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[ccp4bb]: Re: Heavy Atom Negative Density

Hi, thanks for the reply. I'll try to answer your questions as best as I can. Attached is a copy of my input file. It's setup just like the example - and I use the in-house data set as the reference. Datasets were scaled using FHSCAL (SCALIT) in CCP4.
How did you calculate these maps? Through the interface or by hand
(using FB/PHIB from eden.mtz)? Are these directly from SHARP or
after solvent flattening?
 
The maps are made in the SHARP interface without solvent flattening. I've read the documentation on maps - so know about care needed in making solvent flattened maps.
 
Do you get negative density on the sites (all, some?) or around the
sites?
 
There is negative density around all the sites.
 
If it's electron density maps we're talking about (before or after
solvent flattening): have you tried the inverted hand?
 
No - I'll give it a try. However, why then do I get nice positive density in the residual map when I use just one Iron atom? Wouldn't these be negative too?
 
The residual maps don't really use phases, so I assume that you used
the residual maps (preferably ANO since it's MAD) from your single
site MAD to find additional sites.
 
Yes.
 
Most important: what do the residual maps look like? Are these fairly
flat?
 
The residual map from the 4Fe phased data set still has alot of strong positive density round the 4Fe-4S cluster . I assumed this was from the 4 sulfurs and noise.
 
How did you treat your f'/f'' values: if from a (good) fluorescence
scan you probably kept them fixed. A look at the ANO residual maps for
each wavlength then is a good test to see if these values are resonable.
 
I used the EXAFS values for inflection and peak - and table values for in-house and remote. They were all kept fixed.
 
Cheers

Clemens

PS: there is even the possibility of a problem wiht your anomalous
differences being (F-) - (F+) instead of (F+) - (F-) ...
 

I'll give anything a try!!!
 
--

***************************************************************
* Clemens Vonrhein, Ph.D.          vonrhein@GlobalPhasing.com
*
*  Global Phasing Ltd.
*  Sheraton House, Castle Park     Tel: +44-(0)1223-353033
*  Cambridge CB3 0AX, UK           Fax: +44-(0)1223-366889
*--------------------------------------------------------------
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***************************************************************

-- 
Paul Hubbard
Dept. of Biochemistry
Medical College of Wisconsin
Phone: 414-456 4305
Fax: 414-456 6510
URL: iris9.biochem.mcw.edu
  
