Re: [ccp4bb] P21, twinned date & NCS refinement

[Pavel Afonine]

Hi Jürgen,

in phenix.refine I would do:

phenix.refine [PDB] [mtz] twin_law="h,-k,-l" main.ncs=true ncs.type=torsion

(or equivalent using the GUI).

Note, "ncs.type=torsion" will use the new NCS handling machinery that takes
care of NCS automatically with no need the user to provide definitions for
NCS groups. For this it is advisable to use a recent Phenix version from
the nightly builds.

If resolution is ~3-2.8A and higher, then automated water update using
ordered_solvent=true is good to use, so no need to waste time doing it
manually.

If it is a final refinement run before PDB deposition then weights
optimization using optimize_xyz_weight=true and optimize_adp_weight=true
should be tried.

That's all I can tell given the information you provided.

All the best,
Pavel

On Tue, Mar 27, 2012 at 1:37 PM, Bosch, Juergen  wrote:

> Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both
> anyhow)
>
> to the experts out there here's my question:
>
> We have a P21 dataset with 2 molecules in the asu and a refined twin
> fraction of 38% according to phenix.refine using a twin law operator.
>
> My gut feeling tells me that I can't use NCS unless I detwin the data, is
> that a correct assumption ?
>
> How does phenix.refine [PDB] [mtz] twin_law="h,-k,-l" main_ncs=true would
> deal with this problem ?
>
> Same question goes to Garib, it's very convenient to just specify twin in
> your Refmac script without further values and magic happens but what if I
> add NCS restrains, will Refmac treat them correctly according to the twin
> operator ?
>
> Twins are confusing in real life and even more in crystals I think.
>
> And no the data can not be processed in a higher space group, P222 results
> in Rmerges of >40% in the lowest resolution shell, the beta angle is 92.4
> degrees in P21. And if processed in P1 we get 4 molecules per asu and a
> refined twin fraction of 50%, which in my eyes clearly indicates it's not
> P1 but really P21.
>
> Hope to get some interesting feedback on this issue.
>
> Thanks,
>
> Jürgen
>
> ..
> Jürgen Bosch
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
> Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
> http://web.mac.com/bosch_lab/
>
>
>
>
>

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Bernhard Rupp (Hofkristallrat a.D.)]

> the liberty being offered is not so dangerous

Parameter-wise, true, - if it is understood that you are now *modeling with
little evidence*.
How much electron density to affirm your model choices would one expect at 
say 7 ideal rotamers - which on top of static split will likely display
dynamic motion?

On the other hand, the side chain HAS to be somewhere, and a distribution of
the most probable rotamers with their respective frequencies may be more
realistic
than all other options (as long as the lack of specific experimental
evidence is acknowledged).

But overall, that model might be better due to the more reasonable prior
expectation term 
although the R-value will be practically unaffected due to the lack of
localized scattering contributions.

Anyhow, if it can be abused, it will be ;-)

Best BR

-Original Message-
From: Ethan Merritt [mailto:merr...@u.washington.edu] 
Sent: Tuesday, March 27, 2012 5:14 PM
To: b...@hofkristallamt.org
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] REFMAC5 residues with bad geometry

On Tuesday, March 27, 2012 04:35:40 pm Bernhard Rupp (Hofkristallrat a.D.)
wrote:
> >phenix.refine allows any number of alternate conformers. 
> 
> Hmm. quoting our old friends from the validation circuit: Where 
> freedom is given, liberties will be taken

True, but...

[warning: back of the envelope calculation]

Consider, for example, an isoleucine sidechain.

It would require 12 positional parameters to refine the position of each
sidechain atom (XYZ * {CB CG1 CG2 CD1}) for a single conformation.

One the other hand there are only 7 rotamers in the library.
So if you limited the refinement to the relative occupancy of the
7 ideal rotamers, that is a more parsimonious model than refining the
individual atoms for one conformation.

Season to taste with arguments about geometry restraints, but still I think
the liberty being offered is not so dangerous.

Ethan

--
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg University of
Washington, Seattle 98195-7742

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Ethan Merritt]

On Tuesday, March 27, 2012 04:35:40 pm Bernhard Rupp (Hofkristallrat a.D.) 
wrote:
> >phenix.refine allows any number of alternate conformers. 
> 
> Hmm. quoting our old friends from the validation circuit: Where freedom
> is given, liberties will be taken

True, but...

[warning: back of the envelope calculation]

Consider, for example, an isoleucine sidechain.

It would require 12 positional parameters to refine the position of
each sidechain atom (XYZ * {CB CG1 CG2 CD1}) for a single conformation.

One the other hand there are only 7 rotamers in the library.
So if you limited the refinement to the relative occupancy of the
7 ideal rotamers, that is a more parsimonious model than refining
the individual atoms for one conformation.

Season to taste with arguments about geometry restraints, but still
I think the liberty being offered is not so dangerous.

Ethan

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Bernhard Rupp (Hofkristallrat a.D.)]

>phenix.refine allows any number of alternate conformers. 

Hmm. quoting our old friends from the validation circuit: Where freedom
is given, liberties will be taken

BR

Re: [ccp4bb] REFMAC5 residues with bad geometry

[Paul Adams]

Hi James,
  
  my understanding is that phenix.refine allows any number of alternate 
conformers. There may have been a limit of 4 some time in the past, but no 
longer. So your idea could be tested.

  Cheers,
Paul

On Mar 27, 2012, at 12:33 PM, James Holton wrote:

> Try this:
> 
> 1) take your favorite PDB file and set all the B factors to ~80 (reduces 
> series-termination errors)
> 2) use sfall/fft in CCP4 to calculate structure factors to 4A resolution
> 3) use sftools to add a "SIGF" column (0.1 will do) to make refmac5 happy
> 4) refine the "perfect" model against these fake data for ~5 cycles (with 
> "solvent no")
> 5) load this up in coot and contour at 1 sigma
> 6) repeat the refinement with a PDB file containing only main chain.
> 7) repeat the refinement after putting all the side chains in their most 
> likely (Ponder-Richards) rotamers.
> 
> Ask yourself these questions:
> 1) can you "see" the side chains?
> 2) can you "see" the waters?
> 3) what are the R factors from these refinements?
> 
> Answers: 1) no, 2) no, 3) ~3% for "perfect", ~50% for "main chain", and ~36% 
> for "likely rotamer"
> 
> Now ask yourself: even though there is "no density" for side chains and 
> waters, is there really "no evidence" that they exist?
> 
> The point I am trying to make here is that you EXPECT side chains to poke out 
> of density at low resolution, even under ideal conditions (perfect phases).  
> For example, the C-deltas of Leu will "breach" the 1-sigma contour at around 
> 2.8A resolution and worse.  You can see this in my old movie:
> http://bl831.als.lbl.gov/~jamesh/movies/index.html#reso
> 
> When it comes to building, yes, once an atom dips below the 1-sigma contour 
> it gets harder and harder to know exactly where it is, but it does have to be 
> somewhere.  Somewhere nearby.  Formally, there is "prior knowledge" of bond 
> lengths, etc. at play.  And if you know that there is one copy of a given 
> atom in every unit cell of the crystal, then occupancy < 1 is inappropriate.  
> Much better to use B = 999, which models the atom as a Gaussian with the 
> electrons spread over an area about 3.5 A wide.  This is roughly the range 
> your average side chain atom has available to it, given that it is attached 
> to the main chain by covalent bonds.
> 
> Of course, a more "Bayesian" model for the "I don't know what the rotamer is" 
> situation would be to build in ALL possible rotamers, with occupancies equal 
> to their Ponder-Richards probabilities.  Some improvement to this initial 
> "guess" would no doubt be made by using constrained occupancy refinement of 
> rigid-body side chains.  Unfortunately, this is impossible with any 
> refinement program I know about, since refmac, phenix.refine, etc. don't 
> support more than 3 or 4 alternate conformers.
> 
> Building in all possible conformers and using the occupancy as a "p-value" 
> would also help solve the problem of the careless and/or uneducated 
> over-interpreting PDB files.  Which is the "right one"?  Good question!  I 
> think its time we started dispelling the myth of the single-conformer protein 
> anyway.
> 
> -James Holton
> MAD Scientist
> 
> On 3/26/2012 7:40 AM, Ed Pozharski wrote:
>> On Mon, 2012-03-26 at 10:17 -0400, Gregory Bowman wrote:
>>> But what about the issue of resolution? As was previously pointed out,
>>> at say 3.2 Å resolution, many side chains will fail to fit, but it
>>> doesn't seem appropriate to trim them all down.
>> Why is it inappropriate to trim them down?  Sometimes at low resolution
>> all one can be confident about is the backbone trace.
>> 
>> Just to be clear, I am talking about atoms whose positions are not
>> supported by electron density, i.e. where difference map in the absence
>> of the side chain is featureless.  I assume that is the likely situation
>> when one would set occupancy to zero.
>> 
>> Cheers,
>> 
>> Ed.
>> 

