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[ccp4bb]: REFMAC5 and heme
hi,
i am trying to use the latest version of REFMAC5 for a protein which
contains a heme. i used residue name "HEM", and even corrected the atom
names of "FE ", " N_A", " N_B", " N_C" and " N_D" to match the PDB
standard, but refmac tells me that it
does not recognise my ligand. it says my ligand resembles existing entry
"MHM" (file mon_lib_10.cif) and that it will create a new dictionary entry
for me based on that. i don't know why it doesn't recognise my heme,
which is surely a commonly used ligand, except perhaps that the dictionary
entry (defined in file mon_lib_1.cif) contains hydrogens while my
coordinates do not.
i took a look at the documentation and there was a lot about creating new
dictionary entries by various means (sketcher, etc) but i didn't notice
anything about using existing dictionary entries, so that i guessed it
should happen automatically.(?)
i would like to know
a) why REFMAC does not recognise my ligand as "HEM"
b) should i try to force it to recognise my heme and
c) how would i go about that?
attached are my REFMAC output, my HEM coordinates, and my script.
thank you,
=======================================================================
"Politicians can survive sex scandals or fiscal mismanagement,
but they cannot survive being laughed at." - Robert Park
=======================================================================
David J. Schuller
modern man in a post-modern world
University of California-Irvine
schuller@uci.edu
<!--SUMMARY_BEGIN-->
1##########################################################
##########################################################
##########################################################
### CCP PROGRAM SUITE: Refmac_5.0 VERSION 4.0: 14/07/00##
##########################################################
User: schuller Run date: 26/10/00 Run time:18:26:55
Please reference: Collaborative Computational Project, Number 4. 1994.
"The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 760-763.
as well as any specific reference in the program write-up.
<!--SUMMARY_END-->
$TEXT:Reference1: $$ comment $$
"Refinement of Macromolecular Structures by the Maximum-Likelihood Method:"
G.N. Murshudov, A.A.Vagin and E.J.Dodson,(1997)
Acta Crystallogr. D53, 240-255
EU Validation contract: BIO2CT-92-0524
$$
$SUMMARY :Reference1: $$ Refmac: $$
:TEXT:Reference1: $$
Comment line--- #
Data line--- LABIN FP=FP11 SIGFP=SIGFP11 FREE=FreeR_flag
Data line--- LABO FC=FC PHIC=PHIC FWT=2FOFCWT PHWT=PH2FOFCWT DELFWT=FOFCWT PHDELWT=PHFOFCWT
Data line--- MAKE HYDR Y
Data line--- REFI TYPE REST
Data line--- REFI RESI MLKF
Data line--- REFI BREF MIXED
Comment line--- #
Comment line--- #
Data line--- SCAL TYPE BULK
Data line--- NCYC 5
Data line--- MONI FEW
Data line--- end
<!--SUMMARY_BEGIN-->
(Q)QOPEN allocated # 1
User: schuller Logical Name: HKLIN
Status: READONLY Filename: ../../mtz/gn11_free.mtz
<!--SUMMARY_END-->
* Input Program Labels :
H K L FP SIGFP FREE FPART1 PHIP1 FPART2 PHIP2 FPART3 PHIP3 HLA HLB HLC HLD FOM
PHIB
* Input File Labels :
H K L FP11 SIGFP11 dum2 FreeR_flag
* Lookup Table : the number indicates the input column no.
