (e.g. telnet lorentz).

Change directories to where your hkl file is.

cd ~sawaya/data/sawaya/fe 

Copy the following script into a file called "hg.ins". The first part of the name corresponds to the hkl file and the extension must end in ".ins", meaning "instruction" file.
----------------------------------------
TITL ucla in P6(1)
CELL 0.71073 80.3490 80.3490 86.4620 90.000 90.000 120.000
ZERR 6.00 0.0100 0.0100 0.0100 0.000 0.000 0.000
LATT -1
SYMM -Y,X-Y,Z+1/3
SYMM Y-X,-X,Z+2/3
SYMM -X,-Y,Z+1/2
SYMM Y,Y-X,Z+5/6
SYMM X-Y,X,Z+1/6
SFAC HG
UNIT 40

PATS
FIND 10 20
NTRY 50
MIND -3.0
SHEL 100 2.6

HKLF 3
END
----------------------------------------
 

To execute SHELXD, (login to lorentz) type 
/joule2/programs/shelx/shelxd/linux/shelxd hg
where "hg" is the name of the .ins file.

The program will finish in about 10 minutes. You can see a summary of the results for each trial as the program runs. Here we see results from try 19, 20, and 21. Note PATFOM and CC as the summary goes by on the screen. The best solution will have the highest PATFOM and CC.

--------------------------------------------------
PSUM 90.71 PSMF Peaks: 62 36 32 27 26 26 14 13 13 12 11 11 10 9 9
Try 19:20 Peaks 99 99 96 87 66 54 53 48 40 33 32 31 29 28 16
R = 0.293, Min.fun. = 0.518, = 0.404, Ra = 0.294
Try 19, CC All/Weak 41.07 / 21.50, best 42.01 / 22.61, best PATFOM 17.42
PATFOM 17.04
--------------------------------------------------
PSUM 76.48 PSMF Peaks: 33 18 17 16 13 11 10 10 10 10 9 9 9 8 8
Try 20:20 Peaks 99 98 92 88 65 58 46 42 40 33 27 25 24 22 16
R = 0.296, Min.fun. = 0.522, = 0.402, Ra = 0.297
Try 20, CC All/Weak 40.98 / 21.65, best 42.01 / 22.61, best PATFOM 17.42
PATFOM 18.22
--------------------------------------------------
PSUM 96.74 PSMF Peaks: 56 38 36 35 32 14 11 11 10 10 10 9 9 9 8
Try 21:20 Peaks 99 99 96 84 63 51 50 47 44 32 28 28 25 23 15
R = 0.284, Min.fun. = 0.516, = 0.406, Ra = 0.294
Try 21, CC All/Weak 41.02 / 21.06, best 42.01 / 22.61, best PATFOM 18.22
PATFOM 16.47
--------------------------------------------------
 

 


CELL -- Wavelength followed by unit cell dimensions. The wavelength has no importance in this calculation.

ZERR -- Number of asymmetric units in the unit cell, followed by estimated errors in the unit cell dimensions. Estimates do not have to be accurate.

LATT -- Primitive is "-1", C centered is "-7", I centered is "-2", F centered is "-4". The negative sign indicates that the structure is non-centrosymmetric.

SYMM -- Symmetry operators. The operator X,Y,Z is always assumed, so may NOT be input. If your structure is C, I, or F centered, do not put the lattice centering operators. These operators are specified by the choice of LATT, above. See this list of symmetry operators for your space group.

SFAC -- The element symbol defining the heavy atom. The first 94 elements of the periodic table are recognized.

UNIT -- The number of heavy atom in the cell. A ball park figure, can be larger than what you expect.

PATS -- Generates starting atoms consistent with Patterson. It is "slightly" better than starting from random positions.

FIND -- Number of atoms to find. Just a guess.

NTRY -- Number of trials. If not specified, the program will run forever. I have found that if you don't get a decent answer in 50 trials, you will probably never get it. Find a better derivative.

MIND -- The shortest distance allowed between atoms for PATS and FIND. Here it is set to 3 Angstroms since it is unlikely that two heavy atom sites would be closer than this. The negative sign is used as a flag to output a crossword table in the log file. The crossword table is important for judging the quality of the solution. Keep MIND negative.

SHEL -- Resolution limits.

HKLF -- Set it to 3 for F or FA or deltaF (e.g. output from xprep). Set it to 4 for F2 in .hkl file. 
 
