Re: [ccp4bb] Extra electron density

2012-11-05 Thread Sangeetha Vedula
You know what? Scratch what I said. As Mark says, there isn't any 2Fo-Fc
density, which is suspicious.

However, I would still model what may fit in it, with appropriate
occupancy, based on the symmetry. To be doubly sure.

I have, in the past, used
ProDRGhttp://davapc1.bioch.dundee.ac.uk/prodrg/submit.htmlfor
obtaining pdf, topology and parameter files for ligands. ARP/wARP can
be used for a first fit of ligand into density.

You may find appropriate fatty acid pdb-top-param files already in the
HIC-UP http://xray.bmc.uu.se/hicup/ database.

I would do a quick search for a fatty acid of an appropriate length.

If it is truly spurious density as I suspect it is, you'll see negative
density lighting up your screen despite using appropriately low occupancy.

Good luck!

Sangeetha.

On Sun, Nov 4, 2012 at 8:17 PM, Sangeetha Vedula sangeetha...@gmail.comwrote:

 Could it be a fatty acid? It looks like it has a (hydrophobic?) tail with
 a (charged?) head. Partially occupied perhaps, as they're so close together.


 On Sun, Nov 4, 2012 at 7:38 PM, yogesh khandokar 
 yogesh.khando...@gmail.com wrote:

 Dear All

 I am working on an enzyme which involved in fatty acid biosynthesis. We
 solved the crystal structure of it. Biological unit of this enzyme is
 Hexamer and showing extra electron density in the center of Hexamer.
 Wincoot software doesn't recognize this extra electron density as water
 molecules/ substrate.

 My question is:Is there any way to find the molecules responsible for
 extra electron density?

 Picture is attached with email.

 Thanks in advance for your comments.

 Regards
 --
 Yogesh Khandokar
 PhD Student
 School of Biomedical Sciences
 Charles Sturt University
 Wagga Wagga, NSW,
 Australia
 http://www.csu.edu.au/faculty/science/biomed/





Re: [ccp4bb] Extra electron density

2012-11-04 Thread Sangeetha Vedula
Could it be a fatty acid? It looks like it has a (hydrophobic?) tail with a
(charged?) head. Partially occupied perhaps, as they're so close together.

On Sun, Nov 4, 2012 at 7:38 PM, yogesh khandokar yogesh.khando...@gmail.com
 wrote:

 Dear All

 I am working on an enzyme which involved in fatty acid biosynthesis. We
 solved the crystal structure of it. Biological unit of this enzyme is
 Hexamer and showing extra electron density in the center of Hexamer.
 Wincoot software doesn't recognize this extra electron density as water
 molecules/ substrate.

 My question is:Is there any way to find the molecules responsible for
 extra electron density?

 Picture is attached with email.

 Thanks in advance for your comments.

 Regards
 --
 Yogesh Khandokar
 PhD Student
 School of Biomedical Sciences
 Charles Sturt University
 Wagga Wagga, NSW,
 Australia
 http://www.csu.edu.au/faculty/science/biomed/




Re: [ccp4bb] protein cleavage

2012-11-04 Thread Sangeetha Vedula
Do you see the same MBP band (or corresponding to the same MW, in case it
isn't MBP) before cleavage, at the same total concentration of protein? If
not, your protein could be crashing out. Even so, if you use SDS and heat
up the sample, I am surprised that your protein just disappeared. TEV
protease has a pretty long recognition sequence. It doesn't appear as is in
your protein, does it?

Is it possible to attach the tag at the other terminus? Perhaps it is more
accessible? Of course, it still doesn't solve the mystery of the
disappearing protein if the band is indeed MBP that appears after the
cleavage reaction.

On Sun, Nov 4, 2012 at 3:07 PM, VAN RAAIJ , MARK JOHAN 
mjvanra...@cnb.csic.es wrote:

 We once had a more-or-less MBP-sized fragment before cleavage, but this
 turned to be a spontaneous mutation. This expression experiment had been
 started from a glycerol stock with an unknown number of growth cycles prior
 to expression.
 Starting from a fresh transformation with the purified and sequenced
 plasmid solved the problem.
 Since then, I insist everybody does a fresh transformation before every
 expression experiment and not generate extra growth/dilution cycles beyond
 the normal transformation, growth on plate, overnight culture, dilution
 into large-scale expression culture.



 Quoting Bosch, Juergen:

  @Cynthia,
 On Nov 4, 2012, at 10:58 AM, Cynthia Kinsland wrote:

 Since you're seeing the MBP band, it sounds as if you're getting some
 cleavage.  If there were no cleave you would only see the fusion and TEV
 bands on the gel.

 I think that is a wrong assumption.
 He did not specify if he sees the MBP band also just before TEV addition
 - it might also be truncation products which we happen to see all the time.
 The ratio varies depending on the construct but it can be as bad as a 1:1
 ratio. You can really only tell if TEV cleaves if you do a time course
 experiment at RT with a defined amount of your protein and see if the
 fusion construct decreases. An alternative for the lack of your 17kDa
 desired band is simply your fusion construct is cleaved but your cleaved
 product might a) not be soluble at that pH or b) aggregates and
 precipitates.
 You might be able to perform the cleavage on the Amylose column keeping a
 constant flow cycling.

 Jürgen


 ..
 Jürgen Bosch
 Johns Hopkins University
 Bloomberg School of Public Health
 Department of Biochemistry  Molecular Biology
 Johns Hopkins Malaria Research Institute
 615 North Wolfe Street, W8708
 Baltimore, MD 21205
 Office: +1-410-614-4742
 Lab:  +1-410-614-4894
 Fax:  +1-410-955-2926
 http://lupo.jhsph.edu








 Mark J van Raaij
 Laboratorio M-4
 Dpto de Estructura de Macromoléculas
 Centro Nacional de Biotecnología - CSIC
 c/Darwin 3, Campus Cantoblanco
 28049 Madrid
 tel. 91 585 4616
 email: mjvanra...@cnb.csic.es



[ccp4bb] determination of oligomerization state of protein

2012-08-13 Thread Sangeetha Vedula
Hello all,

I am working with a protein that is probably a hexamer based on homology
with other proteins but when I ran it on an analytical size gel filtration
column, I see multiple peaks. I would like to determine the exact
oligomerization state of the mixture and have considered blue native gel
electrophoresis and gradient centrifugation. The monomer is about 54 kDa.
All of the peaks are active enzymatically. I wish I could test higher
concentrations to see if the equilibrium will shift towards a hexamer (or
whatever the naturally highest state is) but the protein crashes out.