-- 
Paul Adams
Deputy Division Director, Physical Biosciences Division, Lawrence Berkeley Lab
Division Deputy for Biosciences, Advanced Light Source, Lawrence Berkeley Lab
Adjunct Professor, Department of Bioengineering, U.C. Berkeley
Vice President for Technology, the Joint BioEnergy Institute
Laboratory Research Manager, ENIGMA Science Focus Area

Building 64, Room 248
Tel: 1-510-486-4225, Fax: 1-510-486-5909
http://cci.lbl.gov/paul

Lawrence Berkeley Laboratory
1 Cyclotron Road
BLDG 64R0121
Berkeley, CA 94720, USA.

Executive Assistant: Louise Benvenue [ lbenve...@lbl.gov ][ 1-510-495-2506 ]
--

Re: [ccp4bb] Substituting zero vs. Fc for unobserved reflections

[Craig Bingman]

I would be concerned about the completeness of the data if adding Fcalc values 
has such a large effect on the appearance of this electron density.

Re: [ccp4bb] P21, twinned date & NCS refinement

[Garib N Murshudov]

Unless you have very good reason it is better to use highest possible space 
group (without going over). Then you do not have problem of related reflections 
and covariances between them, If you  go to P1 then reflection will be related 
with crystallographic as well as NCS as well as twin "symmetries". When you go 
to p21 then you at least do not have to deal with reflections related with 
crystallographic symmetry.  I am not sure going to P1 would solve problem of 
split reflections. It is a fundamental problem in data integration programs 
that assume there is only one lattice with one orientations. Your example shows 
that reality is a little bit more complicated. Split spots will cause problems 
in profile generations and in theory will affect quality of the data also.

If you could you should try to inegrate split spots as one (merge them) then 
the problem becomes similar to merohedral twinning (when spots overlap 
exactly). 

Regards
Garib


On 27 Mar 2012, at 22:00, Bosch, Juergen wrote:

> Thanks Garib for your input. And yes we do see some split spots. We used XDS 
> to overcome (I hope) most problems but still intensities of perfectly 
> overlapping reflections will be too large. Would you think it's safer to 
> integrate the data in P1 as symmetry mates will not be merged and then solve 
> in P1 and convert into P21 cell for further refinement afterwards ?
> 
> Jürgen
> 
> 
> On Mar 27, 2012, at 4:53 PM, Garib N Murshudov wrote:
> 
>> I would say that you should use ncs restraints in any case. NCS relates 
>> atoms and twin relates intensities. In some sense presence of twinning 
>> reduces information contents (in the limiting case the number of (effective) 
>> observervations becomes twice less) of the data and using NCS decreases the 
>> effective number of adjustable parameters. 
>> Without NCS some of the stats (like RvR) cannot be trusted. However you 
>> should remember that if ncs is very close to twin operators then in 
>> reciprocal space they relate two reflections exactly and in most of the 
>> cases you do not gain much and differences between ncs related molecules 
>> might be more important than their similarity. I would suggest using local 
>> ncs then global differences are retained and molecules are made locally 
>> similar.
>> 
>> If bet angle is 92.4 degrees (I would also check obliquity in refmac output 
>> if you are using that. In your case this number should be non-zero, meaning 
>> that twin related reflections will not coincide exactly) then I would expect 
>> splitting of spots in some directions at high resolutions. One should be 
>> careful when integrating, if only one of these spots are integrated then you 
>> may end up have several classes of reflections: low resolution and in some 
>> directions when spots overlap perfectly you have contributions from two 
>> crystals, in split spot cases each spot has contribution from single crystal.
>> 
>> 
>> 
>> regards
>> Garib
>> 
>> 
>> On 27 Mar 2012, at 21:37, Bosch, Juergen wrote:
>> 
>>> Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both 
>>> anyhow)
>>> 
>>> to the experts out there here's my question:
>>> 
>>> We have a P21 dataset with 2 molecules in the asu and a refined twin 
>>> fraction of 38% according to phenix.refine using a twin law operator.
>>> 
>>> My gut feeling tells me that I can't use NCS unless I detwin the data, is 
>>> that a correct assumption ?
>>> 
>>> How does phenix.refine [PDB] [mtz] twin_law="h,-k,-l" main_ncs=true would 
>>> deal with this problem ?
>>> 
>>> Same question goes to Garib, it's very convenient to just specify twin in 
>>> your Refmac script without further values and magic happens but what if I 
>>> add NCS restrains, will Refmac treat them correctly according to the twin 
>>> operator ?
>>> 
>>> Twins are confusing in real life and even more in crystals I think.
>>> 
>>> And no the data can not be processed in a higher space group, P222 results 
>>> in Rmerges of >40% in the lowest resolution shell, the beta angle is 92.4 
>>> degrees in P21. And if processed in P1 we get 4 molecules per asu and a 
>>> refined twin fraction of 50%, which in my eyes clearly indicates it's not 
>>> P1 but really P21.
>>> 
>>> Hope to get some interesting feedback on this issue.
>>> 
>>> Thanks,
>>> 
>>> Jürgen
>>> 
>>> ..
>>> Jürgen Bosch
>>> Johns Hopkins University
>>> Bloomberg School of Public Health
>>> Department of Biochemistry & Molecular Biology
>>> Johns Hopkins Malaria Research Institute
>>> 615 North Wolfe Street, W8708
>>> Baltimore, MD 21205
>>> Office: +1-410-614-4742
>>> Lab:  +1-410-614-4894
>>> Fax:  +1-410-955-2926
>>> http://web.mac.com/bosch_lab/
>>> 
>>> 
>>> 
>>> 
>> 
>> Garib N Murshudov 
>> Structural Studies Division
>> MRC Laboratory of Molecular Biology
>> Hills Road 
>> Cambridge 
>> CB2 0QH UK
>> Email: ga...@mrc-lmb.cam.ac.uk 
>> Web http://www.mrc-lmb.cam.ac.uk
>> 
>> 
>> 
> 
> ..
> Jürge

Re: [ccp4bb] P21, twinned date & NCS refinement

[Garib N Murshudov]

I would say that you should use ncs restraints in any case. NCS relates atoms 
and twin relates intensities. In some sense presence of twinning reduces 
information contents (in the limiting case the number of (effective) 
observervations becomes twice less) of the data and using NCS decreases the 
effective number of adjustable parameters. 
Without NCS some of the stats (like RvR) cannot be trusted. However you should 
remember that if ncs is very close to twin operators then in reciprocal space 
they relate two reflections exactly and in most of the cases you do not gain 
much and differences between ncs related molecules might be more important than 
their similarity. I would suggest using local ncs then global differences are 
retained and molecules are made locally similar.

