Re: [ccp4bb] rigid body vs restrained refinement

[Pavel Afonine]

Norman,

the value of free-R alone doesn't tell a whole lot, though the numbers you
quote seem good given the resolution. What's Rwork?
The behavior of free-R you tell is also totally expected: in rigid-body
refinement there are way less refinable parameters!
Most likely you should proceed with individual coordinate refinement, may
be using SA.
If you send me the input model and data files (off-list of course!), I
might be able to suggest the optimal refinement strategy.

Pavel

On Thu, Jul 19, 2012 at 7:24 PM, Norman zhu  wrote:

> hello everyone
>
> I just noticed my model would give two very different Rfree
> values from refmac depending which mode I use to refine it.  Rigid
> body it gives a Rfree value of 24.57% while restrained refinement
> gives a value of 27.96%.  Both are processed at 3.1angstrom
> resolution.  I am confused as to which number to believe.  Should I
> keep refining with RB refinement or go back to RR even though it give
> me a higher Rfree value?  Thanks.
>
>
> Norman Zhu
> Department of Chemistry
> San Diego State University
> norman...@gmail.com
>

[ccp4bb] Nucleophilic attack by the side-chain carboxyl group of Asp?

[Crystal Xu]

Hi everyone,

I am studying the catalytic mechanism of an enzyme. My structural data
indicates that an Asp is of most possibility to sever as a general base.
The Asp residue is coordinated to a zinc ion. Would it be possible
that the side-chain
carboxyl group of Asp attacks a carbon atom of the substrate? Does anyone
know any examples?

Thanks very much.

Best regards.

[ccp4bb] rigid body vs restrained refinement

[Norman zhu]

hello everyone

I just noticed my model would give two very different Rfree
values from refmac depending which mode I use to refine it.  Rigid
body it gives a Rfree value of 24.57% while restrained refinement
gives a value of 27.96%.  Both are processed at 3.1angstrom
resolution.  I am confused as to which number to believe.  Should I
keep refining with RB refinement or go back to RR even though it give
me a higher Rfree value?  Thanks.


Norman Zhu
Department of Chemistry
San Diego State University
norman...@gmail.com

Re: [ccp4bb] Large unit cell, overlaps

[Kay Diederichs]

Hi Jason,

I looked at the part of FRAME.cbf you posted, and I'd say it looks ok (i.e. 
nothing to worry about). 

Some overlap of a reflection doesn't matter much; if its full profile is not 
available then INTEGRATE replaces the missing intensity by an estimate based on 
the known fraction of the intensity. Below a cutoff of MINPK (default is 75 %) 
however the estimate is considered inaccurate and the reflection is discarded. 
To judge whether and how many reflections are affected by overlaps, you should 
consult the table in XDSSTAT.LP; it has no graphical representation and it is a 
little dense (sorry).

If many reflections fail the MINPK test, you might get better completeness if 
you re-integrate with lower values of the mosaicity - see 
http://strucbio.biologie.uni-konstanz.de/xdswiki/index.php/Optimisation . If 
the data are for molecular replacement and refinement, you might want to 
decrease MINPK somewhat (check the table in XDSSTAT.LP!). As this is at the 
cost of data quality, I'd not recommend this for experimental phasing.

HTH,

Kay

Re: [ccp4bb] RE : [ccp4bb] Large unit cell, overlaps

[Kay Diederichs]

On Tue, 17 Jul 2012 06:26:39 +, LEGRAND Pierre 
 wrote:

> Hi Jason,
> 
> To answer your initial question about overlaps versus finer slicing, you can 
> get a good description of the problem in Fig10 
> of Z. Dauter article "Data-collection strategies"  (open access article here: 
> http://journals.iucr.org/d/issues/1999/10
> /00/ba0020/ba0020.pdf).
> 
> From the initial cell parameters, XDS calculates the "Maximum oscillation 
> range to prevent angular overlap at high 
> resolution limit" (bottom of the IDXREF.LP file). This calculation assumes 
> zero mosaicity, and crystal in the worst orientation:
>  with the longest axis perpendicular to the spindle axis.

It is not true that this list in IDXREF.LP assumes the crystal to be in the 
worst orientation. Rather, it refers to the crystal in its _current_ 
orientation! I think this is a more useful piece of information once the 
crystal is indexed.

best,

Kay

Re: [ccp4bb] resolution limit

[Kay Diederichs]

Hi Narayan,

there's nothing wrong with using data with I/sigmaI 2.5, Rsym 224.3 % for 
multiplicity 7.8 and completeness 98.2 %. 

However, when you discarded frames you might have made the data worse - one 
should only reject data if they deviate systematically (e.g. from radiation 
damage). Weak data should not be rejected, and Rmerge is the wrong measure to 
judge about data quality.

best,

Kay

On Wed, 18 Jul 2012 02:41:48 -0700, narayan viswam  wrote:

>Hello CCP4ers,
>
> In my data, the highest reolution shell 2.8-3.0 A has I/sigmaI 2.5 & Rsym
>224.3 % for multiplicity 7.8 and completeness 98.2 %. I solved the
>structure by MAD & refined it to Rfree 27.3 %. Ths crystal belongs to P622
>space group and it is not twinned. The water content is 68%. I loweredthe
> multiplicity to 4.1 by excluding few images but still the Rsym is > 200 %
>and I/sigmaI > 2.0. My rudimentary crystallography knowledge makes me
>believe it's quite Ok to use data upto 2.8 A and report the statistics.
>Could I request people's views. Thanks very much.
>Narayan
>

Re: [ccp4bb] aimless, anisotropy, and resolution cutoff?