The protein was expressed in baculovirus and the multitude of states could
be an artifact but I'd like to know what I am working with. Also, down the
line, I'll need to pick one of the states for crystallization trials.

Has anyone encountered this problem? How did you determine the
oligomerization state(s)?

I have basic biorad gel apparatus available and no gradient mixer.

Thanks for any input.

Best regards,

Sangeetha.


Re: [ccp4bb] Enhancing Crystal Quality

2012-08-13 Thread Sangeetha Vedula
Lucas, if your crystals diffract worse at room temp than cryoprotected, it
looks like there may be radiation damage at room temperature, so you may
need to cryoprotect. The change in osmotic pressure may be too much for
your crystals when you're cryoprotecting the crystals by a sudden dunk.
Also, did you just add PEG 400 to the reservoir solution already in the
wells or did you make it afresh with higher precipitant concentration to
compensate for removal into a solution without any soluble protein or
crystalline protein to be in equilibrium with? I always made solutions
afresh unless the crystal was cryoprotected and if it diffracted just fine
by dragging the crystal through a reservoir+cryoprotectant solution.

My crystals did not tolerate sudden changes in osmotic pressure.

Harvest the crystal into 15 uL of reservoir solution with excess
precipitant to compensate for loss of protein to be in equilibrium with (if
I had a crystal from 8% PEG 8000, I used to use 12% PEG8000 with all of the
other components having the same concentration. It was random but I found
one that worked and stuck to it. Sometimes, there may be a solubility
issue, so I had to cut back on a salt but just enough to keep everything
soluble. So I made the solution fresh because my crystals didn't always
appear at exactly the same precipitant concentration. I always set up a
small range of concentrations.

Add 2.5 uL of cryoprotectant (reservoir solution with excess precipitant
concentration AND required concentration of precipitant present in it).
Remove 2.5 uL from a different part of the drop.

Wait 6 min (again, random. 5 min didn't work well, 6 min did).

Repeat with 2.5 uL, 3.75 uL, 5 uL, 7.5 uL. By that time, I achieved enough
cryoprotectant concentration for it to do its job.

I never mixed the drop because my crystals were very fragile and obviously,
I was always looking at it under the microscope to make sure I wasn't
smashing or sucking up my crystal.

Have you tried additive screens? I had tremendous improvement in my
crystals with that. They were very mosaic and overall resolution was only
about 3.7 A or so without additives. With 3% DMSO (from an additive
screen), the size was great, still mosaic, but overall resolution with good
outer shell completeness (I don't remember the exact numbers for data
quality but it was much better than without) improved to about 2.8-ish A.

Good luck!

S.

On Sun, Aug 12, 2012 at 3:31 PM, Yi-Liang (Lucas) Liu
yiliang...@gmail.comwrote:

 Hi CCP4ers,

 I tested my crystal under room temperature. It still only has low
 resolution (7A). Are there any way to improve this? I attached the
 diffraction as well. Thanks.

 Lucas



 On Aug 2, 2012, at 5:27 PM, Roger Rowlett wrote:

 Mitegen makes a nice little product that is a plastic tube that will slide
 over one of their magnetic cap/loops. If you put some well solution in the
 tube and seal the base with apiezon, you can collect quite a bit of data on
 the loop mounted crystal before it dries out.

 Cheers,

 ___
 Roger S. Rowlett
 Gordon  Dorothy Kline Professor
 Department of Chemistry
 Colgate University
 13 Oak Drive
 Hamilton, NY 13346

 tel: (315)-228-7245
 ofc: (315)-228-7395
 fax: (315)-228-7935
 email: rrowl...@colgate.edu






 On Thu, Aug 2, 2012 at 2:14 PM, Yi-Liang Liu yiliang...@gmail.com wrote:

 Hi Herman and other CCP3BBers,

 Thanks for your suggestions. I didn't see any cracks in the crystal drops
 initially. I will certainly try to shot crystals under room temperature and
 see what happens. Does the plastic loops fit into the cryo stands Molecular
 Dimension sells?

 LUcas
 On Aug 2, 2012, at 2:24 AM, herman.schreu...@sanofi.com wrote:

  Hi Lucas,
 
  The funky diffraction pattern is most likely due to a cracked crystal,
  resulting in a mixture of slightly differently aligned diffraction
  patterns. Were the cracks there before you added the cryprotectant? If
  not, the cryoprotectant is definitively to blame. As has mentioned
  before, you have to take a shot at room temperature without any
  cryoprotectant added, to make sure the bad quality is not due to the
  cryoprotectant. Mitegen sells plastic capillaries, which you can slide
  over your loop to prevent the crystal from drying out.
 
  Good luck!
  Herman
 
  -Original Message-
  From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
  Yi-Liang Liu
  Sent: Thursday, August 02, 2012 4:15 AM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: Re: [ccp4bb] Enhancing Crystal Quality
 
  Hi,
 
  Thanks for the kindly answers from everyone. I actually haven't tried
  different cryoprotectants. I might will give a try next time. I usually
  only use mother liquor+30% PEG400. It is noticeable that it has some
  patterns (cracks (?)) on the crystal. However, it didn't form icy
  rings or etc. The diffraction pattern looks funky too. It looks like it
  is twin and the diffraction spot has tails. Does this indicate the
  

[ccp4bb] Desalting columns

2012-02-27 Thread Sangeetha Vedula
Dear bb users,

I am trying to crystallize a ~320 kDa protein that crashes out if
concentrated past about 3 mg/mL.

I would like to try to exchange it into various buffer-salt-additive
combinations to see which buffer works. For a starting point, I'd like to
use desalting colums.

Does anyone have suggestions for good buffer exchange and sample recovery?
I woud like to load about 250 uL onto each column.

Thanks a lot!

Best regards,

Sangeetha.


[ccp4bb] Very high B-factors of some atoms of ligand in Refmac

2010-01-28 Thread Sangeetha Vedula
Dear all,

I am refining a crystal structure with two enantiomers of the ligand lying
on a two-fold crystallographic axis (making the density an average of 4
orientations/optical identity). The ligand fits pretty well over all in the
density, but some atoms stick out of density. While B-factors for the atoms
that are within the density, the B-factors are in the range of 20-40. For
the ones sticking out, the B-factors are 200-400. Which doesn't make
chemical sense, as clearly, one atom isn't jumping around so much more than
the others.