If bet angle is 92.4 degrees (I would also check obliquity in refmac output if 
you are using that. In your case this number should be non-zero, meaning that 
twin related reflections will not coincide exactly) then I would expect 
splitting of spots in some directions at high resolutions. One should be 
careful when integrating, if only one of these spots are integrated then you 
may end up have several classes of reflections: low resolution and in some 
directions when spots overlap perfectly you have contributions from two 
crystals, in split spot cases each spot has contribution from single crystal.



regards
Garib


On 27 Mar 2012, at 21:37, Bosch, Juergen wrote:

> Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both 
> anyhow)
> 
> to the experts out there here's my question:
> 
> We have a P21 dataset with 2 molecules in the asu and a refined twin fraction 
> of 38% according to phenix.refine using a twin law operator.
> 
> My gut feeling tells me that I can't use NCS unless I detwin the data, is 
> that a correct assumption ?
> 
> How does phenix.refine [PDB] [mtz] twin_law="h,-k,-l" main_ncs=true would 
> deal with this problem ?
> 
> Same question goes to Garib, it's very convenient to just specify twin in 
> your Refmac script without further values and magic happens but what if I add 
> NCS restrains, will Refmac treat them correctly according to the twin 
> operator ?
> 
> Twins are confusing in real life and even more in crystals I think.
> 
> And no the data can not be processed in a higher space group, P222 results in 
> Rmerges of >40% in the lowest resolution shell, the beta angle is 92.4 
> degrees in P21. And if processed in P1 we get 4 molecules per asu and a 
> refined twin fraction of 50%, which in my eyes clearly indicates it's not P1 
> but really P21.
> 
> Hope to get some interesting feedback on this issue.
> 
> Thanks,
> 
> Jürgen
> 
> ..
> Jürgen Bosch
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
> Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
> http://web.mac.com/bosch_lab/
> 
> 
> 
> 

Garib N Murshudov 
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road 
Cambridge 
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk 
Web http://www.mrc-lmb.cam.ac.uk

Re: [ccp4bb] P21, twinned date & NCS refinement

[Bosch, Juergen]

Thanks Garib for your input. And yes we do see some split spots. We used XDS to 
overcome (I hope) most problems but still intensities of perfectly overlapping 
reflections will be too large. Would you think it's safer to integrate the data 
in P1 as symmetry mates will not be merged and then solve in P1 and convert 
into P21 cell for further refinement afterwards ?

Jürgen


On Mar 27, 2012, at 4:53 PM, Garib N Murshudov wrote:

I would say that you should use ncs restraints in any case. NCS relates atoms 
and twin relates intensities. In some sense presence of twinning reduces 
information contents (in the limiting case the number of (effective) 
observervations becomes twice less) of the data and using NCS decreases the 
effective number of adjustable parameters.
Without NCS some of the stats (like RvR) cannot be trusted. However you should 
remember that if ncs is very close to twin operators then in reciprocal space 
they relate two reflections exactly and in most of the cases you do not gain 
much and differences between ncs related molecules might be more important than 
their similarity. I would suggest using local ncs then global differences are 
retained and molecules are made locally similar.

If bet angle is 92.4 degrees (I would also check obliquity in refmac output if 
you are using that. In your case this number should be non-zero, meaning that 
twin related reflections will not coincide exactly) then I would expect 
splitting of spots in some directions at high resolutions. One should be 
careful when integrating, if only one of these spots are integrated then you 
may end up have several classes of reflections: low resolution and in some 
directions when spots overlap perfectly you have contributions from two 
crystals, in split spot cases each spot has contribution from single crystal.



regards
Garib


On 27 Mar 2012, at 21:37, Bosch, Juergen wrote:

Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both 
anyhow)

to the experts out there here's my question:

We have a P21 dataset with 2 molecules in the asu and a refined twin fraction 
of 38% according to phenix.refine using a twin law operator.

My gut feeling tells me that I can't use NCS unless I detwin the data, is that 
a correct assumption ?

How does phenix.refine [PDB] [mtz] twin_law="h,-k,-l" main_ncs=true would deal 
with this problem ?

Same question goes to Garib, it's very convenient to just specify twin in your 
Refmac script without further values and magic happens but what if I add NCS 
restrains, will Refmac treat them correctly according to the twin operator ?

Twins are confusing in real life and even more in crystals I think.

And no the data can not be processed in a higher space group, P222 results in 
Rmerges of >40% in the lowest resolution shell, the beta angle is 92.4 degrees 
in P21. And if processed in P1 we get 4 molecules per asu and a refined twin 
fraction of 50%, which in my eyes clearly indicates it's not P1 but really P21.

Hope to get some interesting feedback on this issue.

Thanks,

Jürgen

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





Garib N Murshudov
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road
Cambridge
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk
Web http://www.mrc-lmb.cam.ac.uk




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

Re: [ccp4bb] Substituting zero vs. Fc for unobserved reflections

[Ethan Merritt]

[Snipped from the full message, which is appended below]
> The program that kept showing me two forms bound was not
> substituting Fcalc for unobserved reflections.  So, I turned on the option
> to substitute Fcalc, and the minor form disappeared � the density looked
> like it did in the second program.  I figured the density that reveals the
> two forms must be correct being that it would be a big coincidence for
> artifactual density to appear that just so happens to fit perfectly our
> added (unmodified)ligand at 1.55 A.  So, I suppose, being that the occupancy
> of the major form is so much higher, by substituting unobserved reflections
> with Fcalc, the major form is being overemphasized, and the minor form
> becomes invisible.

A weighted difference map (mFo - DFc) that does not include the ligand
atoms in Fc at all would be a better guide.  It would not be biased by
the current ligand model [or at least much less biased] and it certainly
would not be sensitive to the modelled occupancies since these atoms
would not be contributing to Fc at all regardless of occupancy.

It is in general more convincing to show difference density from 
an Fo-Fc map with ligands omitted from Fc than it is to show density 
from some variant of 2Fo-Fc with ligands included in Fc.

How complete is your data set?  
Are you trying to deal with more than a few per cent of missing reflections?
If it is only the highest resolution shell that has poor completeness,
have you tried truncating the map calculation to a shell that is complete?

