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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
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