[ccp4bb] Career opportunity at Ambry Genetics

[Ccp4 No-Reply]

Hi,

We would like to draw your attention to a new career opportunity in 
structural biology at Ambry Genetics. Please see the link below:


https://recruiting.ultipro.com/AMB1001AMGEN/JobBoard/bcdd10b1-192e-e264-1203-f18eb6dd40c1/OpportunityDetail?opportunityId=6a66793e-789c-4857-b14a-cc50cbdd1d1f] 



Igor Petrik, PhD

Structural Biologist II

Ambry Genetics




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

Re: [ccp4bb] AW: [ccp4bb] Cleaved peptide density!

[CCP4]

Once thing I don't fully understand: do you have a cleaved density or a cleaved 
peptide? If your peptide is sitting in a binding site, and that you can see 
only the part inside the pocket, with the rest supposably sitting in a 'more 
opened' part of the molecule, or else outside of the molecule, than the cleaved 
density could be due to non-specificity of this  part of the peptide and high 
flexibility / non-recognition. This would obviously have great biological 
interest and implications. 

Leo

Sent from my iPad (most likely in conference)

 On 21 Apr 2015, at 10:25, herman.schreu...@sanofi.com wrote:
 
 Dear Dipankar,
  
 as Bonsor mentioned, 2-3 weeks is something completely different from the few 
 hours normally used for enzyme assays. A lot may happen during the long 
 crystallization that does not happen in the assay. Also, your enzyme will 
 still have the remainder of the catalytic machinery (oxy-anion hole, Hisp-Asp 
 “proton shuttle”) intact and a water molecule in the cavity left by the 
 removed sulfur may function as nucleophile.
  
 One question: do you incubate overnight and then set up crystallizations, or 
 do you grow apo-crystals and incubate fully grown crystals with peptide?
 In the first case, I would freeze the crystals after overnight incubation 
 with fresh peptide.
 In the second case, I would incubate only 30 minutes and then freeze the 
 crystals.
 In both cases, I would add as much intact peptide as I can, since it gets 
 cleaved during incubation.
  
 One other question: do you seen both ends of the peptide, or only half of it. 
 If you see only half of the peptide, it might be that the full peptide is not 
 compatible with the crystal packing and only protein molecules with half a 
 peptide bound may enter the crystal lattice. In this case, you may have to 
 screen for completely new crystallization conditions in the presence of some 
 protease inhibitor(s) like PMSF to see if you can get a different crystal 
 form. You may also want to scan the literature to see if some non-cleavable 
 substrate-analog (inhibitor) is available.
  
 On the positive side: your structure with the cleaved peptide is also 
 interesting, since it represents the product complex!
  
 Good luck!
 Herman
  
  
 Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
 Dipankar Manna
 Gesendet: Montag, 20. April 2015 21:22
 An: CCP4BB@JISCMAIL.AC.UK
 Betreff: Re: [ccp4bb] Cleaved peptide density!
  
 Dear Bonsor,
  
 Thanks for your suggestions!
  
 It need 2-3 weeks to get the fully grown crystals. And harvest, freeze and 
 data collection just take usually next 3-5 days. I usually incubated 
 substrate overnight. Initially I was purifying with the same column as the WT 
 but in the next batch I used new beads to purify the mutant as you 
 categorically pointed out. But results are the same, I mean I got the same 
 cleaved peptide density! I tried soaking with different time frames and with 
 different peptide concentrations as well but in this case I can't see any 
 peptide density at all.
  
 Best,
  
 Dipankar 
  
 On Mon, Apr 20, 2015 at 9:06 PM, D Bonsor dbon...@ihv.umaryland.edu wrote:
 First of all, you don't say how long it took to first set up crystals, for 
 them to grow, harvest, freeze and collect data on. Secondly how long did 
 leave the peptide/substrate for your SDS PAGE experiment? If they are of a 
 different time scale e.g. 6 hours v.s. 30 days, it may be that your enzyme is 
 not totally dead.
 
 Also how did you purify the alanine mutant? If you purified it on the same 
 columns/beads as the WT protein you may have a residual amount of active 
 protein which could cleave your peptide over the course of crystallization. 
 You may want to use fresh beads, or treat columns with pepsin or sodium 
 hydroxide.
 
 Not real answers I am afraid, more like suggestions.
 
 
  
 --
 Dipankar Manna
 Research Scholar
 Department of Chemistry
 University of Oslo
 Oslo, Norway

Re: [ccp4bb] Best method for weighted averaging of Friedel pairs?

[ccp4]

This is a case where it is really helpful to keep some record of the 
unmerged integrated data.

And again rejecting the odd outlier does no harm to most analyses..

I like to use scala to check for outliers looking at all i+ I- 
measurements; if there is a wild discrepancy for weak anomalous signals 
you have probably found an outlier which is best rejected.


If you have a huge anomalous signal with good redundancy you probably 
shouldnt use Imean and the anomalous difference will be I= -I- with SD = 
sqrt (VarI+ VarI-).


Most software which uses that signal will check for outliers in the 
Anom difference lists too, and it is usually safest to exclude them from 
anom site searches, and phase calculations.

Eleanor
  On 14.02.2012 06:30, Tim Gruene wrote:

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Markus,

why don't you reintegrate the data with hkl2000 telling the program 
to

treat them as non-anomalous data-set? This should give you scalepack
output with the Bijvoet pairs merged and overcome the problem you 
describe.


Cheers,
Tim

On 02/13/2012 06:19 PM, Markus Meier wrote:

On 11/02/12 02:52 PM, Bryan Lepore wrote:

did you ever get a response on this? it is interesting but nobody
posted publicly.

-Bryan



Dear Bryan,

so far no one replied ... so please find my answer below. If someone
disagrees, please post.

None of the methods I have described are appropriate.

If the negative Bijvoet mates and the positive Bijvoet mates have 
been
merged separately to one intensity value for each (i.e. I+ or I-) 
plus
the associated standard deviation (sigI+ or sigI-), any weighted 
method
to calculate the mean will bias the intensity to either the I+ or 
the I-.


