Re: [ccp4bb] Room temperature change from 25ºC to 20ºC

[Katherine Sippel]

In the Southern realms of the US at least, academic centers maintain room temperature at WSRT year round because “77°, ew gross.” In fact, the only time those air conditioners turn off is when we are struck by errant polar vertices and our power grids fail. Postdocs described the experience as sweltering. Cheers,KatSent from my iPhoneOn Apr 1, 2024, at 8:20 AM, David J. Schuller  wrote:






Looking forward to feedback from the Southern realms as to which should be higher; SSRT or WSRT.







===
 All Things Serve the Beam
 ===
 David J. Schuller
 modern man in a post-modern world
 MacCHESS, Cornell University
 schul...@cornell.edu




From: CCP4 bulletin board  on behalf of Nukri Sanishvili 
Sent: Monday, April 1, 2024 10:47 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] Room temperature change from 25ºC to 20ºC
 



Excellent idea, Mark!
I think the solution to your dilemma is rather straightforward: we introduce a Winter Standard Room Temperature (WSRT) and a Summer Standard Room Temperature (SSRT). This way, the folks from locales closer to the equator can always use SSRT, while those
 from more temperate climates can take advantage of using both WSRT and SSRT.
The added benefit of this approach will be inclusion of researchers closer to either of the poles (which, forgive me for saying it, you so egregiously omitted from your considerations). Similar to the tropics, they could use just one standard - WSRT.
Hope it helps your and so many young scientists' careers.
Best wishes,
Nukri

 






On Mon, Apr 1, 2024 at 9:29 AM Mark  wrote:



Room temperature change from 25ºC to 20ºC


As a member of the inter-society standards commission St-Incent I have been asked to take the bearings of the structural biology community regarding a proposal to lower the universally understood room temperature from 25ºC (77º Fahrenheit) to 20ºC (68º
 Fahrenheit). Obvious advantages would be less heating necessary for experiments at this standard temperature. Given that laboratories nowadays are not commonly heated to this high temperature anyway, it does appear to make sense.


Members of tropical and subtropical countries have already expressed opposition to the proposal, because they have to reach room temperature by cooling rather than heating, so for them the proposal would mean more CO2 emissions, not less.


Please express opinions to this list today, so that I have time to collate them before the local deadline of 28 December.






Mark J van Raaij
Dpto de Estructura de Macromoleculas, lab 20B
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain






To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1





To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1






To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1

Re: [ccp4bb] Calculation of RSRZ Score in PDB Validation Reports

[Katherine Sippel]

I started a ccp4 thread a few years ago about RSRZ score calculations
favoring trimmed side chains because they produce better scores. Based on
what I could find at that time, it looked like the density of your
structure was compared to the density of that residue type in submitted
PDBs of similar resolutions. However, I could be seriously mistaken.

Cheers,
Katherine

On Mon, Nov 28, 2016 at 11:46 PM, Ethan Merritt 
wrote:

> On Monday, 28 November 2016 08:35:44 PM Pavel Afonine wrote:
> > I find Lothar's comments regarding H and RSRZ excellent! I would think of
> > it as a pretty much bug report. I hope developers at that end listen.
> This
> > goes very well in line with Phoebe's comment earlier today.
>
> I guess I'm a bit surprised that adding or subtracting hydrogens from the
> model
> without re-refining or at least re-calculating Fc would affect RSRZ at all.
> I had thought that RSRZ was obtained by comparing density in an Fc map
> (or probably mFo-DFc) with the corresponding density in an Fo map.
> I thought that the coordinates were used only to determine the per-residue
> region of the map to be compared.
>
> Going back to the 2004 Kleywegt paper that the PDB cites for calculation of
> RSRZ I see that it's a bit ambiguous exactly what maps are being compared.
> So maybe I'm wrong and the current coordinates are used directly to get
> local "Fc density" by expanding 3D Gaussians without reference to a
> previously
> calculated map from refined phases.
>
> Can anyone clarify exactly what maps are being compared during wwPDB
> validation?
>
> Ethan
>
> >
> > Pavel
> >
> > On Mon, Nov 28, 2016 at 2:51 PM, Dale Tronrud 
> wrote:
> >
> > > On 11/28/2016 12:52 PM, esse...@helix.nih.gov wrote:
> > > >> I found that one can get RSRZ to go way down by loosening the
> geometry
> > > >> restraints.  The result is a crappy structure and I don't recommend
> > > doing
> > > >> that, but it does get all the atoms crammed into some sort of
> density.
> > > >
> > > >   Your observation is quite interesting. I can add this: when we were
> > > working
> > > > with low to medium resolution structures, deleting the hydrogen atoms
> > > from
> > > > the model after refinement moved the very bad RSRZ statistic to
> about the
> > > > average in the given resolution range! Note, no re-refinement was
> done
> > > just
> > > > a simple deletion of the riding H-atoms. I find this to be odd given
> the
> > > > fact that, say the phenix developers favor the inclusion of H-atoms
> on
> > > > riding positions even in cases of low resolution structures. (I
> assume
> > > the
> > > > refmac5 and BUSTER-TNT developers have also a favorable opinion about
> > > > including H-atoms in the final model - and during refinement).
> > > >
> > > > In my mind, it may be tempting to delete H-atoms to improve this
> > > statistic but
> > > > when you use them in refinement they should be included regardless
> of the
> > > > outcome of the RSRZ analysis.
> > >
> > >Of course, if you trick a validation statistic like this you haven't
> > > accomplished anything.  All you are saying is that one should rank RSRZ
> > > scores with and without hydrogen atoms separately.  Perhaps you should
> > > suggest that to the PDB validation people.
> > >
> > > Dale Tronrud
> > > >
> > > >>
> > > >> RSRZ, in my most humble of opinions, seems like one of those
> statistics
> > > that
> > > >> is far more useful in theory than reality.   Particularly for
> > > >> medium-resolution structures, the fit of each entire side chain to
> the
> > > density
> > > >> is likely to be imperfect because the density is imperfect,
> especially
> > > toward
> > > >> the tips of those side chains.
> > > >>
> > > >> Then again, it can be a good flag for bits of the structure worth a
> > > second
> > > >> look in rebuilding.
> > > >
> > > >   The latter is certainly true. It may mean that the developers of
> RSRZ
> > > > analysis need to tune it a bit to make it fully useful.
> > > >
> > > > L.
> > > >
> > > >>
> > > >> 
> > > >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of
> Matthew
> > > >> Bratkowski [mab...@cornell.edu]
> > > >> Sent: Tuesday, November 22, 2016 10:12 AM
> > > >> To: CCP4BB@JISCMAIL.AC.UK
> > > >> Subject: [ccp4bb] Calculation of RSRZ Score in PDB Validation
> Reports
> > > >>
> > > >> Hello all,
> > > >>
> > > >> I was wondering if anyone knew how the RSRZ score was calculated in
> the
> > > >> protein data bank validation reports and how useful of a metric this
> > > actually
> > > >> is for structure validation?  I am trying to improve this score on a
> > > structure
> > > >> that I am working on, but I'm not really sure where to begin.  From
> my
> > > >> understanding, the score is based on the number of RSRZ outliers
> with a
> > > score
> > > >>> 2.  In my case, I have several residues with scores between 2 and
> 4,
> > > but at
> > > >> least 

Re: [ccp4bb] Guard columns from FPLC

[Katherine Sippel]

I think the typical Akta configuration has the prefilter installed after
the mixing pump, but before the sample loop. That is where it is on mine at
least. It wouldn't stop sample precipitate from loading on the column.
Technically, it would probably still work if you installed the prefilter
after the sample loop, but that would add to the dilution problem stated
earlier.

As mentioned by a previous commenter, I personally spin filter everything
right before I load it. That is usually sufficient unless the buffer change
is causing the protein to crash out. Of course if that is the case a guard
column not the solution.

Cheers,
Katherine


On Tue, Sep 16, 2014 at 12:46 PM, Scott Pegan scott.d.pe...@gmail.com
wrote:

 Anita,

 From your description, you most likely are looking for a prefilter6000.


 http://www.gelifesciences.com/webapp/wcs/stores/servlet/productById/en/GELifeSciences/18458201

 Scott


 Scott D. Pegan, Ph.D.
 Associate Professor
 Pharmaceutical and Biomedical Sciences
 College of Pharmacy
 University of Georgia
 240 W. Green St.
 Athens, GA, USA, 30602
 phone: 706-542-3435

 On Tue, Sep 16, 2014 at 4:29 AM, Anita P crystals...@gmail.com wrote:

 Hi All,
 Sorry for this off topic.

 I have heard that there are these little columns called guard columns
 which can be attached to AKTA purifiers. These columns prevent the incoming
 huge aggregates to be deposited and blocking of the gel filtration columns.

 Can any one advice me regarding where to purchase these columns. I could
 not find them in GE website. We have Superdex 16/60 on AKTA purifier.

 Thanks in advance.
 Have a good day

 Anita





-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] ligand mismathces in pdb deposit

[Katherine Sippel]

Hi Bing,

This is the result of the new PDB deposition system being buggy. I had a
similar problem with imidazole recently. This is the solution given to me
by the PDB annotators.

Please choose from the instance page:

type of ligand: heterogen,

then give alternate ligand ID as IMD, (in your case HEM)

then click finish button address the issue.

It should solve the problem, if not, please let us know.

Hope this helps,
Katherine


On Mon, Sep 15, 2014 at 3:03 PM, Wang, Bing bingw...@ou.edu wrote:

 Forgot the picture! Here is it!

 
 From: Paul Emsley [pems...@mrc-lmb.cam.ac.uk]
 Sent: Sunday, September 14, 2014 6:16 PM
 To: Wang, Bing
 Cc: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] ligand mismathces in pdb deposit

 On 12/09/14 23:28, Wang, Bing wrote:
  Hi guys,

 Hello.

 
  A quick question about pdb deposition! My protein has a common ligand
  'heme' which mismatches with the ligand in pdb CCD (/Chemical
  Component Dictionary/).

 What makes you think so?

  However i didn't find any differences in it. Is that because of the
  positions of double bonds or hydrogen atoms,

 unlikely.

  since my model don't have hydrogen atoms. Actually i replace the heme
  with exact one from CCD and run refmac which give me a new pdb file.
  Unfortunately, CCD still didn’t recognize the heme.

 It is not clear to me what test you are performing.  I do note that the
 SMILES for HEM in the CCD doesn't contain Fe - that might be related.

 HTH,

 Paul.





-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] reporting rotational and translational interface shifts

[Katherine Sippel]

Hi all,

I am trying to figure out the best way of reporting changes in dimer
orientations between three structures. I have an apo, a drug-bound, and a
homolog dimer structure. I had originally decided to calculate the angle
from the rotation matrix generated by LSQKAB, but the translational
component is oriented in opposite directions (translation vector of -22.9,
3.2, 10.8 versus 22.4, -3.4, -17.1). I've only ever seen these types of
orientation shifts reported in degrees, so I was wondering how other people
dealt with accurately reporting these numbers in their manuscripts. Did you
include the translation vectors in the text or just rely on figures and
arrow to make your point?

Thanks for your time,
Katherine

-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] New PDB validation reports

[Katherine Sippel]

Hi all,

I've been playing with the new PDB validation service. It is very pretty
and kudos to all the hard work that has clearly gone into it. I did notice
however that the way the information is presented, there seems to be a bias
towards truncating side chains versus modeling them with higher b-factors.
The disordered side chains have higher RSRZs (rightfully so), but there
doesn't seem to be any indicator for missing atoms. As a results I can make
my validation report prettier by truncating versus modeling with high Bs.

I don't want to kick an ant pile here, but given this rather significant
difference in quality reporting, I was wondering if the community had
reached a consensus on this issue that I had missed.

Cheers,
Katherine

-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] Lysine coordinated ions

[Katherine Sippel]

Hi all,

My google-fu has failed me once again so I am turning to the collective
knowledge of the bb. I'm working on a blobology challenge at the moment and
have hit a wall. Is anyone aware of an ion that coordinates to lysine and
prefers octahedral geometry. The mystery ion seems to have perfect
octahedral geometry with bond distances of ~2.1 angstrom, but the only
direct side chain interaction is to a lysine NZ, the rest are waters.

I've been through the various and sundry metal coordination resources
including the Harding website and the Minor paper on datamining metal
binding sites. These unsurprisingly indicate that a cation is unlikely,
especially in the absence of any other, more coordination friendly side
chain or backbone atoms. I manually attempted chlorine but the density said
no. I have this protein in four other space groups, many from the same
batch of protein, and none of them have similar density at this residue so
it isn't a covalent modification. I have Mg, Na, and Cl in the crystal
solution/storage buffer, but as mentioned above the cations don't make
chemical sense and chlorine was a no go. I'm not ruling out an adventitious
tag-along. Any suggestions would be appreciated.