DATAFILES       /XXXXX/XXXXXX/sharp/sushi/base/users/paul/datafiles
LEVEL           STA
TITLE           APS DCR data ; all 4 Fe - unlocked coords
MODE            ESTIMATE REFINE RESIDUAL ELECTRON_DENSITY 
CYCLES          10 3
CEN/ACEN        ALL
1_OUT_OF        1
REJECT          YES 5
NUM_BINS        8
INTEG_MODE      ALL 
CELL            66.308 107.968 111.286 90.000 90.000 90.000
SYMMETRY        P212121
ATOMS {
 C 3200
 N 950
 O 950
 S 26
}
G-SITES {
   G-SITE-01 X  0.875003 NOREFINE Y  0.386650 NOREFINE Z  0.308084 NOREFINE
   G-SITE-02 X  0.465436 NOREFINE Y  0.204235 NOREFINE Z  0.050625 NOREFINE
   G-SITE-03 X  0.197307 NOREFINE Y  0.189428 NOREFINE Z  0.203150 NOREFINE
   G-SITE-04 X  0.582323 NOREFINE Y  0.643011 NOREFINE Z  0.172828 NOREFINE
   G-SITE-05 X  0.172700   REFINE Y  0.168400   REFINE Z  0.150900   REFINE
   G-SITE-06 X  0.158600   REFINE Y  0.154700   REFINE Z  0.133100   REFINE
   G-SITE-07 X  0.163200   REFINE Y  0.145000   REFINE Z  0.155900   REFINE
   G-SITE-08 X  0.133400   REFINE Y  0.161100   REFINE Z  0.149640   REFINE
}
COMPOUND {
   COMPOUND-TEXT {
   }
   C-SITES {
      C-SITE-01  G-SITE-05  Fe
      C-SITE-02  G-SITE-06  Fe
      C-SITE-03  G-SITE-07  Fe
      C-SITE-04  G-SITE-08  Fe
   }
   CRYSTAL {
      CRYSTAL-TEXT {
      }
      T-SITES {
         T-SITE-01 {
            C-SITE-01
            HAT_OCC           1.00000 NOREFINE
            HAT_B            17.15927   REFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
         T-SITE-02 {
            C-SITE-02
            HAT_OCC           1.00000 NOREFINE
            HAT_B            46.02629   REFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
         T-SITE-03 {
            C-SITE-03
            HAT_OCC           1.00000 NOREFINE
            HAT_B            15.49853   REFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
         T-SITE-04 {
            C-SITE-04
            HAT_OCC           1.00000 NOREFINE
            HAT_B           162.85841   REFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
      }
      WAVELENGTH {
         WAVELENGTH-TEXT {
         }
         RESOLUTION   28.483    3.000
         BATCH {
            BATCH-TEXT {
            }
            SCAL_K            0.52750 NOREFINE
            SCAL_B            0.00000 NOREFINE
            SCAL_B6_ADD       0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
            NISO_BGLO         0.00000 NOREFINE
            NISO_CLOC         0.00000 NOREFINE
            NANO_BGLO         0.16615   REFINE
            NANO_CLOC         0.46759   REFINE
            HATOM_LABEL {
               Fe    ATOM_f'  -1.17900 NOREFINE ATOM_f"   3.20400 NOREFINE 
            }
            OBSFILE dcrall.data.mtz
            COLUMNS H=H K=K L=L FMID=F_NAT SMID=SIGF_NAT DANO=SIGF_NAT SANO=SIGDANO_NAT ISYM=ISYM_NAT
         }
      }
      WAVELENGTH {
         WAVELENGTH-TEXT {
         }
         RESOLUTION   28.483    3.000
         BATCH {
            BATCH-TEXT {
            }
            SCAL_K            0.53904   REFINE
            SCAL_B           -1.41089   REFINE
            SCAL_B6_ADD       0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
            NISO_BGLO         2.79862   REFINE
            NISO_CLOC         0.02512   REFINE
            NANO_BGLO         0.26706   REFINE
            NANO_CLOC         0.19850   REFINE
            HATOM_LABEL {
               Fe    ATOM_f'  -1.17900 NOREFINE ATOM_f"   3.20400 NOREFINE 
            }
            OBSFILE dcrall.data.mtz
            COLUMNS H=H K=K L=L FMID=F_INF SMID=SIGF_INF DANO=DANO_INF SANO=SIGDANO_INF ISYM=ISYM_INF
         }
      }
      WAVELENGTH {
         WAVELENGTH-TEXT {
         }
         RESOLUTION   28.483    3.000
         BATCH {
            BATCH-TEXT {
            }
            SCAL_K            0.54345   REFINE
            SCAL_B           -1.60807   REFINE
            SCAL_B6_ADD       0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