Therefore the only appropriate method is to use the unweighted mean:

Imean = 0.5*( I+ + I- )
sigImean = 0.5 * sqrt( sigI+^2 + sigI-^2 )

The only CCP4 program I found that actually does this is mtzMADmod. 
This
method also has the advantage that the original intensity values of 
I+
and I- can be reconstructed from the mean and the anomalous 
difference

(albeit with the loss of the original standard deviations).

Method 1 (scalepack2mtz)
should not be used. The resulting value is not the best estimate
(maximum likelihood)

Method 2 (in book by B. Rupp)
gives the maximum likelihood average in case that the reflections 
are
equivalent and is thus appropriate for the merging of the negative 
(or
positive) set of Bijvoet mates, centric reflections (where the 
anomalous

differences are zero) or in the case of an non-anomalous dataset the
merging of symmetry equivalent reflections.

Method 3
gives a more realistic sigma value in the case that the individual
intensity values are far apart and their individual standard 
deviations

are small. Consider the example I have posted:

I+: 23841.50 sigI+: 634.01 I-: 9628.57, sigI-: 264.75
Method 2: Imean=11738.95, sigIMean=244.31
Method 3: Imean=11738.95, sigIMean=7106.47

If the I+ and I- values above actually were symmetry equivalent
reflections in an non-anomalous dataset, the sigImean from method 2 
is

ridiculously small and method 3 gives a far more realistic value. If
method 3 is the best mathematical solution to this problem I am not 
able

to judge and I have to trust the statistician (or programmer) who
implemented this solution.

Cheers,
Markus

On 10/02/12 01:47 PM, Markus Meier wrote:

Dear all,
I have a anomalous dataset, processed in HKL2000. Scalepack outputs 
a
file containing the separately merged sets of the Friedel pairs I- 
and
I+ and their standard deviations sigI+ and sigI-. Scalepack does 
not

output the averaged intensities (Imean) and the standard deviations
(sigIMean).

The CCP4 program truncate that I use to convert the intensities to
amplitudes requires Imean, I- and I+ and the respective standard
deviations in its input file.

I have now found at least three different methods to generate the
averaged intensities from the Friedel pairs:

1) scalepack2mtz

   uses standard deviations for the weights:
   weights w = 1/sigI

   Imean = (w+*I+ + w-*I- ) / (w+ + w-)
   sigImean = 1 / (w+ + w-)

2) Method described in Biomolecular crystallography by Bernhard 
Rupp, p.

332/333
   to average symmetry equivalent reflections

   uses variances for the weights:
   weight w = 1/sigI^2

   Imean = (w+*I+ + w-*I- ) / (w+ + w-)
   sigImean = 1 / sqrt(w+ + w-)

3) Method used in cctbx
   function miller.set.average_bijvoet_mates() that calls generic
merge.merge_equivalent_obs():

   same as methods 2, except that

   sigImean is the larger of either
 a) sigImean = 1 / sqrt(w+ + w-)
 or
 b) sigImean = sqrt( wvariance )

   where wvariance =
 (w+ + w-) / [ (w+ + w-)^2 - (w+^2 + w-^2) ] *
 [ w+*(F+ - Imean)^2 + w-*(F- - Imean)^2 ]

What are the advantages and disadvantages of each method? Should 
method

1 be used at all?

Some example from my dataset:
Reflection (1, 1, 0), space group P3 2 1

I+: 23841.50 sigI+: 634.01 I-: 9628.57, sigI-: 264.75
Method 1: Imean

Re: [ccp4bb] chirality problem

[ccp4]

Wont coot fix the nomenclature issue, then you can check whether you have
a real chirality problem - eg a squashed flattened VAL..

Eleanor


On Fri, 6 Jan 2012 09:47:15 -0600, Katherine Sippel
katherine.sip...@gmail.com wrote:
 Incidentally the PDB validation server will spit out similar errors if
you
 have hydrogens on lysine side chains (also not a chiral center) should
they
 get swapped upon regularization during refinement. It makes the chemist
in
 me cringe a little bit.
 
 Katherine
 
 On Fri, Jan 6, 2012 at 8:30 AM, Robbie Joosten
 robbie_joos...@hotmail.comwrote:
 
  Hi Afshan,

 I assumed, because you mentioned only VAL and LEU, that you were
refering
 to the CB (VAL) and CG (LEU) as problematic chiral centers. Paul is
right
 that these atoms are not chiral in a chemical sense, but they are in a
 computational sense because every connected atom has a unique name. The
 PDB
 is pretty strict in this sense (as it should be), but they could/should
 call it a nomenclature error. They could also just swap the atom names
 like
 I described and solve the problem for you. Anyway, please give a bit
more
 details about your problem.

 Computational chirality problem can be a serious problem for
refinement:
 if the chirality is wrong due to swapped atom names, the chiral volume
 restraint will try to invert your chiral center. This can lead to
 malformed
 geometry, typically flattening of of the group. This means that a
 computational chirality problem can lead to a 'real' chirality problem.
 In
 Refmac, this will not happen for LEU or VAL, but it will happen for
 things
 like SO4, GOL, and a whole lot of other more interesting hetero
 compounds.

 @ Paul, I don't think it will be a CCP4 program that reported the
 problem.
 Does the 'fix nomenclature problems' option in Coot also do VAL and
LEU?

 Cheers,
 Robbie

  --
 Date: Fri, 6 Jan 2012 10:44:58 +
 From: paul.ems...@bioch.ox.ac.uk
 Subject: Re: [ccp4bb] chirality problem

 To: CCP4BB@JISCMAIL.AC.UK

 Hi Afshan,

 This is not the solution if you are right about the problem being one
of
 chirality (and it is if it is not and is merely an issue of
nomenclature
 (as I suspect is the case)).  So the question is, if the problem is
 indeed
 one of nomenclature, what software (if any) described it as a chirality
 issue?  If it is one of ours we should fix that.

