Re: [ccp4bb] Tools for measuring RMSD of entire molecule when a single domain is aligned

[Sabine Schneider]

Hi Kyle

DynDom gives you the angle of the relative domain movement, which I also 
find a good quantitative measure.


http://dyndom.cmp.uea.ac.uk/dyndom/runDyndom.jsp

Best Sabine

On 12/11/2021 15:13, Edwin Pozharski wrote:
https://pymolwiki.org/index.php/Rms_cur 



On Fri, Nov 12, 2021, 9:05 AM Kyle Gregory 
<3632e92fcc15-dmarc-requ...@jiscmail.ac.uk 
> wrote:


Dear CCP4 bulletin board,

I was wondering if there are any tools to determine the RMSD of
the entire molecule (two domains) when only aligning one domain,
the point i'm trying to make is a variation in domain positioning
relative to one another but I'd like to quantify it. LSQ alignment
in coot reports RMSD for the aligned porition only, as does the
alignment tools on ccp4 cloud (unless i've missed a feature)

Any advice would be greatly appreciated.

Kind regards,
Kyle



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[ccp4bb] cell discrepancies and stuck refinement using different XDS-versions

[Sabine Schneider]

Dear all,

We are encountering a differences in indexing using different XDS 
versions, which results in either 2 or 4 mols/asu (SG P1; structure 
determination via MR (30% seq ID model)) and either successful 
refinement or stuck R/Rfree


- the data were collected at ESRF ID30A3 (Aiger detector) and extend to 
about 2.3A resolution (CC1/2 ~50%)


The XDS version build 20180126 (and or autoprocessing at the ESRF via 
autoproc, dials, xdsapp or grenade -> here also xds version from 
20180126 used) gives us:


P1  38.65    50.76    61.11 110.057  99.945  90.197
XDS complains and I need to use "DEFPIX INTEGRATE CORRECT"
-> 2mols/asu, structure refines to R/Rfree of 22/25

In contrast the actual XDS-Version BUILT=20180409 results in:
P1  39.2   51.3  116.8  86.3  82.3  89.8
but XDS runs smoothly.
-> 4mols/asu, tNCS, R/Rfree stuck at 28/32
If I feed XDS with the smaller cell above, it fails.

(The smaller cell is also found by Xia2/dials via the CCP4i2 interface.)

Thus I am wondering, what are the differences between the two 
XDS-versions? (I remember vaguely that there was a tread about different 
XDS-versions, but couldn't find it..)


Cheers Sabine


--
------
Dr. Sabine Schneider
Research Group Leader
Technical University of Munich
Department of Chemistry
Chair of Biochemistry
Lichtenbergstr. 4
85748 Garching
Germany
Tel.: +49 (0) 89 289 13759
Fax: +49 (0) 89 289 13363
http://www.biochemie.ch.tum.de/index.php?id=919

--
------
Dr. Sabine Schneider
Research Group Leader
Technical University of Munich
Department of Chemistry
Chair of Biochemistry
Lichtenbergstr. 4
85748 Garching
Germany
Tel.: +49 (0) 89 289 13759
Fax: +49 (0) 89 289 13363
http://www.biochemie.ch.tum.de/index.php?id=919



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Re: [ccp4bb] Problem with lattice disorder / tNCS / whatever - SOLVED!

[Sabine Schneider]

Hello everyone,

Maybe of interest/help to others:
The solution of my problem is a totally unexpected structural change of 
2 out of 4 protein molecules in the asu! The straight forward solution 
was additionally obscured through the presence of tNCS.


What I did:
- placed as many molecules as possible in P1 using phaser
- deleted clashing regions (pretty much half of half of the proteins in 
the asu!)
- run refmac, corrected the model and build as many residues as possible 
in the residual density, etc
-> here it became apparent that the loop of a beta hairpin structure 
shifted, (the loop became now the beta-strand!!!) with a cystein forming 
a disulfide bond with the neighbouring, short protein! And one could see 
that a two-fold axis has to run directly through that region.
- Than I run phaser again, with the orthorombic cell. First I let it 
place two full proteins and in the second round I gave it the short 
protein as search model (with the .sol file from the first run)

- cycling through building in the residual density, refinement etc...

So the symmetry is apparently P2 21 21, with two protein mols folded as 
expected and two more mols, where almost half of the residues of the 
protein go somewhere else, with a shift in a beta-hairpin and disulfide 
bond formation with the now symmetry related molecule! (despite heaps of 
reducing agent in the buffer...)


Sabine

(I also gave the initial P1 solution to Phenix autobuild and Buccaneer, 
but I didn't do much tweaking here, and in the meantime I'd figured it 
out by hand)





Hello everybody,

I got a problem with solving and refining a structure of a small protein
of 20kDa, where we just swapped two residue at the surface by mutation
(AF vs FA).

For the FA variant we got three crystal forms, where we had no problem
whatsoever with MR and refinement
- P6522 cell 63 63 184 ~2.9A
- P21212 cell: 53 62 109 ~3A
- P212121 cell 40 43 96 ~1.8A

However with the AF-variant we only got one crystal (out of 50) that
diffracted reasonable well to ~2.4A at the synchrotron. And now the
problem starts: initially the data appear to be orthorhombic, but:

- SG18/19: cell 59 91 109 (I noticed that the b-axis is longer by 1/2b
compared to the other variant?!)
-> running phaser checking all possible alternative SGs found initally 2
mols/AUS in SG18, but since there was still space (and density!) I let
it search for another one (increased the clash tolerance)
SOLU SET RFZ=7.7 TFZ=11.8 PAK=0 LLG=141 TFZ==13.9 RFZ=3.0 TFZ=11.2 PAK=0
LLG=280 TFZ==17.1 LLG=288 TFZ==17.3 RFZ=5.9 TFZ=16.0 PAK=16 LLG=979
TFZ==15.3 LLG=1479 TFZ==17.4

- However the third molecule than clashes partly

- running refmac: R/Rfree stays in the high 40

- analysing the data with Xtriage indicates tNCS

- I also tried P212121, P21 and P1, always with the same result: space
as well as density between the proteins; placing more protein molecules
results in clashes (i.e. refinement in P1 gives R/Rfree of 38/41)

- data were processed with XDS as well as Dials/Aimless

- the protein is generally well folded, quite stable, without any loose
ends or flexible regions

- calculation of selfrotation functions in P21 and P21221 gives peak at
90dg (kappa=180dg)

Anyone an idea what could be going on or how to solve that?