Is there any way I can force refmac to make the B-factors more reasonable? I
also notice that some side chain atoms that stick out of density have
horrendous B-factors.

Thanks in advance,

Sangeetha.


[ccp4bb] Biorad Profinia vs GE AKTAPrime Plus, for affinity tagged proteins

2010-01-28 Thread Sangeetha Vedula
Dear all,

My lab is looking into buying a protein purification system. The AKTA is
more versatile, but does anyone know if the Profinia is in any way superior
to the AKTA for purification of affinity-tagged proteins? If it is, I would
really appreciate if you can tell me in what way it is superior.

Thanks,

Sangeetha.


Re: [ccp4bb] Biorad Profinia vs GE AKTAPrime Plus, for affinity tagged proteins

2010-01-28 Thread Sangeetha Vedula
Thanks for your response, Edward. Yes, that is what I thought. For the most
part, people plan to tag their proteins in my lab. I'll possibly be the only
one purifying non-tagged proteins in my lab. I love the AKTA. Another was
all praise of the Profinia. To my knowledge, tagged proteins don't always
get fully purified with just an affinity column. An additional size
exclusion step helps. That is, if the tag is even available at the surface
for binding the affinity column. My vote is AKTA. So, I wanted to find out
what the world thought about both.

On Thu, Jan 28, 2010 at 11:16 AM, Edward Collins edward_coll...@med.unc.edu
 wrote:

 personally, I don't know why you would want to limit yourself to just using
 affinity tags to purify your proteins.  For my money the flexibility of the
 AKTA is critical.
 good luck
 -ed

 On Jan 28, 2010, at 11:08 AM, Sangeetha Vedula wrote:

  Dear all,
 
  My lab is looking into buying a protein purification system. The AKTA is
 more versatile, but does anyone know if the Profinia is in any way superior
 to the AKTA for purification of affinity-tagged proteins? If it is, I would
 really appreciate if you can tell me in what way it is superior.
 
  Thanks,
 
  Sangeetha.





Re: [ccp4bb] Very high B-factors of some atoms of ligand in Refmac

2010-01-28 Thread Sangeetha Vedula
You know what, decarboxylation could be an issue. But that'll explain the
B-factors only for the side chains and some of the atoms in the ligand.

I don't see in what other orientation/conformation the ligand can fit the
density, not clash with itself or with the protein, lie on a 2-fold symmetry
axis, and have two enantiomers in each asymmetric unit. The atoms that don't
have density are terminal atoms. When the density is an average of four
different species on a symmetry axis, that some atoms of a 16 atom-ligand
stick out of density is frustrating but not entirely surprising, I think.
What do you all think?

The protein was co-crystallized with a racemic mixture of the ligand and I
haven't been able to find any evidence that one binds better than the other,
or one binds exclusively, not the other.

On Thu, Jan 28, 2010 at 11:32 AM, Clemens Vonrhein 
vonrh...@globalphasing.com wrote:

 Dear Sangeetha,

 before worrying about the B-factor column in your PDB file I would be
 worrying about atoms not having density. This should tell you that
 your model (atoms and/or parametrisation) isn't correct.

 One possible reason for those high-B side-chain atoms could be a
 sequence shift: sometimes the difference between neighbouring residues
 could be very small in terms of atoms and constellation. Just putting
 the B-factors into a numerical range you are happy with doesn't make
 your model better - it still doesn't fit your density.

 Same for your ligand: if you have one atom inside density with a B of
 40 and the next atom bonded to that one sticks out with a B-factor of
 200 ... are you sure your ligand is actually in there and has bound in
 that orientation?

 So I would say these high B-factors are a good thing: they show you
 where your model is poor. But then: you already see those atoms
 sticking out of density, so you know where you still have to improve
 your model, right?

 There could also be some issue with radiation damage (are those high-B
 atoms on the ligand maybe I or Br?) ... decarboxylation on side-chains
 etc etc.

 Just some ideas ...

 Cheers

 Clemens

 On Thu, Jan 28, 2010 at 11:04:52AM -0500, Sangeetha Vedula wrote:
  Dear all,
 
  I am refining a crystal structure with two enantiomers of the ligand
 lying
  on a two-fold crystallographic axis (making the density an average of 4
  orientations/optical identity). The ligand fits pretty well over all in
 the
  density, but some atoms stick out of density. While B-factors for the
 atoms
  that are within the density, the B-factors are in the range of 20-40. For
  the ones sticking out, the B-factors are 200-400. Which doesn't make
  chemical sense, as clearly, one atom isn't jumping around so much more
 than
  the others.
 
  Is there any way I can force refmac to make the B-factors more
 reasonable? I
  also notice that some side chain atoms that stick out of density have
  horrendous B-factors.
 
  Thanks in advance,
 
  Sangeetha.

 --

 ***
 * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
 *
 *  Global Phasing Ltd.
 *  Sheraton House, Castle Park
 *  Cambridge CB3 0AX, UK
 *--
 * BUSTER Development Group  (http://www.globalphasing.com)
 ***



Re: [ccp4bb] hairpin formation in gene

2009-11-22 Thread Sangeetha Vedula
Thank you, everyone, for your replies. I did go ahead and order it as is
because expression is a strange beast. One never knows what may happen, I
feel. It is around the 600th bp or so.

Yes, it was detected by computational means. I am hesitant to change codon
usage at this stage just in case the gene as is works better.

I'll give it a shot, and if it doesn't work, I'll take your recommendations
and post back about the result.

Thanks again,

Sangeetha.

On Sat, Nov 21, 2009 at 9:12 AM, Artem Evdokimov ar...@xtals.org wrote:

  Dear Sangeetha,



 Can I assume that you detected the hairpin by computational means? In
 general a single hairpin may not necessarily doom your experiment or reduce
 expression – however you could reduce the hairpin stability via silent codon
 changes just to be on the safe side. Keep in mind that in some cases the
 presence of specific secondary structure of RNA can be important for proper
 expression and folding. Alternatively you could try the gene as is and if
 you encounter trouble you can always change the nucleotides later.