 Paul


 On 05/01/12 11:44, Robbie Joosten wrote:

 Hi Afshan,

 Just swap the (names of) the CD and CG atoms, no need for refinement.
The
 CCP4 dictionary allows both chiralities for LEU and VAL, so Refmac
won't
 detect the problem. The problem is still very real to many programs so
it
 should be fixed.

 Cheers,
 Robbie Joosten

 --
 Date: Thu, 5 Jan 2012 02:46:30 -0800
 From: afshan...@yahoo.com
 Subject: [ccp4bb] chirality problem
 To: CCP4BB@JISCMAIL.AC.UK

  Dear Users,

 I am facing difficulties to validate my structure according to PDB
 server.
 I have solved my structure and now want to submit in PDB but during
 validation process i have  some chirality problem specially   VAL and
LEU
 amino acids there are total 18 amino acids which deviated from the
 chirality so how can i solve this problem.

 Any suggestion would be highly appreciated.


 Best Regards

 AFSHAN

Re: [ccp4bb] From non-twinned to twinned?

[ccp4]

I wouldnt expect radiation damage to cause twinning!

1) maybe you have different indexing for the 2 passes? Have you used
pointless to check if this could happen (depends on SG and cell dimensions)


Ask for match reference set in input and give it sca1 as input 1, then
check sca2

2) use pointless to examine quality of symmetry matches before merging -
maybe you have the wtrong SG?

Eleanor


On Wed, 4 Jan 2012 08:54:38 -0600, Jacob Keller
j-kell...@fsm.northwestern.edu wrote:
 Need more info: how many degrees per frame? Also, on integration, do
 various stats change over the 'sets?
 
 JPK
 
 On Wed, Jan 4, 2012 at 7:42 AM, Zhiyi Wei ustcwebri...@gmail.com
wrote:
 Dear all,

 I recently collected a dataset (~2000 frames) from a single crystal.
 If merge first 600 frames (sca1) or last 600 frames (sca2), Rmerge
 values from scalepack seem to be ok (~10%) though rejection ratios are
 high (~5%). But if I merge all frames together, Rmerge value goes up
 to ~20% and rejection is extremely high (~20%). Then, I checked sca1
 and sca2 by xtriage in phenix. Surprisingly, the logfiles told me that
 sca1 is no twining while sca2 is very likely to be twinned. I never
 met this case before. So, I am wondering if it is possible from a
 non-twinned structure to a twinned structure just due to radiation
 damage. If the answer is yes, does it mean that I should not collect a
 large number of frames to amplify anomalous signals by using this
 crystal?

 Thanks a lot!

 Best,
 Zhiyi

Re: [ccp4bb] Help with deleting reflections from an mtz file

[ccp4]

Thanks Clemens - I knew it should be possible! Documentation didnt exactly
help..

Eleanor

On Wed, 17 Aug 2011 19:20:21 +0100, Clemens Vonrhein
vonrh...@globalphasing.com wrote:
 Dear Eleanore,
 
 Ooops - the ccp4bb rejects attachements with *.sh ending? Let's try it
 again with *.sh.txt ...
 
 The attached script works for me - fantastic what one can do with
 SFTOOLS :-)
 
 Cheers
 
 Clemens
 
 On Wed, Aug 17, 2011 at 04:55:59PM +0100, Eleanor Dodson wrote:
 There are 2 rogue reflections in a data set I have here. How can I
 eliminate them?
 I thought sftools did this but i cant seem to get the syntax right.
 Short of dumping the whole file, using an editor, then
 reconstructing it I am stuck..
 
 Eleanor

Re: [ccp4bb] No reflections in resolution bin???

[ccp4]

Oh dear - how did you lose it! It isworth hunting back through the data
files - all data processing software I know of outputs both F and SigF and
I and SigI ..
nt
Remember - it is good practice to ALWAYS use that first merged data set as
the input to refinement

Eleanor

On Thu, 4 Aug 2011 08:10:19 -0400, james09 pruza james09x...@gmail.com
wrote:
 Dear CCP4BBers,
 
 Refmac is giving the error  No reflections in resolution bin??? It
seems
 there is no SigFP column. I wonder how to fix the problem.
 
 Thanks in advance.
 James

Re: [ccp4bb] adding side-chains to a backbone model (without introducing clashes in the process)

[ccp4]

You are asking quite a lot! 

But one of the auto building tools should help. It depends on the
resolution - Arp-Warp)  2.2A or higher - Buccaneer below that. 

Get the best phases you can acquire - experimental I guess combined with
the PHIC/FOM, then see what Buccaneer / or Arp-Warp can do. 
It is always worth adding a csymmatch step at the end to move the model
back to the starting symmetry equivalent. (This is automatic in Buccaneer I
think now)

Eleanor

On Fri, 5 Aug 2011 13:11:27 +0900, Francois Berenger beren...@riken.jp
wrote:
 Hello,
 
 Recently I wa
s advised PULCHRA on phenixbb for such task
 (http://www.pirx.com/pulchra/index.shtml).
 
 I wonder if there is another tool in the CCP4 treasure chest
 for the same kind of task (preferably open source).
 
 Regards,
 F.

Re: [ccp4bb] cTruncate fails after Scala - clipper::Message_fatal

[ccp4]

This has been reported and I thought fixed??