Thanks a lot in advance!

Sabine

Re: [ccp4bb] Problem with lattice disorder / tNCS / whatever ?!

[Sabine Schneider]

. -5.00 -0.00  0.00
   57 -0.0025795 0.0119525-0.0241882 2025.  2.00 -1.00  1.00
   58  0.0396384-0.0041547 0.0204763 2025. -1.00 4.00  0.00
   59  0.0196131-0.0152142 0.0141121 2020. -2.00 2.00 -0.00
   60 -0.0241198 0.0076707 0.0246372 2014. -0.00 -1.00 -2.00

 PARAMETERS OF THE REDUCED CELL (ANGSTROEM & DEGREES)
 59.59 91.21109.44 90.03 90.05 90.09

   #  COORDINATES OF REC. BASIS VECTORREDUCED CELL INDICES

1   0.0001394 0.0087644-0.0025818 0.00   -0.00   -1.00
2   0.0099426 0.0011533 0.0044729 0.001.000.00
3   0.0070894-0.0044146-0.0145563 1.00   -0.000.00



  LATTICE-  BRAVAIS-   QUALITY  UNIT CELL CONSTANTS (ANGSTROEM & DEGREES)
 CHARACTER  LATTICE OF FIT  a  b  c   alpha  beta gamma

 *  44aP  0.0  59.9   91.6  110.0  90.0  90.0 90.0
 *  31aP  0.4  59.9   91.6  110.0  90.0  90.0 90.0
 *  33mP  0.9  59.9   91.6  110.0  90.0  90.0 90.0
 *  34mP  1.1  59.9  110.0   91.6  90.0  90.0 90.0
 *  35mP  1.2  91.6   59.9  110.0  90.0  90.0 90.0
 *  32oP  1.6  59.9   91.6  110.0  90.0  90.0 90.0
37mC249.7 227.9   59.9   91.6  90.0  90.0 74.8
36oC250.1  59.9  227.9   91.6  90.0  90.0 105.2
39mC250.3 192.7   59.9  110.0  90.0  90.0 71.9
28mC250.3  59.9  227.9   91.6  90.0  90.0 74.8
38oC250.7  59.9  192.7  110.0  90.0  90.0 108.1


------
Dr. Sabine Schneider
Research Group Leader
Technical University of Munich
Department of Chemistry
Chair of Biochemistry
Lichtenbergstr. 4
85748 Garching
Germany
Tel.: +49 (0) 89 289 13336
Fax: +49 (0) 89 289 13363
http://www.biochemie.ch.tum.de/index.php?id=919

On 11/17/2016 08:56 AM, Tim Gruene wrote:

Hi Sabine,

your reduced cell angles are exactly 90 degree. This seems unusual to me. Do
you specify the unit cell constants in XDS.INP, including a space group, or do
you leave the space group undefined? In the former case I am unsure if your
list of clusters may be biased, although it might contain half integers in
that case.

Best,
Tim

On Thursday, November 17, 2016 08:37:07 AM Sabine Schneider wrote:

Hello Kay,

I'd say yes, but I might miss something.

Here is the IDXREF.LP output:

Sabine


   * DETERMINATION OF THE RECIPROCAL LATTICE BASIS **
   NUMBER OF DIFFERENCE VECTOR CLUSTERS USED 200
   MAXIMUM RADIUS OF DIFFERENCE VECTOR CLUSTERS (pixels)   3
   MINIMUM DISTANCE BETWEEN DIFFRACTION SPOTS (pixel)6.0
   MINIMUM ALLOWED DISTANCE BETWEEN REC. LATTICE POINTS 0.2682E-02
   OBSERVED BASIS CELL VOLUME 0.6021E+06
   DIMENSION OF SPACE SPANNED BY DIFFERENCE VECTOR CLUSTERS   3

 #  COORDINATES OF REC. BASIS VECTORLENGTH   1/LENGTH

  1   0.0070481-0.0043879-0.0144998  0.0167084  59.85
  2   0.0099022 0.0011565 0.0044633  0.0109230  91.55
  3  -0.0001404-0.0087276 0.0025729  0.0091000 109.89

   CLUSTER COORDINATES AND INDICES WITH RESPECT TO REC. LATTICE BASIS VECTORS

 #  COORDINATES OF VECTOR CLUSTER   FREQUENCY   CLUSTER INDICES
  1 -0.0198775-0.0023086-0.0089462 3033. -0.00 -2.01 -0.00
  2  0.0170296-0.0032635-0.0100896 2931.  1.01 1.00  0.00
  3 -0.0028512-0.0055653-0.0190257 2769.  1.00 -1.00  0.00
  4  0.0001345 0.0087600-0.0025802 2756. -0.00 -0.00 -1.00
  5 -0.0099406-0.0011518-0.0044669 2748. -0.00 -1.00 -0.00
  6  0.0369226-0.0009571-0.0011409 2572.  1.01 3.01  0.00
  7 -0.0027157 0.0031989-0.0215974 2517.  1.00 -1.00 -1.00
  8  0.0102242 0.0186700-0.0006904 2503.  0.00 1.00 -2.01
  9 -0.0098018 0.0076072-0.0070529 2471. -0.00 -1.00 -1.00
 10  0.0096649-0.0163634 0.0096361 2465.  0.00 1.00  2.01
 11 -0.0171648-0.0055039 0.0126657 2460. -1.00 -1.00  1.00
 12 -0.0070927 0.0044138 0.0145582 2456. -1.00 -0.00 -0.00
 13  0.0072273 0.0043496-0.0171271 2446.  1.00 0.00 -1.00
 14  0.0068016-0.0219329-0.0093878 2437.  1.00 0.00  2.01
 15  0.0197453-0.0064538 0.0115256 2428.  0.00 2.01  1.00
 16  0.0100947 0.0099143 0.0018904 2413.  0.00 1.00 -1.00
 17  0.0073643 0.0131126-0.0197118 2403.  1.00 0.00 -2.01
 18  0.0397690 0.0046107 0.0178903 2388.  0.00 4.01  0.00
 19  0.0227406 0.0078707 0.0279747 2383. -1.00 3.01  0.00
 20 -0.0141824 0.0088224 0.0291127 2383. -2.01 -0.00 -0.00
 21  0.0103597 0.0274362-0.0032718 2361.  0.00 1.00 -3.01
 22 -0.0269727 0.0021092 0.0056176 2346. -1.01 -2.01 -0.00
 23  0.0340618-0.0065201-0.0201693 2329.  2.01 2.01  

Re: [ccp4bb] Problem with lattice disorder / tNCS / whatever ?!