* Array element n corresponds to the nth program label
1 2 3 4 5 7 0 0 0 0 0 0 0 0 0
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
0 0 0 0
**** Input and Default parameters# ****
Input coordinate file. Logical name - XYZIN actual file name - start.pdb
Output coordinate file. Logical name - XYZOUT actual file name - refm01.pdb
Input reflection file. Logical name - HKLIN actual file name - ../../mtz/gn11_free.mtz
Output reflection file. Logical name - HKLOUT actual file name - refm01.mtz
Cell from mtz : 63.167 63.167 100.378 90.000 90.000 90.000
Space group from mtz: number - 96; name - P43212
Refinement type : Restrained
**** Makecif parameters ****
Dictionary files for restraints : /usr/share/local/ccp4_master/refmac5/dic/mon*cif
Parameters for new entry and VDW: /usr/share/local/ccp4_master/refmac5/dic/ener_lib.cif
Apart from amino acids and DNA/RNA all monomers will be checked to see if atom names and connectivity is correct
hydrogens in input coordinate file will be kept
Symmetry related links be analysed and used
Cis peptides will be found and used automatically
Residual : Rice Maximum Likelihood for Fs
**** Least-square scaling parameters ****
Overall scale
Overall B value
Overall anisotropic B with tr(B) = 0.0
Bulk solvent based on Babinet"s principle
Constant bulk solvent in non protein region
Probe radii for non-ions 1.400
Probe radii for ions 0.800
Shrinkage of the mask by 0.800
Method of minimisation : Sparse Matrix
Experimental sigmas used for weighting
Number of Bins and width: 20 0.0222
Refinement of mixed isotropic and anisotropic Bfactors
Refinement resln : 53.4522 1.4997
Estimated number of reflections : 39642
Free R exclusion - flag equals: 0
Weighting by comparison of trace of matrix
Weighting parameters : 0.5000
Refinement cycles : 5
Scaling type :
Bulk solvent using using Babinet principle
using working set of reflns but not experimental sigmas
Estimation of SigmaA Using 2 Gaussians:
using free set of reflns with experimental sigmas
using free set of reflns with experimental sigmas
Scaling and SigmaA resln: 53.4522 1.4997
Damping factors: 1.0000 1.0000
**** Geometry restraints and weights ****
Sigma:
Bonding distances
Weight = 1.00
Bond angles
Weight = 1.00
Planar groups
WEIGHT= 1.00
Chiral centers
Weight= 1.00
NON-BONDED CONTACTS
Overall weight = 1.00
Sigma for simple VDW = 0.30
Sigma for VDW trhough torsion angle = 0.50
Sigma for HBOND = 0.50
Sigma for metal-ion = 0.50
Sigma for DUMMY and other atom = 0.30
Distance for donor-accepetor = vdw1+vdw2+(-0.30)
Distance for acceptor - H = vdw1+vdw2+( 0.20)
VDW distance through torsion = vdw1+vdw2+(-0.75)
Distance for DUMMY-others = vdw1+vdw2+(-0.70)
TORSION ANGLES
Weight= 1.00
THERMAL FACTORS
Weight= 1.00
Main chain bond (1-2 neighbour) 1.50A**2
Main chain angle (1-3 neighbour) 2.00A**2
Side chain bond 3.00A**2
Side chain angle 4.50A**2
RESTRAINTS AGAINST EXCESSIVE SHIFTS
Positional parameters 0.03A
Thermal parameters 0.03A
Occupancy parameters 0.20A
Monitoring style is "FEW". Minimum information will be printed out
---------------------------------------------------------------
Input file : start.pdb
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
------------------------------
--- LIBRARY OF MONOMERS ---
_lib_name mon_lib
_lib_version 3.1
_lib_update 16/07/00
------------------------------
NUMBER OF MONOMERS IN THE LIBRARY : 2406
with complete description : 335
NUMBER OF MODIFICATIONS : 37
NUMBER OF LINKS : 62
I am reading libraries. Please wait.
- energy parameters
- monomer"s description (links & mod )
FORMATTED OLD file opened on unit 45
Logical name: ATOMSF, Full name: /usr/share/local/ccp4_master/ccp4/lib/data/atomsf.lib
FORMATTED OLD file opened on unit 8
Logical name: SYMOP, Full name: /usr/share/local/ccp4_master/ccp4/lib/data/symop.lib
WARNING: Chain_list is not correct. Program changed it.
Number of atoms : 1812
Number of residues : 391
Number of chaines : 3
I am reading library. Please wait.
mon_lib.cif
WARNING : monomer looks like MHM program will use this one
WARNING : monomer has the minimal description
program will create complete description
WARNING : MHM : default angle value will used
chem_type : C -O -H 120.000 (CGD -O2D -DHO2)
WARNING : MHM : default bond legnth will used
chem_type: H -O 0.890 (DHO2-O2D )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -H 120.000 (C4D -CHA -HHA )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -CR5 120.000 (C4D -CHA -C1A )
WARNING : MHM : default angle value will used
chem_type : H -C -CR5 120.000 (HHA -CHA -C1A )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HHA -CHA )
WARNING : MHM : default angle value will used
chem_type : C -O -H 120.000 (CGA -O1A -AHO1)
WARNING : MHM : default bond legnth will used