 Cheers,



 Artem
  --

 *From:* CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] *On Behalf Of 
 *Sangeetha
 Vedula
 *Sent:* Friday, November 20, 2009 8:03 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] hairpin formation in gene



 Dear bb-ers,


 I am trying to have a gene synthesized and found out that it forms an 11-bp
 hairpin. Does that complicate expression? Would it be better to try and
 disrupt it by altering codon usage to improve expression?

 Thank you in advance,

 Sangeetha.



[ccp4bb] hairpin formation in gene

2009-11-20 Thread Sangeetha Vedula
Dear bb-ers,

I am trying to have a gene synthesized and found out that it forms an 11-bp
hairpin. Does that complicate expression? Would it be better to try and
disrupt it by altering codon usage to improve expression?

Thank you in advance,

Sangeetha.


Re: [ccp4bb] xds undefined data_range error

2009-06-11 Thread Sangeetha Vedula
Thank you.

Unfortunately, it doesn't work even now.

On Thu, Jun 11, 2009 at 11:47 AM, Jürgen Bosch jubo...@jhsph.edu wrote:

 Hi,you should remove the following lines:

  SPOT_RANGE=7  18  !First and last data image number for finding spots
  SPOT_RANGE=20 20  !First and last data image number for finding spots

 and instead define the spot range only in one line e.g. SPOT_RANGE=1  50

 That should fix your problem I think.

 Jürgen

 On 11 Jun 2009, at 11:27, Sangeetha Vedula wrote:

 I checked:

 1. I did name it XDS.INP; no problem there.
 2. The path along with file name template is only about 30 characters.
 3. No. of files = 156 (DATA_RANGE=1 156)
 4. Edited Nx, Ny, Qx, Qy (unless the image header is wrong, which could
 cause problems, but why does it have a problem with data range?).

 I am attaching the file, XDS.INP that I am using. Thank you so much in
 advance for taking the time.

 Regards,

 Sangeetha.

 On Thu, Jun 11, 2009 at 3:23 AM, Kay Diederichs 
 kay.diederi...@uni-konstanz.de wrote:

 Sangeetha Vedula schrieb:

  Hello all,

 Apologies for the non-CCP4 question.

 I am trying to process some data collected at X6A in March. I edited the
 XDS.inp file for ADSC detector. When I try to run xds, however, it gives an
 error immediately:

 !!! ERROR !!! UNDEFINED DATA_RANGE=

 I checked the data range and the image path and template I gave. Doesn't
 appear to be anything wrong. What could be causing the error, though?

 Thanks for any input and suggestions!

 Sangeetha.


 Hello Sangeetha,

 please post your XDS.INP for us to check.

 Kay
 --
 Kay Diederichs http://strucbio.biologie.uni-konstanz.de
 email: kay.diederi...@uni-konstanz.de Tel +49 7531 88 4049 Fax 3183
 Fachbereich Biologie, Universitaet Konstanz, Box M647, D-78457 Konstanz


 XDS.INP


 -
 Jürgen Bosch
 Johns Hopkins Bloomberg School of Public Health
 Department of Biochemistry  Molecular Biology
 Johns Hopkins Malaria Research Institute
 615 North Wolfe Street, W8708
 Baltimore, MD 21205
 Phone: +1-410-614-4742
 Lab:  +1-410-614-4894
 Fax:  +1-410-955-3655
 http://web.me.com/bosch_lab/




Re: [ccp4bb] xds undefined data_range error

2009-06-11 Thread Sangeetha Vedula
I am just now editing it again using nedit. I edited it before in textedit.
That was probably my problem! And no, when I look at it using more, it is
all messed up.

I edited Chris's file. New error.

 !!! ERROR !!! CANNOT READ XPARM.XDS

So now, I followed Jurgen's advice of JOB=ALL and it is running. But will
JOB = ALL try to make me strategize data collection after the fact?

I am yet to figure out how to work XDS viewer to see how it is progressing.
*Is it possible to see progress by looking at images in real time like for
denzo and d*trek?*

To Mark Brooks: SMV may not be required because it did say that XDS figures
it out on its own.

This may be entirely different issue or, I don't know (yet) what it is.
Doesn't running xds generate these files for use in successive steps?

I did consider the editor problem but (idiot!) dismissed it.

On Thu, Jun 11, 2009 at 11:53 AM, Tim Gruene t...@shelx.uni-ac.gwdg.dewrote:

 Dear Sangeetha,

 the attached screenshot is what the XDS.INP looks like when I open it with
 vi. Are you sure you did not edit it with e.g. a windows-editor, or maybe
 some other editor which might have caused this apperance?

 Tim


 --
 Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen

 GPG Key ID = A46BEE1A


 On Thu, 11 Jun 2009, Sangeetha Vedula wrote:

  I checked:

 1. I did name it XDS.INP; no problem there.
 2. The path along with file name template is only about 30 characters.
 3. No. of files = 156 (DATA_RANGE=1 156)
 4. Edited Nx, Ny, Qx, Qy (unless the image header is wrong, which could
 cause problems,
 but why does it have a problem with data range?).

 I am attaching the file, XDS.INP that I am using. Thank you so much in
 advance for taking
 the time.

 Regards,

 Sangeetha.

 On Thu, Jun 11, 2009 at 3:23 AM, Kay Diederichs 
 kay.diederi...@uni-konstanz.de wrote:
  Sangeetha Vedula schrieb:

  Hello all,

  Apologies for the non-CCP4 question.

  I am trying to process some data collected at X6A in March. I edited
 the
  XDS.inp file for ADSC detector. When I try to run xds, however, it
 gives
  an error immediately:

  !!! ERROR !!! UNDEFINED DATA_RANGE=

  I checked the data range and the image path and template I gave.
 Doesn't
  appear to be anything wrong. What could be causing the error, though?

  Thanks for any input and suggestions!

  Sangeetha.


 Hello Sangeetha,

 please post your XDS.INP for us to check.

 Kay
 --
 Kay Diederichs http://strucbio.biologie.uni-konstanz.de
 email: kay.diederi...@uni-konstanz.de Tel +49 7531 88 4049 Fax 3183
 Fachbereich Biologie, Universitaet Konstanz, Box M647, D-78457 Konstanz






Re: [ccp4bb] XDS Viewer

2009-06-11 Thread Sangeetha Vedula
I moved the program to /Applications as well.

