[sharp-discuss] Re: Heavy Atom Negative Density
Paul Hubbard
phubbard@post.its.mcw.edu
Wed, 30 Jan 2002 09:39:05 -0600
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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
> *--------------------------------------------------------------
> * BUSTER Development Group (http://www.globalphasing.com)
> ***************************************************************
--
Paul Hubbard
Dept. of Biochemistry
Medical College of Wisconsin
Phone: 414-456 4305
Fax: 414-456 6510
URL: iris9.biochem.mcw.edu
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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.
<blockquote TYPE=CITE>How did you calculate these maps? Through the interface
or by hand
<br>(using FB/PHIB from eden.mtz)? Are these directly from SHARP or
<br>after solvent flattening?
<br> </blockquote>
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.
<blockquote TYPE=CITE>
<br>Do you get negative density on the sites (all, some?) or around the
<br>sites?
<br> </blockquote>
There is negative density around all the sites.
<blockquote TYPE=CITE>
<br>If it's electron density maps we're talking about (before or after
<br>solvent flattening): have you tried the inverted hand?
<br> </blockquote>
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?
<blockquote TYPE=CITE>
<br>The residual maps don't really use phases, so I assume that you used
<br>the residual maps (preferably ANO since it's MAD) from your single
<br>site MAD to find additional sites.
<br> </blockquote>
Yes.
<blockquote TYPE=CITE>
<br>Most important: what do the residual maps look like? Are these fairly
<br>flat?
<br> </blockquote>
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.
<blockquote TYPE=CITE>
<br>How did you treat your f'/f'' values: if from a (good) fluorescence
<br>scan you probably kept them fixed. A look at the ANO residual maps
for
<br>each wavlength then is a good test to see if these values are resonable.
<br> </blockquote>
I used the EXAFS values for inflection and peak - and table values
for in-house and remote. They were all kept fixed.
<blockquote TYPE=CITE>
<br>Cheers
<p>Clemens
<p>PS: there is even the possibility of a problem wiht your anomalous
<br>differences being (F-) - (F+) instead of (F+) - (F-) ...
<br> </blockquote>
I'll give anything a try!!!
<blockquote TYPE=CITE>
<br>--
<p>***************************************************************
<br>* Clemens Vonrhein, Ph.D.
vonrhein@GlobalPhasing.com
<br>*
<br>* Global Phasing Ltd.
<br>* Sheraton House, Castle Park Tel: +44-(0)1223-353033
<br>* Cambridge CB3 0AX, UK
Fax: +44-(0)1223-366889
<br>*--------------------------------------------------------------
<br>* BUSTER Development Group (<a href="http://www.globalphasing.com">http://www.globalphasing.com</a>)
<br>***************************************************************</blockquote>
<pre>--
Paul Hubbard
Dept. of Biochemistry
Medical College of Wisconsin
Phone: 414-456 4305
Fax: 414-456 6510
URL: iris9.biochem.mcw.edu</pre>
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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
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