Thanks for your time,
Katherine


-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] Aimless and multiple crystals

[Katherine Sippel]

Hi all,

I have two nice, but a hair under-complete data sets that I am trying to
merge together. If I run aimless on them separately everything looks
beautiful. When I merge them together the CC1/2 is still good but all the
various and sundry Rs sky rocket. When I go to the log file and look at the
Rs in respect to batch there is a jump between the first and second data
set, so clearly they are not scaling well in respect to one another. I
suspect that there is some obvious scripting command that I am missing, but
I couldn't find it in the Aimless documentation or the bb archives. Any
help would be appreciated.

Cheers,
Katherine

-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] Aimless and multiple crystals

[Katherine Sippel]

That is probably the culprit. The two unit cell dimensions are...

dataset #1: 68.8295   75.9597   83.3998   65.4000   88.8500   64.9000
dataset #2: 68.3005   75.7100   83.1299   65.3601   88.8900   65.2600

I can't believe I didn't look at that. I know better. Thanks for all of
your quick responses.

Katherine



On Thu, May 8, 2014 at 12:18 PM, Craig Bingman cbing...@biochem.wisc.eduwrote:

 How close are the cell constants?

 On May 8, 2014, at 12:17 PM, Katherine Sippel katherine.sip...@gmail.com
 wrote:

 I forgot to mention the space group is P1. Sorry about that.

 Thanks,
 Katherine


 On Thu, May 8, 2014 at 12:02 PM, Katherine Sippel 
 katherine.sip...@gmail.com wrote:

 Hi all,

 I have two nice, but a hair under-complete data sets that I am trying to
 merge together. If I run aimless on them separately everything looks
 beautiful. When I merge them together the CC1/2 is still good but all the
 various and sundry Rs sky rocket. When I go to the log file and look at the
 Rs in respect to batch there is a jump between the first and second data
 set, so clearly they are not scaling well in respect to one another. I
 suspect that there is some obvious scripting command that I am missing, but
 I couldn't find it in the Aimless documentation or the bb archives. Any
 help would be appreciated.

 Cheers,
 Katherine

 --
 Nil illegitimo carborundum* - *Didactylos




 --
 Nil illegitimo carborundum* - *Didactylos





-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] High Salt Cryo

[Katherine Sippel]

-biology/faculty/blaine-mooers-ph-d- [1]

 X-ray lab webpage: http://www.oumedicine.com/department-of-biochemistry-
 and-molecular-biology/department-facilities/macromolecular-
 crystallography-laboratory [2]

 Small Angle Scattering webpage: http://www.oumedicine.com/
 docs/default-source/ad-biochemistry-workfiles/small-
 angle-scattering-links.html?sfvrsn=0 [3]

 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Katherine Sippel [katherine.sip...@gmail.com]
 Sent: Tuesday, February 18, 2014 12:08 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] High Salt Cryo

 Hi all,

 I'm looking for a cryo condition for high NaCl (3+ M) crystallization
 condition. I would do it the proper way, but our beam/cryostream is down.

 I've tried a bunch of things at the moment. Ethylene glycol and PEG 400
 nuke the crystals immediately even at low concentrations. Prolonged
 exposure to glycerol and sucrose starts to break them down so I'm thinking
 that the diffraction will probably suffer. I can't find any reports of
 NaCl's viability as a cryosalt. I've got Paratone/Paraffin oil/Mitegen's LV
 cryo oil on tap but I was hoping to not put all my eggs in one basket.

 I tried the ISRDB database through archive.comhttps://
 urldefense.proofpoint.com/v1/url?u=http://archive.comk=
 7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4
 V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%
 2FzyGkSwsbIoq92CSnOk%3D%0As=3cfbf18821b5b59934971bf583cf3d
 d6e2ded91923c614e670857e10916c687e [4] without any luck (no search
 function). I've gone to the PDB searching for similar crystallization
 conditions and looked up the papers for their cryos, but they are all
 glycerol. Google gives me the same.


 I thought I'd see if anyone on the bb has an anecdotal this worked for
 us story. I would love to hear it.

 Thank you for your time,
 Katherine

 --
 Nil illegitimo carborundum - Didactylos



 Links:
 --
 [1]
 http://www.oumedicine.com/department-of-biochemistry-
 and-molecular-biology/faculty/blaine-mooers-ph-d-
 [2]
 http://www.oumedicine.com/department-of-biochemistry-
 and-molecular-biology/department-facilities/macromolecular-
 crystallography-laboratory
 [3]
 http://www.oumedicine.com/docs/default-source/ad-
 biochemistry-workfiles/small-angle-scattering-links.html?sfvrsn=0
 [4]
 https://urldefense.proofpoint.com/v1/url?u=http://archive.comamp;k=
 7DHVT22D9IhC0F3WohFMBA%3D%3D%0Aamp;r=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4
 V0aIwH4RJhyZU%3D%0Aamp;m=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%
 2FzyGkSwsbIoq92CSnOk%3D%0Aamp;s=3cfbf18821b5b59934971bf583cf3d
 d6e2ded91923c614e670857e10916c687ehttps://urldefense.proofpoint.com/v1/url?u=http://archive.comk=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=Vjr4m%2Fds%2FdLGOVQoQ0x8PApF%2FzyGkSwsbIoq92CSnOk%3D%0As=3cfbf18821b5b59934971bf583cf3dd6e2ded91923c614e670857e10916c687e



 --
 Enrico A. Stura D.Phil. (Oxon) ,Tel: 33 (0)1 69 08 4302 Office
 Room 19, Bat.152, Tel: 33 (0)1 69 08 9449Lab
 http://www-dsv.cea.fr/ibitecs/simopro/ltmb/cristallogenese
 LTMB, SIMOPRO, IBiTec-S, CE Saclay, 91191 Gif-sur-Yvette,   FRANCE
 http://scholar.google.com/citations?hl=enuser=Kvm06WIoPAsCpagesize=100;
 sortby=pubdate
 http://www.chem.gla.ac.uk/protein/mirror/stura/index2.html
 e-mail: est...@cea.fr Fax: 33 (0)1 69 08 90 71




-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] High Salt Cryo

[Katherine Sippel]

Hi all,

I'm looking for a cryo condition for high NaCl (3+ M) crystallization
condition. I would do it the proper way, but our beam/cryostream is down.

I've tried a bunch of things at the moment. Ethylene glycol and PEG 400
nuke the crystals immediately even at low concentrations. Prolonged
exposure to glycerol and sucrose starts to break them down so I'm thinking
that the diffraction will probably suffer. I can't find any reports of
NaCl's viability as a cryosalt. I've got Paratone/Paraffin oil/Mitegen's LV
cryo oil on tap but I was hoping to not put all my eggs in one basket.

I tried the ISRDB database through archive.com without any luck (no search
function). I've gone to the PDB searching for similar crystallization
conditions and looked up the papers for their cryos, but they are all
glycerol. Google gives me the same.

I thought I'd see if anyone on the bb has an anecdotal this worked for us
story. I would love to hear it.

Thank you for your time,
Katherine

-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] Room temperature data collection

[Katherine Sippel]

To answer Mark's question, if your crystals are capillary mounted then
planes are no problem. My protocol is to mount them, wrap them in cotton
batting, put them in a 50 ml falcon tube, and pack that in bubble wrap. I
managed to get them through security in my carry-on with no issues. I
suggest using a long, thin strip of paper across the bottom of the batting
before putting it in the falcon tube so you can get it out without putting
any lateral pressure on the capillary. Also, grey hair is not a
prerequisite for capillary mounting. Mine haven't shown up yet and I still
manage just fine. It's actually quite simple once you get the hang of it.

Cheers,
Katherine


On Thu, Feb 6, 2014 at 6:03 AM, Mark J van Raaij mjvanra...@cnb.csic.eswrote:

 on a related matter, what are current experiences with taking crystals at
 room temperature by plane?
 (we are interested in trying the HC1 at a synchrotron but the closest are
 at ESRF, which is quite a drive or train-ride from Madrid)

 Mark J van Raaij
 Lab 20B
 Dpto de Estructura de Macromoleculas
 Centro Nacional de Biotecnologia - CSIC
 c/Darwin 3
 E-28049 Madrid, Spain
 tel. (+34) 91 585 4616
 http://www.cnb.csic.es/~mjvanraaij





 On 6 Feb 2014, at 11:52, Marco Mazzorana wrote:

  Hi Theresa,
  Diamond Light Source currently offers this capability at all of its
 beamlines (all equipped with Pilatus detectors).
  This can be performed either standard pins under controlled humidity
 conditions, via the HC1 (Sanchez-Weatherby et al. Acta Cryst. (2009). D65,
 1237-1246 http://scripts.iucr.org/cgi-bin/paper?S0907444909037822 )
 available on request at all beamlines.
  The alternative is using the in-situ capabilities of three of our
 beamlines, where diffraction datasets can be collected from crystals within
 the 96well format crystallization plates.
  Fast data collection and Pilatus detectors help enormously (as a
 reference read the paper from Owen et al Acta Cryst. (2012). D68, 810-818
 http://scripts.iucr.org/cgi-bin/paper?S0907444912012553 )
  A dedicated software for merging and clustering partial datasets is also
 available to all users (Foadi et al. Acta Cryst. (2013). D69, 1617-1632
 http://scripts.iucr.org/cgi-bin/paper?S0907444913012274 ).
  If you want to know more, please check http://diamond.ac.uk/mx-home/
  Hope this helps.
  Ciao,
 
  Marco
 
 
 
 
  2014-02-06 9:51 GMT+00:00 Theresa Hsu theresah...@live.com:
  Dear crystallographers
 
  Just out of curiosity, is it possible to collect datasets from crystals
 at room temperature at synchrotron? Are fast detectors like Pilatus useful
 for this?
 
  Thank you.
 
  Theresa
 




-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] preparation of citrate buffer pH3-6.5

[Katherine Sippel]

Alternatively you could make a stock solution of citric acid (say 1 M for
example) and stock solution of sodium citrate (also 1 M). Mix them in the
appropriate ratio to ballpark the right pH and just adjust up or down with
the stock solution. The concentration of citrate will be the same no matter
the final volume. You can then dilute that down to whatever your final
concentration of citrate needs to be.

If you are looking for the actual method to do the calculations I would
suggest finding a chemistry textbook and looking at the chapter on
buffering and the Henderson-Hasselbalch equation.

Cheers,
Katherine


On Thu, Jan 30, 2014 at 9:31 AM, Daniel Picot daniel.pi...@ibpc.fr wrote:

  But you have to be aware that pH depends on the concentration  of the
 buffer. This is especially the case for phosphate and citrate buffer.
 Daniel

 Le 30/01/2014 15:51, Schnicker, Nicholas J a écrit :

 It's a pain but I usually just make each pH of whatever buffer I'm using
 (if you make it concentrated then you'll only have to do it once).  Also,
 if you haven't already found it, Hampton has a nice link to calculate
 volume of components while designing a tray as long as you tell it the
 concentrations.

  http://hamptonresearch.com/make_tray.aspx

  Nick

   From: Roger Rowlett rrowl...@colgate.edu
 Reply-To: Roger Rowlett rrowl...@colgate.edu
 Date: Thursday, January 30, 2014 at 7:23 AM
 To: CCP4BB@JISCMAIL.AC.UK CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] preparation of citrate buffer pH3-6.5

   The easiest way to produce repeatable conditions is to titrate a stock
 solution (say 1M) of citric acid with NaOH to the desired pH and use that
 to mix your screen. That's what Hampton does anyway.

 If fine sampling pH, you can mix various ratios of pH 3 and 6.5 buffers.
 The pH won't be linear with mixing ratio, but will be easily repeatable.
 The actual pH of the final, magic solution can be directly measured if
 desired. Calculations will never be exactly right; pKa values are ionic
 strength dependent. Better to measure.

 Roger Rowlett
 On Jan 30, 2014 2:37 AM, sreetama das somon_...@yahoo.co.in wrote:

  Dear All,
 We have obtained many tiny protein crystals in a condition
 containing 0.1M citric acid pH 3.5, 2M ammonium sulfate. The crystals are
 too small for mounting in loops.

 We intend to vary the salt concentration  pH to obtain
 larger crystals.

 Could anyone direct us to some links, or provide us with a
 method (with calculations) to calculate the amounts of citric acid 
 trisodium citrate required to obtain buffers in a range of pH 3 - 6.5?
 I have come across online buffer calculators and links where
 the amounts of the components required are mentioned in grams, but none
 explaining how those values were arrived at.

  Thanks  regards,
  sreetama





-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] Crystallography Comics

[Katherine Sippel]

Hi all,

I thought the bulletin board might be interested to know that one of the
contributors on Popular Science is featuring a webcomic series on X-ray
crystallography. The first post went up yesterday and can be found here (
http://www.popsci.com/blog-network/boxplot/x-tallographers ). If nothing
else it may provide amusing material for the undergrad lectures.

