## 1 SPANCSI - GENERAL INFORMATION

Program : SPANCSI
Version : 080625
Author : Gerard J. Kleywegt & T. Alwyn Jones, Dept. of Cell and Molecular Biology, Uppsala University, Biomedical Centre, Box 596, SE-751 24 Uppsala, SWEDEN
E-mail : gerard@xray.bmc.uu.se
Purpose : SPAnish NCS Inquisition; can also be used for averaging with variance scaling for each of the NCS-related molecules (substitute for AVE)
Package : RAVE

## 2 REFERENCES

Reference(s) for this program:

* 1 * T.A. Jones (1992). A, yaap, asap, @#*? A set of averaging programs. In "Molecular Replacement", edited by E.J. Dodson, S. Gover and W. Wolf. SERC Daresbury Laboratory, Warrington, pp. 91-105.

* 2 * G.J. Kleywegt & T.A. Jones (1994). Halloween ... Masks and Bones. In "From First Map to Final Model", edited by S. Bailey, R. Hubbard and D. Waller. SERC Daresbury Laboratory, Warrington, pp. 59-66. [http://xray.bmc.uu.se/gerard/papers/halloween.html]

* 3 * G.J. Kleywegt & R.J. Read (1997). Not your average density. Structure 5, 1557-1569. [http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Retrieve&list_uids=9438862&dopt=Citation]

* 4 * R.J. Read & G.J. Kleywegt (2001). Density modification: theory and practice. In: "Methods in Macromolecular Crystallography" (D Turk & L Johnson, Eds.), IOS Press, Amsterdam, pp. 123-135.

* 5 * Kleywegt, G.J., Zou, J.Y., Kjeldgaard, M. & Jones, T.A. (2001). Around O. In: "International Tables for Crystallography, Vol. F. Crystallography of Biological Macromolecules" (Rossmann, M.G. & Arnold, E., Editors). Chapter 17.1, pp. 353-356, 366-367. Dordrecht: Kluwer Academic Publishers, The Netherlands.

## 3 VERSION HISTORY

970522 - 0.1 - first version (options A and C)
970523 - 0.2 - added option B
010122 - 1.0 - use C routines to do dynamic memory allocation and port to Linux
040701 -1.0.1- changed checks of dynamic memory allocation to allow for pointers with negative values as returned by some recent Linux versions
080625 -1.0.2- suppress error messages if more than 10 of them

## 4 MAP AND MASK SIZE

SPANCSI allocates memory for maps and masks dynamically. This means that you can increase the size of maps and masks that the program can handle on the fly:

1 - through the environment variables MAPSIZE and MASKSIZE (must be in capital letters !), for example put the following in your .cshrc file or your RAVE script:


----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
setenv MAPSIZE 8000000
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----


2 - by using command-line arguments MAPSIZE and MASKSIZE (need not be in capitals), for example in your RAVE script:


----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
run spancsi -b mapsize 10000000 masksize 5000000 < spancsi.inp >& spancsi.out
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----


Note that command-line arguments take precedence over environment variables. So you can set the environment variables in your .cshrc file to "typical" values, and if you have to deal with a map and/or mask which is bigger than that, you can use the command-line argument(s).

If sufficient memory cannot be allocated, the program will print a message and quit. In that case, increase the amount of virtual memory (this will not help, of course, if you try to allocate more memory than can be addressed by your machine (for 32-bit machines, something like 2**32-1 bytes, I think), or reduce the size requirements.

SPANCSI needs space for 3 maps and 1 mask.

## 5 PURPOSE

SPANCSI can be useful if you have indications that (of want to investigate if) your NCS-related molecules have very different average temperature factors, for example due to differences in the packing. For example, in the structure of P2 myelin protein (PDB code 1PMP), molecule "C" has a considerably higher average temperature factor than the other two molecules, "A" and "B". This is visible in the skeletons, but was also verified using Rfree by refining an overall B-factor shift for each of the three molecules with X-PLOR. R and Rfree both dropped, and the average B-factor for the "C" molecule ended up being ~10 Å**2 higher than those of the other two molecules.

SPANCSI allows you to:

- calculate density statistics for each of your NCS-related molecules separately

- calculate correlation coefficients etc. for each pair of NCS-related molecules

- average and expand density using variance weighting for each of the NCS-related molecules. For example, suppose you have two molecules for which the variance of the molecular density is 100 and 50, respectively. Now in the averaging, the contribution of the second molecule will be upweighted by a factor of 100/50 = 2.0, and when the averaged density is expanded back into the unit cell or asymmetric unit, the density for the second molecule will be downweighted by a factor of 50/100 = 0.5. This hopefully leads to better convergence and better phases and maps.

If you find that there are differences between the molecules, change your averaging script so it uses SPANCSI instead of AVE.