Eleanor


On Mon, 8 Aug 2011 16:01:27 +0100, Charles Ballard
charles.ball...@stfc.ac.uk wrote:
 Hi Chris
 
 In Stephen's case the problem was that the column names were too long. 
 They are limited to 30 characters by the mtz standard
 
 running through it looks like the problem is name truncation on the
output
 columns.  Whether it is the 
 total name, /crystal/dataset/column , or just the column name I do not
 know.  If I reduce the length of 
 colout it runs through successfully
 Charles Ballard
 CCP4
 
 On 8 Aug 2011, at 15:27, Chris Richardson wrote:
 
 Dear all,
 
 We have a problem where cTruncate generates an error message when
trying
 to process data from Scala as part of a Scale and Merge Intensities
 task in the GUI.  Scala appears to run ok.  cTruncate produces
 

***
 * Information from CCP4Interface script

***
 The program run with command: /sw/share/xtal/ccp4-6.2.0/bin/ctruncate
 -hklin /tmp/foop/Scala_1_1_mtz.tmp -hklout
 /tmp/foop/Scala_1_3_mtz_MPS1-1624-118.tmp -colin
 /*/*/\[IMEAN,SIGIMEAN\] -colano /*/*/\[I(+),SIGI(+),I(-),SIGI(-)\]
 -colout MPS1-1624-118
 has failed with error message
 CCP4MTZfile - internal error
 terminate called after throwing an instance of
'clipper::Message_fatal'

***
 
 #CCP4I TERMINATION STATUS 0 CCP4MTZfile - internal error terminate
 called after throwing an instance of 'clipper::Message_fatal'
 
 We're running CCP4 6.2.0 compiled by 32-bit fink on OS X 10.6.8.  The
 GUI task runs to completion with other input MTZ files that I have
tried.
 If I run the ctruncate command in a shell, it generates the same error.
 
 Dr Stephen Carr posted a similar query back in March 2011, but didn't
 get any replies on the bb.  Does anyone know what's going on here?
 
 Regards,
 
 Chris
 --
 Dr Chris Richardson :: Sysadmin, structural biology, icr.ac.uk
 
 
 The Institute of Cancer Research: Royal Cancer Hospital, a charitable
 Company Limited by Guarantee, Registered in England under Company No.
 534147 with its Registered Office at 123 Old Brompton Road, London SW7
 3RP.
 
 This e-mail message is confidential and for use by the addressee only. 
 If the message is received by anyone other than the addressee, please
 return the message to the sender by replying to it and then delete the
 message from your computer and network.

Re: [ccp4bb] Sodium ion vs. Water

[ccp4]

Search for Metal Cordination sites in Proteins.

It takes you to a site set up by Marjorie Harding which gives a table of
known metal coordination extracted from the PDB including Na.
Of course there may be errors in the PDB entries but it is very helpful I
find


Eleanorr


On Tue, 2 Aug 2011 13:31:09 -0500, Jacob Keller
j-kell...@fsm.northwestern.edu wrote:
 I asked the CCP4 experts this question a while back--maybe try
 searching the archive? There were some very helpful comments.
 
 Jacob
 
 On Tue, Aug 2, 2011 at 1:24 PM, Young-Jin Cho yj...@brandeis.edu
wrote:
 Dear CCP4 experts,

 I would like to ask if there is a clear way to distinguish Na+ and HOH
 molecules in the electron density map.  The table I have suggests Na
 to O distance lies between 2.35 and 2.45 that is very hard to discern
 the difference of these two candidates.  Na+ may have several
 coordination though (5, 6).

 This started from if where the divalent metal locates can be
 substituted by water or sodium.  I would appreciate any valuable
 suggestions.

 Best,
 Young-Jin

Re: [ccp4bb] I/sigma for h+k=2n+1 and h+k=2n

[ccp4]

From memory - check the documentation

mtzutils hklin1 data.mtz
RZONE 1 1 0   2 0   lists reflections with h+k = 2n
END

mtzutils hklin1 data.mtz
RZONE 1 1 0   2 1   lists reflections with h+k = 2n+1
END

Eleanor


This will list all On Tue, 26 Jul 2011 09:51:47 -0400, zhang yu
ccp4f...@gmail.com wrote:
 Hi,
 
 I had a dataset which is P21 but with a pseudo-translational symmetry of
 (1/2, 1/2 ,0). Theoretically the dataset should show systematic weak
spots
 of h+K= 2n+1 compared to h+k= 2n. Is that correct?
 
 I would like to have a close look at the reflections. for example, the
 average I/sigma for reflections with h+k=2n+1 and reflections with
h+k=2n.
 Which software could do this job?  A brief tutorial is appreciated.
 
 Yu

Re: [ccp4bb] ARP/wARP install on 6.2.0 RHEL 6?

[ccp4]

A plea from West Australia too.
I was sitting with someone yesterday who was trying to install it on a Mac
, and finding it a nightmare. 
He finally got it set up as a local installation, whereupon it promtly
failed. 
The message said See refmac-last.log but that told us nothing, and indeed
refmac seemed to have worked..

Can someone - either CCP4 or  arp-warp people check this out?

Eleanor

On Wed, 27 Jul 2011 20:35:50 -0700, Ethan Merritt
merr...@u.washington.edu wrote:
 On Wednesday, 27 July 2011, you wrote:
 Hi Jonathan,
 seems to be a UW centered day today on the BB (Eric, Jan, you, me).
 Have the permissions changed ? I assume you are installing as root ?
 Wouldn't be surprised if Ethan replies soon :-)
 
 Sure.
 
 I hit the same problem trying to install Arp/wARP on Mandriva.
 The specific error message you quote comes because the install
 script fails to create the temp directory before trying to unpack
 into it.  You can fix that by creating the directory by hand first.
 Unfortunately, that doesn't help very much.  The next thing that
 happens is that the ccp4i installer complains that the tarball
 is not recognized as a ccp4i install tarball.  I gave up at that
 point.
   
   Ethan
 
 
 
 
 Jürgen 
 
 ..
 Jürgen Bosch
 Johns Hopkins Bloomberg School of Public Health
 Department of Biochemistry  Molecular Biology
 Johns Hopkins Malaria Research Institute
 615 North Wolfe Street, W8708
 Baltimore, MD 21205
 Phone: +1-410-614-4742
 Lab:  +1-410-614-4894
 Fax:  +1-410-955-3655
 http://web.mac.com/bosch_lab/
 
 On Jul 27, 2011, at 20:31, Jonathan Kay jp...@u.washington.edu wrote:
 
  Hi all,
  
  I have a RHEL 6 x86_64 machine I recently installed CCP4-6.2.0 onto;
  the install went through fine, but when I went to install the
ARP/wARP
  GUI (via System Administration - Install/uninstall task), I received
  the following error in the shell window I started ccp4i from:
  
  UnpackTaskArchive: uncompress failed to create
  /tmp/user/install_ARP_wARP_CCP4I6/ARP_wARP_CCP4I6.tar
  ExamineTaskArchive: failed to unpack temporary copy of
  /usr/local/arp_warp_7.1/ARP_wARP_CCP4I6.tar.gz
  
  /tmp is not at all full and has plenty of inodes left.
  (running the install.sh from the arp_warp_7.1 directory doesn't
  install it either)
  
  I have searched around for some solutions, but haven't found anything
  really relevant.
  The odd thing I have another x86_64 machine running RHEL 5 that I can
  do the exact same install method and it works (and using the
install.sh
  from arp_warp_7.1/ works too), so I wonder if something changed with
  RHEL6 that might be causing problems?
  