Cheers,
Katherine

-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] A question on protein microheterogenity for crystalization

[Katherine Sippel]

Hi Acoot,

There are a lot of great suggestions here already. I've also run into this
phenomenon in couple of cases. The first was a binding protein in mixed
bound and unbound forms. The second was a case of heterogeneous
post-translational modification.  In both cases I could only get crystals
from purified peaks and not pooled protein. If protein is precious I'd
second Mark's suggestion to try screening pooled protein while you scale up
your prep.

Cheers,
Katherine


On Sat, Dec 14, 2013 at 6:16 AM, Acoot Brett acootbr...@yahoo.com wrote:

 Dear All,

 When I purified my protein by ion exchange chromatography for
 crystallization, there were several peaks containing the target protein as
 analyzed by SDS-PAGE. All these peaks have the same MW as determined by gel
 filtration coupled MALLS.

 For crystallization purpose, can I merge the corresponding ion exchange
 chromatography peaks together? Otherwise the protein yield will be too low.
 And how to explain the heterogeneity by ion exchange chromatography in this
 situation?

 I am looking forward  to getting a reply from you.

 Acoot




-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] Data mining interactions in the PDB

[Katherine Sippel]

Hi all,

I was wondering if anyone knew of a software or server to mine the PDB for
a specific class of interactions? I've tried PDBeMotif without much luck
and I thought I'd check to see if there was an alternative before I go
re-inventing the wheel.

Cheers,
Katherine

-- 
Nil illegitimo carborundum* - *Didactylos

[ccp4bb] OT: Ribosomal processivity

[Katherine Sippel]

Denizens of the bulletin board,

I would like to tap into your vast knowledge of all things protein
expression. Is anyone aware of a cell line or even a
transformable/transfectable construct that produces high processivity
ribosomes. I.E. one that can translate to the end of difficult mRNA without
aborting prematurely? It doesn't have to be for E. coli. Yeast or Insect
cells would be acceptable too.

Any help would be appreciated.

Cheers,
Katherine

-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] OT: Ribosomal processivity

[Katherine Sippel]

I forgot to mention that in this case gene optimization and eukaryotic
expression don't help. It's a product of the protein sequence itself.


On Tue, Sep 24, 2013 at 10:29 AM, Katherine Sippel 
katherine.sip...@gmail.com wrote:

 Denizens of the bulletin board,

 I would like to tap into your vast knowledge of all things protein
 expression. Is anyone aware of a cell line or even a
 transformable/transfectable construct that produces high processivity
 ribosomes. I.E. one that can translate to the end of difficult mRNA without
 aborting prematurely? It doesn't have to be for E. coli. Yeast or Insect
 cells would be acceptable too.

 Any help would be appreciated.

 Cheers,
 Katherine

 --
 Nil illegitimo carborundum* - *Didactylos




-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] Glutathione rsin

[Katherine Sippel]

Thermo Scientific/Pierce has gotten into the protein purification game
recently. The resin is slightly cheaper (USD $975/100 ml versus $1656 for
GE) and the binding capacity is equivalent. I haven't rigorously run any
regeneration tests though. Our local sales rep actually sent us a 1 ml spin
column free of charge to give it a test drive.

As for the binding issues I've given up on flow through purification with
GST. I lose too much protein. Nowadays I'll throw the lysate + extra
protease inhibitor in a 50 ml tube with the resin and leave it on an
extremely low speed rocking plate in the cold room overnight then load it
into a column the next morning. The flow through is a cloudy mess and you
have to wash it really well but all of the fusion protein is bound to the
resin and my samples are stable after cleavage. Whatever works right?

Cheers,
Katherine


On Wed, Jun 5, 2013 at 4:55 AM, Barbara Giabbai 
barbara.giab...@elettra.trieste.it wrote:

 Hi all.
  I don't have any experience with Clontech and Fisher resins, but about
 the GE one I faced the same problem as Sebastiano indicated.
 But I have to say that the problem was not general, but protein (or family
 of protein) related: the low binding depends on the oligomerization state
 of the chimeric protein. When it behaves as dimer (GST dimerizes) it's
 fine, when the oligomeric state increases (in my case hexamer) I really
 can't manage to purify the protein on GST sepharose. GST not exposed...I
 guess...

 I'm waiting for the replies Mirek's question...I'm interested too.

 Ciao,
  Barbara





 On Wed, 5 Jun 2013 09:32:28 +0200
  Sebastiano Pasqualato 
 sebastiano.pasqualato@GMAIL.**COMsebastiano.pasqual...@gmail.com
 wrote:


 Hi Mirek, hi all,
 I'm also very interested in the topic, so please keep me up with the
 replies, or make sure to post a summary, please.

 In addition to the price, the problem we're facing with GSH-beads from GE
 (although we haven't tried others yet) is that we can't manage to deplete
 our lysates.
 We are always left with a large amount of unbound GST-tagged protein in
 the flow through, that is eventually captured by a second, third and
 sometime fourth  incubation with fresh beads.
 Using larger beads volume won't help.
 Has anybody faced and/or overcame this problem?

 Thanks a lot in advance,
 ciao,

 Sebastiano


 On Jun 4, 2013, at 8:54 PM, Cygler, Miroslaw miroslaw.cyg...@usask.ca
 wrote:

  Hi,
 I would like to ask the bb faithful for their experience with the
 glutathione affinity resins. We have been using so far the Glutathione
 Sepharose fast flow from GE but the price is getting steeper. We found
 Glutathione Superflow resin from Clontech to be significantly less
 expensive and Glutathione agarose from Fisher somewhere in-between. We have
 no experience with the latter two resins and I wonder what is the
 experience of other people with these resins? Do they have decent binding
 capacity? Can they be efficiently regenerated or are they a single use only?
 Thanks for your help,

 Mirek




 --
 Sebastiano Pasqualato, PhD
 Crystallography Unit
 Department of Experimental Oncology
 European Institute of Oncology
 IFOM-IEO Campus
 via Adamello, 16
 20139 - Milano
 Italy

 tel +39 02 9437 5167
 fax +39 02 9437 5990







 Barbara Giabbai

 Elettra-Sincrotrone Trieste S.C.p.A.
 SS 14 - km 163,5 AREA Science Park
 34149 Basovizza, Trieste ITALY
 Office:   +39 040 375 8840
 Lab:  +39 040 375 8537




-- 
Nil illegitimo carborundum* - *Didactylos

Re: [ccp4bb] list/library of most commonly co-crystallized ligands/solvents and/or their electron density shapes

[Katherine Sippel]

I'm not sure that a library of ligand electron density would be an entirely
good thing. I guess something small like an acetate or DMSO would be
relatively consistent but larger things like PEGS or even glycerols are
going to be dependent on the nature of the binding site. A good example
would be to look at something that binds ATP as an enzymatic co-factor
versus a binding protein. Completely different geometry.

For a beginner I'd suggest to always check your difference peaks and all
your waters between rounds of refinement. If the positive density peaks are
covalent bond distance apart, it's not a water. If you've got giant green
smears between or around your waters it might not be a water. If you've
got good enough density and can guess at a basic framework you can plug a
rough sketch into the chemical structure search function in PubChem that
can give you some possible options. You can cross-reference the hits to the
HIC-Up server or the PDB for restraints or density examples keeping in mind
that your ligand-protein interactions may be different. Also do a sanity
check and ask yourself does this make chemical sense? If you've modeled
something in there is TWILIGHT to see if your density supports your model.
Of course there is also experimental evidence but that's a different can of
worms. Basically really look at your data and if something seems hinky then
look for alternatives.

My $0.02,
Katherine




On Tue, May 21, 2013 at 3:05 PM, Jeffrey, Philip D.
pjeff...@princeton.eduwrote:

  Top 20 HETNAM entries based on 58,469 PDB entries at better than 2.5
 Angstrom resolution (arbitrary cut):

  Number of entries in histogram: 14864
 Total number of instances : 195481

 0 14502 0.0742 GOL(glycerol)
1 10952 0.0560 SO4
2  8064 0.0413  ZN
3  7628 0.0390  MG
4  6930 0.0355 MSE (SeMet)
5  6685 0.0342  CA
6  6555 0.0335 EDO (Ethylene glycol)
7  6315 0.0323  CL
8  5856 0.0300 HEM
9  3922 0.0201  NA
   10  3647 0.0187 NAG
   11  3148 0.0161 PO4
   12  2360 0.0121 ACT(Acetate)
   13  1874 0.0096  MN
   14  1561 0.0080 NAP
   15  1387 0.0071   K
   16  1338 0.0068 FAD
   17  1277 0.0065 PLP (PYRIDOXAL-5'-PHOSPHATE)
   18  1228 0.0063 TRS(Tris buffer)
   19  1205 0.0062 FMN

  (numeric columns are ranking; count; frequency)
 No electron density, sorry.
   Clearly I should be adding more glycerols.

  Phil Jeffrey
 Princeton

   *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 孙庆祥 [
 baby_ten...@163.com]
 *Sent:* Tuesday, May 21, 2013 3:29 PM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] list/library of most commonly co-crystallized
 ligands/solvents and/or their electron density shapes

   hi all,

  Sorry if this has been asked before. I wonder if there is an list or
 library of most commonly co-crystallized ligands(or solvent molecules)
 available? Better if the electron density maps of the ligands are also
 shown with different resolutions. That could help a lot for an
 inexperienced crystallographer (like me) to quickly identify extra electron
 densities in a new structure, by simply comparing the electron density
 shapes.

  I remember a few days ago somebody asked about a PEG electron density,
 which looks like a string of beads. If I knew that earlier, I could have
 modeled it in, instead of waters...

  Thanks,
 Jeremy

Re: [ccp4bb] Philosophical question

[Katherine Sippel]

On Tue, Mar 19, 2013 at 9:34 AM, Jacob Keller 
j-kell...@fsm.northwestern.edu wrote:

 One might argue that since basically all organisms share the convention
 (are there exceptions, even?), that it must be the best of all possible
 conventions.


There are actually lots of exceptions. For example the UGA stop codon in E.
coli codes for Trp in Mycoplasma species. A fairly comprehensive list of
the codon variations as annotated by the NCBI can be found here
http://www.bioinformatics.org/JaMBW/2/3/TranslationTables.html#SG4

Cheers,
Katherine

[ccp4bb] Intein tag cleavage

[Katherine Sippel]

Hi all,

I was wondering if anyone out there has any anecdotal evidence of intein
tag cleavage buffer compatibilities outside those suggested in the IMPACT
manual (pH between 8 and 9, 0.1-2M NaCl or 0.1% Triton X-100). Specifically
I'm looking for whether I can reduce the pH to 8, acceptable additives
like glycerol or xylitol, or crystallization friendly detergents (i.e. not
Triton/Tween). This is the Sma VA Intein tag if that helps.

Telling me what doesn't work would be as useful as what does. Any
assistance is appreciated.

Cheers,
Katherine

Re: [ccp4bb] archival memory?

[Katherine Sippel]

You know if you took a dremel to an insulated benchtop cold box to make USB
shaped holes, lined the bottom with a layer of desiccant, and used a little
vacuum grease to seal it up you might actually have a workable, long term,
freezer storage system.

Wow, the things you think up when you're avoiding grant writing.

On Wed, Dec 12, 2012 at 3:45 PM, Roger Rowlett rrowl...@colgate.edu wrote:

  Maybe the memory chips will retain their bits for 100 years, but what
 about the driver hardware or internal power supply? Anyone had an
 electrolytic capacitor last for 100 years? Just sayin...

 I like the image of the USB sticks in the -80 freezer, though. :)
 ___
 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 12/12/2012 4:38 PM, Artem Evdokimov wrote:

 Or... (gasp) store a regular USB drive in a freezer, yes? If the
 relationship between data decay rate and temperature indeed follows the
 same good old Arrhenius formula then any old USB drive is virtually endless
 at -80C and safe for human life span at -20 (i.e. kitchen freezer, sans
 defrost cycles (so pack your USB in some ice packs so defrost doesn't kill
 it).

 If this works, feel free to send me money, SanDisk...

 Artem

  On Wed, Dec 12, 2012 at 3:02 PM, Richard Gillilan r...@cornell.eduwrote:

 SanDisk advertises a Memory Vault disk for archival storage of photos
 that they claim will last 100 years.

 (note: they do have a scheme for estimating lifetime of the memory,
 Arrhenius Equation ... interesting. Check it out:
 www.sandisk.com/products/usb/memory-vault/ and click the Chronolock
 tab.).

 Has anyone here looked into this or seen similar products?