[Sabine Schneider]

 -2.01
   58  0.0396384-0.0041547 0.0204763 2025.  0.00 4.02  1.01
   59  0.0196131-0.0152142 0.0141121 2020.  0.00 2.01  2.01
   60 -0.0241198 0.0076707 0.0246372 2014. -2.01 -1.01 -0.00

 PARAMETERS OF THE REDUCED CELL (ANGSTROEM & DEGREES)
 59.85 91.55109.89 90.00 90.00 90.00

   #  COORDINATES OF REC. BASIS VECTORREDUCED CELL INDICES

1   0.0070481-0.0043879-0.0144998 1.000.000.00
2   0.0099022 0.0011565 0.0044633 0.001.000.00
3  -0.0001404-0.0087276 0.0025729 0.000.001.00


  LATTICE- BRAVAIS-   QUALITY  UNIT CELL CONSTANTS (ANGSTROEM & DEGREES)
 CHARACTER  LATTICE OF FIT  a  b  c   alpha beta gamma

 *  44aP  0.0  59.9   91.6  110.0  90.0 90.0  90.0
 *  31aP  0.4  59.9   91.6  110.0  90.0 90.0  90.0
 *  33mP  0.9  59.9   91.6  110.0  90.0 90.0  90.0
 *  34mP  1.1  59.9  110.0   91.6  90.0 90.0  90.0
 *  35mP  1.2  91.6   59.9  110.0  90.0 90.0  90.0
 *  32oP  1.6  59.9   91.6  110.0  90.0 90.0  90.0
37mC249.7 227.9   59.9   91.6  90.0 90.0  74.8
36oC250.1  59.9  227.9   91.6  90.0 90.0 105.2
39mC250.3 192.7   59.9  110.0  90.0 90.0  71.9
28mC250.3  59.9  227.9   91.6  90.0 90.0  74.8
38oC250.7  59.9  192.7  110.0  90.0 90.0 108.1
29mC250.7  59.9  192.7  110.0  90.0 90.0  71.9
27mC500.2 192.8   59.9  143.1  90.1 127.5  71.9


On 11/17/2016 08:17 AM, Kay Diederichs wrote:

Hi Sabine,

my first guess would be that you are overlooking half of the reflections 
meaning the  cell has really twice the volume. Is the list of difference 
vectors in IDXREF.LP clean in this respect?

Best,

Kay

On Wed, 16 Nov 2016 17:26:59 +0100, Sabine Schneider 
<sabine.schnei...@cup.uni-muenchen.de> wrote:


Hello everybody,

I got a problem with solving and refining a structure of a small protein
of 20kDa, where we just swapped two residue at the surface by mutation
(AF vs FA).

For the FA variant we got three crystal forms, where we had no problem
whatsoever with MR and refinement
- P6522 cell 63 63 184 ~2.9A
- P21212 cell: 53 62 109 ~3A
- P212121 cell 40 43 96 ~1.8A

However with the AF-variant we only got one crystal (out of 50) that
diffracted reasonable well to ~2.4A at the synchrotron. And now the
problem starts: initially the data appear to be orthorhombic, but:

- SG18/19: cell 59 91 109 (I noticed that the b-axis is longer by 1/2b
compared to the other variant?!)
-> running phaser checking all possible alternative SGs found initally 2
mols/AUS in SG18, but since there was still space (and density!) I let
it search for another one (increased the clash tolerance)
SOLU SET RFZ=7.7 TFZ=11.8 PAK=0 LLG=141 TFZ==13.9 RFZ=3.0 TFZ=11.2 PAK=0
LLG=280 TFZ==17.1 LLG=288 TFZ==17.3 RFZ=5.9 TFZ=16.0 PAK=16 LLG=979
TFZ==15.3 LLG=1479 TFZ==17.4

- However the third molecule than clashes partly

- running refmac: R/Rfree stays in the high 40

- analysing the data with Xtriage indicates tNCS

- I also tried P212121, P21 and P1, always with the same result: space
as well as density between the proteins; placing more protein molecules
results in clashes (i.e. refinement in P1 gives R/Rfree of 38/41)

- data were processed with XDS as well as Dials/Aimless

- the protein is generally well folded, quite stable, without any loose
ends or flexible regions

- calculation of selfrotation functions in P21 and P21221 gives peak at
90dg (kappa=180dg)

Anyone an idea what could be going on or how to solve that?


Thanks a lot in advance!

Sabine

[ccp4bb] Problem with lattice disorder / tNCS / whatever ?!

[Sabine Schneider]

Hello everybody,

I got a problem with solving and refining a structure of a small protein 
of 20kDa, where we just swapped two residue at the surface by mutation 
(AF vs FA).