  Anyone have any suggestions?
  
  Thanks!
  Jonathan

Re: [ccp4bb] Non-crystallography symmetry operator

[ccp4]

superpose has two options undfer CCP4i.
Choose match numbered residues ( not SSM) 

Then you must give two files to match - in your case that means naming
your model twice.
Then say you want to match residues 1 to 20 Chain A to residues 1 to 20
CHAIN B tever span you want to fit.

It will give you the rotation matrix and translatioor n vector
Eleanor


or wha

On Mon, 25 Jul 2011 14:31:33 -0400, zhang yu ccp4f...@gmail.com wrote:
 Thanks for all the reply. I am able to use phenix.find_ncs find NCS
 operators for all the chains. But it doesn't give a NCS matrix between
 whole
 molecules in ASU. Is that right or I missed something?
 
 In SUPERPOSE of CCP4, How to ask the program to calculate the NCS
 operator? It asks me to input two structures and will output another pdb
 file.
 
 Yu
 
 2011/7/25 Frederic VELLIEUX frederic.velli...@orange.fr
 
 You need to specify which format (i.e. for which program) since the
 conventions used are different. I personally use suppos
 which provides a rotation matrix plus translation vector. Row by row
for
 the matrix. Some programs use a column by column approach, the
transpose
 of
 the rotation
 matrix, or a set of 3 angles (with several conventions possible)

  Message du 25/07/11 19:24
  De : zhang yu
  A : CCP4BB@JISCMAIL.AC.UK
  Copie à :
  Objet : [ccp4bb] Non-crystallography symmetry operator
 
  Hi,
 
  Does any one know which software could calculate and output
  non-crystallography symmetry operator?
 
  I am working on a structure with two molecules in one ASU, and two
 identical
  subunits in each of one molecule. I would like to know the
  non-crystallography operator of the two molecules, and also NCS
  operator
 of
  two subunits in the same molecules.
 
  Thanks
 
  Yu
 
  --
  Yu Zhang
  HHMI associate
  Waksman Institute, Rutgers University
  190 Frelinghuysen Rd.
  Piscataway, NJ, 08904
 

Re: [ccp4bb] Phaser and Molrep gave different solutions

[ccp4]

That looks very hopeful. Do you know how the two molecules are related to
each other? Do they form a dimer or have some interesting relationship?
I would keep on improving the model - possibly use COOT to average the
density and build into that for a start. I presume there are still side
chains to mutate and fit, and other corrections to make? You can then save
molecule 1, and fit the molecule 1 model back over molecule 2 and save that
again.
You need to edit the two chains into one PDB in some way and refine again.
You might begin to see DNA at a low sigma level in those maps..

(By the way - I dont understand 2) mutate the protein sequence back to my
 protein and refine the solution in Coot. Chainsaw should output the
model with the correct sequence but with atoms missing where there are
changes. You would need to rebuild that but not change the sequence? ) 

And you would hope then that PHASER might find your 2 protein chains with
a very high LLG then perhaps place the DNA. but in my limited experience
DNA is usually more mobile and therefore harder to pick up than protein.

good luck Eleanor
 

On Mon, 25 Jul 2011 15:38:02 +0800, Hubing Lou louhub...@gmail.com
wrote:
 Hi,
 
 Thanks for those who replied to this thread.
 I have been trying all the means that people suggested: search protein
 alone, DNA alone. However, both not working out.
 One thing Ray Brown suggested MR works if the molecules have identical
 sequences. So I just played around with the following way: 1) use the
 chainsaw editted model in MR; 2) mutate the protein sequence back to my
 protein and refine the solution in Coot; 3) use the partially refined
 protein-DNA as the new search model and run Phaser again then I get the
 following results for the two copies in ASU:
 RFZ=6.5 TFZ=10.7 LLG=903 RFZ=6.2 TFZ=17.4 LLG=1130.
 After one round of rigid body and restraint refinement in Refmac5, the
Rfac
 and Rfree drops from 0.50/0.51 to 0.46/0.51. I did not refine the DNA
 sequences yet.
 
 I am not sure if this way I can further refine the structure, or do I
bring
 two much bias into the structure. Please correct me if any. Thanks.
 
 Best,
 Hubing
 
 
 
 
 On Thu, Jul 21, 2011 at 11:31 PM, ray-br...@att.net wrote:
 
 I have also tried for years to solve a protein-DNA complex without
 sucess.
 If you have a lot more DNA than protein in the AU then MR will not
work.
 You
 always get a good RFZ score but you cannot solve the translation if the
 DNA molecules are forming long stacks. With a plausible packing you
will
 of
 course get model phases and a nice map but refinement will not work and
 you
 will get stuck at 40-50% Rf.

 You may have a chancewith MR if you only have a small DNA and a much
 bigger
 protein molecule or if the search models and the molecules have
identical
 sequences.

 To solve this structure you probably have to do Se-labeled protein and
 SAD
 etc. or collect anomalous from metal ions if present.

 Cheers.

 Ray Brown

  --
 *From:* Hubing Lou louhub...@gmail.com

 *To:* CCP4BB@JISCMAIL.AC.UK
 *Sent:* Thu, July 21, 2011 6:39:49 AM
 *Subject:* Re: [ccp4bb] Phaser and Molrep gave different solutions

 I was worried as well with the low TFZ score. Usually successful cases
 with
 score 8. I am still puzzled why Phaser and Molrep gave different
 solutions.
 Does this mean molecular replacement do not work out in this case so
more
 crystals have to be prepared?