[Kay Diederichs]

Hi Vincent,

my advice is: if in doubt, scale at the highest of those resolution choices. 
This way you don't have to go back to scaling later. There is no harm in using 
the highest resolution. Don't let yourself be fooled by Rmerge.

best,
Kay

On Thu, 19 Jul 2012 17:52:14 +0200, vincent Chaptal  
wrote:

>Dear all,
>
>I'm dealing with anisotropic diffraction of a membrane protein.
>I've read from previous threads that it is better to not cut off any 
>data, and that aimless doesn't anisotropically scale the data any way.
>So my question is: given my anisotropic diffraction, what resolution 
>cutoff do i give aimless for scaling, and what statistics can i report? 
>(log bellow)
>
>- Do I scale at 2.85A knowing that there is at least data in one 
>direction to this resolution? (The different Rs are all over the place, 
>but i can site the Karplus Science paper for the use of CC for 
>resolution cutoff)
>- Do I scale at 3.47A as it is "the best overall estimate" (with decent 
>R statistics).
>
># Aimless log #
>
>Estimates of resolution limits: overall
>from half-dataset correlation CC(1/2) >  0.50: limit = 3.47A
>from Mn(I/sd) >  2.00: limit = 3.83A
>
>Estimates of resolution limits in reciprocal lattice directions:
>   Along 0.98 h + 0.18 l
>from half-dataset correlation CC(1/2) >  0.50: limit = 3.97A
>from Mn(I/sd) >  2.00: limit = 3.65A
>   Along k axis
>from half-dataset correlation CC(1/2) >  0.50: limit = 5.12A
>from Mn(I/sd) >  2.00: limit = 5.23A
>   Along -0.25 h + 0.97 l
>from half-dataset correlation CC(1/2) >  0.50: limit = 2.85A
>from Mn(I/sd) >  2.00: limit = 3.17A
>
>
>
>
>Thank you for your input.
>vincent
>
>
>
>Le 4/25/12 5:40 PM, George Sheldrick a �crit :
>> I think that anything that irrevocably modifies the experimental data 
>> should be avoided whenever possible. Since anisotropic scaling is a 
>> relatively fast calculation and there are several ways of doing it, it 
>> is better to apply it locally when it is needed, e.g. in phasing 
>> (where it is applied by phaser and shelxe etc.) and refinement (with 
>> refmac or phenix_refine etc.). Provided that the standard deviations 
>> of the observed intensities are properly taken into account, 
>> anisotropic data truncation is not so important (i.e. as usual I agree 
>> with Garib and Phil).
>>
>> George
>>
>> On 04/25/2012 06:19 PM, Phil Evans wrote:
>>> You can get the aimless documentation from
>>>
>>> ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/aimless.html
>>>
>>> pending its official release through CCP4
>>>
>>> No it does not do anisotropic scaling as such. That needs some sort 
>>> of model of the "ideal" intensity, probably best calculated from a model
>>>
>>> I'm not sure that anisotropic cutoffs are a good idea. I believe 
>>> Garib thinks they are not and I generally defer to him
>>>
>>> Phil
>>>
>>> On 25 Apr 2012, at 17:00, Bryan Lepore wrote:
>>>
 wondering if aimless performs anisotropic scaling or "elliptical"
 rejections lately.

 I ask because:

 [1] last I knew, scala did not
 [2] I can't seem to google up the aimless manual as readily as scala

 ... also, what consesquence would mosflm anisotropic resolution limits
 have on scaling (if aimless anisoscaling were true).

 -Bryan
>>
>>
>
>-- 
>
>Vincent Chaptal, PhD
>
>Institut de Biologie et Chimie des Prot�ines
>
>Drug-resistance modulation and mechanism Laboratory
>
>7 passage du Vercors
>
>69007 LYON
>
>FRANCE
>
>+33 4 37 65 29 01
>
>http://www.ibcp.fr
>
>
>

Re: [ccp4bb] Protein concentration vs Molecular wt...

[Tom Murray-Rust]

Hi James,

You can get the PCT (Pre-Crystallisation Test) from one of the more
famous manufacturers of crystallography products - essentially you can
quickly screen your protein to get an idea if it is too dilute, too
concentrated, or somewhere in the middle. Of course, the results are
representative only (I've had samples which looked great on the PCT
but then gave me ~90% clear drops even at 40 mg/ml, and an 80 kDa
protein which crystallised at 2 mg/ml) so as with anything they should
be taken with the appropriate vessel full of sodium chloride...

If you have enough protein, then I agree with the previous comments
that it is more useful to set up some trays and see what % of
conditions give hits, and work from there.

Cheers,

Tom


On 19 July 2012 20:58, james09 pruza  wrote:
> Dear Crystallographers,
> Is there any rule of thumb for Protein concentration and molecular weight
> for crystallization trials of a soluble protein? Looking for high molecular
> wt. protein ~50kDa.
> James.
>



-- 
Skype: tom.murray.rust
Twitter: tmurrayrust
http://twitpic.com/photos/tmurrayrust
+44 7970 480 601 (UK)

Re: [ccp4bb] Protein concentration vs Molecular wt...

[Roger Rowlett]

This is almost exactly our basic approach, too. Before we got a 
dropsetter, we did 24 wells (1/2 screen) to get a feel for the correct 
protein concentration. Some additional rules of thumb we use:


1. We usually start at 10 mg/mL protein and go up or down from there
   depending on the results of the initial screen
2. If we observe a high frequency of precipitation in a 10 mg/mL
   protein screen, we will usually set a 1/2 concentration screen by
   diluting the screen solutions 1:1 with water. This frequently
   uncovers additional hits in wells that were heavily precipitated in
   the original screen. Empirically, proteins we study seem to
   crystallize better in the higher protein/lower precipitant zone of
   the phase diagram than the lower protein/higher precipitant zone. YMMV.