## 6 NOTES

NOTE: this program is sensitive to the environment variable CCP4_OPEN. If this variable has *not* been set, you will not be able to create any CCP4 maps. If this happens, the program will abort execution on startup. To fix this, put the following line in your .login or .cshrc file: setenv CCP4_OPEN UNKNOWN

NOTE: this program is sensitive to the environment variable OSYM. It should point to your local copy of $ODAT/symm, the directory which contains the spacegroup symmetry operators in O format. When asked for a file with spacegroup operators in O format, you may either provide a filename, or the name of a sapcegroup (including blanks if you like, case doesn't matter). The program will try to open the following files, assuming that STRING is the what you input: (1) open a file called STRING (2) if this fails, check if OSYM is defined and open$OSYM/STRING
(3) if this fails, open $OSYM/string.sym (4) if this fails, open$OSYM/string.o
Hint: if you make soft links in the OSYM directory, you can also type spacegroup numbers (e.g.: \ln -s p212121.sym 19.sym).

NOTE: you may choose to enter NCS operators either one by one, or all in one go (by putting them all in one file), or a mixture of this.

## 7 EXAMPLE

The input consists of:
- name of the input map (CCP4 format)
- name of the mask file (any MAMA format)
- name of the output map (only if the program is used for doing variance-scaled averaging and expansion)
- spacegroup symmetry operators (O format)
- file(s) with NCS operators (O format; include the unit operator; signal end of input by an empty line)

The use of the program is demonstrated using the unaveraged MIR map (2.7 Å) of P2 myelin protein:


----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Select one of the following options:

A = Analyse density for individual NCS molecules
C = Correlate density for pairs of NCS molecules
B = Both average and expand using variance scaling
Q = Quit this program

Option (A/C/S/Q) ? (A)
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----


The result for option A (note that the variance for the third, "C" molecule is quite a bit lower than that for the other two molecules):


----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Option (A/C/S/Q) ? (A) a

NCS  Aver. dens.     St. dev.     Variance      Minimum      Maximum    Sum dens.
1   2.7474E-01   1.9177E+01   3.6777E+02  -6.4726E+01   7.6205E+01   8.4767E+03
2   2.0014E-01   1.9173E+01   3.6762E+02  -7.0932E+01   7.9255E+01   6.1751E+03
3   2.1734E-01   1.6254E+01   2.6421E+02  -5.9129E+01   8.4527E+01   6.7057E+03

----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----


The result for option C (again, the first two molecules are better correlated than either is with the third molecule):


----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Option (A/C/S/Q) ? (A) c

NCSi  NCSj         RMSD  Corr.coeff. R (wrt NCSi) R (wrt NCSj)
2     1   1.5185E+01        0.686        0.624        0.623
3     1   1.4988E+01        0.653        0.715        0.609
3     2   1.5236E+01        0.641        0.731        0.624

----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----


The result for option B:


----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----
Option (A/C/S/Q) ? (C) b

Add density for operator : (          1)
Scale factor : (   1.000)
Add density for operator : (          2)
Scale factor : (   1.000)
Add density for operator : (          3)
Scale factor : (   1.392)
Average density
Zero output map
Expand for operator : (          1)
Scale factor : (   1.000)
Expand for operator : (          2)
Scale factor : (   1.000)
Expand for operator : (          3)
Scale factor : (   0.718)
Flatten solvent
Nr of points in map  : (     704000)
Nr of masked points  : (     398001)
Nr of solvent points : (     305999)
Solvent fraction (%) : (  43.466)
Average density inside masks : (  2.050E-01)
Average density in solvent   : ( -2.667E-01)
Setting background ...
Average density overall      : (  1.769E-05)

Stamp : (Created by SPANCSI V. 970523/0.2 at Fri May 23 17:56:11 1997 for
user gerard)
(Q)QOPEN allocated #  1
User:   gerard               Logical Name: p2_new.E
Status: UNKNOWN    Filename: p2_new.E

File name for output map file on unit   4 : p2_new.E
logical name p2_new.E

Minimum density in map  =      -53.21290   Maximum density         =       67.12795
Mean density            =        0.00002
Rms deviation from mean =       11.66415

Map written out
----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE ----- EXAMPLE -----


## 8 ERRORS

- if you get "severe FRCSYM errors" during the averaging step, your map probably does not encompass N asymmetric units PLUS ONE POINT ON ALL SIDES

- if you get "interpolation errors" during the expansion step, your mask probably extends too close to the border(s) of its grid (use MAMA to test and remedy this)

## 9 KNOWN BUGS

None, at present.

Created at Thu Jun 26 11:57:42 2008 by MAN2HTML version 070111/2.0.8 . This manual describes SPANCSI, a program of the Uppsala Software Factory (USF), written and maintained by Gerard Kleywegt. © 1992-2007.