 A little more information might be helpful to dissolve the problem
here.
 The model I used is a protein-DNA complex. The protein was Chainsaw
 editted
 but the DNA sequence was directly borrowed from the original model.

 Best,
 Hubing

 On Thu, Jul 21, 2011 at 3:30 PM, Vellieux Frederic 
 frederic.velli...@ibs.fr wrote:

 Hi,

 It's not a bad idea to read the Phaser manual for molecular
replacement;
 see http://www.phaser.cimr.cam.ac.uk/index.php/Molecular_Replacement

 Soon after the start, in a table on the right hand side, there is: TFZ
 score  5, have I solved it ? No.

 Hence with a TFZ score of 3.8 you do not have a solution using Phaser.

 Fred.

 Hubing Lou wrote:

  Dear all,

 I am stuck in a molecular replacement case and looking for advices.
 I have been working on a protein-DNA complex structure.
 Data was processed by HKL2000 to 2.6Ang and some of the data
statistics
 are shown below:

 Space group: P21,
 Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90
 Redundancy: 2.8 (2.7)
 Completeness: 94.8 (93.1)
 Linear R-fac: 0.051 (0.442)

 Data quality was checked by Phenix.xtriage and there's no problem. I
 then
 prepared a model by Chainsaw. Our protein shares only 30% of sequence
 similarity with the model, but structurally they are in the same
group
 and
 almost identical in apo form. Matthrews Coeff indaced two monomers in
 AU. I
 then ran Phaser in automated search mode and there's a solution
with
 RFZ
 score 4.8, TFZ score 3.8. The electron density map was not bad with
DNA
 double helix clearly seen. However Refmac5 couldn't 

Re: [ccp4bb] Phaser and Molrep gave different solutions

[ccp4]

The self rotation isnt the correct input to the translation function -
just run the Auto-Molrep option..
Eleanor

On Thu, 21 Jul 2011 20:57:02 +0800, Hubing Lou louhub...@gmail.com
wrote:
 I also processed with Imosflm and ran Pointless, it was P21. Also it was
 indicated by the Intensity systematic absences in HKL2000 scale.log
file.
 
 Best,
 Hubing
 
 On Thu, Jul 21, 2011 at 8:44 PM,
 herman.schreu...@sanofi-aventis.comwrote:
 
 **
  Dear Hubing,

 One maybe stupid question: Your are sure the space group is P21 and not
 P2
 or even something else? Did you test other possible space groups?
 Choosing
 the wrong space group could exactly lead to the results you observe.

 Best,
 Herman


  --
 *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf
Of
 *Hubing
 Lou
 *Sent:* Thursday, July 21, 2011 6:46 AM
 *To:* CCP4BB@JISCMAIL.AC.UK
 *Subject:* [ccp4bb] Phaser and Molrep gave different solutions

 Dear all,

 I am stuck in a molecular replacement case and looking for advices.
 I have been working on a protein-DNA complex structure.
 Data was processed by HKL2000 to 2.6Ang and some of the data statistics
 are
 shown below:

 Space group: P21,
 Unit Cell: 54.73, 104.91, 78.40, 90, 100.2, 90
 Redundancy: 2.8 (2.7)
 Completeness: 94.8 (93.1)
 Linear R-fac: 0.051 (0.442)

 Data quality was checked by Phenix.xtriage and there's no problem. I
then
 prepared a model by Chainsaw. Our protein shares only 30% of sequence
 similarity with the model, but structurally they are in the same group
 and
 almost identical in apo form. Matthrews Coeff indaced two monomers in
 AU. I
 then ran Phaser in automated search mode and there's a solution with
 RFZ
 score 4.8, TFZ score 3.8. The electron density map was not bad with DNA
 double helix clearly seen. However Refmac5 couldn't get Rfree lower
than
 50%.

 I then changed to MolRep, ran self rotation function first then used
 the
 first 10 peaks for translation search. Again there's a solution but it
is
 different from that from Phaser. I attached a picture here. Checking in
 coot, the packing is the same. But, the refinement couldn't get Rfree
 lower
 than 50%.

 I have tried to include NCS, TLS refinement in Refmac, both not
working.
 Hope someone out there can help.
 Thanks very much for your time.

 Hubing

Re: [ccp4bb] large R-Rfree difference in final structure

[ccp4]

 Hi, Ian Tickle has asked the most pertinent question - there are many
reasons for highish FreeR factors. Only some are due to errors or missing
molecules or other problems. How many Free reflections are there? When
the sample is very small ( 500 say) you can get different results from a
different Free R set.

And remember that the numbers you quote are overall values, 
It is worth looking at the plot of R and Free R against resolution. They
should stay reasonably parallel, but if the data becomes very weak at the
resolution limit the the FreeR can shoot up relative to the overall Rvalue.

 
I use COOT validation to search the difference maps for peaks and holes. 
If there is something missing from your model you should see it in that
list..
Eleanor
 
On Wed, 13 Jul 2011 20:37:51 +0100, Matthias Zebisch
matthias.zebi...@bbz.uni-leipzig.de wrote:
 Hi Careina,
 
  our lab we once had the problem, that the asymmetric unit contained 8 
 molecules,
 whereas 7 had only been modeled. Somehow the 8th monomer had evaded 
 detection.
 So be careful not to miss density.
 
 Matthias
 
 
 On 7/13/2011 7:54 PM, Robbie Joosten wrote:
 Hi Careina,



 Assuming you don't suffer from a very poor data parameter ratio that
 would lead to such a large R-free/R, you need to improve your
refinement.
 If you have NCS you should use local NCS restraints. You could also try
 jelly-body restraints, although they may not work at your resolution.