 Richard Gillilan
 MacCHESS

Re: [ccp4bb] low-resolution and zinc

[Katherine Sippel]

As a follow up to Roger's statement if you are doing any sort of analytical
metal analysis be careful with the controls (metal-free water/acid washed
glassware). Also most AC/heating systems include galvanized steel in the
duct work that spits out Zn like crazy and can screw up your measurements.
If you have access to a neurotically OCD analytical chemist to assist I'd
suggest plying them with coffee and complements.

Cheers,
Katherine

On Wed, Nov 7, 2012 at 3:41 PM, Felix Frolow mbfro...@post.tau.ac.ilwrote:

 As far as sigma level is concerned, and if I remember that you are working
 at 3.4 angstrom resolution - this 6 sigma is VERY STRONG.
 I am sure it is a metal atom. But you can re-process your  data preserving
 anomalous signal and calcule anomalous map easily done in
 PHENIX, less so in CCP4 and then displaying anomalous map as a difference
 map in COOT you MUST see strong peak on this map in the heavy atom location.
 Dr Felix Frolow
 Professor of Structural Biology and Biotechnology, Department of
 Molecular Microbiology and Biotechnology
 Tel Aviv University 69978, Israel

 Acta Crystallographica F, co-editor

 e-mail: mbfro...@post.tau.ac.il
 Tel:  ++972-3640-8723
 Fax: ++972-3640-9407
 Cellular: 0547 459 608

 On Nov 7, 2012, at 20:13 , SD Y ccp4...@hotmail.com wrote:

 Dear Prof. Frolow,

 During sample development I have not used anything related to Zn but could
 be from traces of contamination from Tris, NaCl, DTT, EDTA, LiSO4, HEPES
 and other salt which were part of auto induction media.

 I am trying to search the refence in google. Are you refering to the Book
 published in 1976 titled protein crystallography, if not could you please
 kindly direct me to right reference.

 I sincerely appreciate your time and suggestion.

 Warm reagrds,
 SDY







 --
 From: mbfro...@post.tau.ac.il
 Subject: Re: [ccp4bb] low-resolution and zinc
 Date: Wed, 7 Nov 2012 19:35:21 +0200
 To: ccp4...@hotmail.com

 Zn is always there as anything else.
 If you have high affinity binding site, it will be filled with Zn (or
 similar)  on the various stages of your
 sample development.
 BTW use old BlandellJohnson popular in my time (70-90's) approach - in
 the correct space group the peak hight of this heavy atom will be the
 highest comparing to other space groups
 FF
 Dr Felix Frolow
 Professor of Structural Biology and Biotechnology, Department of
 Molecular Microbiology and Biotechnology
 Tel Aviv University 69978, Israel

 Acta Crystallographica F, co-editor

 e-mail: mbfro...@post.tau.ac.il
 Tel:  ++972-3640-8723
 Fax: ++972-3640-9407
 Cellular: 0547 459 608

 On Nov 7, 2012, at 19:29 , SD Y ccp4...@hotmail.com wrote:

 Dear all,

 I have a related question to the one I have posted low resolution and
 SG, on which I am still working based on the suggestions I have got.

 The model I have used, has Zn co-ordinated well in tetrahydral fashion by
 3 cys and 1 His residues. They have  add Zn in to their experiment.
 In my 3.4 A structure  (I am still working on right SG), initial maps
 show very strong positive density (sigma=6.5) at the place of Zn (
 https://www.dropbox.com/s/4jd6gdor87ab9lj/Zn-coordination.png). I have
 not used Zn in my experiment. I could only suspect Tryptone and
 yeast extract which I used to make media.

 I would like to know how likely  this positive density belongs to Zn? How
 to reason the presence of Zn when its not been used?
 Is there is any way to confirm if its Zn. If this is not Zn, what else
 could it be? Any thing I could try to rule out or in Zn or other ions.
 I appreciate your help and suggestions.

 Sincerely,
 SDY

Re: [ccp4bb] [OT]: Purification in ArcticExpress

[Katherine Sippel]

Apologies for revisiting this topic but I wanted to post a summary of the
responses for the compulsive archive searchers like me.

Several people suggested the addition of ATP (5 mM) to the buffers either
at lysis or once the protein is bound to the column (see ref: R. E. Joseph;
A. H. Andreotti, Protein Expr. Purif. 2008, 60, 194–197.). I've attempted
both options and both are pretty effective (~80% removal). Due to protein
quirks I've have to drop the IMAC and go old school using straight ion
exchange which actually cleans up all the residual Cpn60-bound protein
(Cpn60 has a theoretical pI of 4.8) so downstream polishing is pretty easy.

Additional suggestions included:

1) I once had a protein like this. From SDS-PAGE it looked like the protein
was completely insoluble, but there was 1-2% which folded properly and
bound to Ni-NTA with 6xHIS. This was enough to purify and crystallize. Take
the supernatant and bind it to your capture resin. See what sticks.

2) Have you considered moving to a eukaryotic expression host like sf9/Tni,
S2, HEK, or Pichia Pastoris?  Can you co-express the protein with another
protein you know it interacts with?

3) Try bumping up the salt and glycerol in your wash buffer to get rid of
it.

4) Maybe you can try some useful ideas for your case listed in this study:
Protein Expr Purif. 2007 April; 52(2): 280–285. *really cool paper BTW*

5) I used a panel of chaperonin plasmids from Takara and found one that
folded my enzyme without co-purification.

Thanks for all your help,

Katherine


On Mon, Sep 24, 2012 at 9:38 AM, Katherine Sippel 
katherine.sip...@gmail.com wrote:

 Hi All,

 I've recently swapped over to expression in the ArcticExpress(DE3) cells
 for a particularly rock-like protein. I've got soluble expression but I'm
 having the issue of Cpn60 (the overexpressed chaperonin) piggybacking along
 with the tagged protein. Google-fu and the CCP4bb archives indicate that
 this is a known issue but I haven't seen a solution thus far. Does anyone
 out there have any tricks up their sleeve?

 Also in case you were wondering in the various BL21 lines, even at
 extremely low temperatures and 10 uM IPTG, it expresses well but produces
 inclusion bodies that are completely insoluble in 8M urea, 6M guanidine,
 high temperature, high pH, high reducing agent, and a number of detergents
 so swapping back to another cell line isn't in the cards.

 Any help would be appreciated.

 Cheers,
 Katherine

[ccp4bb] [OT]: Purification in ArcticExpress

[Katherine Sippel]

Hi All,

I've recently swapped over to expression in the ArcticExpress(DE3) cells
for a particularly rock-like protein. I've got soluble expression but I'm
having the issue of Cpn60 (the overexpressed chaperonin) piggybacking along
with the tagged protein. Google-fu and the CCP4bb archives indicate that
this is a known issue but I haven't seen a solution thus far. Does anyone
out there have any tricks up their sleeve?

Also in case you were wondering in the various BL21 lines, even at
extremely low temperatures and 10 uM IPTG, it expresses well but produces
inclusion bodies that are completely insoluble in 8M urea, 6M guanidine,
high temperature, high pH, high reducing agent, and a number of detergents
so swapping back to another cell line isn't in the cards.

Any help would be appreciated.

Cheers,
Katherine

Re: [ccp4bb] loading maps in coot using EDS

[Katherine Sippel]

The 3tvn coordinates/SF were released today. I'm not sure what the lag time
is between the PDB and EDS but you'd probably need to download the
structure factors and generate the map yourself.

If you're not in a super rush I know the person who refined that specific
PDB and I may be able to get you a copy of her final maps to send you
off-board once she gets back from vacation.

Cheers,
Katherine


On Wed, Aug 8, 2012 at 3:39 PM, Shya Biswas shyabis...@gmail.com wrote:

 Hi all,

 I was trying to get maps using the *fetch PDB and Map using EDS option*in 
 coot, however the map would not open I am using coot version 0.6.2 was
 wondering if anybody else had similar problems and how to fix this, the
 following is the error message I get. It used to work fine with a previous
 version of coot.



 CCP4MTZfile: open_read - File missing or corrupted:
 coot-download/3tvn_sigmaa.mtz
 INFO:: not an mtz file: coot-download/3tvn_sigmaa.mtz
 ERROR: no f_cols!
 ERROR: no phi_cols!
 valid_labels(coot-download/3tvn_sigmaa.mtz,FOFCWT,PHFOFCWT,,0) returns 0
  CCP4 library signal library_file:End of File (Error)
  raised in ccp4_file_raw_read 
  System signal 0:Success (Error)
  raised in ccp4_file_rarch 
  CCP4 library signal library_file:End of File (Error)
  raised in ccp4_file_raw_read 
  System signal 0:Success (Error)
  raised in ccp4_file_readchar 
  CCP4 library signal mtz:Read failed (Error)
  raised in MtzGet 
 CCP4MTZfile: open_read - File missing or corrupted:
 coot-download/3tvn_sigmaa.mtz
 INFO:: not an mtz file: coot-download/3tvn_sigmaa.mtz
 ERROR: no f_cols!
 ERROR: no phi_cols!
 WARNING:: label(s) not found in mtz file coot-download/3tvn_sigmaa.mtz
 FOFCWT PHFOFCWT
 WARNING:: -1 is not a valid molecule in set_scrollable_map

 thanks,
 Shya

Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

[Katherine Sippel]

From personal and recent experience I've solved a structure using only
iodine anomalous at Cu K-alpha from a RT crystal (a capillary mounted one
at that). The anomalous signal from iodine is surprisingly robust on a home
source even at room temp.

Katherine

As an aside for those who feel that capillary mounting

On Wed, Jun 6, 2012 at 2:34 PM, Jacob Keller j-kell...@fsm.northwestern.edu
 wrote:

 But the edges for I and Hg are pretty far from CuKa (see attached). I
 am familiar with their being extra signal (white lines) very close to
 the peak, but not so far away

 JPK



 On Wed, Jun 6, 2012 at 2:15 PM, Bernhard Rupp (Hofkristallrat a.D.)
 hofkristall...@gmail.com wrote:
  There is also a relevant point from the physics of the absorption
 spectra -
  the XANES white lines (near edge peaks higher than the continuum
 transition
  or edge step) depend on the chemical environment of the anomalous atom in
  terms of available unoccupied states (which n. b. is something entirely
  different that the local neighbor environment/geometry which can be
  backtransformed - although with quite some uncertainty - from the EXAFS
  wiggles).
 
  Any argument about absolute f peak values in absence of experimental
  evidence (scan) might want to consider that.
 
  Best, BR
 
  -Original Message-
  From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Jacob
  Keller
  Sent: Wednesday, June 06, 2012 11:30 AM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement
 an
  obsolete technique?
 
  No offense taken (we all have our dour moments!), but grant me a sincere
  question: the f occupancy value would have been just as close at 11 as
 5 if
  the true value were 8, am I correct? In other words, do you imply by
 saying
  doing well that you got as *much* as 5, or that you got as *close* as
 5? I
  am just trying to see whether I understand these things correctly.
 
  Jacob
 
 
 
  On Wed, Jun 6, 2012 at 12:21 PM, Gerard Bricogne g...@globalphasing.com
 
  wrote:
  Dear Jacob and all,
 
  I realise that my last statement sounds awfully dour and
  dismissive, in a way I really didn't intend. Especially as Stefan's
  original posting was a Fun Question.
 
  Apologies to all for this over-the-top statement. I enjoyed a lot
  of the replies.
 
 
  With best wishes,
 
   Gerard.
 
  --
  On Wed, Jun 06, 2012 at 06:09:33PM +0100, Gerard Bricogne wrote:
  Dear Jacob,
 
   I thought that getting 5 for each iodine was doing pretty well,
  given the circumstances - e.g. the noisy measurements, the primitive
  software running on slow computers with tiny amounts of memory, etc. .
 
   In any case my main point, directed at the original poster, was
  that reading the early Acta Cryst. issues (RTFL) might be an
  alternative and perhaps more enlightening way of getting a picture of
  the evolution of phasing methods than finding some clever filter
 settings
  in the RCSB ;-) .
 
 
   With best wishes,
 
Gerard.
 
  --
  On Wed, Jun 06, 2012 at 11:08:37AM -0500, Jacob Keller wrote:
   ...Even with such primitive techniques, I can remember an HgI4
derivative in which you could safely refine the anomalous
  occupancies
(i.e. f values) for the iodine atoms of the beautiful planar
HgI3 anion to
5 electrons.
  
   I am surprised--f's of I and Hg are supposed to be around 8 for
   CuKa (or maybe you weren't using CuKa)?
  
   JPK
  
  
   --
   ***
   Jacob Pearson Keller
   Northwestern University
   Medical Scientist Training Program
   email: j-kell...@northwestern.edu
   ***
 
  --
 
  ===
  * *
  * Gerard Bricogne g...@globalphasing.com  *
  * *
  * Global Phasing Ltd. *
  * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
  * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
  * *
  ===
 
 
 
  --
  ***
  Jacob Pearson Keller
  Northwestern University
  Medical Scientist Training Program
  email: j-kell...@northwestern.edu
  ***
 



 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***

Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

[Katherine Sippel]

It would be helpful if I finished my own sentences. As an aside for those
who feel that capillary mounting is a lost art among the newer generation I
assure you it isn't. All you need is a busted cryo system and a crystal
backlog to get past the intimidation factor.

