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[ccp4bb]: Estimated error or occupancy (again)



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Hi all,

First, thank you for the previous suggestions about calculating error
estimates for occupancy values.  We are trying a couple of the suggested
methods, and I am having some trouble with the method suggested by
Eleanor Dodson, which uses Refmac5 and Mlphare. 

I am having trouble getting mlphare to run, and I suspect that I am
making some mistake in the input. I hope that someone can see the
problem and help me to correct it.  I am only slightly familiar with
Refmac5, and not at all familiar with mlphare, (although I have done a
little MIR work with a different program).  Anyway, in trying to follow
the procedure that was described, I believe that I have successfully run
refmac5 on the protein with the ion removed, and created the HA file for
the ion coordinates. (I actually have >10 different datasets on which to
try this...I've only tried one of them so far.)  The mlphare step seems
to be the problem.

In the ccp4i mlphare setup window, I have chosen "Add phase info from
externally calculated phases", "Use centric data only", and FC=FC,
SigmaFP=Unassigned, FPH1=FP, SIGFPH1=SigFP, PHIC=PHIC, Weight=FOM, and
FC=Unassigned.  Is this right?

When I run mlphare, this is what I see at the end of the output file: 

    *********************************************************
    *               START OF MLPHARE PROCEDURE              *
    *********************************************************


 Title : "Run MLPHARE to refine Calcium occupancy in pelc 955 dataset"


 ====== CYCLE   1
===========================================================

  Compound  1
  -----------
  Rms isomorphous error:
          0.00     0.00     0.00     0.00     0.00     0.00     0.00
0.00
 check       0.00      2.00      7.00
 Reflection HKL:   0   2   7 has inconsistent phase likelihood, log
Pmax=  0.18E+05
 check       0.00      3.00     12.00
 Reflection HKL:   0   3  12 has inconsistent phase likelihood, log
Pmax=  0.12E+05
 check       0.00      4.00      5.00
 Reflection HKL:   0   4   5 has inconsistent phase likelihood, log
Pmax=  0.24E+05
 check       0.00      7.00      7.00
 Reflection HKL:   0   7   7 has inconsistent phase likelihood, log
Pmax=  0.36E+05
 check       2.00      0.00      7.00
 Reflection HKL:   2   0   7 has inconsistent phase likelihood, log
Pmax=  0.74E+05
 check       5.00      1.00      0.00
 Reflection HKL:   5   1   0 has inconsistent phase likelihood, log
Pmax=  0.13E+05
 check       6.00      0.00      3.00
 Reflection HKL:   6   0   3 has inconsistent phase likelihood, log
Pmax=  0.11E+05
 check       8.00      7.00      0.00
 Reflection HKL:   8   7   0 has inconsistent phase likelihood, log
Pmax=  0.12E+05

  *** Finished refinement cycle   1
  5032 reflections were rejected for refinement
 **** IN SUBROUTINE MATSOL: DET IS LESS THAN 10E-30****
 **** Possible causes: *****
 You have an atom on a special position where one parameter must be
fixed?;

 You have not fixed one  coordinate  in a polar spacegroup? eg: one XYZ
in P1, one Y in P21; one Z in P3

 You are refining a property whch is indeterminate?
  eg: AX or AOCC without setting DPH; AX when AOCC equals zero
<B><FONT COLOR="#FF0000"><!--SUMMARY_BEGIN-->
 MLPHARE:   Execution Halted 30
============================================================================

I also see these strange characters listed for program and file labels:

 * Output Program Labels :
 
 H K L FC O?ÇÃO?ÇÃO?ÇÃO?ÇÃO?ÇÃO?ÇÃO?ÇÃO? PHIB FOM FP SIGFP PHIC WC
FreeRflag FWT
 PHWT DELFWT PHDELWT
 
 * Output File Labels :
 
 H K L FC O?ÇÃO?ÇÃO?ÇÃO?ÇÃO?ÇÃO?ÇÃO?ÇÃO? PHIB_mlphare1 FOM_mlphare1 FP
SIGFP PHIC
 WC FreeRflag FWT PHWT DELFWT PHDELWT
============================================================================

Here is the input script, (commands copied from the ccp4i window):

#!/bin/sh

mlphare HKLIN /home/heffron/pels/ccp4/955_wil_cnsfreer_refmac1.mtz \
HKLOUT /home/heffron/pels/ccp4/955_wil_cnsfreer_mlphare6.mtz \
<< END-mlph >> mlphare6_pelc_955.log
title Run MLPHARE to refine ion occupancy in pelc 955 dataset
labin  FP=FC -
   FPH1=FP SIGFPH1=SIGFP -
   PHIC=PHIC WC=FOM
labout -
    ALLIN -
    PHIB = PHIB_mlphare1 -
    FOM = FOM_mlphare1
cycle 10
CENTRIC
print AVE
deriv CA+2
dcycle -
    phase all -
    refcyc all -
    kbov all
ATOM  CA+2   0.38223   0.26070  -0.06327    0.89 0.0 BFAC     16.210
atref -
     X ALL  Y ALL  Z ALL -
     OCC ALL
RSIZE 80
USECWD
end
END-mlph

Sorry for this lengthy message... Any help you can give will be greatly
appreciated!  BTW, I'm running CCP4 version 4.1.1 of mlphare using a
script, because the recently installed version 4.2 exits with a memory
fault for this particular program.  My ccp4i is the 4.2 version.

                            Susan Heffron


"Eleanor J. Dodson" wrote:
> 
> ***  For details on how to be removed from this list visit the  ***
> ***          CCP4 home page http://www.ccp4.ac.uk         ***
> 
> >
> >  I have done this with MLPHARE - you run REFMAC5 for the structure
> > without the ions, and that outputs  FC/PHIC and FOM;
> >
> > Then you can run mlphare
> >  LABI FP=FC PHIB=PHIC FOM=FOM FPH=Fobs SIGFPH=SIGFobs  ( Maybe you
have
> > to assign a SIGFP too, but it can be a dummy SIGFP=DELFWT or
something..
> >
> > Then the "heavy" atoms are the ions, and you need only refine the
> > occupancies against the centric data. You will get and SD as well..
> > You can us the the "convert coordinate utilities to chanfge the
ions in
> > the pdb to the ha format used by mlphare.
> > Eleanor
> >  It is pretty reliable I think - good enough for a reviewer!
> > Eleanor
> 
>  After reading all the other contributions I will extend this a bit.
>  1) MLPHARE is a full matrix method, you will refine the ions without
> restraints, so the SDs are quite reliable
>  2) Since you only need an SD for the ion occupancy, amd maybe
> (anisotropic?) B factor if the resolution allows both occupancy and
Bs
> to be refined. you only need limited resolution
> (If the sites are very anisotropic you may need to refine xyz, but
prob.
> not..)
> 
>  You do need a decent observation/parameter ratio, but by restricting
> the refinement to the ions alone you will have this at lowish
> resolution, and in many space groups using the centric data alone..
> 
> 3) Partitioned refinement like this (ie taking the protein as fixed
and
> refining ions alone)  is quite acceptable practice.
> 
> If you have an anomalous signal this refinement could be carried out
for
> the ions ( and any other anom scatters) alone. However that depends
on
> the accuracy of the ano diffs..
> 
> Eleanor

-- 
------------------------------------------------------------------
        Susan Heffron
Dept. of Physiology and Biophysics                               
University of California, Irvine                
 Irvine, CA  92697-4560   U.S.A.                         
    phone:  (949) 824-4625
    FAX:    (949) 824-8540
------------------------------------------------------------------