Cheers,

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

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

On 7/19/2012 4:31 PM, mjvdwo...@netscape.net wrote:
I don't think there is such a rule, but in the old days, when we only 
had Hampton Screen I and II, the rule was:


- Set up screen 1, look at the drops and you should expect some kind 
of precipitation in 50% of the drops. If much less than that, increase 
your protein concentration. If much more than that, decrease protein 
concentration.

- Set up screen 2, look and expect 30% precipitation.

I used to cut corners and do the statistics at 1/2 of a screen (one 
24-well plate). You can probably use this method to get within a 
factor of 2 of the optimal concentration.


There are probably good statistics in the papers for the screens that 
you may use. One of the advantages of structural genomics efforts is 
that these things are known (and hopefully published).


Even older trick is to take a drop of protein and look under a 
microscope, record how much AmSO4 it takes to cause precipitation. Do 
the same with PEG. Keep adding a little at a time and look 
immediately. This will give you an idea if you are near a reasonable 
concentration. I think that this latter method does not tell you much 
more than "physics-information" - which is how many zeroes there are: 
whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable.


Mark


-Original Message-
From: james09 pruza 
To: CCP4BB 
Sent: Thu, Jul 19, 2012 1:59 pm
Subject: [ccp4bb] Protein concentration vs Molecular wt...

Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular 
weight for crystallization trials of a soluble protein? Looking for 
high molecular wt. protein ~50kDa.

James.

Re: [ccp4bb] Protein concentration vs Molecular wt...

[mjvdwoerd]

I don't think there is such a rule, but in the old days, when we only had 
Hampton Screen I and II, the rule was:

- Set up screen 1, look at the drops and you should expect some kind of 
precipitation in 50% of the drops. If much less than that, increase your 
protein concentration. If much more than that, decrease protein concentration.
- Set up screen 2, look and expect 30% precipitation.

I used to cut corners and do the statistics at 1/2 of a screen (one 24-well 
plate). You can probably use this method to get within a factor of 2 of the 
optimal concentration. 

There are probably good statistics in the papers for the screens that you may 
use. One of the advantages of structural genomics efforts is that these things 
are known (and hopefully published).

Even older trick is to take a drop of protein and look under a microscope, 
record how much AmSO4 it takes to cause precipitation. Do the same with PEG. 
Keep adding a little at a time and look immediately. This will give you an idea 
if you are near a reasonable concentration. I think that this latter method 
does not tell you much more than "physics-information" - which is how many 
zeroes there are: whether 1 mg/ml or 10mg/ml or 100mg/ml is reasonable. 

Mark



-Original Message-
From: james09 pruza 
To: CCP4BB 
Sent: Thu, Jul 19, 2012 1:59 pm
Subject: [ccp4bb] Protein concentration vs Molecular wt...


Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular weight for 
crystallization trials of a soluble protein? Looking for high molecular wt. 
protein ~50kDa. 
James.


 

[ccp4bb] Local service for AKTA explorer in Maryland or east coast

[Yong-Fu Li]

Colleagues,

Does anyone use a local service on an AKTA explorer (or other models)
machine ideally in the east coast or in Maryland? GE Healthcare will stop
supporting this model in a few years, and I am not sure if they are willing
to cover an old machine. I remember someone recommended a local service
company in UK some time ago, so I am wondering if such a reliable service
exists in the US or better near Maryland.

Your suggestions are much appreciated.

Yong-Fu Li

[ccp4bb] Protein concentration vs Molecular wt...

[james09 pruza]

Dear Crystallographers,
Is there any rule of thumb for Protein concentration and molecular weight
for crystallization trials of a soluble protein? Looking for high molecular
wt. protein ~50kDa.
James.

Re: [ccp4bb] Regarding refinement in Refmac5

[Garib N Murshudov]

1) Check free R flag. It may be related with free flag being 1 instead of 0. If 
it is the case then you can use newer version of refmac:
http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/

or from ccp4 v 6.3

2) Try arp/warp

Garib
On 19 Jul 2012, at 18:29, Deepthi wrote:

> Hi
> 
> Yes i did check my MTZ file header. It shows p3221. For the same reason i 
> reprocessed the data again in both p3221, p3121 and p321. Except for p3221 
> none of the other space groups fit the model. The packing looks good and the 
> map shows side chains very clearly. When i add the respective side chain its 
> just not accepting. May be it is not the correct solution? Is it possible?
> 
> Thank You
> Deepthi
> 
> On Thu, Jul 19, 2012 at 7:04 AM, Eleanor Dodson  
> wrote:
> It isn't that your space group is wrong, but are you sure that your mtz file 
> has that space group in its header? 
> MR will test all possible alternatives - in this case P3121 or P3221 - but 
> won't change the symmetry information in the input mtz. 
> You need to do that with a utility like 
> mtzutils hklin1 p3121.mtz hklout p3221.mtz
> SYMM P3221
> end
> 
> And there are various options in the REFLECTION UTILITIES.
> 
> The integration and data processing are exactly the same for either space 
> group, but REFMAC does not check that your mtz and pdb have the SAME symmetry 
> information, and by default uses that in the mtz file. So you could have a 
> perfectly good solution in P3221 but be running refinement in P3121, which is 
> NOT GOOD!
> Eleanor.
> 
> 
> On 18 Jul 2012, at 17:50, Deepthi wrote:
> 
>> I tried opening the model with other spacegroups MTZ file. The map doesn't 
>> fit well for other spacegroups. The initial model was refined using Phenix 
>> Autobuild software. I tried MR with every spacegroup possible in primitive 
>> hexagonal. Only p3221 worked. There is no twinning in the crystal. I will 
>> try using other softwares for refinement but this is annoying. I also tried 
>> mutating the  model to poly alanines and refine but this made it worse. The 
>> R-free went up to 0.546. 
>> I initially thought it might be a space group problem but trying other space 
>> groups doesn't work either.
>> 
>> Thank youvery much  for the help
>> Deepthi 
>> 
>> On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic 
>>  wrote:
>> Hi there,
>> 
>> Not much information provided. How was the initial model refined ? Phenix ? 
>> It could be a problem with the Refmac refinement protocol (difficult to say 
>> with so little information) if you switched from Phenix to Refmac.
>> 
>> How certain are you 1 - of the space group; 2 - that the crystal wasn't 
>> twinned ? You can have both and it can be "annoying".
>> 
>> Further, at this resolution I think you could use one of the SHELXes (forgot 
>> the terminology) for refinement, that could be more appropriate.
>> 
>> F.V.
>> 
>> 
>> Deepthi wrote:
>> Hi all
>> 
>> I am working with a small mutant protein which is 56 amino acids long. The 
>> crystal diffracted at 1.4A0 and the space group is  p3221. I did molecular 
>> replacement using Phenix software with all the data (1.4A0) and got a 
>> solution. Phenix did auto building with waters and R-free was 0.3123.
>> 
>> I mutated some residues which don't align with the model protein  to 
>> Alanines. When i change the residues back to their respective side chains 
>> Refmac5 won't  refine it well. The maps looks clear( you can guess its 1.4A0 
>> data) but R-free is shooting up to 0.41. It is not accepting any changes to 
>> the Phenix generated model. I have no idea what is going on. Can anyone help 
>> me?
>> 
>> Thank You in advance
>> Deepthi
>> 
>> 
>> 
>> 
>> 
>> -- 
>> Deepthi
> 
> 
> 
> 
> -- 
> Deepthi

Dr Garib N Murshudov
Group Leader, 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] Regarding refinement in Refmac5

[Deepthi]

Hi

Yes i did check my MTZ file header. It shows p3221. For the same reason i
reprocessed the data again in both p3221, p3121 and p321. Except for p3221
none of the other space groups fit the model. The packing looks good and
the map shows side chains very clearly. When i add the respective side
chain its just not accepting. May be it is not the correct solution? Is it
possible?

Thank You
Deepthi

On Thu, Jul 19, 2012 at 7:04 AM, Eleanor Dodson
wrote:

> It isn't that your space group is wrong, but are you sure that your mtz
> file has that space group in its header?
> MR will test all possible alternatives - in this case P3121 or P3221 - but
> won't change the symmetry information in the input mtz.
> You need to do that with a utility like
> mtzutils hklin1 p3121.mtz hklout p3221.mtz
> SYMM P3221
> end
>
> And there are various options in the REFLECTION UTILITIES.
>
> The integration and data processing are exactly the same for either space
> group, but REFMAC does not check that your mtz and pdb have the SAME
> symmetry information, and by default uses that in the mtz file. So you
> could have a perfectly good solution in P3221 but be running refinement in
> P3121, which is NOT GOOD!
> Eleanor.
>
>
> On 18 Jul 2012, at 17:50, Deepthi wrote:
>
> I tried opening the model with other spacegroups MTZ file. The map doesn't
> fit well for other spacegroups. The initial model was refined using Phenix
> Autobuild software. I tried MR with every spacegroup possible in primitive
> hexagonal. Only p3221 worked. There is no twinning in the crystal. I will
> try using other softwares for refinement but this is annoying. I also tried
> mutating the  model to poly alanines and refine but this made it worse. The
> R-free went up to 0.546.
> I initially thought it might be a space group problem but trying other
> space groups doesn't work either.
>
> Thank youvery much  for the help
> Deepthi
>
> On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic <
> frederic.velli...@ibs.fr> wrote:
>
>> Hi there,
>>
>> Not much information provided. How was the initial model refined ? Phenix
>> ? It could be a problem with the Refmac refinement protocol (difficult to
>> say with so little information) if you switched from Phenix to Refmac.
>>
>> How certain are you 1 - of the space group; 2 - that the crystal wasn't
>> twinned ? You can have both and it can be "annoying".
>>
>> Further, at this resolution I think you could use one of the SHELXes
>> (forgot the terminology) for refinement, that could be more appropriate.
>>
>> F.V.
>>
>>
>> Deepthi wrote:
>>
>>> Hi all
>>>
>>> I am working with a small mutant protein which is 56 amino acids long.
>>> The crystal diffracted at 1.4A0 and the space group is  p3221. I did
>>> molecular replacement using Phenix software with all the data (1.4A0) and
>>> got a solution. Phenix did auto building with waters and R-free was 0.3123.
>>>
>>> I mutated some residues which don't align with the model protein  to
>>> Alanines. When i change the residues back to their respective side chains
>>> Refmac5 won't  refine it well. The maps looks clear( you can guess its
>>> 1.4A0 data) but R-free is shooting up to 0.41. It is not accepting any
>>> changes to the Phenix generated model. I have no idea what is going on. Can
>>> anyone help me?
>>>
>>> Thank You in advance
>>> Deepthi
>>>
>>>
>>>
>
>
> --
> Deepthi
>
>
>


-- 
Deepthi

Re: [ccp4bb] Stereo Microscope

[Sampson, Jared]

Our setup is an Olympus SZX-10 with both 1x and 2x objectives and 10x 
eyepieces.  It's nice having the different objectives for different things--a 
close up look at a tiny crystal requires the 2x, but the 1x is less bulky and 
has better depth-of-field, so it's better for freezing crystals.