Katherine

On Wed, Jun 6, 2012 at 2:34 PM, Jacob Keller j-kell...@fsm.northwestern.edu
 wrote:

 But the edges for I and Hg are pretty far from CuKa (see attached). I
 am familiar with their being extra signal (white lines) very close to
 the peak, but not so far away

 JPK



 On Wed, Jun 6, 2012 at 2:15 PM, Bernhard Rupp (Hofkristallrat a.D.)
 hofkristall...@gmail.com wrote:
  There is also a relevant point from the physics of the absorption
 spectra -
  the XANES white lines (near edge peaks higher than the continuum
 transition
  or edge step) depend on the chemical environment of the anomalous atom in
  terms of available unoccupied states (which n. b. is something entirely
  different that the local neighbor environment/geometry which can be
  backtransformed - although with quite some uncertainty - from the EXAFS
  wiggles).
 
  Any argument about absolute f peak values in absence of experimental
  evidence (scan) might want to consider that.
 
  Best, BR
 
  -Original Message-
  From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
 Jacob
  Keller
  Sent: Wednesday, June 06, 2012 11:30 AM
  To: CCP4BB@JISCMAIL.AC.UK
  Subject: Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement
 an
  obsolete technique?
 
  No offense taken (we all have our dour moments!), but grant me a sincere
  question: the f occupancy value would have been just as close at 11 as
 5 if
  the true value were 8, am I correct? In other words, do you imply by
 saying
  doing well that you got as *much* as 5, or that you got as *close* as
 5? I
  am just trying to see whether I understand these things correctly.
 
  Jacob
 
 
 
  On Wed, Jun 6, 2012 at 12:21 PM, Gerard Bricogne g...@globalphasing.com
 
  wrote:
  Dear Jacob and all,
 
  I realise that my last statement sounds awfully dour and
  dismissive, in a way I really didn't intend. Especially as Stefan's
  original posting was a Fun Question.
 
  Apologies to all for this over-the-top statement. I enjoyed a lot
  of the replies.
 
 
  With best wishes,
 
   Gerard.
 
  --
  On Wed, Jun 06, 2012 at 06:09:33PM +0100, Gerard Bricogne wrote:
  Dear Jacob,
 
   I thought that getting 5 for each iodine was doing pretty well,
  given the circumstances - e.g. the noisy measurements, the primitive
  software running on slow computers with tiny amounts of memory, etc. .
 
   In any case my main point, directed at the original poster, was
  that reading the early Acta Cryst. issues (RTFL) might be an
  alternative and perhaps more enlightening way of getting a picture of
  the evolution of phasing methods than finding some clever filter
 settings
  in the RCSB ;-) .
 
 
   With best wishes,
 
Gerard.
 
  --
  On Wed, Jun 06, 2012 at 11:08:37AM -0500, Jacob Keller wrote:
   ...Even with such primitive techniques, I can remember an HgI4
derivative in which you could safely refine the anomalous
  occupancies
(i.e. f values) for the iodine atoms of the beautiful planar
HgI3 anion to
5 electrons.
  
   I am surprised--f's of I and Hg are supposed to be around 8 for
   CuKa (or maybe you weren't using CuKa)?
  
   JPK
  
  
   --
   ***
   Jacob Pearson Keller
   Northwestern University
   Medical Scientist Training Program
   email: j-kell...@northwestern.edu
   ***
 
  --
 
  ===
  * *
  * Gerard Bricogne g...@globalphasing.com  *
  * *
  * Global Phasing Ltd. *
  * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
  * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
  * *
  ===
 
 
 
  --
  ***
  Jacob Pearson Keller
  Northwestern University
  Medical Scientist Training Program
  email: j-kell...@northwestern.edu
  ***
 



 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***

[ccp4bb]

[Katherine Sippel]

Hi Ed,

I've actually run that exact test in phenix as an exercise to prove to my
PI the validity of occupancy refinement. Though as a disclaimer it was a
1.2 angstrom data set and this was an alternate conformation situation. I
ran different input occupancies without occupancy refinement and measure
the difference density peak values and average B-factors and ended up with
the same occupancy ratio that the program's occupancy refinement spit out.
Of course this might not hold true if someone is refining the occupancy of
a ligand that is partially bound without an alternate option (i.e. total
occupancy 1). I haven't tested that one systematically yet though I
suspect Pavel has probably already done this at some point.

Cheers,

Katherine

On Mon, Jun 4, 2012 at 7:35 AM, Ed Pozharski epozh...@umaryland.edu wrote:

  Is it reasonable to refine occupancy in phenix at 2.2 A resolution?

 Implementations may differ, but imgo refining occupancy at 2.2A
 resolution is not very reasonable under most circumstances, as it will
 correlate strongly with the B-factor.  A reasonable approach might be to
 fix occupancy at different levels and get a series of refined models.
 Then you look at (i) B-factor behavior and see at what occupancy it
 matches the surrounding atoms and (ii) difference density (my
 unsubstantiated theory is that if you plot it against occupancy it
 should have a central flat region where B-factors are capable of
 compensating and two linear regions on extreme ends which should allow
 to extrapolate the true value.

 Refmac does occupancy refinement.  It's quite fast, so you may try
 randomizing the initial value and get some idea about convergence.

 Cheers,

 Ed.



 --
 Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] zinc with HEPES

[Katherine Sippel]

That is probably because you pH Tris with HCl rather than HEPES with NaOH.
The Ksp for Zn(OH)2 is 3x10^17 so the excess hydroxides are probably what
are killing your solution.

Cheers,

Katherine

On Fri, May 11, 2012 at 11:43 AM, Rajesh Kumar ccp4...@hotmail.com wrote:

  If there is no cure , then fine.
 pH may not be the answer as it doesn't Happen with TRIS buffer pH 7.6.
 Thanks to every one
 Rajesh


 --
 Date: Fri, 11 May 2012 12:35:45 -0400
 From: dj...@cornell.edu

 Subject: Re: [ccp4bb] zinc with HEPES
 To: CCP4BB@JISCMAIL.AC.UK



  There is no cure for HEPES.




 --
 ===
 All Things Serve the Beam
 ===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu

Re: [ccp4bb] zinc with HEPES

[Katherine Sippel]

You are correct. My Friday frazzled brain is stuck in pharmacology mode so
I was thinking not of free hydroxide concentration but in terms of
potential exchangable groups being sequestered by the buffer. No more
posting on Friday. My apologies.

Katherine

On Fri, May 11, 2012 at 12:23 PM, Jacob Keller 
j-kell...@fsm.northwestern.edu wrote:

 Just to make sure I understand pH correctly: isn't it true that the [OH-]
 should always be the same at a given pH (by definition)?

 JPK

 On Fri, May 11, 2012 at 11:48 AM, Katherine Sippel 
 katherine.sip...@gmail.com wrote:

 That is probably because you pH Tris with HCl rather than HEPES with
 NaOH. The Ksp for Zn(OH)2 is 3x10^17 so the excess hydroxides are probably
 what are killing your solution.

 Cheers,

 Katherine


 On Fri, May 11, 2012 at 11:43 AM, Rajesh Kumar ccp4...@hotmail.comwrote:

  If there is no cure , then fine.
 pH may not be the answer as it doesn't Happen with TRIS buffer pH 7.6.
 Thanks to every one
 Rajesh


 --
 Date: Fri, 11 May 2012 12:35:45 -0400
 From: dj...@cornell.edu

 Subject: Re: [ccp4bb] zinc with HEPES
 To: CCP4BB@JISCMAIL.AC.UK



  There is no cure for HEPES.




 --
 ===
 All Things Serve the Beam
 ===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu





 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 email: j-kell...@northwestern.edu
 ***

Re: [ccp4bb] negative difference density around sulphur and oxygen atoms

[Katherine Sippel]

On Wed, Apr 4, 2012 at 10:31 AM, Roger Rowlett rrowl...@colgate.edu wrote:

  I have also seen a recent paper where radiation damage of a bound protein
 ligand was apparently observed in a synchrotron beam.


That was a manuscript were I would have happily given the coordinates and
structure factors to the reviewers with my blessing. Learned a valuable
lesson about adopting orphaned data sets though.

Cheers,
Katherine


 I look forward to hearing from others how best to handle this in
 refinement.

 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 4/4/2012 11:16 AM, Chris Meier wrote:

 Dear all,
 I am refining the X-ray structure of a protein:
 Data to ~2A were collected at a latest-generation synchrotron.
 The 2fo-Fc maps are crisp, the model of the protein is complete and I am
 reasonably happy with the stats (R below 20%, Rfree below 25% in Refmac
 5.5).
 However, I am seeing a lot of negative difference density,
 especially around sulphur atoms (negative density around -9 sigma)
 and oxygen atoms (e.g. side-chain oxygens of Glu, Asp, etc. residues with
 negative density around -6 sigma).
 Has anyone observed this before?
 I have found CCP4bb postings discussing radiation damange of suplphur
 atoms
 (e.g. http://www.dl.ac.uk/list-**archive-public/ccp4bb/2004-07/**
 msg00532.htmlhttp://www.dl.ac.uk/list-archive-public/ccp4bb/2004-07/msg00532.html).
 Can this also happen with oxygen atoms?
 What would be an appropriate way to deal with this issue during
 refinement?
 Suggestions greatly appreciated.
 Thanks,
 Chris

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Katherine Sippel]

Might I suggest looking to Sean Seaver and the P212121.com as an example of
a a successful crystallographer science blogger though the site has shifted
more towards a consumable supplier in recent years.

I would also consider looking into adding an RSS feed to your site so that
those people interested in your articles can be informed without spamming
the boards. The gods of the interwebz have blessed us with the gift of RSS
so that we may be made aware of when someone might be yelling something
potentially interesting into the void (that and to bring us silly pictures
of cats covered in phonetically spelled captions when we have a failed
experiment).

It is my hope that this will not discourage you from taking every
opportunity to improve your writing skills but help you find a more
appropriate means of disseminating your product.

Cheers,

Katherine

On Tue, Apr 3, 2012 at 7:22 PM, Kevin Jin kevin...@gmail.com wrote:

 Thanks of your education. I got it.

 By the way, what does Orcus mean here?

 Regards,

 Kevin

 On Tue, Apr 3, 2012 at 5:11 PM, Bernhard Rupp (Hofkristallrat a.D.) 
 hofkristall...@gmail.com wrote:

 Orcus,

 ** **

 if you put yourself persistently into the face of guys who play hard, you
 need to learn to

 take a few hits and shake it off. Maybe a little retrospection on why
 your postings might

 perhaps possibly maybe perceived as somewhat self-promoting and
 ungracious could be helpful.

 ** **

 The skill of presentation is at least as important in Science as being
 right.

 ** **

 Best, BR 

 ** **

 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of
 *Kevin Jin
 *Sent:* Tuesday, April 03, 2012 3:34 PM

 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] very informative - Trends in Data Fabrication

 ** **

 Dear All, 

  Here may be another example for the importance of  image storage. 

  

 http://www.jinkai.org/DERA/DERA_1O0Y_3R12.html

  

 Regards,

  

 Kevin

 ** **




 --
 Kevin Jin

 Sharing knowledge each other is always very joyful..

 Website: http://www.jinkai.org/

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Katherine Sippel]

I agree with Herman about personal preference but it also boils to our job
as crystallographers to educate non-structural end-users. The fact of the
matter is that a lot of researchers use structures without looking at the
nuances of the PDB. It's actually pretty common among biologists to
download a PDB file and build hypothesis or throw the coordinates into a
downstream program like APBS or AutoDock without looking. They don't even
realize that density exists or that they should check it, which makes the
odds of them reading a header file for missing atoms or understanding the
concept of b-factors and occupancy almost nil. Realizing that renders the
argument moot until crystallographic data is demystified across the other
sciences.

I will say that in principle I do like the idea of a data model + a
projected model because it seems like something an end-user could wrap
their head around, but in practice this would probably refuel the what
constitutes modelable density debate all over again.

Cheers,

Katherine

On Mon, Mar 26, 2012 at 10:19 AM, herman.schreu...@sanofi.com wrote:

 Dear Ed,

 In the end it boils down to personal preferences. With the number of
 crystal structures I refine each year, I am not going to go over every
 flexible surface residue to decide whether to truncate the side chain or
 whether there may be some low level density justifying to keep the side
 chain, so I opt for the biochemical evidence. For me the added advantage is
 that I only have a single pdb file to take care of. And again, I see no
 problem in having a model with some atoms with a larger error bar.