When I start XDSviewer giving it the whole path, it errors out, saying:

/Applications/XDS-Viewer.app/Contents/MacOS/XDS-Viewer: line 6:   818 Bus
error
  $CURRENT_DIR/xds-viewer-bin

If I double-click on XDS-Viewer.app icon, it begins to start and quits
immediately.

Could someone tell me what this is, and how to fix it?

Thanks,
Sangeetha.


On Thu, Mar 5, 2009 at 9:21 AM, Derek Logan derek.lo...@mbfys.lu.se wrote:

 Hi,

 I very recently downloaded and installed the Mac OS X executable of the new
 XDS Viewer program to inspect diffraction images. I have moved the program
 to /Applications. It starts up fine, but when I try to load an image it
 complains:

 Cannot open file! For image formats other than .cbf you need to install
 the 2cbf script. Please make sure you have added it to the executable
 path.

 Now 2cbf is in /usr/local/bin, which is in my path. I use /bin/tcsh as
 shell. Is there some confusion between the shell used by XDS Viewer (if any)
 and my preferred shell? Looking in the XDS Viewer.app directory I can't find
 any obvious clues.

 Thanks
 Derek

 __

 Derek Logan   tel: +46 46 222 1443

 Associate Professor   fax: +46 46 222 4692

 Molecular Biophysics  mob: +46 76 8585 707

 Centre for Molecular Protein Science

 Lund University, Box 124, 221 00 Lund, Sweden




[ccp4bb] libcheck merge monomer library

2009-06-03 Thread Sangeetha Vedula
Hello all:

I am trying to merge library files for 2 molecules using libcheck dialogue.
But it only writes out the first library file that I read in.

FILE_L 1.cif
FILE_L2 2.cif

It writes out a file called libcheck.lib which contains only lines from
1.cif.

What am I doing wrong? Is there something I should be doing differently?

Thanks in advance for your advice!

Sangeetha.


Re: [ccp4bb] libcheck merge monomer library

2009-06-03 Thread Sangeetha Vedula
Thank you, Sridhar.

For some reason, that gives an error, saying that it couldn't execute
libcheck. Instead, I tried using the keyworded input format. Do you know
what could be wrong with that?

I tried to install libcheck but apparently my computer can't figure out what
the make function is (make makecif_all errors out). So, I went for running
libcheck in the terminal. I just used elbow to merge the cif files, and the
file looks like one can merge the files manually.

On Wed, Jun 3, 2009 at 5:14 PM, Sridhar Prasad prasadsrid...@hotmail.comwrote:


 Hi,
 G
  If you are using CCP4i, go to refmac and use Merge monomer libraries.

 Then in the input column give the name of Library file 1, select add file
 and input file 2. Give an output file name in output library file.

 This should work.

 Best
 Sridhar
 --
 Date: Wed, 3 Jun 2009 16:40:31 -0400
 From: sangeetha...@gmail.com
 Subject: [ccp4bb] libcheck merge monomer library
 To: CCP4BB@JISCMAIL.AC.UK


 Hello all:

 I am trying to merge library files for 2 molecules using libcheck dialogue.
 But it only writes out the first library file that I read in.

 FILE_L 1.cif
 FILE_L2 2.cif

 It writes out a file called libcheck.lib which contains only lines from
 1.cif.

 What am I doing wrong? Is there something I should be doing differently?

 Thanks in advance for your advice!

 Sangeetha.


 --
 Insert movie times and more without leaving Hotmail®. See 
 how.http://windowslive.com/Tutorial/Hotmail/QuickAdd?ocid=TXT_TAGLM_WL_HM_Tutorial_QuickAdd_062009



[ccp4bb] arp/warp ligand

2009-03-16 Thread Sangeetha Vedula
Hi all,

I am trying to fit a ligand into density using ARP/wARP 7.0.1 in CCP4 suite
6.0.2 on CCP4interface 1.4.4.2.

I get an error message telling me to look for the error in a
##_warp_ligand_details.log.

_

Running Refmac5 to refine the protein PDB without the search ligand.

 After refmac, R = 0.177 (Rfree = 0.000)


 The difference electron density map has been calculated.


Segmentation fault

QUITTING ... ARP/wARP module stopped with an error message:
MAPREAD_MODE_GRIDMAKER

*** Look for error message in the file:
29_warp_ligand_details.log


#CCP4I TERMINATION STATUS 0 All done
#CCP4I TERMINATION TIME 16 Mar 2009  14:43:30
#CCP4I MESSAGE Task failed

***
* Information from CCP4Interface script
***
 Error during script execution.
***

When I look at the details file, all I see at the end is (no error message):


 ## COORDINATE READING ##

 Reading apo protein ... done.
 Identifying N and O atoms for h-bond investigations ... done.
 Reading clean search ligand(s) ... (PDBfmt)  done

___

The details file ends thus, regardless of whether I read in a library file
for the ligand or not, the library is one generated from ProDRG or from
refmac.

Funnily enough, the program ends the same way even using input files that I
had used previously, with a previous version of ARP/wARP; input files that
worked before.

Help, please!

Thanks a ton!

Sangeetha.


Re: [ccp4bb] VOIDOO average volume report

2008-08-20 Thread Sangeetha Vedula
Hi Manish,

But then volume is biased by the plot grid size one chooses and not the
converged volume. In short, one isn't even using the results of volume
refinement because one'd get the same answer from the same grid size for the
same orientation (given the same probe radius). Using the final converged
volume (ie, the last value in the table above Nr of cycles/average/sigma
line in output file), as Dr. Mayer says, should be more correct.

If it is only a % change of cavity volume on binding of ligand one is
looking for, it MAY not make a difference. I am not too sure about that
though. I haven't averaged the volumes both ways and compared the ratios.

Thanks to everyone who replied and everyone who read the exchanges!

Sangeetha.

On Wed, Aug 20, 2008 at 10:24 AM, [EMAIL PROTECTED] wrote:

 Hi,

 After running VOIDOO, you will get the log file with cavity volume
 information.  It has the average volume and the sigma volume information,
 but along with that If you look at the end of the log file, you will find
 the Cavity volume on plot grid (A3) provided separately.  This is the
 volume you report.