For the FA variant we got three crystal forms, where we had no problem 
whatsoever with MR and refinement

- P6522 cell 63 63 184 ~2.9A
- P21212 cell: 53 62 109 ~3A
- P212121 cell 40 43 96 ~1.8A

However with the AF-variant we only got one crystal (out of 50) that 
diffracted reasonable well to ~2.4A at the synchrotron. And now the 
problem starts: initially the data appear to be orthorhombic, but:


- SG18/19: cell 59 91 109 (I noticed that the b-axis is longer by 1/2b 
compared to the other variant?!)
-> running phaser checking all possible alternative SGs found initally 2 
mols/AUS in SG18, but since there was still space (and density!) I let 
it search for another one (increased the clash tolerance)
SOLU SET RFZ=7.7 TFZ=11.8 PAK=0 LLG=141 TFZ==13.9 RFZ=3.0 TFZ=11.2 PAK=0 
LLG=280 TFZ==17.1 LLG=288 TFZ==17.3 RFZ=5.9 TFZ=16.0 PAK=16 LLG=979 
TFZ==15.3 LLG=1479 TFZ==17.4


- However the third molecule than clashes partly

- running refmac: R/Rfree stays in the high 40

- analysing the data with Xtriage indicates tNCS

- I also tried P212121, P21 and P1, always with the same result: space 
as well as density between the proteins; placing more protein molecules 
results in clashes (i.e. refinement in P1 gives R/Rfree of 38/41)


- data were processed with XDS as well as Dials/Aimless

- the protein is generally well folded, quite stable, without any loose 
ends or flexible regions


- calculation of selfrotation functions in P21 and P21221 gives peak at 
90dg (kappa=180dg)


Anyone an idea what could be going on or how to solve that?


Thanks a lot in advance!

Sabine

Re: [ccp4bb] The modified DNA could not be linked to the downstream DNA in the COOT?

[Sabine Schneider]

Hello Wei,

is the modified base defined as DNA in the cif? (see below)
Is the O3* or O3'? You need the latter.
And check whether there are three hydrogens on the O3. You need to 
either remove one from the cif file manually, or name them according to 
a normal DNA base. (In the latter case, by making the phosphodiester 
bond, one should be removed, if coot/refmac/phenix know that this is DNA...)


Hope that helps!
Sabine


# ---   LIST OF MONOMERS ---
#
data_comp_list
loop_
_chem_comp.id
_chem_comp.three_letter_code
_chem_comp.name
_chem_comp.group
_chem_comp.number_atoms_all
_chem_comp.number_atoms_nh
_chem_comp.desc_level
GdX  G   'Guanosine   ' DNA
35  23 .

#

On 15/07/14 11:14, Wang, Wei wrote:

Hi,

There is a problem about modified DNA refinement.

I generated one CIF file of a modified DNA base using eLBOW software 
of PHENIX. However I found the modified DNA could not be linked to the 
downstream DNA when I refined it in the COOT. Then I generated another 
CIF file using sketcher of CCP4, but the problem still existed.


Is there any expert to help me?

Thanks!!
Best

Wei

[ccp4bb] 3D Monitors

[Sabine Schneider]

Hello everyone,

I know there already has been discussion about 3D monitors on the 
Coot/CCP4bb.
However since there are a few more out there now and I am currently 
thinking about buying one, I thought to get a few opinions from 
crystallographers would be nice! Especially if there are people happily 
model building with the cheaper LG 3D monitors? :-)


 So I am looking at the moment at:
- Zalman ZM-M215W 21,5in
- Zalman ZM-M240W 24in
- Samsung SyncMaster S27A750D 27in
- LG D2342P 23in / LG D2542P 25in

Thanks a lot!

Sabine

Re: [ccp4bb] Stabilization of crystals and ligand exchange

[Sabine Schneider]

Thanks a lot for all the excellent suggestions!
Lots more things to try now!

Cheers,
Sabine


On 10/18/2012 12:18 PM, Boaz Shaanan wrote:

Hi Sabine,

On top of the excellent suggestions of Herman, I was just wondering.   Do you 
have a structure of the ligand-bound protein? If you do and the ligand bound at 
crystal contact with another molecule, I would think that it would be hard to 
get it out without harming the crystals (although not impossible). This may 
help you decide which way to go for obtaining the ligand-free structure.

  Cheers,

 Boaz

Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 
herman.schreu...@sanofi.com [herman.schreu...@sanofi.com]
Sent: Thursday, October 18, 2012 10:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Stabilization of crystals and ligand exchange

Hi Sabine,

The easy experiment to start with, is to take your best conditions (nice 
looking crystals, no diffraction) and instead of overnight wait 4-8 weeks. 
Sometimes ligand exchange is slow, or the ligand induces a conformational 
change which takes a long time to complete in the crystals. There are cases 
that after a number of weeks, diffraction came back.

However, even if the above experiment works, there will be the nagging 
uncertainty that the crystal packing may have prevented some completely 
unexpected, nature or science publication worthy conformational change. There 
will be no way around at least trying to cocrystallize your ligand. Your chance 
of success will depend on how your protein and ligand behave:

-your ligand causes your protein to precipitate. Here your chances are slim. 
You could try to use the ligand as a precipitant by slowly diffusing it in 
(e.g. in a capillary with some gel to separate the protein and ligand 
solutions).
-your ligand is poorly soluble. Here you have better chances. As you mentioned, 
one can add the dilute ligand to a dilute protein solution and then concentrate 
the complex. The amount of ligand needed depends on the affinity of the ligand 
for the protein. To get 90% occupancy, you need a free ligand concentration at 
least 10 times over the Kd (or ~IC50).

To give an example: if you use for crystallization 10 mg/ml of a 30 kDa 
protein, your protein concentration is ~0.33 mM. If your ligand has an affinity 
of 100 nM, you need a free ligand concentration of 1 µM, which is 300 fold less 
than what you need to saturate all binding sites in the protein. To account for 
uncertainties in protein concentrations, I would add 0.5 - 1.0 mM Ligand. If 
you dilute 10 fold, you have 33 µM protein and I would add 50-100 µM ligand, 
which is still well above the 1 µM free concentration needed. Even with 100 
fold dilution, you still just can dilute the ligand with the same factor as the 
protein and still be well above the required free concentration and you do not 
need more ligand.

Of course, this only works for high-affinity ligands, for low affinity ligands 
it is quite a different story. I also would try to use the highest possible 
ligand concentration, since in many cases, although the ligand should bind in 
theory, in practise it is quite a different story.