            NISO_BGLO         3.49398   REFINE
            NISO_CLOC         0.01011   REFINE
            NANO_BGLO         0.37147   REFINE
            NANO_CLOC         0.38918   REFINE
            HATOM_LABEL {
               Fe    ATOM_f'  -1.17900 NOREFINE ATOM_f"   3.20400 NOREFINE 
            }
            OBSFILE dcrall.data.mtz
            COLUMNS H=H K=K L=L FMID=F_PK1 SMID=SIGF_PK1 DANO=DANO_PK1 SANO=SIGDANO_PK1 ISYM=ISYM_PK1
         }
      }
      WAVELENGTH {
         WAVELENGTH-TEXT {
         }
         RESOLUTION   28.483    3.000
         BATCH {
            BATCH-TEXT {
            }
            SCAL_K            0.53928   REFINE
            SCAL_B           -2.08127   REFINE
            SCAL_B6_ADD       0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
            NISO_BGLO         3.55573   REFINE
            NISO_CLOC         0.00837   REFINE
            NANO_BGLO         0.19373   REFINE
            NANO_CLOC         0.17383   REFINE
            HATOM_LABEL {
               Fe    ATOM_f'  -1.17900 NOREFINE ATOM_f"   3.20400 NOREFINE 
            }
            OBSFILE dcrall.data.mtz
            COLUMNS H=H K=K L=L FMID=F_REM SMID=SIGF_REM DANO=DANO_REM SANO=SIGDANO_REM ISYM=ISYM_REM
         }
      }
   }
}
COMPOUND {
   COMPOUND-TEXT {
      *mt*
   }
   C-SITES {
      C-SITE-01  G-SITE-01  Os
   }
   CRYSTAL {
      CRYSTAL-TEXT {
      }
      T-SITES {
         T-SITE-01 {
            C-SITE-01
            HAT_OCC           1.00000 NOREFINE
            HAT_B           299.95453   REFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
      }
      WAVELENGTH {
         WAVELENGTH-TEXT {
         }
         RESOLUTION   28.483    4.000
         BATCH {
            BATCH-TEXT {
            }
            SCAL_K            0.52377   REFINE
            SCAL_B           -0.37381   REFINE
            SCAL_B6_ADD       0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
            NISO_BGLO         0.00000   REFINE
            NISO_CLOC         0.07722   REFINE
            NANO_BGLO         0.00000 NOREFINE
            NANO_CLOC         0.00000 NOREFINE
            HATOM_LABEL {
               Os    ATOM_f'  -5.58100 NOREFINE ATOM_f"   6.22100 NOREFINE 
            }
            OBSFILE dcrall.data.mtz
            COLUMNS H=H K=K L=L FMID=F_OS1 SMID=SIGF_OS1
         }
      }
   }
}
COMPOUND {
   COMPOUND-TEXT {
      *mt*
   }
   C-SITES {
      C-SITE-01  G-SITE-02  Hg
      C-SITE-02  G-SITE-03  Hg
      C-SITE-03  G-SITE-04  Hg
   }
   CRYSTAL {
      CRYSTAL-TEXT {
      }
      T-SITES {
         T-SITE-01 {
            C-SITE-01
            HAT_OCC           1.00000 NOREFINE
            HAT_B            20.60098 NOREFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
         T-SITE-02 {
            C-SITE-02
            HAT_OCC           0.60005 NOREFINE
            HAT_B            83.48477 NOREFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
         T-SITE-03 {
            C-SITE-03
            HAT_OCC           0.64331 NOREFINE
            HAT_B            84.55530 NOREFINE
            HAT_B6_ADD        0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
         }
      }
      WAVELENGTH {
         WAVELENGTH-TEXT {
         }
         RESOLUTION   28.483    4.000
         BATCH {
            BATCH-TEXT {
            }
            SCAL_K            0.54984   REFINE
            SCAL_B           -0.59595   REFINE
            SCAL_B6_ADD       0.00000    0.00000    0.00000    0.00000    0.00000    0.00000 NOREFINE
            NISO_BGLO         5.94281   REFINE
            NISO_CLOC         0.06424   REFINE
            NANO_BGLO         0.00000 NOREFINE
            NANO_CLOC         0.00000 NOREFINE
            HATOM_LABEL {
               Hg    ATOM_f'  -4.99000 NOREFINE ATOM_f"   7.68600 NOREFINE 
            }
            OBSFILE dcrall.data.mtz
            COLUMNS H=H K=K L=L FMID=F_HG2 SMID=SIGF_HG2
         }
      }
   }
}
END