 Example:-  in the output log file, scroll down to find the following line
 in the end:

 Nr voxels/Voxel volume/Cavity volume on plot grid (A3) 860   0.342
  294.980

 and the cavity volume you report in this case is 294.980.

 Hope this helps.

 Manish





  Hello all
 
  I am comparing the probe-occupied volumes of a protein cavity with and
 without several ligands. Which cavity volume is used for the
 comparisons?
 
  I found an FAQ link:
  http://www.imsb.au.dk/~mok/o/ofaq/Q.507.htmlhttp://www.imsb.au.dk/%7Emok/o/ofaq/Q.507.html
 http://www.imsb.au.dk/%7Emok/o/ofaq/Q.507.html
 
  Q.507 - Which VOIDOO cavity volume should I quote ? *Category: S.507 -
 cavity voidoo *
 
  The question: Should I use the average cavity volume, or the last
 calculated one ? Sometimes those values are not very stable.
 
  If the cavity volumes fluctuate rather wildly:
 
 1. start with a finer grid (e.g., 0.7 instead of 1 A, or 0.5 instead
 of
 0.7 A)
 2. use a slightly larger probe radius
 3. repeat the calculation N times (e.g., N=10) with randomly oriented
 copies of your molecule  average those (see the manual and the
 paper)
 
 
  However, the reply does not indicate which volume to report.
 
  When the program runs, I see a summary that looks thus:
 
   Summary :
   Nr of cavities found : (  1)
   Nr of original grid points in cavities : (506)
   Total cavity volume (A3)   : (  3.387E+02)
 
  This is the last calculated volume with the finest grid size. However,
 this
  summary does not appear in the log file that VOIDOO writes out.
 
  So, which volume should be used?
 
  A. Run VOIDOO for, say 25 cycles to convergence for 10 different random
 orientations, and average the final converged volumes (volumes with the
 finest grid size) for all.
 
  or
 
  B. Run VOIDOO for 10 cycles for 10 different random orientations, and
 average the average volume for all grid sizes reported in the log file
 for
  10 different orientations regardless of convergence.
 
  or
 
  C. Run VOIDOO till volumes converge for 10 different random
 orientations,
  and average the average volume for all grid sizes reported in the log
 file
  for 10 different orientations.
 
  I am rooting for A. This seems to make most sense. But why then does the
 log
  file not give this value separately although it gives average volume for
 all
  grid sizes and standard deviation? I understand that this tells us how
 sensitive the volume calculation is to grid size. Is that the only
 reason
  these numbers are listed?
 
  Any input will be appreciated!
 
  Thanks,
  Sangeetha.
 









Re: [ccp4bb] VOIDOO average volume report

2008-08-20 Thread Sangeetha Vedula
Hi Manish and everyone,

If one uses the volume on the plot grid size, volume is biased by the plot
grid size one chooses and not the converged volume. In short, one isn't even
using the results of volume refinement because one'd get the same answer
from the same grid size for the same orientation (given the same probe
radius) always. Using the final converged volume (ie, the last value in the
table above Nr of calcns/average/sigma volume line in output file), as Dr.
Mayer says, should be more correct.

If it is only a % change of cavity volume on binding of ligand one is
looking for, it MAY not make a difference. I am not too sure about that
though. I haven't averaged the volumes both ways and compared the ratios.

Thanks to everyone who replied and everyone who read the exchanges!

Sangeetha.

On Wed, Aug 20, 2008 at 10:24 AM, [EMAIL PROTECTED] wrote:

 Hi,

 After running VOIDOO, you will get the log file with cavity volume
 information.  It has the average volume and the sigma volume information,
 but along with that If you look at the end of the log file, you will find
 the Cavity volume on plot grid (A3) provided separately.  This is the
 volume you report.

 Example:-  in the output log file, scroll down to find the following line
 in the end:

 Nr voxels/Voxel volume/Cavity volume on plot grid (A3) 860   0.342
  294.980

 and the cavity volume you report in this case is 294.980.

 Hope this helps.

 Manish





  Hello all
 
  I am comparing the probe-occupied volumes of a protein cavity with and
 without several ligands. Which cavity volume is used for the
 comparisons?
 
  I found an FAQ link:
  http://www.imsb.au.dk/~mok/o/ofaq/Q.507.htmlhttp://www.imsb.au.dk/%7Emok/o/ofaq/Q.507.html
 http://www.imsb.au.dk/%7Emok/o/ofaq/Q.507.html
 
  Q.507 - Which VOIDOO cavity volume should I quote ? *Category: S.507 -
 cavity voidoo *
 
  The question: Should I use the average cavity volume, or the last
 calculated one ? Sometimes those values are not very stable.
 
  If the cavity volumes fluctuate rather wildly:
 
 1. start with a finer grid (e.g., 0.7 instead of 1 A, or 0.5 instead
 of
 0.7 A)
 2. use a slightly larger probe radius
 3. repeat the calculation N times (e.g., N=10) with randomly oriented
 copies of your molecule  average those (see the manual and the
 paper)
 
 
  However, the reply does not indicate which volume to report.
 
  When the program runs, I see a summary that looks thus:
 
   Summary :
   Nr of cavities found : (  1)
   Nr of original grid points in cavities : (506)
   Total cavity volume (A3)   : (  3.387E+02)
 
  This is the last calculated volume with the finest grid size. However,
 this
  summary does not appear in the log file that VOIDOO writes out.
 
  So, which volume should be used?
 
  A. Run VOIDOO for, say 25 cycles to convergence for 10 different random
 orientations, and average the final converged volumes (volumes with the
 finest grid size) for all.
 
  or
 
  B. Run VOIDOO for 10 cycles for 10 different random orientations, and
 average the average volume for all grid sizes reported in the log file
 for
  10 different orientations regardless of convergence.
 
  or
 
  C. Run VOIDOO till volumes converge for 10 different random
 orientations,
  and average the average volume for all grid sizes reported in the log
 file
  for 10 different orientations.
 
  I am rooting for A. This seems to make most sense. But why then does the
 log
  file not give this value separately although it gives average volume for
 all
  grid sizes and standard deviation? I understand that this tells us how
 sensitive the volume calculation is to grid size. Is that the only
 reason
  these numbers are listed?
 
  Any input will be appreciated!
 
  Thanks,
  Sangeetha.
 