The DP21 camera is also nice, and provides for simple save-to-USB-drive photos 
and movies and scale bars, and also displays the field of view on an LCD 
monitor, which is nice for teaching and discussion among lab members.  (To get 
the right value for the scale with a zoom microscope, though, it needs to be 
set up with the "objectives" as the zoom knob stop values and the "adapter" 
setting as the actual objective lens magnification.  A bit of a workaround, but 
it's been fine for us.)

Cheers,

--
Jared Sampson
Xiangpeng Kong Lab
NYU Langone Medical Center
550 First Ave MSB 398
New York, NY 10016
212-263-7898
http://kong.med.nyu.edu/

On Jul 18, 2012, at 2:26 PM, Roger Rowlett wrote:

We have an Olympus SZX-12 microscope and are running a 1X objective (with 
polarizer) and a 10 X eyepiece. The scope will zoom from approximately 10X-90X 
magnification. At 90X a 400 nL drop in a 96-well plate will nearly fill the 
field.

Cheers,

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

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

On 7/18/2012 1:03 PM, PRASENJIT BHAUMIK wrote:
Hello,
I am planning to purchase a stereo microscope for visualizing crystallization 
drops. I would be very grateful if someone let me know the “objective” and 
“binocular eyepiece” specifications for SZX-7 or SZ-61 (Olympus) to get good 
magnification.

Thank you.

Regards,

Prasenjit

[ccp4bb] aimless, anisotropy, and resolution cutoff?

[vincent Chaptal]

Dear all,

I'm dealing with anisotropic diffraction of a membrane protein.
I've read from previous threads that it is better to not cut off any 
data, and that aimless doesn't anisotropically scale the data any way.
So my question is: given my anisotropic diffraction, what resolution 
cutoff do i give aimless for scaling, and what statistics can i report? 
(log bellow)


- Do I scale at 2.85A knowing that there is at least data in one 
direction to this resolution? (The different Rs are all over the place, 
but i can site the Karplus Science paper for the use of CC for 
resolution cutoff)
- Do I scale at 3.47A as it is "the best overall estimate" (with decent 
R statistics).


# Aimless log #

Estimates of resolution limits: overall
   from half-dataset correlation CC(1/2) >  0.50: limit = 3.47A
   from Mn(I/sd) >  2.00: limit = 3.83A

Estimates of resolution limits in reciprocal lattice directions:
  Along 0.98 h + 0.18 l
   from half-dataset correlation CC(1/2) >  0.50: limit = 3.97A
   from Mn(I/sd) >  2.00: limit = 3.65A
  Along k axis
   from half-dataset correlation CC(1/2) >  0.50: limit = 5.12A
   from Mn(I/sd) >  2.00: limit = 5.23A
  Along -0.25 h + 0.97 l
   from half-dataset correlation CC(1/2) >  0.50: limit = 2.85A
   from Mn(I/sd) >  2.00: limit = 3.17A




Thank you for your input.
vincent



Le 4/25/12 5:40 PM, George Sheldrick a écrit :
I think that anything that irrevocably modifies the experimental data 
should be avoided whenever possible. Since anisotropic scaling is a 
relatively fast calculation and there are several ways of doing it, it 
is better to apply it locally when it is needed, e.g. in phasing 
(where it is applied by phaser and shelxe etc.) and refinement (with 
refmac or phenix_refine etc.). Provided that the standard deviations 
of the observed intensities are properly taken into account, 
anisotropic data truncation is not so important (i.e. as usual I agree 
with Garib and Phil).


George

On 04/25/2012 06:19 PM, Phil Evans wrote:

You can get the aimless documentation from

ftp://ftp.mrc-lmb.cam.ac.uk/pub/pre/aimless.html

pending its official release through CCP4

No it does not do anisotropic scaling as such. That needs some sort 
of model of the "ideal" intensity, probably best calculated from a model


I'm not sure that anisotropic cutoffs are a good idea. I believe 
Garib thinks they are not and I generally defer to him


Phil

On 25 Apr 2012, at 17:00, Bryan Lepore wrote:


wondering if aimless performs anisotropic scaling or "elliptical"
rejections lately.

I ask because:

[1] last I knew, scala did not
[2] I can't seem to google up the aimless manual as readily as scala

... also, what consesquence would mosflm anisotropic resolution limits
have on scaling (if aimless anisoscaling were true).