 You are right that terminii are often truncated. In contrast to a missing
 side chain, here we really have no reasonable hypothesis where the missing
 residues are. They may even have been removed by a protease.

 Cheers,
 Herman

 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ed
 Pozharski
 Sent: Monday, March 26, 2012 4:50 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] REFMAC5 residues with bad geometry

 On Mon, 2012-03-26 at 16:30 +0200, herman.schreu...@sanofi.com wrote:
  It is like with Heisenbergs uncertainty principle. Either one has a
  complete model with a number of atoms having a coordinate uncertainty
  of 4-6 Å, or one has a model where the uncertainty of all atoms is
  below say 0.5 Å, but with a lot of truncated side chains with clearly
  contradict available biochemical evidence.

 Excellent analogy.  I am not sure why truncated arginine (as long as it is
 not renamed to alanine) contradicts biochemical evidence though.
 Termini are routinely truncated, no problem there. I have plenty of
 biochemical evidence that there are more waters in the crystal than I model.

 If the truncated model contradicts biochemical evidence, the projected
 model contradicts crystallographic evidence. I agree that a truncated model
 may lead to interpretation problems, and thus the option of depositing a
 projected model resolves that.

 Cheers,

 Ed.

 --
 Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

Re: [ccp4bb] All-D World

[Katherine Sippel]

Well if you think about it technically God produced plants within three
days which if far too little work for a thesis. Maybe it could count as
preliminary results for one aim of a thesis proposal so it might be enough
to get PhD candidacy depending on how demanding your committee is. You
could always propose the remaining 5999 years and 362 days to do a massive
data mining initiative.

Thanks for the laugh. I needed it.

Katherine

On Wed, Feb 15, 2012 at 4:48 PM, William G. Scott wgsc...@ucsc.edu wrote:

 Hi Jacob:

 After giving this a great deal of reflection …..
 I realized that you would face the same paradox that
 God had to resolve six thousand years ago at the Dawn of
 Creation, i.e., He needed D-deoxyribose DNA to code for L-amino acid
 proteins, and vice versa.  Likewise, you would probably be faced
 with a situation where you need L-deoxyribose DNA to code for D-amino
 acid proteins, so once again, you need a ribozyme self-replicase to
 escape the Irreducible Complexity(™).  (The Central Dogma at least is
 achiral.)

 At least it can be done six thousand years, which isn't unreasonable for
 a Ph.D. thesis project (especially when combined with an M.D.), and you,
 unlike Him, have access to a Sigma catalogue.

 All the best,

 Bill


 William G. Scott
 Professor
 Department of Chemistry and Biochemistry
 and The Center for the Molecular Biology of RNA
 228 Sinsheimer Laboratories
 University of California at Santa Cruz
 Santa Cruz, California 95064
 USA





 On Feb 15, 2012, at 10:28 AM, Jacob Keller wrote:

  So who out there wants to start an all-D microbial culture by total
  synthesis, a la the bacterium with the synthetic genome a while back?
  Could it work, I wonder? I guess that would be a certain benchmark for
  Man's conquest of nature.
 
  JPK
 
  ps maybe if there is a broadly-acting amino-acid isomerase or set of
  isomerases of appropriate properties, this could be helpful for
  getting the culture started--or even for preying on the L world?
 
 
 
  On Wed, Feb 15, 2012 at 12:17 PM, David Schuller dj...@cornell.edu
 wrote:
  On 02/15/12 12:41, Jacob Keller wrote:
 
  Are there any all-D proteins out there, of known structure or
  otherwise? If so, do enantiomer-specific catalyses become inverted?
 
  JPK
 
  What do you mean by Out There? If you mean in the PDB, then yes.  As
 of
  two weeks ago, there are ~ 14 racemic structures deposited; most in
 space
  group P -1, with one outlier in space group I -4  C 2. This includes
 RNA,
  DNA, and PNA, but 6 entries are actually protein. The longest is over 80
  residues.
 
  Theoretically, enantiomer-specific catalysis ought to be inverted, but
 most
  of the structures solved are not enzymes. kaliotoxin, plectasin,
 antifreeze
  protein, monellin, villin, and a designed peptide.
 
  On the other hand, if by out there you meant in nature outside of
  biochemistry and organic chemistry labs; then no, I am not aware of any
  all-D proteins. There are a few protein/peptides which include a small
  number of D-residues, which is marked up to nonribosomal synthesis.
 
  The first paper I managed to Google:
  http://jb.asm.org/content/185/24/7036.full
  Learning from Nature's Drug Factories: Nonribosomal Synthesis of
 Macrocyclic
  Peptides
  doi: 10.1128/JB.185.24.7036-7043.2003 J. Bacteriol. December 2003 vol.
 185
  no. 24 7036-7043
 
  If racemic crystallization isn't exciting enough for you, look into
  quasi-racemic crystallization.
 
 
  On Wed, Feb 15, 2012 at 8:05 AM, David Schuller dj...@cornell.edu
 wrote:
 
  Wukovitz  Yeates (1995) Nature Struc. Biol. 2(12): 1062-1067
  predicts that the most probable space group for macromolecular
  crystallization is P -1 (P 1-bar). All you have to do to try it out is
  synthesize the all-D enantiomer of your protein and get it to fold
 properly.
 
 
  On 02/14/12 18:36, Prem Kaushal wrote:
 
 
  Hi
 
  We have a protein that crystallized in P21212 space group. We are
 looking
  for some different crystal forms. We tried few things did not work. Now
 we
  are thinking to mutate surface residues. Anybody aware of any software
 which
  can predict the mutations that might help in crystallizing protein in
  different space group, please inform me.
 
  Thanks in advance
 
  Prem
 
 
  --
  ===
  All Things Serve the Beam
  ===
David J. Schuller
modern man in a post-modern world
MacCHESS, Cornell University
schul...@cornell.edu
 
 
 
  --
  ***
  Jacob Pearson Keller
  Northwestern University
  Medical Scientist Training Program
  email: j-kell...@northwestern.edu
  ***

Re: [ccp4bb] No diffraction

[Katherine Sippel]

Might I suggest consulting the CCP4 user community wiki on the topic:

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Improving_crystal_quality

Good luck,

Katherine


On Thu, Jan 26, 2012 at 9:33 AM, Theresa H. Hsu theresah...@live.comwrote:

 Dear crystallographers

 I have a protein of 90 kDa forming dimers. Crystals formed with microbatch
 and vapor diffusion method in 24 hours but no diffraction at home source.
 Dissolved crystals was confirmed to be the protein with mass spec.

 Any suggestions to improve diffraction would be welcome.

 Thanking you in advance.

 Theresa

Re: [ccp4bb] native gels

[Katherine Sippel]

Hi Rashmi,

In my experience native (even blue native) on proteins around that pI is
sketchy at best. The electrophoretic mobility once it gets past the
stacking gel goes to crap meaning long electrophoresis times and it needs
to be done on a chillable system or in a cold room. If this is a multimer
issue I'd suggest trying analytical ultracentrifugation, analytical size
exclusion (with the caveat that buffer, temperature and protein shape will
affect the output/interpretation), or SAXS first. If native is the only
alternative you'll probably get better results changing up the buffer
system from traditional tris-glycine or those listed in the blue native
protocol keeping in mind that you'll still need to stack the bands.

Good luck,

Katherine

On Thu, Jan 19, 2012 at 7:03 AM, anita p crystals...@gmail.com wrote:

 Hi All,
 Has anyone run a native gel for proteins at pI8 .
 I want to pour my own native gel. Do I run a discontinuous page or a
 continuous one?? Please help with regards to the buffer system to be used,
 and the dye to be used.
 With regards
 Rashmi

[ccp4bb] Metal won't strip from IMAC

[Katherine Sippel]

Hi all,

I've run into a bit of a protein purification conundrum and wondered if
anyone had encountered a similar situation. I've exercised all of my
google-fu and can't find anything. It's a fairly straightforward setup;
His-tagged protein and Talon Co2+ resin, load lysate, wash with 5 mM
imidazole, elute with 150 mM imidazole. There is protein in the elution
fractions as would be expected. The strangeness occurs when I try to
regenerate the column. Using the standard protocol of 25 mM MES, 100 mM
NaCl pH 5 doesn't change the color of the resin back to light pink the way
it should with a regenerated column. I try stripping with the suggested
0.2M EDTA, still pink, 0.5M EDTA, still pink, 8 M urea plus 4% CHAPS and
then EDTA, still pink, 1 M NaOH then EDTA, still pink. I've checked the
resin using a Western (with a really specific monoclonal Ab) and it seems
that my protein has somehow irreversibly bound to the column and is
preventing the metal from releasing the sepharose. I've even tried
competing the protein off with excess Co2+ and Mg2+ (the endogenous
divalent bound cation).

Clearly the solution is swapping to a Ni column, but this is really bugging
me now. Has anyone run into this problem with IMAC before?

Background: The protein does bind divalent cations (Mg and Mn) with low
affinity (~1 mM) and has a ridiculous number of cysteines (10 in 416
residues total). There is 1 mM BME and 1 mM MgCl2 in all of the buffers.

Thanks,

Katherine

Re: [ccp4bb] PHENIX vs REFMAC refinement had me fooled

[Katherine Sippel]

In a non-computational capacity would also suggest perhaps resequencing
your clone. Occasionally the published sequences are off, the specific base
is polymorphous or there is also the possibility that you introduced a
mutation somewhere. That would be the cheap and easy way to definitively
answer the question.

Cheers,
Katherine

On Thu, Dec 8, 2011 at 1:43 PM, Petr Leiman petr.lei...@epfl.ch wrote:

 Dear Tim,

 I agree with you completely. The question then becomes why does the
 automatic weighting scheme in refmac allow R and R-free to run away from
 each other by 8% in a 1.1 A resolution structure?

 Petr

 On Dec 8, 2011, at 6:50 PM, Tim Gruene wrote:

  -BEGIN PGP SIGNED MESSAGE-
  Hash: SHA1
 
  Dear Christopher,
 
  if your R/Rfree from Refmac5 really are 10% vs. 18%, you might simply be
  looking at an electron density map with strong model bias, i.e. the map
  shows the features of the model and not of the data. Although at 1.1A
  resolution this seems quite unlikely, but that's what might explain this
  great gap between R and Rfree.
 
  Tim
 
  On 12/08/2011 06:36 PM, Christopher Browning wrote:
  Dear All,
 
  Question: Has anybody ever refined the same structure using PHENIX and
  then tried REFMAC to see what happens?
 
  I did and I stumbled on something funny. I'm refining a structure at
  1.1A resolution which was solved with Iodine phasing using PHENIX
  AutoSolve. Got a great map and the structure was built almost
  completely. I had to build a few residues myself, and using the
  published sequence, I started filling in the residues, but as I came
  nearer the N-terminus, it looked like the density did not match residues
  from the sequence. I kept the residues as in the sequence, but as you
  can see from the PHENIX refined picture (below is the link) it still
  looks like the amino acid sequence in the crystal does not match the
  published protein sequence.
 
  Out of interest I refined the same file in REFMAC, and now the electron
  density is correct, and the sequence of the amino acids in the crystal
  matches the published sequence (see link for picture below). Not only
  that. my R/Rfree improved (16.5/19 for PHENIX, 10/18 for REFMAC).
 
  I've also refined the occupancies of the iodide, however the the output
  FO-FC map from PHENIX complains and the REFMAC map is fine.
 
  How can this be and what causes this?
 
  Link for the pictures:
  Both maps are at identical Sigma levels in both pictures.
  PHENIX: http://dl.dropbox.com/u/51868657/PHENIX_refined.png
  REFMAC: http://dl.dropbox.com/u/51868657/REFMAC_refined.png
 
  Cheers,
 
  Chris Browning
 
 
 
 
  - --
  Dr Tim Gruene
  Institut fuer anorganische Chemie
  Tammannstr. 4
  D-37077 Goettingen
 
  GPG Key ID = A46BEE1A
  -BEGIN PGP SIGNATURE-
  Version: GnuPG v1.4.10 (GNU/Linux)
  Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
 
  iD8DBQFO4PjgUxlJ7aRr7hoRAszXAKCNmTZvCVaDUm6v3lQjp051H+ilDgCgxd1l
  as9CcWEseq9uEV8qMZsOfsg=
  =KKUr
  -END PGP SIGNATURE-

Re: [ccp4bb] Description of a newly solved structure

[Katherine Sippel]

For drawing a 2D fold topology I'd suggest TopDraw (
http://www.ccp4.ac.uk/html/topdraw.html). To see if you structure looks
like other structures I'd look at the DALI server (
http://ekhidna.biocenter.helsinki.fi/dali_server/). Dali is pretty good for
digging out results/discussion fodder because it gives you another
structure(s) to compare. PISA would be my suggestion for roughly
identifying any oligimerization though it's best to back those claims up
with experimental data. If you've got ligands bound Gerard Kleywegt and the
PDBe staff have put together a lovely means of searching and representing
motifs which is also useful for comparative purposes (
http://www.ebi.ac.uk/pdbe-site/pdbemotif/). There are a panoply of good
figure generating softwares available but my personal preference is Pymol
for it's flexibility, comprehensive wiki, and supportive users groups (
http://www.pymol.org/).