[ccp4bb] VOIDOO average volume report

2008-08-19 Thread Sangeetha Vedula
Hello all

I am comparing the probe-occupied volumes of a protein cavity with and
without several ligands. Which cavity volume is used for the comparisons?

I found an FAQ link:
http://www.imsb.au.dk/~mok/o/ofaq/Q.507.htmlhttp://www.imsb.au.dk/%7Emok/o/ofaq/Q.507.html

Q.507 - Which VOIDOO cavity volume should I quote ? *Category: S.507 -
cavity voidoo *

The question: Should I use the average cavity volume, or the last
calculated one ? Sometimes those values are not very stable.

If the cavity volumes fluctuate rather wildly:

   1. start with a finer grid (e.g., 0.7 instead of 1 A, or 0.5 instead of
   0.7 A)
   2. use a slightly larger probe radius
   3. repeat the calculation N times (e.g., N=10) with randomly oriented
   copies of your molecule  average those (see the manual and the paper)


However, the reply does not indicate which volume to report.

When the program runs, I see a summary that looks thus:

 Summary :
 Nr of cavities found : (  1)
 Nr of original grid points in cavities : (506)
 Total cavity volume (A3)   : (  3.387E+02)

This is the last calculated volume with the finest grid size. However, this
summary does not appear in the log file that VOIDOO writes out.

So, which volume should be used?

A. Run VOIDOO for, say 25 cycles to convergence for 10 different random
orientations, and average the final converged volumes (volumes with the
finest grid size) for all.

or

B. Run VOIDOO for 10 cycles for 10 different random orientations, and
average the average volume for all grid sizes reported in the log file for
10 different orientations regardless of convergence.

or

C. Run VOIDOO till volumes converge for 10 different random orientations,
and average the average volume for all grid sizes reported in the log file
for 10 different orientations.

I am rooting for A. This seems to make most sense. But why then does the log
file not give this value separately although it gives average volume for all
grid sizes and standard deviation? I understand that this tells us how
sensitive the volume calculation is to grid size. Is that the only reason
these numbers are listed?

Any input will be appreciated!

Thanks,
Sangeetha.


Re: [ccp4bb] Preventing close contact between protein and ligand

2008-07-30 Thread Sangeetha Vedula
On Wed, Jul 30, 2008 at 8:17 AM, Garib Murshudov [EMAIL PROTECTED]wrote:

 Dear Snageetha
 1) Could you check please if specified atoms have zero occupancy. Atoms
 with zero occupancy are considered as absent and there are not restraints on
 them


They do not have zero occupancy.


 2) symm y at the end of instructions means that the program check all
 possible symmetry operators and finds minimal distance. Most probably 5.024
 is the distance between symmetry related atoms


Thanks.


 3) to remove antibumping between different chains there is an undocumented
 keyword. It can be used. the keyword is (as an example)

 vdwrestraints exclude between chains A B


But this seems to remove 'anti'bumping, not bumping.

I tried it. No effect. The ligand fits the density well but a methyl carbon
in the ligand is 2.74 A from a Ser O-gamma. I would think that I should have
the opposite problem; ligand being pushed out of the density because of
clashes.

The line I added was:

vdwrestraints exclude between chains A Y

(I renamed the ligand chain Y, not X as I had in the previous email).



 Please let me know if this instruction does not work.
 NB: This option should not be used unless you know what you are doing (that
 is the reason why it has not been documented). If there are clashes between
 chains then there are reasons for that. For example
 if ligand has half occupancy then it is very likely that surrounding atoms
 also have multiple conformation and you should model them.


In some of my the complexes that I am working with, that is, indeed the
case. But in this one, I don't see evidence for an alternate conformation of
the serine.

I would be happy to know if you could suggest anything else. I'll try
anything!

Thanks,

Sangeetha.



 regards
 Garib


 On 29 Jul 2008, at 18:24, Sangeetha Vedula wrote:

 Dear bb users,

 I am refining a protein-ligand complex (at 1.68 A resolution) in which the
 ligand lies on a 2-fold crystallographic symmetry axis. The ligand occupancy
 is, therefore, 0.5 in each asymmetric unit.

 I am almost at the end of the refinement but one problem has me stumped.
 Refmac keeps moving a carbon in the ligand too close to a serine OG and an
 oxygen too close to an arginine CD. Given that the ligand is at the
 interface, the density is not perfect. However, I rebuild the ligand to
 eliminate close contacts and still be within density and refmac pulls it
 right back close to the protein. The refined position does not even look
 better than the rebuilt one! It almost always looks worse! Would refmac put
 less weight on close contacts with the ligand because it is only partially
 occupied?

 I tried to use external restraints between the ligand and the residues so
 that they are kept further away.

 Upon searching the net, I found this command line:

 *external distance first chain [ch] residue [res] insertion [ins] -
 atom [n] [altcode [a]] second chain [ch] residue [res] insertion [ins]-
 atom [n] [altcode [a] ] value [v] sigma [s] [symm y/n]

 *I thought (hoped) that the distance herein is the minimum distance of
 approach between the specified atoms, I added these lines from within
 Developer options in refmac interface:

 exte dist first chain A resi 59 atom CD seco chain X resi 2001 atom O1 valu
 3.2 sigm 0.02 symm Y
 exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10
 valu 3.2 sigm 0.02 symm Y

 It didn't recognize these restraints at all.

 However, when I change these lines to:

 exte dist first chain A resi 59 atom CA seco chain X resi 2001 atom O1 valu
 3.2 sigm 0.02 symm Y
 exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10
 valu 3.2 sigm 0.02 symm Y

 Refmac recognizes the first line but not the second - lines from log file:

 Bond distance deviations from the ideal 10.000Sigma will be monitored

 A 59 ARG CA  . - X   2001 DIE O1  . mod.= 5.024 id.= 3.200 dev= -1.824
 sig.= 0.020

 This raises two concerns:

 Concern 1: From the first line of output: the restraints here don't seem to
 be minimizing close contact at all; it seems to think they are bonded
 somehow (the distance between these atoms is not 5.024; it is 6.26 A; I
 don't know what 5.024 A is!).

 I am missing something here. It'd be great if someone can tell me what that
 is!

 Concern 2: This command only works when the first atom specified is a
 C-alpha atom (or maybe a main chain atom; I didn't try using other main
 chain atoms). Why is that?

 AND ULTIMATELY,

 is there some way I can tell refmac not to make the ligand and protein
 clash?