Good luck!
Herman




-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Sabine 
Schneider
Sent: Wednesday, October 17, 2012 6:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Stabilization of crystals and ligand exchange

Hi everyone,

I am trying to get the structure of a protein-ligand complex were I need to 
exchange the ligand which it co-crystallises nicely with.
Problem: either they crack, disolve, turn brown,...  OR they still look very 
nice, well shaped but do not show a single reflection at the synchrotron!!!


Here is what I tried so far:

1) initially stabilising with higher precipitant (here PEG1500) before slowly 
transferring (*) it to the ligand-removal solution (= artifical mother liquor 
with higher PEG, ethylen glycol or glucose, but without initial ligand)

(*) by slow exchange I mean : initially mixing drop solution with 
stabilising/ligand-removal solution and adding it back to the drop stepwise 
before fully transferring it. Or calculation wise I have fully exchange the 
solution to the new solution

2) here I let them ist over night (if they did not disolve, crack or
whatever)
3) slow exchange transfer to the artificial ML with the new ligand (10mM), left 
them over night and directly froze them

'Best' so far (crystals still looking nice but no reflection...) was slow 
exchange into higher PEG, than to higher PEG with ethylenglycol (30% and also 
adding ethylenglycol to the reservoir), let them sit for over night, before 
again slow exchange to the solution

[ccp4bb] Stabilization of crystals and ligand exchange

[Sabine Schneider]

Hi everyone,

I am trying to get the structure of a protein-ligand complex were I need 
to exchange the ligand which it co-crystallises nicely with.
Problem: either they crack, disolve, turn brown,...  OR they still look 
very nice, well shaped but do not show a single reflection at the 
synchrotron!!!



Here is what I tried so far:

1) initially stabilising with higher precipitant (here PEG1500) before 
slowly transferring (*) it to the ligand-removal solution (= artifical 
mother liquor with higher PEG, ethylen glycol or glucose, but without 
initial ligand)


(*) by slow exchange I mean : initially mixing drop solution with 
stabilising/ligand-removal solution and adding it back to the drop 
stepwise before fully transferring it. Or calculation wise I have fully 
exchange the solution to the new solution


2) here I let them ist over night (if they did not disolve, crack or 
whatever)
3) slow exchange transfer to the artificial ML with the new ligand 
(10mM), left them over night and directly froze them


'Best' so far (crystals still looking nice but no reflection...) was 
slow exchange into higher PEG, than to higher PEG with ethylenglycol 
(30% and also adding ethylenglycol to the reservoir), let them sit for 
over night, before again slow exchange to the solution with the new 
ligand in higher PEG and 30% ethylen glycol.


As I said here the crystals keep shape, but don't diffract at all 
anymore. Just freezing them with 30% ethylen glycol they diffract nicely 
to 2.5A on a home source. But already after step one they are sometimes 
not happy anymore.


Co-crystallisation failed since when I add the ligand, which is not that 
soluble to the purified protein, everything crashed out of solution. I 
am thinking about to test adding the ligand to the diluted protein and 
concentrate it together. But I don't have that much ligand, since the 
synthesis is quite tedious The ligand can be dissolved in 30% 
ethylenglycol to ~50mM


Thus I was wondering if someone has done successfully ligand exchange 
with glutaraldehyd stabilised xtals?

Or any ideas how to stabilise them? I appreciate any ideas or comments!

Sorry for the lengthy email!

Best,
Sabine

Re: [ccp4bb] idiffdisp fails from CCP4 gui in ccp4-6.2.0

[Sabine Schneider]

Hi Boaz,

I get the same thing when I try to run it from the command line. Well as 
a workaround I used mosflm instead...


Best
Sabine



On 08/09/2011 11:33 PM, Boaz Shaanan wrote:

Hi Sabine,

What happens if you try to run it from command line? Just curious. It's a 
useful program (though not as good as ipdisp in some options). I hope it'll be 
fixed soon.

Cheers,

   Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sabine Schneider 
[sabine.schnei...@mytum.de]
Sent: Tuesday, August 09, 2011 3:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] idiffdisp fails from CCP4 gui in ccp4-6.2.0

Hello everyone,

I installed new ccp4 about two weeks ago. When I try to open idiffdisp
from the CCP4 gui it fails with:

Impossible to load Diffraction Image library
/usr/local/share/Xray/ccp4new/ccp4-6.2.0/lib/libDiffractionImage.so!!!.

if I look into /ccp4-6.2.0/lib it looks like that

lrwxrwxrwx  1 13003 10602  29 2011-07-26 16:33
lib_DiffractionImage.so -  lib_DiffractionImage.so.0.0.0
lrwxrwxrwx  1 13003 10602  28 2011-07-26 16:33
libDiffractionImage.so -  libDiffractionImage.so.0.0.0
lrwxrwxrwx  1 13003 10602  29 2011-07-26 16:33
lib_DiffractionImage.so.0 -  lib_DiffractionImage.so.0.0.0
lrwxrwxrwx  1 13003 10602  28 2011-07-26 16:33
libDiffractionImage.so.0 -  libDiffractionImage.so.0.0.0
-rwxr-xr-x  1 13003 10602 7708504 2011-06-16 23:09
lib_DiffractionImage.so.0.0.0
-rwxr-xr-x  1 13003 10602 7678200 2011-06-16 23:09
libDiffractionImage.so.0.0.0


Anyone an idea what's going on, or how I can fix that problem?

Thanks for your help!