If your just looking for a description of what to put into a results and
discussion section for a new structure you might try browsing through the
Acta Cryst D archives for some good examples.

Hope this helps,

Katherine

On Wed, Nov 30, 2011 at 10:49 PM, sadaf iqbal sadaf_che...@yahoo.comwrote:

 Hello everyone,

 I have submitted one cysteine protease structure in PDB and now i have to
 describe the structure completely for my PhD thesis. I know some web
 servers who are good to provide knowledge about protein but i would like to
 ask expert persons of this field as i am quite new in describing a
 structure. Which softwares/web servers you prefer when you are going to
 describe one complete native structure? I am a chemist basically.

 Thanks in advance

 Sadaf Iqbal
 PhD Scholar
 ICCBS, University of Karachi, Pakistan.
  Visiting Scientist
 University of Hamburg, Germany.

[ccp4bb] Off topic: Kendrew Model (Resolved)

[Katherine Sippel]

I wanted to let you all know with the gracious assistance of Bernhard Rupp
the model has found a new home with Katherine Kantardjieff in the Center
for Molecular Structure were it can be appreciated as it deserves.

I also wanted to extend a thank you to the whole community. I was amazed
and touched by the response which ended up spanning three continents.  In
the few years I've been subscribed this bulletin board it has been reliably
helpful and immensely educational. It has raised my awareness of potential
issues and allowed me to make informed decisions as to the direction I
proceed. I have watched an entire field evolve in a matter of weeks and
months rather than trying to fumble with policy in a week at yearly
conferences. And while there are always contentious issues and heated
debates you never fail to lend your expertise and advice. You guys are one
of the reasons I love this job. Again, thanks.

Cheers,

Katherine


2011/11/1 Katherine Sippel katherine.sip...@gmail.com

 Hi Andreas,

 It is much larger and looks slightly less like Chthulu cut himself shaving
 (I'm going to crystallographer hell for that comment). For those interested
 I've put the picture on Photobucket so as to not trash people's inbox.
 http://s1085.photobucket.com/albums/j422/KatherineSippel/?action=viewcurrent=ABP_pic.jpg

 Katherine


 2011/11/1 Andreas Förster docandr...@gmail.com

 Is it like this one:

 http://www.sciencemuseum.org.**uk/images/I053/10321094.aspxhttp://www.sciencemuseum.org.uk/images/I053/10321094.aspx

 Not sure I would want to put it into the dining room...


 Andreas




 On 01/11/2011 2:34, Katherine Sippel wrote:

 Hi all,

 I'm going to interject into the middle of this rousing though protracted
 debate to pick your brains. I am in possession of a rather large and
 intact brass scale Kendrew model (sans mirrors). Due to facility
 restructuring we no longer have room for it. I have approached the local
 health science and natural science museums but have gotten nothing but
 the run around. This amazing model is in need of a forever home and I'm
 stumped as far as alternative ideas. I am seriously considering
 suspending a Mars bars in the sugar binding cleft, calling it MBP, and
 trying to spin it to the art museum as a modernist piece commenting on
 the diets in Western civilization. Either that or putting it in my
 dining room if I can get it in the door. Any suggestions would be
 appreciated.

 Cheers,

 Katherine


 --
Andreas Förster, Research Associate
Paul Freemont  Xiaodong Zhang Labs
 Department of Biochemistry, Imperial College London
http://www.msf.bio.ic.ac.uk

[ccp4bb] Off topic: Kendrew Model

[Katherine Sippel]

Hi all,

I'm going to interject into the middle of this rousing though protracted
debate to pick your brains. I am in possession of a rather large and intact
brass scale Kendrew model (sans mirrors). Due to facility restructuring we
no longer have room for it. I have approached the local health science and
natural science museums but have gotten nothing but the run around. This
amazing model is in need of a forever home and I'm stumped as far as
alternative ideas. I am seriously considering suspending a Mars bars in the
sugar binding cleft, calling it MBP, and trying to spin it to the art
museum as a modernist piece commenting on the diets in Western
civilization. Either that or putting it in my dining room if I can get it
in the door. Any suggestions would be appreciated.

Cheers,

Katherine

Re: [ccp4bb] Off topic: Kendrew Model

[Katherine Sippel]

Hi Andreas,

It is much larger and looks slightly less like Chthulu cut himself shaving
(I'm going to crystallographer hell for that comment). For those interested
I've put the picture on Photobucket so as to not trash people's inbox.
http://s1085.photobucket.com/albums/j422/KatherineSippel/?action=viewcurrent=ABP_pic.jpg

Katherine

2011/11/1 Andreas Förster docandr...@gmail.com

 Is it like this one:

 http://www.sciencemuseum.org.**uk/images/I053/10321094.aspxhttp://www.sciencemuseum.org.uk/images/I053/10321094.aspx

 Not sure I would want to put it into the dining room...


 Andreas




 On 01/11/2011 2:34, Katherine Sippel wrote:

 Hi all,

 I'm going to interject into the middle of this rousing though protracted
 debate to pick your brains. I am in possession of a rather large and
 intact brass scale Kendrew model (sans mirrors). Due to facility
 restructuring we no longer have room for it. I have approached the local
 health science and natural science museums but have gotten nothing but
 the run around. This amazing model is in need of a forever home and I'm
 stumped as far as alternative ideas. I am seriously considering
 suspending a Mars bars in the sugar binding cleft, calling it MBP, and
 trying to spin it to the art museum as a modernist piece commenting on
 the diets in Western civilization. Either that or putting it in my
 dining room if I can get it in the door. Any suggestions would be
 appreciated.

 Cheers,

 Katherine


 --
Andreas Förster, Research Associate
Paul Freemont  Xiaodong Zhang Labs
 Department of Biochemistry, Imperial College London
http://www.msf.bio.ic.ac.uk

Re: [ccp4bb] raw data deposition

[Katherine Sippel]

Generally during these rigorous bb debates I prefer to stay silent and
absorb all the information possible so that I can make an informed decision
later on. I fear that I am compelled to contribute in this instance. In
regards to the does this make a difference in the biological interpretation
stage issue, I can state that it does. In my comparatively miniscule career
I have run into this issue three times. The first two address Adrian's
point...

And (b) even if they do, is this continual improvement even worthwhile?  I
  am always depressed at how little a model changes from an initial build to
 the final one, even when the rfree drops from 35 to 23. All that work! - and
 my biological interpretation would have been almost the same at the
 beginning as at the end.


In one instance I adopted an orphaned structure and ran it through a
slightly more advanced refinement protocol (on the same structure factors)
and ended up with a completely different story than the one I started with
[1]. Another researcher in my grad lab identified mis-oriented catalytic
residues in an existing structure from EDS server maps which affects the
biochemistry of the catalytic mechanism [2].

In another case I decided that I would reprocess some images that I had
originally indexed and scaled in my Ooo buttons clicky clicky stage of
learning crystallography and the improved structure factors revealed a
alternate conformations for both a critical loop and ligand orientation [3].

And this was all in the last 4 years. So I would posit that the answer is
yes there are significant biological insights to be had with the capacity to
reassess data in any form.

Katherine

[1] J Phys Chem Lett. 2010 Oct 7;1(19):2898-2902
[2] Acta Crystallogr D Biol Crystallogr. 2009 Mar;65(Pt 3):294-6.
[3] Manuscript in progress


Katherine Sippel, PhD
Postdoctoral Associate
Baylor College of Medicine

Re: [ccp4bb] raw data deposition

[Katherine Sippel]

Hi Boaz,

I see your point in regards to making a database of all diffraction
images. The argument I clearly failed to make effectively was that
improvement of structures can frequently yield useful biological information
which is why I believe that, at the least, images of deposited structures
should be archived. The availability of the structure factors alone can
allow crystallographers to improve models significantly, but there is always
the question of whether there was more data lost to button smashing despite
developers efforts to make data processing idiot-proof.

If I am going to invest years of my life and millions of tax dollars on a
hypothesis derived from a structure I personally would be willing to take a
day to reprocess the images and put the model through it's paces to ensure
that I'm not wasting my time and/or other people's money.

Yes experimenters (including myself) make mistakes, but the joy of
crystallography is that we can effectively backtrack, identify where the
mistake was made, fix it and learn from it. All it costs us is a couple of
hours and perhaps a little pride whereas the result is stronger more
effective science for our field as a whole. In my mind that equalizes out
the cost:benefit ratio considerably.

Of course this is all just my opinion,

Katherine



2011/10/28 Boaz Shaanan bshaa...@exchange.bgu.ac.il

  Hi Katherine,

 It sounds as if you had all you needed to correct other people's (and your
 own) errors, as you described,  in the existing database (EDS, PDB) or your
 own data, right? That hardly justifies establishing a new database of which
 at least 80-90% is worthless. Furthermore, since much of the non-indexable
 data arise from experimenter's faults, is it not the time to start a
 discussion (preferably prior to setting up a committee) on deposition of
 crystals so that anybody can have a go at them to detect problems if they
 wish?

  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 Katherine
 Sippel [katherine.sip...@gmail.com]
 *Sent:* Friday, October 28, 2011 8:06 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* Re: [ccp4bb] raw data deposition

  Generally during these rigorous bb debates I prefer to stay silent and
 absorb all the information possible so that I can make an informed decision
 later on. I fear that I am compelled to contribute in this instance. In
 regards to the does this make a difference in the biological interpretation
 stage issue, I can state that it does. In my comparatively miniscule career
 I have run into this issue three times. The first two address Adrian's
 point...

  And (b) even if they do, is this continual improvement even worthwhile?
  I  am always depressed at how little a model changes from an initial build
 to the final one, even when the rfree drops from 35 to 23. All that work! -
 and my biological interpretation would have been almost the same at the
 beginning as at the end.


 In one instance I adopted an orphaned structure and ran it through a
 slightly more advanced refinement protocol (on the same structure factors)
 and ended up with a completely different story than the one I started with
 [1]. Another researcher in my grad lab identified mis-oriented catalytic
 residues in an existing structure from EDS server maps which affects the
 biochemistry of the catalytic mechanism [2].

 In another case I decided that I would reprocess some images that I had
 originally indexed and scaled in my Ooo buttons clicky clicky stage of
 learning crystallography and the improved structure factors revealed a
 alternate conformations for both a critical loop and ligand orientation [3].

 And this was all in the last 4 years. So I would posit that the answer is
 yes there are significant biological insights to be had with the capacity to
 reassess data in any form.

 Katherine

 [1] J Phys Chem Lett. 2010 Oct 7;1(19):2898-2902
 [2] Acta Crystallogr D Biol Crystallogr. 2009 Mar;65(Pt 3):294-6.
 [3] Manuscript in progress

 
 Katherine Sippel, PhD
 Postdoctoral Associate
 Baylor College of Medicine

Re: [ccp4bb] raw data deposition

[Katherine Sippel]

I have said my piece of the issue of depositing but there is one comment I
would like to address.

Besides, I thought that by now there are some standards on how data should
 be processed (this has been discussed on this BB once every few months, if
 I'm not mistaken). Isn't that part of the validation process that so many
 good people have established? Also, to the best of my knowledge (and
 experience) referees (at least of some journals) are instructed to look into
 those issues these days and comment about them, aren't they?

 There is a big difference between data that is processed correctly and data
that is processed well. I'm reminded of a Yogi Berra quote In theory,
theory and practice are the same. In practice, they are not.  Every year
our grad level crystallography course would take the same lysozyme data set
and break into groups of two to process them independently in HKL2000 and
every year we'd get a vastly different array of statistics. All of the
processing would produce valid structure factors that were essentially the
same and all would pass SFCHECK. The difference in the numbers varied for
many reasons including the choice of reference image, initial spot number
picking, profile fitting radius, spot size, the use of 3D profile fitting,
choice of scaling factors and whether appropriate sacrifices had been made
to the Denzo gods.  And though the overall backbone and structure remained
mostly the same there were clearly some who had better maps than the others.


So yes there is a standard protocol in place and it can identify and correct
gross error but by no means does that indicate the data was processed well.

Sincerely,
Katherine

[ccp4bb] Microcrystal cryo

[Katherine Sippel]

Hi all,

I was wondering with the all of the advances in microbeam technology and
hardware, what is the best methods for cryoprotecting microcrystals? Are
oils better than solutions? How do you address the excess liquid issues? Is
there a way to test the conditions prior to shipping it off to the
synchrotron? Tips and tricks would be appreciated and I'll make a summary of
the replies for future archive searchers like myself.