Ethan


On Tuesday, March 27, 2012 01:06:16 pm Gregg Crichlow wrote:
> Please excuse me for bringing up an old issue.  I have an interesting
> example of a difference seen when DFc was substituted for missing
> reflections versus when it wasn�t. Maybe others had this experience.  I had
> a structure in which the electron density showed two �overlapping� ligands
> bound in the same active site.  One was the ligand that was co-crystallized
> with the protein.  The other was the same ligand but with an unintentional
> modification (presumably due to radiation dose).  I was able to discern the
> two forms in the electron density (1.55 A) being that they did not
> completely overlap.  Based on occupancy refinement, the occupancies were
> 0.12 and 0.88 (unmodified and modified forms, respectively).  Then one time
> I calculated the map using a second program, and the lower occupancy ligand
> disappeared!  When I calculated maps in the first program, there were again
> two forms visible.  I thought that the difference may be due to the
> difference between substituting unobserved reflections with Fc (or rather
> DFc because of sigma-A weighting) versus omitting them from the Fourier
> transform.  The program that kept showing me two forms bound was not
> substituting Fcalc for unobserved reflections.  So, I turned on the option
> to substitute Fcalc, and the minor form disappeared � the density looked
> like it did in the second program.  I figured the density that reveals the
> two forms must be correct being that it would be a big coincidence for
> artifactual density to appear that just so happens to fit perfectly our
> added (unmodified)ligand at 1.55 A.  So, I suppose, being that the occupancy
> of the major form is so much higher, by substituting unobserved reflections
> with Fcalc, the major form is being overemphasized, and the minor form
> becomes invisible.
>   There may be many cases in which substituting Fcalc (or
> DFc) for missing reflections is beneficial. I don�t know the mathematical or
> theoretical arguments behind it.  I�m not arguing for one way being
> generally superior to the other, or for one program over another.  However,
> this is one empirical example of it being advantageous not to make this
> substitution.
>   When calculating experimentally phased maps, we multiply
> our structure factors by a figure of merit to down-weight reflections with
> less certain phases. Could one consider leaving missing reflections as zero
> analogous to multiplying Fcalc by FOM = 0? (just asking � maybe this is
> faulty logic.) Of course, this would be for the sake of the amplitude
> instead of the phase in this case. If an intensity is not observed, we have
> the ultimate uncertainty regarding its value.
> Maybe some developers will want to use this structure and the corresponding
> data to test DFc vs. �0� vs. DFc multiplied by a specific FOM only used for
> the missing reflections, varying from 0 to 1.  Unfortunately, this structure
> is not yet published (we needed to wait for other experiments to be
> finished) so I cannot yet provide it or the structure factors. However, if
> anyone is interested, feel free to contact me, and when it is published I
> would be happy to let you know the PDB code, if you still want it.
> 
> 
> 
> ***
> Gregg Crichlow
> Dept. of Pharmacology
> Yale University
> P.O. Box 20806

[ccp4bb] P21, twinned date & NCS refinement

[Bosch, Juergen]

Dear CCP4BBers and PhenixBBers (cross posting here, since we all read both 
anyhow)

to the experts out there here's my question:

We have a P21 dataset with 2 molecules in the asu and a refined twin fraction 
of 38% according to phenix.refine using a twin law operator.

My gut feeling tells me that I can't use NCS unless I detwin the data, is that 
a correct assumption ?

How does phenix.refine [PDB] [mtz] twin_law="h,-k,-l" main_ncs=true would deal 
with this problem ?

Same question goes to Garib, it's very convenient to just specify twin in your 
Refmac script without further values and magic happens but what if I add NCS 
restrains, will Refmac treat them correctly according to the twin operator ?

Twins are confusing in real life and even more in crystals I think.

And no the data can not be processed in a higher space group, P222 results in 
Rmerges of >40% in the lowest resolution shell, the beta angle is 92.4 degrees 
in P21. And if processed in P1 we get 4 molecules per asu and a refined twin 
fraction of 50%, which in my eyes clearly indicates it's not P1 but really P21.

Hope to get some interesting feedback on this issue.

Thanks,

Jürgen

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

[ccp4bb] Substituting zero vs. Fc for unobserved reflections

[Gregg Crichlow]

Please excuse me for bringing up an old issue.  I have an interesting
example of a difference seen when DFc was substituted for missing
reflections versus when it wasn¹t. Maybe others had this experience.  I had
a structure in which the electron density showed two Œoverlapping¹ ligands
bound in the same active site.  One was the ligand that was co-crystallized
with the protein.  The other was the same ligand but with an unintentional
modification (presumably due to radiation dose).  I was able to discern the
two forms in the electron density (1.55 A) being that they did not
completely overlap.  Based on occupancy refinement, the occupancies were
0.12 and 0.88 (unmodified and modified forms, respectively).  Then one time
I calculated the map using a second program, and the lower occupancy ligand
disappeared!  When I calculated maps in the first program, there were again
two forms visible.  I thought that the difference may be due to the
difference between substituting unobserved reflections with Fc (or rather
DFc because of sigma-A weighting) versus omitting them from the Fourier
transform.  The program that kept showing me two forms bound was not
substituting Fcalc for unobserved reflections.  So, I turned on the option
to substitute Fcalc, and the minor form disappeared ­ the density looked
like it did in the second program.  I figured the density that reveals the
two forms must be correct being that it would be a big coincidence for
artifactual density to appear that just so happens to fit perfectly our
added (unmodified)ligand at 1.55 A.  So, I suppose, being that the occupancy
of the major form is so much higher, by substituting unobserved reflections
with Fcalc, the major form is being overemphasized, and the minor form
becomes invisible.
  There may be many cases in which substituting Fcalc (or
DFc) for missing reflections is beneficial. I don¹t know the mathematical or
theoretical arguments behind it.  I¹m not arguing for one way being
generally superior to the other, or for one program over another.  However,
this is one empirical example of it being advantageous not to make this
substitution.
  When calculating experimentally phased maps, we multiply
our structure factors by a figure of merit to down-weight reflections with
less certain phases. Could one consider leaving missing reflections as zero
analogous to multiplying Fcalc by FOM = 0? (just asking ­ maybe this is
faulty logic.) Of course, this would be for the sake of the amplitude
instead of the phase in this case. If an intensity is not observed, we have
the ultimate uncertainty regarding its value.
Maybe some developers will want to use this structure and the corresponding
data to test DFc vs. ³0² vs. DFc multiplied by a specific FOM only used for
the missing reflections, varying from 0 to 1.  Unfortunately, this structure
is not yet published (we needed to wait for other experiments to be
finished) so I cannot yet provide it or the structure factors. However, if
anyone is interested, feel free to contact me, and when it is published I
would be happy to let you know the PDB code, if you still want it.



***
Gregg Crichlow
Dept. of Pharmacology
Yale University
P.O. Box 208066
New Haven, CT 06520-8066
***

 

Re: [ccp4bb] REFMAC5 residues with bad geometry

[James Holton]

Try this:

1) take your favorite PDB file and set all the B factors to ~80 (reduces 
series-termination errors)

2) use sfall/fft in CCP4 to calculate structure factors to 4A resolution
3) use sftools to add a "SIGF" column (0.1 will do) to make refmac5 happy
4) refine the "perfect" model against these fake data for ~5 cycles 
(with "solvent no")

5) load this up in coot and contour at 1 sigma
6) repeat the refinement with a PDB file containing only main chain.
7) repeat the refinement after putting all the side chains in their most 
likely (Ponder-Richards) rotamers.


Ask yourself these questions:
1) can you "see" the side chains?
2) can you "see" the waters?
3) what are the R factors from these refinements?

Answers: 1) no, 2) no, 3) ~3% for "perfect", ~50% for "main chain", and 
~36% for "likely rotamer"


Now ask yourself: even though there is "no density" for side chains and 
waters, is there really "no evidence" that they exist?