 
 
 

 

Log file from SHELXD 
Here is an exerpt from a SHELXD log file obtained using the input above (i.e. from the Hg derivative of T7 helicase domain in space group P61). This is Try 20, which has the best PATFOM of all 50 trials.

Some definitions:

PATFOM- Patterson figure of merit, useful for selecting the best solution. Higher is better.

CC -Correlation coefficient. Higher is better.

Crossword Table -In this table the rows correspond to the potential atoms. They are listed in order of peak height. Reading across a row, peak height is given first, then x,y,z for the atom, then the distance (D) between this atom and its crystallographic symmetry mate. Just below D is the PMF (Patterson minimum function) calculated from all vectors between the this atom and its crystallographic symmetry mate. When this number is large and positive there is a good correlation between the heavy atom position and the Patterson map. When this number is 0, then there is no correlation, and the heavy atom site should be discarded. For each successive row, there is an additional column to the right which describes the cross peaks between the heavy atom and its neighbors. Look at the PMF values. In general wrong sites can be recognized in this table by the presence of several zero PMF values.

In the example below, the first five sites look quite convincing, PMF values are large and positive for both self peaks and cross peaks. The sixth and seventh sites look weeker, the self vectors are 0.0, suggesting that these sites should be discarded. Site eight looks OK and site nine should definitely be discarded.
 

---------------------------------------

PSUM 76.48 PSMF Peaks: 33 18 17 16 13 11 10 10 10 10 9 9 9 8 8
Try 20:20 Peaks 99 98 92 88 65 58 46 42 40 33 27 25 24 22 16
R = 0.296, Min.fun. = 0.522, = 0.402, Ra = 0.297
Try 20, CC All/Weak 40.98 / 21.65, best 42.01 / 22.61, best PATFOM 17.42

PATFOM 18.22 
       x           y           z        sof    U  height
0.39223 0.60700 0.05429 20.0 1.0 99.90
0.67147 0.81235 0.10274 20.0 1.0 99.24
0.45693 0.59164 0.13364 20.0 1.0 96.09
0.78883 0.23500 0.12360 20.0 1.0 94.31
0.59833 0.79134 0.03881 20.0 1.0 81.02
0.78874 0.08715 0.07110 20.0 1.0 76.61
0.33154 0.47636 0.14833 20.0 1.0 68.61
0.62743 0.81091 0.09937 20.0 1.0 65.10
0.74727 0.38372 0.06416 20.0 1.0 63.64
0.13704 0.29890 0.08135 20.0 1.0 57.66
0.74709 0.10291 0.05963 20.0 1.0 52.38
0.15316 0.51651 0.10570 20.0 1.0 50.64
0.80244 0.67429 0.04994 20.0 1.0 49.22
0.68119 0.43611 0.06406 20.0 1.0 47.35
0.92481 0.62717 0.15297 20.0 1.0 40.70

Minimum distances (top row, 0 if special position) and PSMF (bottom row)

CROSSWORD TABLE
Peak        x        y        z        self        cross-vectors

99.9 0.3922 0.6070 0.0543 32.2
                                            25.2

99.2 0.6715 0.8123 0.1027 27.1 20.6
                                            79.4 63.4

96.1 0.4569 0.5916 0.1336 37.6 9.1 17.7
                                            38.9 38.3 47.7

94.3 0.7888 0.2350 0.1236 34.2 15.3 18.4 19.3
                                            24.3 41.7 38.8 30.4

81.0 0.5983 0.7913 0.0388 31.5 15.8 7.6 16.5 22.0
                                            18.0 22.9 42.0 32.9 24.9

76.6 0.7887 0.0872 0.0711 25.8 26.8 12.3 20.2 12.7 19.1
                                            0.0 19.3 30.5 5.6 21.0 7.8

68.6 0.3315 0.4764 0.1483 36.9 12.2 27.4 9.8 17.9 25.4 27.5
                                            0.0 21.9 2.7 3.6 15.1 8.2 4.9

65.1 0.6274 0.8109 0.0994 29.7 18.2 3.5 16.3 17.3 5.6 13.9 25.8
                                            16.6 26.8 21.6 24.4 33.4 8.7 9.6 2.4

63.6 0.7473 0.3837 0.0642 30.4 13.8 25.2 19.4 14.8 19.7 25.7 21.8 22.3
                                            0.0 3.5 20.9 4.1 13.1 0.0 0.0 0.0 12.5
 


 


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