-Bryan





--

Vincent Chaptal, PhD

Institut de Biologie et Chimie des Protéines

Drug-resistance modulation and mechanism Laboratory

7 passage du Vercors

69007 LYON

FRANCE

+33 4 37 65 29 01

http://www.ibcp.fr

Re: [ccp4bb] off topic Thermal shift assay

[Dr. Lorenzo Finci]

Anita, In terms of the basics:Thermodynamic stability of a protein is related 
to Gibbs Free Energy of unfolding. The Gibbs Free Energy is made of 
temperature, enthalpy and entropy (Delta G =  Delta H - T Delta S). At Delta G 
equivalent to zero, the concentration of folded protein is equivalent to the 
unfolded protein. Thus, proteins are most stable at conditions where their 
melting temperature (Tm) is highest.  Protein solubility and stability are 
prerequisites for subsequent biochemical and biophysical analysis and 
characterization. The preparation of concentrated, soluble, and stable protein 
sample can often be a difficult task as proteins aggregate, precipitate, or 
denature. Protein stability and solubility is directly correlated to 
temperature, pH, buffer composition, salt composition, additives, and ligands. 
The fluorophore ( ANS, SYPRO) has a high quantum yield in a low dielectric 
medium. Protein unfolding exposes the hydrophobic core corresponding to the low 
dielectric medium, and the fluorophore is quenched in the solution. Melting a 
proteins internal amino acid side chains is a first order phase transition 
associated with absorption heat. The Differential Scanning Fluorimetric (DSC, 
or Thermofluor) assay gives a direct measurement of a proteins melting 
temperature (Tm).In terms of Applications:The Thermofluor assay is used for the 
optimization of solution for protein stabilization. This is utilized for 
protein preparations and biochemical assays, crystallization, as well as 
optimization of ligand binding affinity, including drug screening, a measure of 
protein cooperatively, and to probe function. In terms of Extrinsic Dyes:SYPRO 
Orange is by far the most popular, along with ANS as they both are utilized to 
bind to the hydrophobic core of the soluble protein. CPM has been recently 
utilized to study membrane proteins as it binds to buried cysteines. I would 
look at the paper by Stevens et al 2008, and for high throughput, I would look 
at the utilization of CPM in the presence of detergents by Fan et al 2011. In 
terms of limitations, there are advantages and disadvantages:Advantages include 
the small of quantity of protein needed, the low concentration needed, the 
reproducibility, and the possibility of simultaneous screening of multiple 
conditions. For disadvantages, the interactions between the dye chosen and 
other compounds may mask stabilization or cause artifacts, as well as difficult 
interpretation effects of oligomeric proteins that show multi-phasic unfolding 
characteristics. Also, most dyes are not applicable to conditions comprising 
hydrophobic additives such as detergents that are necessary for membrane 
proteins (Hence, applications utilizing CNS). I hope this helps, good luck with 
your studies!Sincerely,lorenzoLorenzo Ihsan FInci, Ph.D.Postdoctoral Scientist, 
Wang LaboratoryHarvard Medical SchoolDana-Farber Cancer InstituteBoston, MA 
Peking UniversityThe College of Life SciencesBeijing, China


Date: Thu, 19 Jul 2012 11:00:28 -0400
From: jubo...@jhsph.edu
Subject: Re: [ccp4bb] off topic Thermal shift assay
To: CCP4BB@JISCMAIL.AC.UK

Yes that is very true. But I assume true membrane proteins exclude high 
throughput :-)
However the cytoplasmic part might be fine in this case and then I would just 
go for Sypro Orange.
Jürgen

On Jul 19, 2012, at 10:39 AM, Edwin Pozharski wrote:My understanding is that 
the advantage of the thermofluor assay is that
you can test many conditions rapidly unless of course you have some kind
of high throughput CD spectrometer in mind.

> If you have a
CD available (not the one with music on it) you don't need a
> dye
just sufficient protein and you can thermal denature your protein
> assuming it contains some secondary structure elements.
>

> Jürgen
> 
> 
> On Jul 19, 2012, at
4:26 AM, anita p wrote:
> 
> Hi All,
> I want to
use a thermofluor for the thermal shift assay. My proteins are
>
cytoplasmic truncations of membrane protein. I have read about ANS,
> sypro-orange and CPM. Which is the once that is popularly used by
the
> crystallographers for condition optimization for
crystallization ??
> 
> I have read that it sypro orange
is not good for hydrophobic proteins and
> CPM can't be used with
DTT or bME in the buffer.
> I am a bit confused .
> Please
help
> thanks in advance
> Anita
> 
>
..
> Jürgen Bosch
> Johns Hopkins
University
> Bloomberg School of Public Health
>
Department of Biochemistry & Molecular Biology
> Johns Hopkins
Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
>
Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
>
http://lupo.jhsph.edu
> 
> 
> 
> 
> 


-- 
Edwin Pozharski, PhD
University of
Maryland, Baltimore

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W870

Re: [ccp4bb] off topic Thermal shift assay

[Bosch, Juergen]

Yes that is very true. But I assume true membrane proteins exclude high 
throughput :-)

However the cytoplasmic part might be fine in this case and then I would just 
go for Sypro Orange.

Jürgen


On Jul 19, 2012, at 10:39 AM, Edwin Pozharski wrote:

My understanding is that the advantage of the thermofluor assay is that you can 
test many conditions rapidly unless of course you have some kind of high 
throughput CD spectrometer in mind.

> If you have a CD available (not the one with music on it) you don't need a
> dye just sufficient protein and you can thermal denature your protein
> assuming it contains some secondary structure elements.
>
> Jürgen
>
>
> On Jul 19, 2012, at 4:26 AM, anita p wrote:
>
> Hi All,
> I want to use a thermofluor for the thermal shift assay. My proteins are
> cytoplasmic truncations of membrane protein. I have read about ANS,
> sypro-orange and CPM. Which is the once that is popularly used by the
> crystallographers for condition optimization for crystallization ??
>
> I have read that it sypro orange is not good for hydrophobic proteins and
> CPM can't be used with DTT or bME in the buffer.
> I am a bit confused .
> Please help
> thanks in advance
> Anita
>
> ..
> Jürgen Bosch
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
> Lab: +1-410-614-4894
> Fax: +1-410-955-2926
> http://lupo.jhsph.edu
>
>
>
>
>


--
Edwin Pozharski, PhD
University of Maryland, Baltimore

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

[ccp4bb] CCP4 Fukuoka workshop 29 Oct - 2 Nov 2012

[Charles Ballard]

Dear All,
 
We are pleased to announce the CCP4 Fukuoka Workshop. All details can be found 
at http://www.ccp4.ac.uk/schools/Japan-2012/
 
Title:
"CCP4 Fukuoka school: From data processing to structure refinement and beyond"
Dates: October 29 to November 2.
Site: Biomedical Research Station, Kyushu University, Kyushu, Japan.
 