The point I am trying to make here is that you EXPECT side chains to 
poke out of density at low resolution, even under ideal conditions 
(perfect phases).  For example, the C-deltas of Leu will "breach" the 
1-sigma contour at around 2.8A resolution and worse.  You can see this 
in my old movie:

http://bl831.als.lbl.gov/~jamesh/movies/index.html#reso

When it comes to building, yes, once an atom dips below the 1-sigma 
contour it gets harder and harder to know exactly where it is, but it 
does have to be somewhere.  Somewhere nearby.  Formally, there is "prior 
knowledge" of bond lengths, etc. at play.  And if you know that there is 
one copy of a given atom in every unit cell of the crystal, then 
occupancy < 1 is inappropriate.  Much better to use B = 999, which 
models the atom as a Gaussian with the electrons spread over an area 
about 3.5 A wide.  This is roughly the range your average side chain 
atom has available to it, given that it is attached to the main chain by 
covalent bonds.


Of course, a more "Bayesian" model for the "I don't know what the 
rotamer is" situation would be to build in ALL possible rotamers, with 
occupancies equal to their Ponder-Richards probabilities.  Some 
improvement to this initial "guess" would no doubt be made by using 
constrained occupancy refinement of rigid-body side chains.  
Unfortunately, this is impossible with any refinement program I know 
about, since refmac, phenix.refine, etc. don't support more than 3 or 4 
alternate conformers.


Building in all possible conformers and using the occupancy as a 
"p-value" would also help solve the problem of the careless and/or 
uneducated over-interpreting PDB files.  Which is the "right one"?  Good 
question!  I think its time we started dispelling the myth of the 
single-conformer protein anyway.


-James Holton
MAD Scientist

On 3/26/2012 7:40 AM, Ed Pozharski wrote:

On Mon, 2012-03-26 at 10:17 -0400, Gregory Bowman wrote:

But what about the issue of resolution? As was previously pointed out,
at say 3.2 Å resolution, many side chains will fail to fit, but it
doesn't seem appropriate to trim them all down.

Why is it inappropriate to trim them down?  Sometimes at low resolution
all one can be confident about is the backbone trace.

Just to be clear, I am talking about atoms whose positions are not
supported by electron density, i.e. where difference map in the absence
of the side chain is featureless.  I assume that is the likely situation
when one would set occupancy to zero.

Cheers,

Ed.

Re: [ccp4bb] Unable to reproduce robot tray hits in hand trays

[Patrick Shaw Stewart]

Hi Matt

Rajesh asked a similar question last week, below

Essentially, you have to *reduce *the protein concentration when you scale
up because you lose proportionally more protein from smaller drops.

This usually works very well and we see no reason to use more than 0.3 +
0.3 for initial screening.

There's a page on our web site which explains this in much more detail, see
http://www.douglas.co.uk/Scaling_Up.htm

Best wishes

Patrick


-- Forwarded message --
From: Patrick Shaw Stewart 
Date: 19 March 2012 22:29
Subject: Re: [ccp4bb] microseeding
To: Rajesh kumar , ccp4bb@jiscmail.ac.uk



Rajesh

If you set up the volumes you suggest you will probably get precipitation.
This is counterintuitive until you realize that (as Ed says) you will be
losing a lot of protein with those small drops.  When you scale up the
surface area to volume ratio is lower, so a smaller proportion of the
protein is lost.  Therefore you go *up* on the phase diagram and get
precipitation or very small crystals.

Normally halving the amount of protein for the hits from 200 nl drops works
(suggesting that half the protein is lost from such small drops).  Try say
500+1000+500 (don't reduce the volume of seed stock because the solution
that you suspended the crystals in may be important).  Or dilute the
protein and use 1000+1000+500.

For the hits from the 450 nl drops you could reduce or dilute the protein
by say 25.%.

Or make plenty of seed-stock and try seeding into a random screen again
with larger drops, say 1.5+1+0.5 ul

Those tiny crystals should be good for seeding, don't worry about that
(provided they are protein of course).

Streak seeding may work but bear in mind that roughly a third of the
precipitant comes from the seed stock in your 250 nl drops.

You can add the seed stock with a syringe and needle if you don't have
suitable robot ;)

Experience and data-mining suggests that increasing the salt precipitant
(in high-salt drops) or salt additive (in PEG drops) by around 50% may be
helpful too when scaling up - I'm not sure why this works.

Good luck

Patrick






For the hits in the 250 nl drops you are probably losing

On 19 March 2012 20:31, Rajesh kumar  wrote:

> Dear All,
>
> I have few papers in hand which  explain me about microseeding, matrix
> microseeding, and cross seeding.
> I have also read few earlier threads and some more literature in google.
> Using Phoenix robot, I did a matrix micro-seeding and matrix cross
> seeding. I have few hits with this.
> In 96 well I used 100+100+50 nL and 200+200+50 nl (protein+screen+seed) in
> separate expts.
> I have hard time to plan to translate this 96 sitting drop well plate to
> 24 well plate to refine the conditions to get better crystals. only 1-2
> hits are small crystals and they are tiny.
>
>  I wonder in 24 well plate, if I should do-
> 1)  for Example 500+500+50nl (I am sure I cant add less that 500
> nL precisely)
> 2) to a drop of 500+500 nL do microseeding/streaking with a hair
>
> I appreciate if you could advise and share some practical ways to further
> my experiment.
>
> Thanks in advance
> Regards,
> Rajesh
>



-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36


-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] COOT Real Space Refinement keyboard shortcut??

[Ed Pozharski]

I suspect Chris is asking for the shortcut to the zone refinement
button, i.e. invoking the manual zone selection.  Not sure if there is a
scripting way to do this, nothing obvious.

On Tue, 2012-03-27 at 17:03 +0100, Debreczeni, Judit wrote:
> Yes, look here:
> 
> http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Coot#Example_11:_Paul_Emsley.27s_Key_Bindings
> 
> and the key bindings for r, x, t, R.
> 
> 
> JED.
> 
> 
> 
> 
> > 
> --
> AstraZeneca UK Limited is a company incorporated in England and Wales with 
> registered number: 03674842 and a registered office at 2 Kingdom Street, 
> London, W2 6BD.
> Confidentiality Notice: This message is private and may contain confidential, 
> proprietary and legally privileged information. If you have received this 
> message in error, please notify us and remove it from your system and note 
> that you must not copy, distribute or take any action in reliance on it. Any 
> unauthorised use or disclosure of the contents of this message is not 
> permitted and may be unlawful.
> Disclaimer: Email messages may be subject to delays, interception, 
> non-delivery and unauthorised alterations. Therefore, information expressed 
> in this message is not given or endorsed by AstraZeneca UK Limited unless 
> otherwise notified by an authorised representative independent of this 
> message. No contractual relationship is created by this message by any person 
> unless specifically indicated by agreement in writing other than email.
> Monitoring: AstraZeneca UK Limited may monitor email traffic data and content 
> for the purposes of the prevention and detection of crime, ensuring the 
> security of our computer systems and checking Compliance with our Code of 
> Conduct and Policies.
> -Original Message-
> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> > Christopher Browning
> > Sent: 27 March 2012 15:45
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [ccp4bb] COOT Real Space Refinement keyboard shortcut??
> > 
> > Hi,
> > 
> > I was wondering whether there was a way to assign a keyboard shortcut
> > to
> > the real space refine button in COOT. Atleast this way one does not
> > have
> > to keep looking back to where the real space refine button is to click
> > it if you want to carry out the refinement.
> > 
> > Cheers,
> > 
> > Chris
> > 
> > 
> > 
> > 
> > --
> > Dr. Christopher Browning
> > Post-Doctor to Prof. Petr Leiman
> > EPFL
> > BSP-416
> > 1015 Lausanne
> > Switzerland
> > Tel: 0041 (0) 02 16 93 04 40

-- 
"Hurry up before we all come back to our senses!"
   Julian, King of Lemurs

Re: [ccp4bb] COOT Real Space Refinement keyboard shortcut??

