[ccp4bb] Available postdoctoral position

[Zheng, Lei]

One postdoctoral position is available immediately at the Center for Membrane 
Biology, Department of Biochemistry and Molecular Biology, The University of 
Texas Houston Medical School (http://www.uth.tmc.edu/cmb/). The fellow will 
focus on structural determination of important membrane proteins involved in 
lipid metabolism and signaling. The fellow will also work with other lipid 
experts in the center for structural and functional characterization.

The ideal candidate should be a self-motivated individual and have a recent PhD 
degree in structural biology or biochemistry. Decent experiences with standard 
molecular biology technique, protein crystallization and structural 
determination approaches are expected.

The laboratory locates in the Texas Medical Center campus which provides an 
excellent environment for academia and research. The lab is fully equipped for 
protein x-ray crystallographic study, including a Graphon LCP crystallization 
robot, a Rigaku rotating anode X-ray home source and immediate access to the 
Berkeley ALS beamline 4.2.2 in the framework of the molecular biology 
consortium.

To apply or request further information, please contact: Dr. Lei Zheng (E-mail: 
lei.zh...@uth.tmc.edu). The application should 
include a current resume and the names and addresses of three referees.

[ccp4bb] Reminder: CCP4 summer school at APS, in USA

[Sanishvili, Ruslan]

Dear Colleagues,

This is a reminder that the deadline for applications for the 5th annual CCP4 
Summer School "From data collection to structure refinement and beyond" is 
April 17, 2012. The school will take place from June 19 through June 26, 2012 
at the Advanced Photon Source (APS) near Chicago.

There is no registration fee for the school. The students will be responsible 
for their own travel and lodging expenses. These and other details (The 
program, the list of speakers, the application process, accommodations, site 
access, contacts etc) can be found at the workshop website at 
http://www.ccp4.ac.uk/schools/APS-2012/index.php
The school will include data collection, processing, structure solution, model 
building, refinement, validation, automation of many steps etc. Participants 
are encouraged to bring their own crystals, raw data or processed data for 
hands-on problem solving under the guidance of software developers and other 
experts.

Garib, Ronan and Nukri



Ruslan Sanishvili (Nukri), Ph.D.

GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Sanishvili, Ruslan
Sent: Monday, December 19, 2011 11:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Annual CCP4 summer school in USA, at APS, June 19-26

Dear Colleagues,

We are pleased to announce the fifth annual CCP4 summer school at Advanced 
Photon Source (APS), Argonne National Laboratory (ANL). All details can be 
found at http://www.ccp4.ac.uk/schools/APS-2012/

Title:
"CCP4 school: From data collection to structure refinement and beyond"
Dates: June 19 to 26.
Site: Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois 
(Near Chicago), USA

The school content:
Data collection workshop the first two days: beamline training and data 
collection on GM/CA-CAT beamlines 23ID-B and 23ID-D. For data collection, only 
the participants' crystals will be used.
Software workshop: The rest of the time after data collection 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 or crystals so the problems can 
be addressed during data collection workshop and/or hands-on sessions.

Application:
Application deadline is April 17. The application form, the program, contact 
info and other details can be found at http://www.ccp4.ac.uk/schools/APS-2012/

Fees: There is no fee for the workshop. The students will be responsible for 
their transportation and lodging. The workshop organizers will arrange 
economical lodging at the Argonne Guest House. The workshop will also cover the 
expenses for all meals and refreshments.

Garib, Ronan and Nukri




Ruslan Sanishvili (Nukri), Ph.D.

GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov

[ccp4bb] research software developer vacancy at EMBL Hamburg

[Victor Lamzin]

Dear all,

There is a staff member vacancy for a Research Software Developer at the 
EMBL Unit in Hamburg, Germany. The post holder will have a leading role 
in technical implementation and scientific development of the ARP/wARP 
software for crystallographic structure determination and the building 
of macromolecular models in 3D electron density maps generated from 
X-ray diffraction and, potentially, from electron microscopy and 
free-electron laser based data.


Application deadline is 22nd April, 2012.

For details see:

http://ig14.i-grasp.com//fe/tpl_embl01.asp?newms=jj&id=48242&aid=15470

With best regards,
Victor

Re: [ccp4bb] sudden drop in R/Rfree

[Ian Tickle]

On 2 March 2012 18:01, Jacob Keller  wrote:
> Can't there be a "group of atoms?"

For sure, but doesn't a given parameter either apply to a single atom
or to a group of atoms?

Cheers

-- Ian

Re: [ccp4bb] sudden drop in R/Rfree

[Jacob Keller]

Can't there be a "group of atoms?"