 I'd really appreciate any help!

 Thanks,

 Sangeetha.





Re: [ccp4bb] Preventing close contact between protein and ligand

2008-07-30 Thread Sangeetha Vedula
I am looking at my ligand library file that ProDRG generated. I do not see
any column id that indicates that it is a restraints weight or such. Could
you tell me exactly which one I should edit?

Thanks,

Sangeetha.

On Tue, Jul 29, 2008 at 3:23 PM, Robert Immormino [EMAIL PROTECTED]wrote:

 You're probably better off trying to change the restraints in the
 ligand .cif file.  It sounds like you have some torsion angle or
 chiral center set wrong.
 -bob

 On Tue, Jul 29, 2008 at 10:24 AM, Sangeetha Vedula
 [EMAIL PROTECTED] wrote:
  Dear bb users,
 
  I am refining a protein-ligand complex (at 1.68 A resolution) in which
 the
  ligand lies on a 2-fold crystallographic symmetry axis. The ligand
 occupancy
  is, therefore, 0.5 in each asymmetric unit.
 
  I am almost at the end of the refinement but one problem has me stumped.
  Refmac keeps moving a carbon in the ligand too close to a serine OG and
 an
  oxygen too close to an arginine CD. Given that the ligand is at the
  interface, the density is not perfect. However, I rebuild the ligand to
  eliminate close contacts and still be within density and refmac pulls it
  right back close to the protein. The refined position does not even look
  better than the rebuilt one! It almost always looks worse! Would refmac
 put
  less weight on close contacts with the ligand because it is only
 partially
  occupied?
 
  I tried to use external restraints between the ligand and the residues so
  that they are kept further away.
 
  Upon searching the net, I found this command line:
 
  external distance first chain [ch] residue [res] insertion [ins] -
  atom [n] [altcode [a]] second chain [ch] residue [res] insertion [ins]-
  atom [n] [altcode [a] ] value [v] sigma [s] [symm y/n]
 
  I thought (hoped) that the distance herein is the minimum distance of
  approach between the specified atoms, I added these lines from within
  Developer options in refmac interface:
 
  exte dist first chain A resi 59 atom CD seco chain X resi 2001 atom O1
 valu
  3.2 sigm 0.02 symm Y
  exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10
 valu
  3.2 sigm 0.02 symm Y
 
  It didn't recognize these restraints at all.
 
  However, when I change these lines to:
 
  exte dist first chain A resi 59 atom CA seco chain X resi 2001 atom O1
 valu
  3.2 sigm 0.02 symm Y
  exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10
 valu
  3.2 sigm 0.02 symm Y
 
  Refmac recognizes the first line but not the second - lines from log
 file:
 
  Bond distance deviations from the ideal 10.000Sigma will be monitored
 
  A 59 ARG CA  . - X   2001 DIE O1  . mod.= 5.024 id.= 3.200 dev=
 -1.824
  sig.= 0.020
 
  This raises two concerns:
 
  Concern 1: From the first line of output: the restraints here don't seem
 to
  be minimizing close contact at all; it seems to think they are bonded
  somehow (the distance between these atoms is not 5.024; it is 6.26 A; I
  don't know what 5.024 A is!).
 
  I am missing something here. It'd be great if someone can tell me what
 that
  is!
 
  Concern 2: This command only works when the first atom specified is a
  C-alpha atom (or maybe a main chain atom; I didn't try using other main
  chain atoms). Why is that?
 
  AND ULTIMATELY,
 
  is there some way I can tell refmac not to make the ligand and protein
  clash?
 
  I'd really appreciate any help!
 
  Thanks,
 
  Sangeetha.
 



[ccp4bb] Preventing close contact between protein and ligand

2008-07-29 Thread Sangeetha Vedula
Dear bb users,

I am refining a protein-ligand complex (at 1.68 A resolution) in which the
ligand lies on a 2-fold crystallographic symmetry axis. The ligand occupancy
is, therefore, 0.5 in each asymmetric unit.

I am almost at the end of the refinement but one problem has me stumped.
Refmac keeps moving a carbon in the ligand too close to a serine OG and an
oxygen too close to an arginine CD. Given that the ligand is at the
interface, the density is not perfect. However, I rebuild the ligand to
eliminate close contacts and still be within density and refmac pulls it
right back close to the protein. The refined position does not even look
better than the rebuilt one! It almost always looks worse! Would refmac put
less weight on close contacts with the ligand because it is only partially
occupied?

I tried to use external restraints between the ligand and the residues so
that they are kept further away.

Upon searching the net, I found this command line:

*external distance first chain [ch] residue [res] insertion [ins] -
atom [n] [altcode [a]] second chain [ch] residue [res] insertion [ins]-
atom [n] [altcode [a] ] value [v] sigma [s] [symm y/n]

*I thought (hoped) that the distance herein is the minimum distance of
approach between the specified atoms, I added these lines from within
Developer options in refmac interface:

exte dist first chain A resi 59 atom CD seco chain X resi 2001 atom O1 valu
3.2 sigm 0.02 symm Y
exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10 valu
3.2 sigm 0.02 symm Y

It didn't recognize these restraints at all.

However, when I change these lines to:

exte dist first chain A resi 59 atom CA seco chain X resi 2001 atom O1 valu
3.2 sigm 0.02 symm Y
exte dist first chain A resi 27 atom OG seco chain X resi 2001 atom C10 valu
3.2 sigm 0.02 symm Y

Refmac recognizes the first line but not the second - lines from log file:

Bond distance deviations from the ideal 10.000Sigma will be monitored

A 59 ARG CA  . - X   2001 DIE O1  . mod.= 5.024 id.= 3.200 dev= -1.824
sig.= 0.020

This raises two concerns:

Concern 1: From the first line of output: the restraints here don't seem to
be minimizing close contact at all; it seems to think they are bonded
somehow (the distance between these atoms is not 5.024; it is 6.26 A; I
don't know what 5.024 A is!).

I am missing something here. It'd be great if someone can tell me what that
is!

Concern 2: This command only works when the first atom specified is a
C-alpha atom (or maybe a main chain atom; I didn't try using other main
chain atoms). Why is that?

AND ULTIMATELY,

is there some way I can tell refmac not to make the ligand and protein
clash?

I'd really appreciate any help!

Thanks,

Sangeetha.