[Debreczeni, Judit]

Yes, look here:

http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Coot#Example_11:_Paul_Emsley.27s_Key_Bindings

and the key bindings for r, x, t, R.


JED.




>
--
AstraZeneca UK Limited is a company incorporated in England and Wales with 
registered number: 03674842 and a registered office at 2 Kingdom Street, 
London, W2 6BD.
Confidentiality Notice: This message is private and may contain confidential, 
proprietary and legally privileged information. If you have received this 
message in error, please notify us and remove it from your system and note that 
you must not copy, distribute or take any action in reliance on it. Any 
unauthorised use or disclosure of the contents of this message is not permitted 
and may be unlawful.
Disclaimer: Email messages may be subject to delays, interception, non-delivery 
and unauthorised alterations. Therefore, information expressed in this message 
is not given or endorsed by AstraZeneca UK Limited unless otherwise notified by 
an authorised representative independent of this message. No contractual 
relationship is created by this message by any person unless specifically 
indicated by agreement in writing other than email.
Monitoring: AstraZeneca UK Limited may monitor email traffic data and content 
for the purposes of the prevention and detection of crime, ensuring the 
security of our computer systems and checking Compliance with our Code of 
Conduct and Policies.
-Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Christopher Browning
> Sent: 27 March 2012 15:45
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] COOT Real Space Refinement keyboard shortcut??
>
> Hi,
>
> I was wondering whether there was a way to assign a keyboard shortcut
> to
> the real space refine button in COOT. Atleast this way one does not
> have
> to keep looking back to where the real space refine button is to click
> it if you want to carry out the refinement.
>
> Cheers,
>
> Chris
>
>
>
>
> --
> Dr. Christopher Browning
> Post-Doctor to Prof. Petr Leiman
> EPFL
> BSP-416
> 1015 Lausanne
> Switzerland
> Tel: 0041 (0) 02 16 93 04 40

Re: [ccp4bb] dea all

[Kevin Jin]

I guess you already run SDS-PAGE to check the pellets before and after
sonication. Not only the media.

On Tue, Mar 27, 2012 at 7:35 AM, rana ibd  wrote:
> Dear all
> I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB
> madia, I am having problems with the expression which shows small amount of
> the protein , I also have problems with purification using NI-NTA by also
> having small amount even after extensive buffer exchange , Is it likely due
> to the small amount of protein in the medium , should I use a different kind
> of media, any sugestions or any kind of details or a paper that might help I
> will be thankful
>
> Best Regards
> Rana



-- 
Kevin Jin

Candle always burns itself out to light up the tunnel .

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

[ccp4bb] FW: For 24 well Netxal plates (non ccp4 question) ==>> SUMMARY

[Mathews, Irimpan I.]

Hi Brad,

See below my attempts on this in 2010.

Regards,
Mathews

-Original Message-
From: Mathews, Irimpan I. 
Sent: Friday, May 21, 2010 8:23 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: RE: For 24 well Netxal plates (non ccp4 question) ==>> SUMMARY

Dear All,

Thank you for the several responses. Please see the summary below.

1. Several of them:  complain to Qiagen (two or three said: protests loudly).  
Unfortunately, my e-mail to the Qiagen marketing manager never got any reply. I 
e-mailed to him on 5-12-10. Let me know if you need his e-mail address.

2.  Molecular dimensions has the following new item. 

a novel 96 well version of a screw top plate for hanging drop which is of 
standard 96 well plate size.
Please see
http://www.moleculardimensions.com/shopdisplayproducts.asp?id=54&cat=Hanging
+drop+

Kind regards,
Mathews


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mathews, 
Irimpan I.
Sent: Tuesday, May 11, 2010 12:41 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] For 24 well Netxal plates (non ccp4 question)

Dear All,

Qiagen has stopped selling the 24 well Netxal trays and changed to 15 well 
trays (almost same price) that doesn't fit with any screens. Do you know of 
some source for the 24 well screw cap trays? 

Thank you very much for your help,
Mathews

Re: [ccp4bb] dea all

[Kelly Daughtry]

When you lyse the cells and spin down cellular debris, is the pellet large
and white (indicating inclusion bodies)? Is your protein soluble or
membrane? What temperature did you use for expression? What vector are you
using? Providing more details allows us to better answer your questions.

Off the top of my head:
Altering expression can include lowered the temperature just prior to
induction (25, 18, or lower) and letting the cells grow overnight.
Induction at increased cell density (1.0 vs 0.6 O.D.).

Anther option to increase the expression of soluble protein is to use the
auto-induction media: http://www.ncbi.nlm.nih.gov/pubmed/15915565

Another option is to try other cells lines, and co-expression with
chaperonins (the arctic express cell line is useful
http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Product&SubPageType=ProductDetail&PageID=467
).

Did you try other tags (GST, MBP, etc)?

Hope this helps,
Kelly Daughtry

***
Kelly Daughtry, Ph.D.
Post-Doctoral Fellow, Raetz Lab
Biochemistry Department
Duke University
Alex H. Sands, Jr. Building
303 Research Drive
RM 250
Durham, NC 27710
P: 919-684-5178
***


On Tue, Mar 27, 2012 at 10:35 AM, rana ibd  wrote:

> Dear all
> I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB
> madia, I am having problems with the expression which shows small amount of
> the protein , I also have problems with purification using NI-NTA by also
> having small amount even after extensive buffer exchange , Is it likely due
> to the small amount of protein in the medium , should I use a different
> kind of media, any sugestions or any kind of details or a paper that might
> help I will be thankful
>
> Best Regards
> Rana
>

Re: [ccp4bb] 24 well screw-cap crystallization plates

[Pascal Egea]

Hi Brad,

I am afraid that there is no alternate source for these plates. The screw
cap system , I believe, was patented by the canadian company NEXTAL that
was then assimilated by Q...N and the patent is probably still holding.

Pascal


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu

Re: [ccp4bb] Clustal omega

[Jayashankar]

Dear Tim,

Thanks, The Jalview, output to textbox in PIR worked thr trick.

S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.


On Tue, Mar 27, 2012 at 3:52 PM, Tim Gruene  wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
> Dear Jayashankar,
>
> sorry, you did explain this in your model and I flipped clustalo with
> clustalw. Even though when I used the option '-convert', clustalw does
> not re-align the input but simply converts it, so in case it can read
> the clustalo output, this should do the job, shouldn't it?
>
> Have you tried reading the output file with clustalx or seaview or
> jalview? These programs should be able convert to various file formats
> without messing up with the alignment.
>
> Cheers,
> Tim
>
> On 03/27/12 15:44, Jayashankar wrote:
> > Dear Tim,
> >
> > I try to model a full length protein based on various domains solved
> > already.
> > In that case, alignment of multiple sequence should approximately locate
> to
> > the corresponding domain region.
> >
> > Clustalw results in an unexpected alignment file.
> > Whereas the output from clustal omega is perfect and the format what I
> need
> > is not given in the output option.
> >
> > Because I will be needing this .pir output file to model my target
> sequence
> > using modeller.
> >
> > thanks
> >
> > S.Jayashankar
> > Research Student
> > Institute for Biophysical Chemistry
> > Hannover Medical School
> > Germany.
> >
> >
> > On Tue, Mar 27, 2012 at 3:26 PM, Tim Gruene 
> wrote:
> >
> > Dear S. Jayashankar,
> >
> > have you tried the clustalw options '-convert -output=PIR'? This should
> > result in a pir-formatted output file.
> >
> > Tim
> >
> > On 03/27/12 10:18, Jayashankar wrote:
>  Dear All,
> 
>  A bit offtopic question,
> 
>  Does any body know how can I get the PIR output of the aligned
> sequence
>  from clustal omega.
>  I have a full length protein solved, to give weightage during
> comparitive
>  modelling, I use domains of the same/similar protein solved before.
>  I expect an alignment something that looks like one below, and
> clustalw
>  does'nt seems to work , but clustal omega does it. But there is no
> .pir
>  format output.
> 
> 
>  1.caa
>  2.-
>  3.c--
>  4.--aaa--
> 
> 
>  Thanks
>  S.Jayashankar
>  Research Student
>  Institute for Biophysical Chemistry
>  Hannover Medical School
>  Germany.
> 
> >
> >>
>
> - --
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
> GPG Key ID = A46BEE1A
>
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.12 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
>
> iD8DBQFPccYEUxlJ7aRr7hoRAqT2AKDyz0fwVnqhp5DjiiocWz7VrI5IOgCgpxhL
> YfM5EJQt5e2WoEU7iILcDbI=
> =QNtR
> -END PGP SIGNATURE-
>