Thanks in advance,

Katherine

Re: [ccp4bb] Off Topic: PDB validation server

[Katherine Sippel]

Ha. That is obviously it. I failed to account for Brownian motion in the pdb
file itself. Properly modeling this is really going to mess with my data to
parameter ratio.

Katherine

On Tue, Jul 12, 2011 at 12:54 AM, James Stroud xtald...@gmail.com wrote:

 On Jul 8, 2011, at 11:13 AM, Katherine Sippel wrote:

  I was shocked to discover that the file with only one questionable
 solvent in April now has 173 of them.

 One word: Diffusion.

 James

[ccp4bb] Off Topic: PDB validation server

[Katherine Sippel]

Hi all,

I am putting the finishing touches on a structure and as a good little
crystallographer I am running it through Molprobity and PDB validation to
make sure everything clears before deposition. Everything was looking
alright until I threw the file into the PDB validation server and suddenly
there are a significant number of solvents that violate the 3.5 angstrom
rule. Concerned that something had gone wrong I put an older file that I had
run through the server in April. I was shocked to discover that the file
with only one questionable solvent in April now has 173 of them.

I know that the PDB updated its validation server in May as described in
their news link but it seemed to indicate an increase in output options
rather than a change in criteria. Is anyone aware of  what changes were made
to the validation server in regards to the preferred geometrical and
stereochemical features?

Thanks for your time,

Katherine

Re: [ccp4bb] Off Topic: PDB validation server

[Katherine Sippel]

Hi again,

I have an update. The nice people at the PDB have gotten in touch and they
think it might be a bug. They are looking into it.

Thank you for all the off-board replies and I hope you all have a wonderful
weekend.

Katherine

On Fri, Jul 8, 2011 at 1:13 PM, Katherine Sippel katherine.sip...@gmail.com
 wrote:

 Hi all,

 I am putting the finishing touches on a structure and as a good little
 crystallographer I am running it through Molprobity and PDB validation to
 make sure everything clears before deposition. Everything was looking
 alright until I threw the file into the PDB validation server and suddenly
 there are a significant number of solvents that violate the 3.5 angstrom
 rule. Concerned that something had gone wrong I put an older file that I had
 run through the server in April. I was shocked to discover that the file
 with only one questionable solvent in April now has 173 of them.

 I know that the PDB updated its validation server in May as described in
 their news link but it seemed to indicate an increase in output options
 rather than a change in criteria. Is anyone aware of  what changes were made
 to the validation server in regards to the preferred geometrical and
 stereochemical features?

 Thanks for your time,

 Katherine

Re: [ccp4bb] No Observed SO4 in AmSO4 Crystallizations

[Katherine Sippel]

Hey Jacob,

If you are looking for a specific example there are numerous carbonic
anhydrase structures that were crystallized in ~2.8M ammonium sulfate that
have no sulfate in the structure. Usually they have a ligand in the active
site which displaces the only ordered sulfate molecule. I have found this to
be the case with 1M concentrations of citrate and acetate as well. Unless
there is a specific binding site to organize the molecule in regards to
the protein it just loiters in the solvent that you are flattening anyways.

Just my two cents,

Katherine

---

Katherine Sippel, PhD
Postdoctoral Associate
Department of Biochemistry
Baylor College of Medicine

On Fri, Jun 3, 2011 at 4:22 PM, Jacob Keller j-kell...@fsm.northwestern.edu
 wrote:

 Dear Crystallographers,

 is it possible not to observe density for any sulfate ions in
 crystallizations done with molar-range sulfate concentrations?
 Beguiles the mind, but I seem to be looking at such a structure...

 Jacob

 --
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 cel: 773.608.9185
 email: j-kell...@northwestern.edu
 ***

[ccp4bb] Ubuntu 10.10 installation Issues

[Katherine Sippel]

Hi all,

I recently upgraded (if you can call it that) to Ubuntu 10.10 and I'm having
some issues with CCP4 and file recognition. I suspect this is an Ubuntu
issue rather than a CCP4 issue but I'm hoping someone will have an idea. I
downloaded the CCP4-Lin-generic.tar and ran the install.sh. It unpacks
beautifully and generates the ccp4.setup file. I source that into my bash
terminal and there are no issues there. However when I try to execute a
command (in this case ccp4i) I get the error...

exec: 4: /usr/local/ccp4/tcltk++/bin/bltwish: not found

The directory and file do, in fact, exist as can be evidenced by cd cut and
paste directory from error. I also know that the issue isn't with the
ccp4.setup file because I've scoured it for misplaced spaces or backslashes
that might have disrupted a path and the aliases seem to all be pointing in
the right place. I thought it might be a permission error but I am running
phenix out of /usr/local/ as well and have no issues. The Ubuntu forums
indicate that there may be an issue with this version and binary executable
files but I haven't found a solution.

If anyone else has run into this problem and come up with an answer I would
be grateful.

Katherine

Re: [ccp4bb] Ubuntu 10.10 installation Issues

[Katherine Sippel]

Bingo! I might suggest adding this library to the list of packages to
install in the platform specific portion of the problems page documentation.


Thanks again.

Katherine

On Fri, Mar 11, 2011 at 4:01 PM, Ben Eisenbraun b...@hkl.hms.harvard.eduwrote:

 Hi Katherine,

  terminal and there are no issues there. However when I try to execute a
  command (in this case ccp4i) I get the error...
 
  exec: 4: /usr/local/ccp4/tcltk++/bin/bltwish: not found

 This happens to be the error you get when you run a 32-bit binary on a
 64-bit Ubuntu machine _without_ the 32-bit compatibility libraries
 installed.

 So are you running a 64-bit installation?  (uname -m)
 Do you have the ia32-libs package installed? (dpkg -l ia32-libs)

 -ben

 --
 | Ben Eisenbraun
 | SBGrid Consortium  | http://sbgrid.org   |
 | Harvard Medical School | http://hms.harvard.edu  |

Re: [ccp4bb] PISA ligand handling: Summary

[Katherine Sippel]

To recap it turns out that the element symbol does supersede the atom and
residue name which seems to provide a sneaky workaround to the unrecognized
ligand issue in PISA.

However this information does come with the disclaimer that it does not
replace electrostatic calculations such as CHARMM or AMBER. To quote Eugene
Krissinel be wary about specific energy values given by PISA, especially
for protein-ligand interactions. They are quite approximate. PISA does
simplified calculations which are very sensitive to the quality of
interface, which differs from structure to structure.

Once again thank you all for the quick responses.

Katherine

On Wed, Feb 23, 2011 at 10:51 AM, Katherine Sippel 
katherine.sip...@gmail.com wrote:

 Hi all,

 I have sort of an oddball question regarding how PISA reads the ligand file
 into the program for calculations. Does the element symbol of the PDB file
 take precedence over the atom name or residue name?

 Specifically, if a ligand contains either atom names or residue names that
 are inconsistent with those in the CCP4 monomer library will the resulting
 energetic calculation be valid?

 Thanks,

 Katherine

[ccp4bb] PISA ligand handling

[Katherine Sippel]

Hi all,

I have sort of an oddball question regarding how PISA reads the ligand file
into the program for calculations. Does the element symbol of the PDB file
take precedence over the atom name or residue name?

Specifically, if a ligand contains either atom names or residue names that
are inconsistent with those in the CCP4 monomer library will the resulting
energetic calculation be valid?

Thanks,

Katherine

[ccp4bb] Off Topic: Fixed point sequence alignments

[Katherine Sippel]

Hi all,

I was wondering if you know of a program that can do protein sequence
alignments around experimentally determined equivalent residues (i.e.
biochemists have determined via mutagenesis and kinetics that residues 3,6
and 9 of protein X are the equivalent of residues 7, 14, 21 in homologous
protein Y). I've got a good sized pool of homologous sequences to improved
the accuracy of the alignments and a few of the homologs have pdbs. I get
close in T-coffee, but it is missing a few theoretically conserved residues
and it is an unwieldy job for manual correction. Any assistance would be
appreciated.

Thanks,

Katherine

Re: [ccp4bb] I3C Phasing kit from Hampton

[Katherine Sippel]

In our hands the Rsymm difference after scaling was 3% and the crystals were
not particularly isomorphous (1.2 degree difference in beta angle). We were
also shooting at room temp and still found a solution. We were using an in
house source to optimize the anomalous signal, so if the data was shot at a
synchrotron the signal won't be as strong.

To comment on Artem's reply the I3C from Hampton is prepared in LiOH (for
solubility purposes) so it is basic already. Our crystallization conditions
were at pH 3 and there was no reactivity issue.

If you are interested here's the reference.
http://www.ncbi.nlm.nih.gov/pubmed/19020356

Cheers,

Katherine

On Sat, Jan 1, 2011 at 11:55 PM, Artem Evdokimov
artem.evdoki...@gmail.comwrote:

 http://shelx.uni-ac.gwdg.de/~tbeck/papers/mad_triangle.pdfhttp://shelx.uni-ac.gwdg.de/%7Etbeck/papers/mad_triangle.pdf

 is the paper where the 'I3C' designation originates. I also recommend
 Dauter's papers on halide phasing - very instructive stuff there.

 5-Amino-2,4,6-triiodoisophthalic acid as such is not supposed to be
 chemically reactive under 'normal' aqueous conditions. This is not to say
 that this compound can't react chemically, but I don't expect it to
 form/break covalent bonds unless there are chemical activators present
 (under right conditions, certain soluble carbodiimides for example may
 conjugate this compound to amines such as lysines or the N-terminus of the
 protein - although the bulky iodine may hinder the reaction considerably).
 So, in strict terms the actual chemical reactivity of this stuff is low.

 I suspect however that you're not really interested in reactivity as such
 but rather in the ability of this compound to form loosely associated (but
 fairly specific) protein-compound complexes. This is highly dependent on the
 nature of the protein you're working with and more specifically on the
 availablilty of appropriately shaped and charged zones on the surface(s)
 presented by packed protein molecules in your crystals. The answer to this
 question in your specific context is 'maybe' - sorry, it's just not very
 certain and there's not enough data available on I3C phasing so far to make
 a better guess. I hazard to suggest that this stuff will work less well at
 basic-ish pH but that's entirely speculative.

 The answer to your second question depends on the nature of your protein
 and the crystal, as well as the processing software and specific processing
 parameter values employed. Expected contribution (deltaFanom /F ratio) can
 be estimated using Hendrickson  Teeter (? or was it earlier than them, so
 sorry if it's someone else's) formula which works well for reasonably strong
 data (i.e. at low-ish resolutions where measured errors in the data do not
 significantly affect the ratio) - but you need to know how many anomalous
 scatterers you might expect to find.   It's so much easier to collect data
 and calculate an anomalous Patterson map, or run some rudimentary phasing
 attempts. Several programs output statistics (SHELX package and SOLVE come
 to mind) that are also quite useful.

 So, I would recommend that you 'start working on the data' rather than
 wonder if it's worth the effort. It's the only way to be sure, and it's not
 like it's usually very hard to do a few quick calculations and find out. You
 might have a structure in there already.

 Artem

 On Sat, Jan 1, 2011 at 10:10 PM, Vijay Pagadala 
 pagada...@niehs.nih.govwrote:

 Hi savers,

 What is the reactivity of the I3C compound- Iodine compound for phasing
 from Hampton Research?
 I was wondering if there is any chance that the crystals soaked at 50mM in
 the drop  for 24 hours at 4 celcius would stay unreacted to the compound
 I3C.
 Another detail question-How much of a difference in R-factor value after
 scaling (normal Vs anamolous ) is significant to say that there is signal
 from Iodine?
 I know it can be found out after phasing but want to have an idea to even
 start working on the data.

 Thanks in advance.

 Vijay

Re: [ccp4bb] Heavy atom salt at low pH

[Katherine Sippel]

Hi Debajyoti,

For my low pH phasing (pH 3) the thing that worked for me was the Magic
Triangle. It works best at Cu K alpha (i.e. home source). There is a Mad
Triangle that works at synchrotron sources as well. You can get it all made
up from Hampton or Jena Biosciences but you can also buy the chemical direct
from Fisher or Sigma and make it up in LiOH or KOH for super cheap. You can
check out the papers here: http://www.ncbi.nlm.nih.gov/pubmed/19020356 or
here: http://www.ncbi.nlm.nih.gov/pubmed/19851024

Best of luck,

Katherine

On Wed, Nov 17, 2010 at 4:31 AM, Debajyoti Dutta 
debajyoti_dutt...@rediffmail.com wrote:


 Hi All,

 Sorry for a non CCP4 question. I have been trying to phase a protein
 structure using different heavy atom derivatives. The problem is the
 crystallization pH is very low (from 2.8 to 3.5). I will be highly benefited
 if anybody kindly suggests me the possible heavy atom salts to try

 sincerely
 Debajyoti


 http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline@middle?