Sabine


--
--
Dr. Sabine Schneider
Technische Universität Munich
Department of Chemistry
Chair of Biochemistry
Lichtenbergstr. 4
D-85747 Garching
Germany
Tel.: +49 (0) 89 289 12891
Fax: +49 (0) 89 289 13363
http://www.biochemie.ch.tum.de/index.php?id=919

[ccp4bb] idiffdisp fails from CCP4 gui in ccp4-6.2.0

[Sabine Schneider]

Hello everyone,

I installed new ccp4 about two weeks ago. When I try to open idiffdisp 
from the CCP4 gui it fails with:


Impossible to load Diffraction Image library 
/usr/local/share/Xray/ccp4new/ccp4-6.2.0/lib/libDiffractionImage.so!!!.


if I look into /ccp4-6.2.0/lib it looks like that

lrwxrwxrwx  1 13003 10602  29 2011-07-26 16:33 
lib_DiffractionImage.so - lib_DiffractionImage.so.0.0.0
lrwxrwxrwx  1 13003 10602  28 2011-07-26 16:33 
libDiffractionImage.so - libDiffractionImage.so.0.0.0
lrwxrwxrwx  1 13003 10602  29 2011-07-26 16:33 
lib_DiffractionImage.so.0 - lib_DiffractionImage.so.0.0.0
lrwxrwxrwx  1 13003 10602  28 2011-07-26 16:33 
libDiffractionImage.so.0 - libDiffractionImage.so.0.0.0
-rwxr-xr-x  1 13003 10602 7708504 2011-06-16 23:09 
lib_DiffractionImage.so.0.0.0
-rwxr-xr-x  1 13003 10602 7678200 2011-06-16 23:09 
libDiffractionImage.so.0.0.0



Anyone an idea what's going on, or how I can fix that problem?

Thanks for your help!

Sabine


--
--
Dr. Sabine Schneider
Technische Universität Munich
Department of Chemistry
Chair of Biochemistry
Lichtenbergstr. 4
D-85747 Garching
Germany
Tel.: +49 (0) 89 289 12891
Fax: +49 (0) 89 289 13363
http://www.biochemie.ch.tum.de/index.php?id=919

[ccp4bb] script for pdbset to apply repetitive rotation and translation

[Sabine Schneider]

Hello,

I want to apply to a small structure a rotation and translation (i.e 10A 
translation and 10dg rotation), write out the coordinates and apply the 
same rotation/translation to the new coordinates and repeat this lets 
say 100 times.


I guess the program of choice would be pdbset but I am not sure how to 
script it to do the repetition?


Thanks a lot for your help!

Sabine

Re: [ccp4bb] Freezing under oil

[Sabine Schneider]

Hello Claudia,

Under what conditions did the crystals grow? If its sodium acetate and 
ammonium sulphate I would try adding sodium malonate as a cryo 
protectant (with 2-2.3M ammonium sulphate adding 0.6-0.8M Na malonate 
should be sufficient) or increasing the ammonium sulphate concentration.


Sabine



Claudia Scotti wrote:
 
Dear list,
 
I'm trying to freeze crystals in cryoconditions containing the following:
 
0.1 M Sodium acetate pH 4.4

2.15-2.3 M Ammonium sulphate
7% n-butanol
15% glycerol
 
The problem is that the crystals (beautiful hexagonal prisms) seem to 
shatter in a random fashion: some are unaffected, some, coming from 
the same drop, disgregate miserably. One even split into three parts, 
of which two disgregated and one survived perfectly well.
 
I've tried both by moving the crystals directly in the cryocondition 
and by progressively increasing the glycerol concentration to no avail.
 
Shall I just select those that survive?
 
I was wondering if anybody has ever had this same problem and 
if freezing under oil could be an alternative. If yes: any suggestions 
on how to fish the crystals? I tried it in the past, but I found it 
very difficult...
 
Experiences and suggestions are welcome.
 
Claudia
 
 
 



Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di 
Patologia Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia 
Italia Tel. 0039 0382 986335/8/1 Facs 0039 0382 303673




Hotmail: Trusted email with powerful SPAM protection. Sign up now. 
https://signup.live.com/signup.aspx?id=60969


--
--
Dr. Sabine Schneider
Ludwig-Maximilians-University
Department of Chemistry
Butenandtstrasse 5-13, Building F
81377 Munich
Germany
Phone: +49 (0)89 2180 77752
Fax: +49 (0)89 2180 77756
http://www.carellgroup.de/

[ccp4bb] Twin refinement - selecting FreeR

[Sabine Schneider]

Hi everyone,

I got good data to 2.4A on a protein-ligand complex. I want to solve the 
structure by MR using a model with 45% seq. ID and 55% similarity.
Initially the data appeared to be P622 (pointless, selfrotation function 
etc). Unit cell: 145 145 65, MW protein is 28kDa
I prepared the MR model with chainsaw, choping of non-conserved residues 
at the C gamma and reset the B-factors. Phaser found a solution, but 
with negativ LLG.


I than processed the data in P3, P321, P312 and P6 and  run Phaser 
searching all possible alternative spacegroups. The best solution is P3 
(4mol/asu) followed by P321 (2mol/asu),both with a LLG of above 400. But 
when trying to refine the models in Refmac the R/Rfree stays at ~48%.


Looking at the truncate output and phenix xtriage, twinning is suggested 
with the merohedral twin law -h, -k, l and a twin fraction of 40.3%
Using twin refine option in Refmac (5.5.0070), the R/Rfree for the P321 
solution drops to around 33%, but the difference between R/Rfree is only 
1%. For the solution found in P3 the R/Rfree stays at around 47%.


So I assumed that P321 is the better solution. Is the difference in 
R/Rfree only 1%, because the free and work reflections are related 
through the twin law?


I used phenix  to assign the free reflections putting in the twin 
operators. Doing simulated annealing in phenix, I get a rather large 
difference in R/Rfree of  ~7%. Well, I guess I need to do some tweaking 
of the parameters in phenix (running the latest phenix and cci_apps). 
When I than use these free reflections assigned by phenix in Refmac I 
still get only 1.5% difference between R and Rfree? 

So is it doable in Refmac, or is my best bet phenix? Any advice what is 
the best way to proceed is much appreciated!