 Cheers,

 Robbie

 
 Date: Wed, 13 Jul 2011 08:38:38 -0700
 From: careinaedgo...@yahoo.com
 Subject: [ccp4bb] large R-Rfree difference in final structure
 To: CCP4BB@JISCMAIL.AC.UK

 Dear ccp4 bulletin board

 I just have a slight concern regarding my Rwork Rfree difference. I
 have a structure that I have solved. I am reasonably content that it
is
 complete because it has refined well, it no longer has bad geometries
 and contacts and all the rotamers, ramachandra, bond lengths etc are
 good. It gives favourable scores on molprobity and procheck. My only
 concern is the R factor difference. The resolution of the structure is
 2.3A. The R factor is 0.24 after refinement but the Rfree is 0.33
which
 seems to me to be rather high. Should I be concerned?

 During refinement Rfree only drops from about 0.36 to 0.33 while the R
 factor drops from 0.31 to 0.24.. I have removed automatic weighting in
 refmac in order to constrain my bond lengths and angles during a
couple
 of rounds of refinement. This did not have any effect on the R
factors,
 however. I am fairly content that the space group I have chosen is
 correct so I am not sure what else could cause the big difference in R
 factors? There is no twinning.

 Can I be satisfied that my structure is correct despite the high R
free
 or should I be doing other checks/ trying other things before I can
 submit this structure?

 Thank you for any help
 Careina

Re: [ccp4bb] occupancy-refinement

[ccp4]

You are right that refining occupancy and B  value at the same time cant
be done with REFMAC, and probably wouldnt work anyway at 2.9A 

However, if you set the ligand occupancy to 0.7 say, and refine the
residue B factors, the occupancy should not be set back to 1.0. It
certainly isnt in the local version of REFMAC. 
You can judge to some extent what occupancy gives the cleanest map by
inspection. 
But at 2.9A the correlation between B and occupancy is very high and it
wilnswer. l be hard to get a definitive answer.
Eleanor
On Wed, 13 Jul 2011 10:31:59 -0500, dhurjati putcha
antharvaah...@gmail.com wrote:
 Dear CCP4ers,ncy and B value 
 
 While trying to refine a protein-ligand structure (reso=2.9A) I notice
that
 the density (2Fo-Fc)for the ligand is discontinuous. I also notice that
the
 density for the residues in the ligand binding pocket (LBP) is also very
 feeble. And when I refine the ligand occupancy the density for the
ligand
 appears and covers almost all the molecule, and the occupancy refines to
 1.0.
 
 However, I cannot refine the occupancy/B factors for the LBP residue
side
 chains because the occupancy stays at 1.0 (and if I change it manually,
it
 returns to 1) and the B factors (group  individual) remain close to the
 average for the remainder of the molecule. If I omit the LBP residue
side
 chains, I get some Fo-Fc density suggesting that these side chains are
 real.
 
 
 Is there previous evidence for such phenomena suggesting multiple
 conformations/dynamics/loose binding? Is there a way to quantify the
 dynamics associated with the side chains (as with the ligand where the
 occupancy is 1.0) so I can argue it out with potential manuscript
 reviewers? The R-factor/R free #s (22%/19%) suggest that the refinement
is
 nearing convergence.
 
 Best regards,
 
 Kumar.

Re: [ccp4bb] Same protein, different molecule numbers per ASU

[ccp4]

They must represent a different packing - the volume of Shape 1 is 10% 
than 2*Vol-Shape2 

But it isnt uncommon to get different forms - check your symmetry contacts
(PISA will do that) and see how the two forms pack.
eleanor

On Mon, 11 Jul 2011 18:09:52 +0800, ferrol shariff ferrol2...@gmail.com
wrote:
 Hi Alenxander,
 Thanks for the reply. Here's the details on the unit cell parameters
 
 [image: image.png]
 
 [image: image.png]
 
 [image: image.png]
 
 Thank you. :)
 
 Regards,
 
 Fairolniza
 On Mon, Jul 11, 2011 at 2:47 PM, Alexandre OURJOUMTSEV
 sa...@igbmc.frwrote:
 
  Dear Ferrol,

 ** **

 Could you let us know the unit cell parameters for both these crystals
?
 (did I miss them somewhere in your previous mails?).  I wonder if in
fact
 this is not the same packing with minor variation.

 Sacha

 ** **

 *De :* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *De la part
 de*ferrol shariff
 *Envoyé :* dimanche 10 juillet 2011 10:10
 *À :* CCP4BB@JISCMAIL.AC.UK
 *Objet :* [ccp4bb] Same protein, different molecule numbers per ASU

 ** **

 Hello and good day to everyone! :)

 ** **

 I have some general questions on crystallography work. I hope you don't
 mind giving me some ideas.

 I have solved my lipase protein both ground-grown crystals and
 space-grown
 crystals with good resolutions (1.4A and 2.2A). They are the same
protein
 from the same source, same purification methods, and produced crystals
 from
 the same crystallization conditions (except the gravity part).

 From the data, it shows that both of them belong to the same space
group
 P212121. But they have different number of molecule per asymmetric
unit.
 Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the
 moment i have problem explaining this issue. Is it normal to have such
 results? Same protein with different number of molecule/ASU?

 I've been trying to get some references on this matter but so far i
don't
 really get anything that can directly explain it. Furthermore, do i
need
 to
 relate this with the gravity effect?

 I hope you don't mind sharing some experiences on crystallography
 especially regarding this matter.

 Thank you very much

 --
 FAIROLNIZA

 

Re: [ccp4bb] abnormal I/Sigma over phi rotation range

[ccp4]

This seems somewhat weird - your Rmerge values increase with more frames
as explained by Tim, but I cant see why the I/SigI should increase sharply
for the 180 degree set then fall off again.
if you have run Scala look at the scaling plots v batch, and resolution to
see if there are any weird outliers.
Eleanor

On Mon, 11 Jul 2011 10:23:00 +0200, Tim Gruene t...@shelx.uni-ac.gwdg.de
wrote:
 Dear Vennila,
 
 my guess is that you had a larger number of images in the batch/set
 rge  starting at
 180 degrees (hence the slightly incereased Rmerge for that batch) and
that
 by
 the end of that run your crystal had suffered from radiation damage
(hence
 the
 large Rmerge for the set starting at 360degrees).
 