chem_type: H -O 0.890 (AHO1-O1A )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -CR5 108.000 (C4D -N_D -C1D )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -FE 108.000 (C4D -N_D -FE )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -FE 108.000 (C1D -N_D -FE )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -H 120.000 (C1D -CHD -HHD )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -CR5 120.000 (C1D -CHD -C4C )
WARNING : MHM : default angle value will used
chem_type : H -C -CR5 120.000 (HHD -CHD -C4C )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HHD -CHD )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -H 120.000 (C3C -CAC -HAC )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -C 120.000 (C3C -CAC -CBC )
WARNING : MHM : default angle value will used
chem_type : H -C -C 120.000 (HAC -CAC -CBC )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HAC -CAC )
WARNING : MHM : default angle value will used
chem_type : C -C -H 120.000 (CAC -CBC -HBC2)
WARNING : MHM : default angle value will used
chem_type : C -C -H 120.000 (CAC -CBC -HBC )
WARNING : MHM : default angle value will used
chem_type : H -C -H 120.000 (HBC2-CBC -HBC )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HBC2-CBC )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HBC -CBC )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -FE 108.000 (C1C -N_C -FE )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -CR5 108.000 (C1C -N_C -C4C )
WARNING : MHM : default angle value will used
chem_type : FE -NR5 -CR5 108.000 (FE -N_C -C4C )
WARNING : MHM : default angle value will used
chem_type : NR5 -FE -NR5 180.000 (N_C -FE -N_A )
WARNING : MHM : default angle value will used
chem_type : NR5 -FE -NR5 90.000 (N_C -FE -N_B )
WARNING : MHM : default angle value will used
chem_type : NR5 -FE -NR5 90.000 (N_C -FE -N_D )
WARNING : MHM : default angle value will used
chem_type : NR5 -FE -NR5 90.000 (N_A -FE -N_B )
WARNING : MHM : default angle value will used
chem_type : NR5 -FE -NR5 90.000 (N_A -FE -N_D )
WARNING : MHM : default angle value will used
chem_type : NR5 -FE -NR5 180.000 (N_B -FE -N_D )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -H 120.000 (C1C -CHC -HHC )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -CR5 120.000 (C1C -CHC -C4B )
WARNING : MHM : default angle value will used
chem_type : H -C -CR5 120.000 (HHC -CHC -C4B )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HHC -CHC )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -CR5 108.000 (C4B -N_B -C1B )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -FE 108.000 (C4B -N_B -FE )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -FE 108.000 (C1B -N_B -FE )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -H 120.000 (C3B -CAB -HAB )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -C 120.000 (C3B -CAB -CBB )
WARNING : MHM : default angle value will used
chem_type : H -C -C 120.000 (HAB -CAB -CBB )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HAB -CAB )
WARNING : MHM : default angle value will used
chem_type : C -C -H 120.000 (CAB -CBB -HBB2)
WARNING : MHM : default angle value will used
chem_type : C -C -H 120.000 (CAB -CBB -HBB )
WARNING : MHM : default angle value will used
chem_type : H -C -H 120.000 (HBB2-CBB -HBB )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HBB2-CBB )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HBB -CBB )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -H 120.000 (C1B -CHB -HHB )
WARNING : MHM : default angle value will used
chem_type : CR5 -C -CR5 120.000 (C1B -CHB -C4A )
WARNING : MHM : default angle value will used
chem_type : H -C -CR5 120.000 (HHB -CHB -C4A )
WARNING : MHM : default bond legnth will used
chem_type: H -C 1.015 (HHB -CHB )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -FE 108.000 (C4A -N_A -FE )
WARNING : MHM : default angle value will used
chem_type : CR5 -NR5 -CR5 108.000 (C4A -N_A -C1A )
WARNING : MHM : default angle value will used
chem_type : FE -NR5 -CR5 108.000 (FE -N_A -C1A )
* Plotfile: /var/tmp/refmac5_temp.04504_new_MHM.ps
INFO: connection is found (not be used) dist= 2.054 ideal_dist= 1.935
ch:XX res: 23 HIS at:NE2 --> ch:Xa res: 300 HEM at:FE
--------------------------------
--- title of input coord file ---
PDB_code:xxxx
PDB_name:----
PDB_date:XX-XXX-XX
--------------------------------
FORMATTED OLD file opened on unit 8
Logical name: SYMOP, Full name: /usr/share/local/ccp4_master/ccp4/lib/data/symop.lib
Number of chains : 3
Total number of monomers : 391
Number of atoms : 1812
Number of missing atoms : 0
Number of rebuilding atoms : 0
Number of unknoun atoms : 0
Number of deleted atoms : 0
Number of bonds restraints : 1669
Number of angles restraints : 2272
Number of torsions restraints : 1174
Number of chiralities : 230
Number of planar groups : 294
I am writing new library:
new.lib
Important, important important!!!!!