The school content:
Software workshop: The workshop will feature many modern crystallographic 
software packages taught by authors and other experts. It will be organized in 
three Sections – lectures, tutorials and hands-on trouble-shooting. There will 
be model data sets available for tutorials but data, provided by participants, 
will have higher priority for the hands-on sessions.
 
Applicants:
Graduate students, postdoctoral researchers and young scientists at the 
assistant professor level are encouraged to apply. Only 20 applicants will be 
selected for participation. Participants of the workshop are strongly 
encouraged to bring their own problem data sets so the problems can be 
addressed during the hands-on sessions.
 
Application:
Application deadline is October 1. The application form, the program, contact 
info and other details can be found at http://www.ccp4.ac.uk/schools/Japan-2012/
 
Fees: 
There is no fee for the workshop, but students are responsible for their travel 
and accommodation costs.
 
Charles Ballard, Daisuke Kohda, Garib Murshudov
-- 
Scanned by iCritical.

Re: [ccp4bb] peptide fragment

[John Hinks]

Hi Leonard,

I would treat this like a MS/MS ion search experiment looking for the peptide 
via MASCOT or similar. Talk to some local Mass Spec people, I'm certain they 
could identify it and then you'd be closer to an explanation.

John.



Dr. John  Hinks
Syngenta
Jealott's Hill International Research Centre
Bracknell
Berkshire
RG42 6EY
United Kingdom

john.hi...@syngenta.com
www.syngenta.com

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Leonard 
Thomas
Sent: 18 July 2012 23:12
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] peptide fragment

Hello All,

I have been working on a structure for one of the research groups here
and have come across a peptide fragment of about 10 residues.  The
protein forms a tetramer and the peptide is found between two of the
monomers.  The protein itself does not require a  peptide for function
or formation for that matter.  Has anyone come across similar things?
and have a reference.  We are trying to figure out where it may have
come from.  My best guess so far is from somewhere in the purification
though I have no idea how long the protein sat around before
crystallization.

Cheers,

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571




This message may contain confidential information. If you are not the 
designated recipient, please notify the sender immediately, and delete the 
original and any copies. Any use of the message by you is prohibited. 

Re: [ccp4bb] off topic Thermal shift assay

[Edwin Pozharski]

My understanding is that the advantage of the thermofluor assay is that
you can test many conditions rapidly unless of course you have some kind
of high throughput CD spectrometer in mind.

> If you have a
CD available (not the one with music on it) you don't need a
> dye
just sufficient protein and you can thermal denature your protein
> assuming it contains some secondary structure elements.
>

> Jürgen
> 
> 
> On Jul 19, 2012, at
4:26 AM, anita p wrote:
> 
> Hi All,
> I want to
use a thermofluor for the thermal shift assay. My proteins are
>
cytoplasmic truncations of membrane protein. I have read about ANS,
> sypro-orange and CPM. Which is the once that is popularly used by
the
> crystallographers for condition optimization for
crystallization ??
> 
> I have read that it sypro orange
is not good for hydrophobic proteins and
> CPM can't be used with
DTT or bME in the buffer.
> I am a bit confused .
> Please
help
> thanks in advance
> Anita
> 
>
..
> Jürgen Bosch
> Johns Hopkins
University
> Bloomberg School of Public Health
>
Department of Biochemistry & Molecular Biology
> Johns Hopkins
Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
>
Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
>
http://lupo.jhsph.edu
> 
> 
> 
> 
> 


-- 
Edwin Pozharski, PhD
University of
Maryland, Baltimore

Re: [ccp4bb] Regarding refinement in Refmac5

[Eleanor Dodson]

It isn't that your space group is wrong, but are you sure that your mtz file 
has that space group in its header? 
MR will test all possible alternatives - in this case P3121 or P3221 - but 
won't change the symmetry information in the input mtz. 
You need to do that with a utility like 
mtzutils hklin1 p3121.mtz hklout p3221.mtz
SYMM P3221
end

And there are various options in the REFLECTION UTILITIES.

The integration and data processing are exactly the same for either space 
group, but REFMAC does not check that your mtz and pdb have the SAME symmetry 
information, and by default uses that in the mtz file. So you could have a 
perfectly good solution in P3221 but be running refinement in P3121, which is 
NOT GOOD!
Eleanor.