Sabine


--
Dr. Sabine Schneider
Ludwig-Maximilians-University
Department of Chemistry and Pharmacy
Butenandtstrasse 5-13, Building F
81377 Munich
Germany
Phone: +49 (0)89 2180 77846
Fax: +49 (0)89 2180 77756
http://www.carellgroup.de/

[ccp4bb] imosflm fails on suse

[Sabine Schneider]

Hi everyone,

Also got a imosflm problem - I installed the ccp4-6.1.0 before Xmas. 
Downloaded the binaries and installed it using the install script. (both 
setup-files are sourced in my .bashrc file). All programs I tried so far 
worked fine. But if I try to start imosflm (from the gui or the 
comandline) I get the following error message:


MOSDIR is /home/schneider/project1/scratch
Error in startup script: unknown namespace in import pattern itcl::*
   while executing
namespace import itcl::*
   (file 
/usr/local/share/CCP4new/ccp4-6.1.0/ccp4i/imosflm/src/imosflm.tcl line 91)

   invoked from within
source $env(IMOSFLM)
   (file 
/usr/local/share/CCP4new/ccp4-6.1.0/ccp4i/imosflm/imosflm.tcl line 111)


Am using a Suse 10.3 x84_64 linux box and I have the old CCP4 version 
somewhere else, but not sourced (just kept it since I thought ie shar 
might get upset). I also installed CCP4 6.1 on a different suse 
linux-box (where there hadn't been CCP4 on it before) and it worked just 
fine?

I got Tkl/tk version 8.4 installed. Any ideas?

Thanks a lot for your help!

Sabine


--
--
Dr. Sabine Schneider
Ludwig-Maximilians-University
Department of Chemistry and Pharmacy
Butenandtstrasse 5-13, Building F
81377 Munich
Germany
Phone: +49 (0)89 2180 77846
Fax: +49 (0)89 2180 77756
http://www.carellgroup.de/

[ccp4bb] Summary - crystals grown from high ammonium sulphate

[Sabine Schneider]

Thanks a lot for all the helpful replies I got!

Here is a summary:
- increase drop size to 4+4ul if protein is not the limiting factor - 
limits skin formation

- add 10-20ul well solution directly to drop
- freeze straight from the drop
- microseeding into less conc. AmSO4 to get more crystals
- add AmSO4 free motherliquor to well solution to equilibrate drop to 
lower AmSO4
- add cryo solution in 1/4 of drop volume steps to drop and freeze 
directly from drop - glucose more gentle than glycerol

- http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Cryo
- reduce evaporation from drop by mounting and freezing crystals in the 
cold room
- try mineral oils - either add small amount directly to drop or move 
crystal to drop with oil first

- test diffraction of crystal at RT
- crystals could be grown in a plastic tube, which can be put directly 
in the cryo stream (Yevgeniy Kalinin and Robert Thorne, Acta Cryst.

(2005). D61, 1528–1532.)
- be quick

Sabine



Sabine Schneider wrote:
Hi everyone,

We got crystals that grew in ~3.2M ammonium sulphate and some 
tris-buffer at 18dgC. Unfortunately the crystals take a while to grow 
(~4-5 weeks) and so far we only have 4-5 xtals.
I tried to freeze the crystals, but as soon as I broke though the skin 
of the drop the ammonium sulphate started to crystallise. I got the 
crystals out, froze them using sort of an artifical mother liquor with 
sodium malonate as cryo and tested the diffraction. The freezing seems 
to be OK and it is definitely a protein crystal. The crystal suffered 
when the ammonium sulphate in the drop started to crystallise, but 
didn't seem to deteriorate anymore in the cryo. Well the corners had 
already more or less disappeared by the time I got them out of the drop...
Since we only have a few xtals at the moment and I can't try out a lot 
of things, I was wondering if anyone has experienced and solved a 
similar problem? My freezing attempt so far was in an airconditioned 
room with 18dgC. I thought about higher humidity and temperature in the 
room, and/or adding the cryo directly to the drop Any ideas are 
very much appreciated!

[ccp4bb] Crystals grown from high ammonium sulphate

[Sabine Schneider]

Hi everyone,

We got crystals that grew in ~3.2M ammonium sulphate and some 
tris-buffer at 18dgC. Unfortunately the crystals take a while to grow 
(~4-5 weeks) and so far we only have 4-5 xtals.
I tried to freeze the crystals, but as soon as I broke though the skin 
of the drop the ammonium sulphate started to crystallise. I got the 
crystals out, froze them using sort of an artifical mother liquor with 
sodium malonate as cryo and tested the diffraction. The freezing seems 
to be OK and it is definitely a protein crystal. The crystal suffered 
when the ammonium sulphate in the drop started to crystallise, but 
didn't seem to deteriorate anymore in the cryo. Well the corners had 
already more or less disappeared by the time I got them out of the drop...
Since we only have a few xtals at the moment and I can't try out a lot 
of things, I was wondering if anyone has experienced and solved a 
similar problem? My freezing attempt so far was in an airconditioned 
room with 18dgC. I thought about higher humidity and temperature in the 
room, and/or adding the cryo directly to the drop 
Any ideas are very much appreciated!


Sabine

--
Dr. Sabine Schneider
Ludwig-Maximilians-University
Department of Chemistry and Pharmacy
Butenandtstrasse 5-13, Building F
81377 Munich
Germany
Phone: +49 (0)89 2180 77846
Fax: +49 (0)89 2180 77756
http://www.carellgroup.de/

[ccp4bb] difference in data processed with xscale and scala

[Sabine Schneider]

Hello everyone,

I am puzzled about differences I see when I refine the very same 
structure against data processed with xscale or scala.


I got data to 2.55A from a protein-ligand complex. The data were 
processed with xds/xscale (1) or xds/scala (2)
Free R was imported from a previously solved structure with a different 
ligand, first to (1) and from there to (2).
After MR and refinement (Refmac/Phenix), I get some difference density 
peaks (a bit pos and neg),  near the ligand when I refine against (1) 
and it converges more or less around R/Rfree of 20.9 and 24.8. I do not 
get these pos and negative density blobs when I refine the same 
coordinates against (2), with a tiny bit higher R/Rfree of 21.6/26.1?
So the data processed with scala, where I get slightly better refinement 
statistics, I end up with some pos/neg density peaks. They don't show up 
when I apply a high resolution cut-off of 2.65A during refinement.


Symmetry is orthorhombic and the statistics are OK (Rsym 0.09 and 0.38 
in highest shell, redundancy ~5, I/sigI =1.9, mean I/sigI  = 5.2 ) and 
in both files appear to be more or less the same number of reflections.