[ccp4bb] granular precipitate

[anita p]

Hi All,
I have set up initial screen in hanging drop trays with a protein of
theoritical pI of 8.5. The protein is in acetate buffer 10mM, KCl 100mM and
2% glycerol
pH 5 . In 85-90% of the conditions I see granular precipitate in 1 day. I
tried to open the coverslip, and touch few drops, They had a skin layer and
the granular looking thing are part of the skin. The protein was set up at
10 and 5 mg/ml concentration. Is this normal?

I have tried to change the protein buffer to Hepes at pH 7 with other
constituents same,and tried to set up few screen trays. I cant see granular
precipitate again. The precipitates look light brown and amorphous in
nature.

Is Na acetate forming some granular precipitate instead of protein?

Kindly suggest me ways to move forwards.
Thanks in advance
Anita

[ccp4bb] 24 well screw-cap crystallization plates

[Brad Bennett]

Hi all-
So far, I have not been a fan of Qiagen's decision to revamp the 24 well
Nextal crystallization plates with screw caps to 15 well plates without a
top cover plate. I know that it is less plastic and now a standard SBS
footprint but IMHO it is rather flimsy and harder to handle than the
original plates. Does anyone know of a distributor of the 24 well version
of these plates or an alternative vendor that is manufacturing plates
similar to these?

Thanking you in advance for any suggestions-
Brad

[ccp4bb] dea all

[rana ibd]

Dear all
I am expressing a 6xHis tagged in a dHBx protein in E.coli BL21 using LB madia, 
I am having problems with the expression which shows small amount of the 
protein , I also have problems with 
purification using NI-NTA by also having small amount even after 
extensive buffer exchange , Is it likely due to the small amount of 
protein in the medium , should I use a different kind of media, any 
sugestions or any kind of details or a paper that might help I will be 
thankful

Best Regards 
Rana

[ccp4bb] COOT Real Space Refinement keyboard shortcut??

[Christopher Browning]

Hi,

I was wondering whether there was a way to assign a keyboard shortcut to
the real space refine button in COOT. Atleast this way one does not have
to keep looking back to where the real space refine button is to click
it if you want to carry out the refinement.

Cheers,

Chris




-- 
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
1015 Lausanne
Switzerland
Tel: 0041 (0) 02 16 93 04 40

Re: [ccp4bb] Coot set-refine-max-residues

[Ed Pozharski]

works here on 0.7-pre-1 rev 3713

so try downloading the latest version if yours is <3713

On Tue, 2012-03-27 at 14:50 +0100, Morten Grøftehauge wrote:
> set-refine-max-residues
-- 
Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
Julian, King of Lemurs

[ccp4bb] Coot set-refine-max-residues

[Morten Grøftehauge]

Hi ccp4bb,

I've been having trouble with Coot. Specifically the scripting to change
the maximum number of residues to refine.
So if I try to refine more than 20 residues I get this neat little warning
message in the terminal:

WARNING:: Hit heuristic fencepost! Too many residues to refine
   FYI: 23 > 20 (which is your current maximum).
Use (set-refine-max-residues 40) to increase limit

but when I try set-refine-max-residues 40 or set-refine-max-residues 30 in
either the terminal or the scheme scripting command window, it doesn't
actually change anything.
I've run the Python version of the hydrogen bond restraints script before
all this.
Hope someone can point me in the right direction.

Cheers,
Morten

-- 
Morten K Grøftehauge, PhD
Pohl Group
Durham University

Re: [ccp4bb] Clustal omega

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Jayashankar,

sorry, you did explain this in your model and I flipped clustalo with
clustalw. Even though when I used the option '-convert', clustalw does
not re-align the input but simply converts it, so in case it can read
the clustalo output, this should do the job, shouldn't it?

Have you tried reading the output file with clustalx or seaview or
jalview? These programs should be able convert to various file formats
without messing up with the alignment.

Cheers,
Tim

On 03/27/12 15:44, Jayashankar wrote:
> Dear Tim,
> 
> I try to model a full length protein based on various domains solved
> already.
> In that case, alignment of multiple sequence should approximately locate to
> the corresponding domain region.
> 
> Clustalw results in an unexpected alignment file.
> Whereas the output from clustal omega is perfect and the format what I need
> is not given in the output option.
> 
> Because I will be needing this .pir output file to model my target sequence
> using modeller.
> 
> thanks
> 
> S.Jayashankar
> Research Student
> Institute for Biophysical Chemistry
> Hannover Medical School
> Germany.
> 
> 
> On Tue, Mar 27, 2012 at 3:26 PM, Tim Gruene  wrote:
> 
> Dear S. Jayashankar,
> 
> have you tried the clustalw options '-convert -output=PIR'? This should
> result in a pir-formatted output file.
> 
> Tim
> 
> On 03/27/12 10:18, Jayashankar wrote:
 Dear All,

 A bit offtopic question,

 Does any body know how can I get the PIR output of the aligned sequence
 from clustal omega.
 I have a full length protein solved, to give weightage during comparitive
 modelling, I use domains of the same/similar protein solved before.
 I expect an alignment something that looks like one below, and clustalw
 does'nt seems to work , but clustal omega does it. But there is no .pir
 format output.


 1.caa
 2.-
 3.c--
 4.--aaa--


 Thanks
 S.Jayashankar
 Research Student
 Institute for Biophysical Chemistry
 Hannover Medical School
 Germany.

> 
>>

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFPccYEUxlJ7aRr7hoRAqT2AKDyz0fwVnqhp5DjiiocWz7VrI5IOgCgpxhL
YfM5EJQt5e2WoEU7iILcDbI=
=QNtR
-END PGP SIGNATURE-

Re: [ccp4bb] Clustal omega

[Jayashankar]

Dear Tim,

I try to model a full length protein based on various domains solved
already.
In that case, alignment of multiple sequence should approximately locate to
the corresponding domain region.

Clustalw results in an unexpected alignment file.
Whereas the output from clustal omega is perfect and the format what I need
is not given in the output option.

Because I will be needing this .pir output file to model my target sequence
using modeller.

thanks

S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.