 You should report Rmeas instead of Rmerge and let us know about the
number
 of
 frames and the frame width in each set.
 
 Cheers, Tim
 
 
 On Sun, Jul 10, 2011 at 01:54:07PM +0100, Vennila Natesan wrote:
 Dear CCP4BB users,
 
 In order to determine the redundancy at which the structure can be
 solved, I divided the master
 data set of my protein into five sets with phi rotations of  45,65,90,
 180 and 360 degrees. I got
 the mean I/Sigma value as 22.4(11.4), 21.7(11.0), 22.7(11.2),
60.9(45.2)
 and 15.6(8.1) respectively.
 I will be very helpful if i get any idea/possible reasons for the
 abnormal value for 180 degree dataset.
 
 For the information, the completeness values are
 75.7(78.5),92.6(92.7),99.5(97.7),99.6(97.7)and 99.5(96.4)
 respectively and values inside brackets are for highest resolution
 shell. There was no noticable change in mosaicity
 value. The Rmerge values are 2.2,2.4,2.7,3.2, and 5.7 (8.7)
 respectively.
 
 Thanks in advance

Re: [ccp4bb] low resolution refinement

[ccp4]

  Roberto steiner has told you how to use these new REFMAC5.6 features,
Rob  Nicholls has suggested how to generate secondary structure restraints,
and Martyn Winn given a page to install a new interface to make it easier
to use them..

But with such limited data it isnt surprising that the FreeR climbs
steadily. Scaling Fcalcs and Fobs together at this resolution is tricky,
and you should look at your plots of Rfactor and Fo/Fc at the end of
refinement. 
There may be anomalies at the top or bottom resolution.. 
There really isnt a general rule for a fix. You need to know your data.
You can still get useful information from the maps.
Eleanor

 

On Sat, 9 Jul 2011 16:59:29 +0800, Qixu Cai caiq...@gmail.com wrote:
 Dear all,
 
 Recently, I refine two low resolution structures in refmac 5.5. Their 
 resolutions are 3A and 3.5A respectively.
 For 3A structure, after MR by phaser and rigidbody refinementrestraint 
 refinement by refmac5.5, I got R factor 25% and R free 35%. And then 
 each time, after my model building in coot and restraint refinement by 
 refmac 5.5, the R factor stays 25%, but R free increases to 38%, even
39%.
 For 3.5A structure, the R factor stays 27%, but R free increases from 
 37% to 42% after my slightly model building in coot.
 Could you help me to find the reason?
 
 Maybe the reason is the overfit of the structure? I found that new 
 version of refmac 5.6 has many new features for low resolution 
 refinement, such as jelly boy, secondary structure restraints. But I 
 don't know how to use these new features in old version ccp4i (6.1.13)?
 
 I also used phenix.refine with the reference model ( I have high 
 resolution model for one domain of the low resolution protein) and 
 secondary structure restraints, but it seams the same. Any suggestion?
 
 BTW, is that simulator annealing not suitable for low resolution 
 structure? I used the simulator annealing method of CNS and 
 phenix.refine, but the geometry of the structure is always destroyed 
 seriously.
 
 Could you help me?
 
 Thank you very much!

[ccp4bb] Stereo 3D LCD is coming for gamers

[CCP4 Sheffield University]

NVIDIA is jumping into stereo games now with its own stereo glasses 
coming out soon.  See:

http://www.maximumpc.com/article/features/everything_you_need_know_about_nvidia%E2%80%99s_3d_goggle_gamble

There's no price available yet for the glasses, nor for the new 
ViewSonic 120 Hz LCD monitor:

http://www.engadget.com/2008/08/26/viewsonic-shows-off-a-120hz-lcd-display-for-computers/

that's supposed to deal with it, so the cost will no doubt be silly, and 
they're only mentioning
Windows Vista (ugh!) drivers so far.  But if the gaming market takes it 
up in a big way
the prices will tumble.  Then maybe us oldies will be able to have 
lovely stereo again, and

even the kiddies may see the light.

Cheers,  Jan

--
*   CCP4 Account (Jan White)  
*   Molecular Biology, Sheffield University  *
*   Phone: +44 114 222 2741  FAX: 222 2800   *
*   E-Mail: [EMAIL PROTECTED]  *

Re: [ccp4bb] Characterization of common salt crystal forms?

[CCP4]

Dear Joe,

working with divalent ions, I often get salt crystals (most surely  
phosphate). I do not have any table available, but can give you the  
commercial kits  numbers going with it. Just let me know.


On Jan 22, 2008, at 6:12 PM, Joe Krahn wrote:


Salt crystals are common in macromolecular crystallography. Has anyone
tried to tabulate salt crystal forms that commonly occur?


HTH

Kind regards.

Leo

Chavas Leonard, Ph.D.
Research Associate

Faculty of Life Sciences
The University of Manchester
The Michael Smith Building
Oxford Road
Manchester Lancashire
M13 9PT

Tel: +44(0)161-275-1586
e-mail: [EMAIL PROTECTED]

Re: [ccp4bb] Cocrystals - should they pop up under native conditions?

[CCP4]

Dear Brenda,

I am running co crystallisation experiments and have thus far been  
trying under

oil screens with some success (i.e. various hits in various conditions
resulting in crystal growth).  These conditions have varied from  
the native

condition up until now.


Do you already have the structure of the native form? If so, you can  
look at the putative binding region of your ligand, and check whereas  
it is involved in crystal packing or not. In any case, you'll have to  
shoot them and look at the density for confirmation; but just to have  
an idea of what worth can be expected.


HTH

Kind regards.

Leo

Chavas Leonard, Ph.D.
Research Associate

Faculty of Life Sciences
The University of Manchester
The Michael Smith Building
Oxford Road
Manchester Lancashire
M13 9PT

Tel: +44(0)161-275-1586
e-mail: [EMAIL PROTECTED]