I also did simulated annealing, omitting the region around the ligand in 
a 15A radius and the density is nicely coming up. But it doesn't make a 
difference: after refinement I always end up with the result as 
discribed above? And there isn't really a difference between the two 
structures in the end.


Has anyone an idea whats going on?

Sabine

[ccp4bb] overlapping DNA in prt-DNA complex structure

[Sabine Schneider]

Hello everyone,

I am working on the structure of a protein-DNA complex with 2 mol/ASU. 
Data are to 2.8A and the symmetry appears to be orthorhombic (P212121; 
a=42.280 b=113.340  c=134.670) I solved the structure by moleculare 
replacement using the coordinates of the protein in phaser. I got 
relative good density for the DNA and started building for the DNA for 
one protein molecule and gave the model back to phaser. The statistics 
for the solution are very good (RFZ=16.7 TFZ=27.8 PAK=0 LLG=820 RFZ=14.9 
TFZ=42.9 PAK=0 LLG=2215 LLG=2215). My problem is now that the ends of 
the DNA strands of the two complexes in the asu are crashing by 
overlapping almost perfectly as well as with the symmetry related 
molecules. So instead of beeing able to fit in a 15mer, which was used 
in the crystallisation, I only can fit in maybe an 8mer (which is also 
not making really sense DNA-sequence wise). I tried reindexing to klh 
and lhk but same problem.


Has anyone an idea how to solve this?

Thanks a lot for your help!

Sabine

[ccp4bb] xds to mtz using pointless

[Sabine Schneider]

Hi everyone,

I have a fine sliced dataset consisting of 720 frames (0.25dg / frame). 
I processed them with Xds and used Pointless to convert it to mtz for 
putting it in Scala. Spacegroup is P212121 and the resolution ~3.2. 
After ~200 frames the Rmerge goes up crazy and after 560 it is OK again. 
Therefore I am trying to process the the data in 3 wedges (1-199, 
200-560, 561-720) in XDS and see how they scale independently and when I 
sort and scale them together with Sortmtz and Scala. When converting the 
.hkl file to mtz with pointless there is no problem with wedge 1-199. 
But for the other two wedges I am getting a Segmentation fault in 
Pointless (example see below). Or for wedge frame 200-560 I also only 
got an mtz file with batches 200 - 360. CORRECT.LP for all three wedges 
looks OK (at least to me)?

Anyone an idea what I am missing?

Thanks for you help!

Sabine

Matrix to transform XDS axis system to CCP4 frame:
|   0.00796, -0.006359,0.|
| -0.001536,-1, -0.006347|
| 1, -0.001485,  -0.00797|

Matrix to transform XDS detector coordinates to CCP4 frame:
|   0.00796, -0.006359,0.|
| -0.001536,-1, -0.006347|
| 1, -0.001485,  -0.00797|

Rotation axis in CCP4 frame: ( 0.000  0.000  1.000)

Incident beam in CCP4 frame: ( 1.000 -0.000 -0.002)


Spacegroup information obtained from library file:
Logical Name: SYMINFO   Filename: 
/usr/local/share/CCP4/ccp4-6.0.2/lib/data/syminfo.lib



  85329 observations accepted
Resolution range   49.1403.016
   6107 accepted incomplete observations with PART  0.98, minimum 0.75
   1214 observations flagged as MISFITS in XDS, kept here

Reconstructing orientation matrix [U] from  199 observations

Orientation matrix [U]:
|0.5847,0.1184,0.8026|
|   -0.4283,   -0.7951,0.4294|
| 0.689,   -0.5948,   -0.4142|
 Determinant = 1.000
Segmentation fault

Re: [ccp4bb] xds to mtz using pointless

[Sabine Schneider]

That solved it! I had an older version (1.2.9). Thanks a lot!

Sabine


Phil Evans wrote:
There was a bug in Pointless for XDS reading due to my 
misunderstanding of the STARTING_FRAME value, which is fixed in 
version 1.2.13, available from


ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/pointless-1.2.13.linux  (eg)

Otherwise if you send me the file I'll investigate

Phil


On 20 Feb 2008, at 09:39, Sabine Schneider wrote:


Hi everyone,

I have a fine sliced dataset consisting of 720 frames (0.25dg / 
frame). I processed them with Xds and used Pointless to convert it to 
mtz for putting it in Scala. Spacegroup is P212121 and the resolution 
~3.2. After ~200 frames the Rmerge goes up crazy and after 560 it is 
OK again. Therefore I am trying to process the the data in 3 wedges 
(1-199, 200-560, 561-720) in XDS and see how they scale independently 
and when I sort and scale them together with Sortmtz and Scala. When 
converting the .hkl file to mtz with pointless there is no problem 
with wedge 1-199. But for the other two wedges I am getting a 
Segmentation fault in Pointless (example see below). Or for wedge 
frame 200-560 I also only got an mtz file with batches 200 - 360. 
CORRECT.LP for all three wedges looks OK (at least to me)?

Anyone an idea what I am missing?

Thanks for you help!

Sabine

Matrix to transform XDS axis system to CCP4 frame:
|   0.00796, -0.006359,0.|
| -0.001536,-1, -0.006347|
| 1, -0.001485,  -0.00797|

Matrix to transform XDS detector coordinates to CCP4 frame:
|   0.00796, -0.006359,0.|
| -0.001536,-1, -0.006347|
| 1, -0.001485,  -0.00797|

Rotation axis in CCP4 frame: ( 0.000  0.000  1.000)

Incident beam in CCP4 frame: ( 1.000 -0.000 -0.002)


Spacegroup information obtained from library file:
Logical Name: SYMINFO   Filename: 
/usr/local/share/CCP4/ccp4-6.0.2/lib/data/syminfo.lib



 85329 observations accepted
   Resolution range   49.1403.016
  6107 accepted incomplete observations with PART  0.98, minimum 0.75
  1214 observations flagged as MISFITS in XDS, kept here

Reconstructing orientation matrix [U] from  199 observations

Orientation matrix [U]:
|0.5847,0.1184,0.8026|
|   -0.4283,   -0.7951,0.4294|
| 0.689,   -0.5948,   -0.4142|
Determinant = 1.000
Segmentation fault