On Tue, Mar 27, 2012 at 3:26 PM, Tim Gruene  wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
> Dear S. Jayashankar,
>
> have you tried the clustalw options '-convert -output=PIR'? This should
> result in a pir-formatted output file.
>
> Tim
>
> On 03/27/12 10:18, Jayashankar wrote:
> > Dear All,
> >
> > A bit offtopic question,
> >
> > Does any body know how can I get the PIR output of the aligned sequence
> > from clustal omega.
> > I have a full length protein solved, to give weightage during comparitive
> > modelling, I use domains of the same/similar protein solved before.
> > I expect an alignment something that looks like one below, and clustalw
> > does'nt seems to work , but clustal omega does it. But there is no .pir
> > format output.
> >
> >
> > 1.caa
> > 2.-
> > 3.c--
> > 4.--aaa--
> >
> >
> > Thanks
> > S.Jayashankar
> > Research Student
> > Institute for Biophysical Chemistry
> > Hannover Medical School
> > Germany.
> >
>
> - --
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
>
> GPG Key ID = A46BEE1A
>
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.12 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
>
> iD8DBQFPccAdUxlJ7aRr7hoRAh2BAJwP6HdQd6t0/TVZBLZCA/S4KEMY9wCeM2WL
> 7R/ULzOB+kq8fltCbQ6EUR4=
> =fzmD
> -END PGP SIGNATURE-
>

Re: [ccp4bb] Clustal omega

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear S. Jayashankar,

have you tried the clustalw options '-convert -output=PIR'? This should
result in a pir-formatted output file.

Tim

On 03/27/12 10:18, Jayashankar wrote:
> Dear All,
> 
> A bit offtopic question,
> 
> Does any body know how can I get the PIR output of the aligned sequence
> from clustal omega.
> I have a full length protein solved, to give weightage during comparitive
> modelling, I use domains of the same/similar protein solved before.
> I expect an alignment something that looks like one below, and clustalw
> does'nt seems to work , but clustal omega does it. But there is no .pir
> format output.
> 
> 
> 1.caa
> 2.-
> 3.c--
> 4.--aaa--
> 
> 
> Thanks
> S.Jayashankar
> Research Student
> Institute for Biophysical Chemistry
> Hannover Medical School
> Germany.
> 

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

GPG Key ID = A46BEE1A

-BEGIN PGP SIGNATURE-
Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFPccAdUxlJ7aRr7hoRAh2BAJwP6HdQd6t0/TVZBLZCA/S4KEMY9wCeM2WL
7R/ULzOB+kq8fltCbQ6EUR4=
=fzmD
-END PGP SIGNATURE-

[ccp4bb] COURSE ANNOUNCEMENT - BIOCRYS 2012

[Colin McVey]

Dear colleagues,

On behalf of the organizers, Maria Armenia Carrondo and Thomas R. 
Schneider I have the following course announcement:


*COURSE ANNOUNCEMENT - BIOCRYS 2012*
FEBS Practical & Lecture Course
Fundamentals of Modern Methods in Biocrystallography


*20th - 27th October 2012 at the Instituto de Tecnologia Química e 
Biológica, Oeiras, Portugal. *


Topics of the course will run from fundamentals such as symmetry, point 
groups and crystal systems, basic diffraction physics, reciprocal space 
and the Ewalds sphere, radiation damage, data processing, symmetry in 
the diffraction pattern, structure factors, Patterson function to modern 
methodologies including molecular replacement, SAD, MAD, MIR and maximum 
likelihood phasing, direct methods, density modification, refinement, 
model building, twinning and structure validation. Main challenges on 
sample preparation and crystallization of proteins will also be covered.


The course will be organized with lectures in the mornings and 
interactive practicals and tutorials in the afternoons. Evening lectures 
will address two main important topics within Structural Biology, one 
covering studies of membrane proteins and the other the beginning of the 
use of free electron lasers in macromolecular Crystallography.


Participants will be limited to 36, aimed primarly at people at the 
beginning of their crystallographic research activity as PhD students or 
others. Selected applicants are invited to present a poster during the 
course.


*Speakers and tutors: *

*Margarida Archer, Isabel Bento, Gabor Bunkoczi, Kevin Cowtan*
*Zbigniew Dauter, Carlos Frazão, Elspeth Garman, Christoph Hermes*
*Edward Hough, Gordon Leonard, Andrew Leslie, Bernhard Lohkamp, Adrian 
Mancuso**

Pedro Matias, Rob Meijers, Poul Nissen, Anastassis Perrakis, Célia Romão*
*Thomas Schneider, Clemens Vonrhein*



A registration fee of*600 Euros* for academic and *1000 Euros *for 
non-academic applicants is requested for full board and accommodation.
Selected applicants will have to pay the registration fee by bank 
transfer before arrival.


A limited number of grants is available from *FEBS*and from *IUCr*. How 
to apply 


For an application, please fill the form on the web page by the 31th of 
July.


For more information visit the course web page 
http://biocrys2012.itqb.unl.pt/


--
*Colin E. McVey, DPhil*

Auxilliary Researcher
Structural Genomics Lab
Macromolecular Crystallography Unit
Instituto de Tecnologia Química e Biológica
Av. da Republica, EAN  | Phone:(351)214469663
Apartado 127  | Fax :(351)214433644
2781-901 Oeiras  | email:mc...@itqb.unl.pt
PORTUGAL

[ccp4bb] Clustal omega

[Jayashankar]

Dear All,

A bit offtopic question,

Does any body know how can I get the PIR output of the aligned sequence
from clustal omega.
I have a full length protein solved, to give weightage during comparitive
modelling, I use domains of the same/similar protein solved before.
I expect an alignment something that looks like one below, and clustalw
does'nt seems to work , but clustal omega does it. But there is no .pir
format output.


1.caa
2.-
3.c--
4.--aaa--


Thanks
S.Jayashankar
Research Student
Institute for Biophysical Chemistry
Hannover Medical School
Germany.

Re: [ccp4bb] DDM

[Daniel Picot]

It is important to distinguish between the solubilisation and the 
purification steps:
1) During the solubisation step you need to care about the 
lipid/detergent ratio. The amount (not the concentration) of detergent 
(i.e. in non monomeric form, the detergent above the cmc) is important. 
You may need a high amount of detergent.
2) During the purification step, you need to keep your protein soluble. 
Here, the concentration is important and you may  keep the concentration 
of detergent around the cmc. But you have to be aware that in the 
initial (and also the not so initial ones!) steps you may have a lot of 
lipids, you need then keep the concentration of detergent fairly high in 
order to keep everything soluble. I like to decrease the detergent 
concentration at each purification steps in order to avoid protein 
denaturation. The protein may sustain a fairly high detergent 
concentration of detergent during the early steps since the 
lipid/detergent mixed micelles will be less denaturing than the pure 
detergent micelles in the later steps. Thin layer chromatography is a 
quick and easy method to check if you have a large amount of lipids in 
your preparation.

HTH
Daniel

Le 26/03/2012 19:17, Katarzyna Rudzka a écrit :

Hi All,
Has anyone had any luck purifying membrane proteins with DDM
(n-dodecyl-D-maltoside) using concentration of detergent ~1-2 x CMC ?
(Its CMC is very low: 0.009%). I would like to keep it as low as
possible, so I don't have too much DDM around when I get to the
crystallization step. I wonder If the amount of detergent sufficient for
the protein extraction has to be determined experimentally for each
protein or maybe there are some good rules of thumb. I appreciate your
help. Thanks.
Kasia

Katarzyna Rudzka, Postdoctoral Fellow
Department of Biophysics and Biophysical Chemistry
Johns Hopkins University, School of Medicine
Baltimore, Maryland 21205 USA