On 18 Jul 2012, at 17:50, Deepthi wrote:

> I tried opening the model with other spacegroups MTZ file. The map doesn't 
> fit well for other spacegroups. The initial model was refined using Phenix 
> Autobuild software. I tried MR with every spacegroup possible in primitive 
> hexagonal. Only p3221 worked. There is no twinning in the crystal. I will try 
> using other softwares for refinement but this is annoying. I also tried 
> mutating the  model to poly alanines and refine but this made it worse. The 
> R-free went up to 0.546. 
> I initially thought it might be a space group problem but trying other space 
> groups doesn't work either.
> 
> Thank youvery much  for the help
> Deepthi 
> 
> On Wed, Jul 18, 2012 at 9:36 AM, Vellieux Frederic  
> wrote:
> Hi there,
> 
> Not much information provided. How was the initial model refined ? Phenix ? 
> It could be a problem with the Refmac refinement protocol (difficult to say 
> with so little information) if you switched from Phenix to Refmac.
> 
> How certain are you 1 - of the space group; 2 - that the crystal wasn't 
> twinned ? You can have both and it can be "annoying".
> 
> Further, at this resolution I think you could use one of the SHELXes (forgot 
> the terminology) for refinement, that could be more appropriate.
> 
> F.V.
> 
> 
> Deepthi wrote:
> Hi all
> 
> I am working with a small mutant protein which is 56 amino acids long. The 
> crystal diffracted at 1.4A0 and the space group is  p3221. I did molecular 
> replacement using Phenix software with all the data (1.4A0) and got a 
> solution. Phenix did auto building with waters and R-free was 0.3123.
> 
> I mutated some residues which don't align with the model protein  to 
> Alanines. When i change the residues back to their respective side chains 
> Refmac5 won't  refine it well. The maps looks clear( you can guess its 1.4A0 
> data) but R-free is shooting up to 0.41. It is not accepting any changes to 
> the Phenix generated model. I have no idea what is going on. Can anyone help 
> me?
> 
> Thank You in advance
> Deepthi
> 
> 
> 
> 
> 
> -- 
> Deepthi

Re: [ccp4bb] off topic Thermal shift assay

[Bosch, Juergen]

If you have a CD available (not the one with music on it) you don't need a dye 
just sufficient protein and you can thermal denature your protein assuming it 
contains some secondary structure elements.

Jürgen


On Jul 19, 2012, at 4:26 AM, anita p wrote:

Hi All,
I want to use a thermofluor for the thermal shift assay. My proteins are 
cytoplasmic truncations of membrane protein. I have read about ANS, 
sypro-orange and CPM. Which is the once that is popularly used by the 
crystallographers for condition optimization for crystallization ??

I have read that it sypro orange is not good for hydrophobic proteins and CPM 
can't be used with DTT or bME in the buffer.
I am a bit confused .
Please help
thanks in advance
Anita

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

[ccp4bb] question about DM

[Vellieux Frederic]

Hi all,

Just a (naive?) question: how does one manage to introduce (and deal 
with) improper non-crystallographic symmetry in DM ? Or does one has to 
go to DMmulti for that (because, by definition, going from one crystal 
form to another crystal form is improper NCS) ?


Ta,

F. Vellieux

Re: [ccp4bb] peptide fragment

[Boaz Shaanan]

Hi,

The closest example I can think of is the extra density that was found in the 
groove of the MHC class I structure (Bjorkman et al., long time ago) which 
turned out to be a peptide that was co-purified. As we all know, it turned out 
to be one of the major findings of this study as it pointed to the way MHC's 
recognize peptides. 

 Cheers,

Boaz

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

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






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Leonard Thomas 
[lmtho...@ou.edu]
Sent: Thursday, July 19, 2012 1:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] peptide fragment

Hello All,

I have been working on a structure for one of the research groups here
and have come across a peptide fragment of about 10 residues.  The
protein forms a tetramer and the peptide is found between two of the
monomers.  The protein itself does not require a  peptide for function
or formation for that matter.  Has anyone come across similar things?
and have a reference.  We are trying to figure out where it may have
come from.  My best guess so far is from somewhere in the purification
though I have no idea how long the protein sat around before
crystallization.

Cheers,

Leonard Thomas Ph.D.
Macromolecular Crystallography Laboratory Manager
University of Oklahoma
Department of Chemistry and Biochemistry
Stephenson Life Sciences Research Center
101 Stephenson Parkway
Norman, OK 73019-5251

lmtho...@ou.edu
http://barlywine.chem.ou.edu
Office: (405)325-1126
Lab: (405)325-7571

Re: [ccp4bb] off topic Thermal shift assay

[Hüsnü Topal]

Dear Anita,

here is a link to a talk with recommended literature: 
http://thermofluor.org/resources/Maksel_Protein-Stability.pdf.

Probably, you will find there the answers you need.

I recommend to try several dyes if available, due to the different 
behaviour of proteins and no general rule for doing thermal shift assays.


good luck

Hüsnü Topal, Dr. rer. nat.

Protein Crystallography & Bioinformatics
Workgroup of Prof. Dr. W. Welte
Universitätsstr. 10
78457 Konstanz







Am 19.07.2012 10:26, schrieb anita p:

Hi All,
I want to use a thermofluor for the thermal shift assay. My proteins 
are cytoplasmic truncations of membrane protein. I have read about 
ANS, sypro-orange and CPM. Which is the once that is popularly used by 
the crystallographers for condition optimization for crystallization ??


I have read that it sypro orange is not good for hydrophobic proteins 
and CPM can't be used with DTT or bME in the buffer.

I am a bit confused .
Please help
thanks in advance
Anita

[ccp4bb] off topic Thermal shift assay

[anita p]

Hi All,
I want to use a thermofluor for the thermal shift assay. My proteins are
cytoplasmic truncations of membrane protein. I have read about ANS,
sypro-orange and CPM. Which is the once that is popularly used by the
crystallographers for condition optimization for crystallization ??

I have read that it sypro orange is not good for hydrophobic proteins and
CPM can't be used with DTT or bME in the buffer.
I am a bit confused .
Please help
thanks in advance
Anita