Your coordinate file has ligand which either have minimum or no description
New ligand description has been added to new.lib
Picture of new ligand can be viewd using postscript file. See above
Check description in that file and if satisfied use it as input library
If not satisfied either change atom types accordingly and rerun refmac or use libcheck
See documentation "how to make new ligand" section for a help
It is strongly recommended that dictionary entry should be checked carfully before using it
Best way of creating dictionary entry is using sketcher whcih is part of ccp4i
If you are sure in the library description then use keyword (MAKE CHECK 0)
I.e. do not check correctness of the coordinates
===> Error: New ligand has been encountered. Stopping now
<!--SUMMARY_BEGIN-->
Refmac_5.0.27: New ligand has been encountered. Stopping now
Times: User: 15.6s System: 0.5s Elapsed: 0:16
<!--SUMMARY_END-->
ATOM 1580 FE HEM 300 14.255 7.860 19.858 1.000 20.79
ATOM 1581 N_A HEM 300 13.553 8.414 18.096 1.000 22.98
ATOM 1582 N_B HEM 300 12.525 7.036 20.336 1.000 20.26
ATOM 1583 N_C HEM 300 14.879 7.624 21.736 1.000 16.67
ATOM 1584 N_D HEM 300 15.939 8.811 19.434 1.000 20.59
ATOM 1585 C1A HEM 300 14.119 9.241 17.133 1.000 23.72
ATOM 1586 CHA HEM 300 15.481 9.537 17.115 1.000 24.14
ATOM 1587 C4D HEM 300 16.334 9.303 18.203 1.000 21.66
ATOM 1588 C1B HEM 300 11.372 7.006 19.567 1.000 23.83
ATOM 1589 CHB HEM 300 11.253 7.601 18.309 1.000 20.76
ATOM 1590 C4A HEM 300 12.343 8.016 17.553 1.000 22.91
ATOM 1591 C1C HEM 300 14.125 7.192 22.808 1.000 20.77
ATOM 1592 CHC HEM 300 12.901 6.535 22.709 1.000 24.48
ATOM 1593 C4B HEM 300 12.135 6.457 21.532 1.000 21.54
ATOM 1594 C1D HEM 300 17.090 8.878 20.205 1.000 19.95
ATOM 1595 CHD HEM 300 17.175 8.462 21.527 1.000 16.27
ATOM 1596 C4C HEM 300 16.084 8.013 22.292 1.000 18.51
ATOM 1597 C2A HEM 300 13.252 9.350 15.980 1.000 24.63
ATOM 1598 CAA HEM 300 13.390 10.278 14.780 1.000 22.74
ATOM 1599 C3A HEM 300 12.166 8.579 16.230 1.000 25.64
ATOM 1600 CMA HEM 300 10.820 8.572 15.514 1.000 28.80
ATOM 1601 CBA HEM 300 12.899 11.701 14.981 1.000 28.80
ATOM 1602 CGA HEM 300 13.158 12.564 13.754 1.000 32.61
ATOM 1603 O1A HEM 300 14.094 12.218 12.999 1.000 36.32
ATOM 1604 O2A HEM 300 12.429 13.563 13.558 1.000 33.95
ATOM 1605 C2B HEM 300 10.295 6.331 20.244 1.000 20.68
ATOM 1606 CMB HEM 300 8.980 5.934 19.567 1.000 27.93
ATOM 1607 C3B HEM 300 10.755 6.018 21.473 1.000 23.64
ATOM 1608 CAB HEM 300 10.042 5.446 22.519 1.000 23.62
ATOM 1609 CBB HEM 300 9.058 4.479 22.524 1.000 21.46
ATOM 1610 C2C HEM 300 14.926 7.105 24.009 1.000 19.04
ATOM 1611 CMC HEM 300 14.331 6.775 25.382 1.000 20.22
ATOM 1612 C3C HEM 300 16.134 7.612 23.681 1.000 19.84
ATOM 1613 CAC HEM 300 17.310 7.684 24.424 1.000 21.83
ATOM 1614 CBC HEM 300 17.528 7.477 25.761 1.000 26.87
ATOM 1615 C2D HEM 300 18.180 9.487 19.483 1.000 25.25
ATOM 1616 CMD HEM 300 19.574 9.790 20.027 1.000 21.36
ATOM 1617 C3D HEM 300 17.699 9.745 18.246 1.000 23.92
ATOM 1618 CAD HEM 300 18.373 10.537 17.144 1.000 27.29
ATOM 1619 CBD HEM 300 18.770 9.706 15.922 1.000 23.29
ATOM 1620 CGD HEM 300 19.599 10.563 14.975 1.000 30.36
ATOM 1621 O1D HEM 300 20.826 10.645 15.168 1.000 23.65
ATOM 1622 O2D HEM 300 18.994 11.155 14.063 1.000 29.19
#!/bin/csh -f
#
refmac5 \
HKLIN ../../mtz/data_free.mtz \
HKLOUT refm01.mtz \
XYZIN start.pdb \
XYZOUT refm01.pdb \
>refmac.out \
<<EOF
#
LABIN FP=FP11 SIGFP=SIGFP11 FREE=FreeR_flag
LABO FC=FC PHIC=PHIC FWT=2FOFCWT PHWT=PH2FOFCWT -
DELFWT=FOFCWT PHDELWT=PHFOFCWT
MAKE HYDR Y
REFI TYPE REST
REFI RESI MLKF
REFI BREF MIXED
#
#
SCAL TYPE BULK
NCYC 5
MONI FEW
end
EOF
#
exit