JPK

On Fri, Mar 2, 2012 at 12:00 PM, Ian Tickle  wrote:

> > I'm aware of this document. Personally I prefer "ADP = Atomic
> Displacement
> > Parameters" over anything ele, because, given that Atomic Displacement
> > Parameters can be parameterized in many different ways, it makes it
> easier
> > to operate with such terms like:
> >
> > - isotropic Atomic Displacement Parameters (isotropic ADP);
> > - anisotropic Atomic Displacement Parameters (anisotropic ADP);
> > - group Atomic Displacement Parameters (group ADP);
> > - group isotropic Atomic Displacement Parameters (group isotropic ADP;
> > example: when refining one isotropic ADP per set of selected atoms);
> > - group anisotropic Atomic Displacement Parameters (group anisotropic
> ADP;
> > example TLS);
> > - ... etc, etc...
> >
> > (no intention to open another "can of worms" ! -:) )
>
> Hi Pavel, but couldn't you simplify it further by omitting the word
> "atomic" throughout?  In fact isn't "group (an)isotropic Atomic
> Displacement Parameter" a contradiction in terms?  Surely it can be
> either a group parameter or an atomic parameter but not both at the
> same time?
>
> I think the worms are already out and making good their escape :).
>
> Cheers
>
> -- Ian
>



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

Re: [ccp4bb] sudden drop in R/Rfree

[Ian Tickle]

> I'm aware of this document. Personally I prefer "ADP = Atomic Displacement
> Parameters" over anything ele, because, given that Atomic Displacement
> Parameters can be parameterized in many different ways, it makes it easier
> to operate with such terms like:
>
> - isotropic Atomic Displacement Parameters (isotropic ADP);
> - anisotropic Atomic Displacement Parameters (anisotropic ADP);
> - group Atomic Displacement Parameters (group ADP);
> - group isotropic Atomic Displacement Parameters (group isotropic ADP;
> example: when refining one isotropic ADP per set of selected atoms);
> - group anisotropic Atomic Displacement Parameters (group anisotropic ADP;
> example TLS);
> - ... etc, etc...
>
> (no intention to open another "can of worms" ! -:) )

Hi Pavel, but couldn't you simplify it further by omitting the word
"atomic" throughout?  In fact isn't "group (an)isotropic Atomic
Displacement Parameter" a contradiction in terms?  Surely it can be
either a group parameter or an atomic parameter but not both at the
same time?

I think the worms are already out and making good their escape :).

Cheers

-- Ian

Re: [ccp4bb] sudden drop in R/Rfree

[Pavel Afonine]

Thanks Ian,

I'm aware of this document. Personally I prefer "ADP = Atomic Displacement
Parameters" over anything ele, because, given that Atomic Displacement
Parameters can be parameterized in many different ways, it makes it easier
to operate with such terms like:

- isotropic Atomic Displacement Parameters (isotropic ADP);
- anisotropic Atomic Displacement Parameters (anisotropic ADP);
- group Atomic Displacement Parameters (group ADP);
- group isotropic Atomic Displacement Parameters (group isotropic ADP;
example: when refining one isotropic ADP per set of selected atoms);
- group anisotropic Atomic Displacement Parameters (group anisotropic ADP;
example TLS);
- ... etc, etc...

(no intention to open another "can of worms" ! -:) )

All the best,
Pavel

On Fri, Mar 2, 2012 at 9:38 AM, Ian Tickle  wrote:

> > (*) ADP = Atomic Displacement Parameters
>
> or "anisotropic displacement parameters"?
>
> See http://ww1.iucr.org/comm/cnom/adp/finrepone/finrepone.html
>
> Section 1.5 Comments about terminology
>
> Cheers
>
> -- Ian
>

Re: [ccp4bb] sudden drop in R/Rfree

[Jacob Keller]

> (*) ADP = Atomic Displacement Parameters

But aren't *isotropic* b-factors subsumed under this TLA (three-letter
acronym?)

JPK




On Fri, Mar 2, 2012 at 11:38 AM, Ian Tickle  wrote:

> > (*) ADP = Atomic Displacement Parameters
>
> or "anisotropic displacement parameters"?
>
> See http://ww1.iucr.org/comm/cnom/adp/finrepone/finrepone.html
>
> Section 1.5 Comments about terminology
>
> Cheers
>
> -- Ian
>



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

Re: [ccp4bb] sudden drop in R/Rfree

[Ian Tickle]

> (*) ADP = Atomic Displacement Parameters

or "anisotropic displacement parameters"?

See http://ww1.iucr.org/comm/cnom/adp/finrepone/finrepone.html

Section 1.5 Comments about terminology

Cheers

-- Ian

Re: [ccp4bb] sudden drop in R/Rfree

[Pavel Afonine]

Hi,

At 3.3A I would recommend trying a TLS model _instead_ of refining
> individual B factors.



may be it is implementation/software/mindset dependent, but in
phenix.refine refining TLS+individual ADP or simply individual ADP(*) is a
better option most of the time at low resolution.
For details see pages 30 and 31 here:
http://phenix-online.org/newsletter/CCN_2010_07.pdf

Pavel

(*) ADP = Atomic Displacement Parameters

Re: [ccp4bb] sudden drop in R/Rfree

[Rajesh kumar]

Dear All,
Thanks for all the suggestions. Lot to learn when its low resolution.
I have few more details5th round of refinement gave R/Rfree 0.2016/0.2767 
after reducing waters and fixing some outliers and difference has gone up to 
7.5%
I have used  Buster 2.10.0 for refinement. First round after molecular 
replacement I used Refmac for refinement. There after I used Buster with 
autoncs_noprune and sim_swap_equiv_plus as it was low resolution in 2nd and 3rd 
round. Fourth round I used -autoncs and -TLS options. Here are the details of 
NCS and  TLS paprameters.
REMARK   3   SIMILARITY.
REMARK   3NCS.  
REMARK   3 NCS REPRESENTATION : RESTRAINT LSSR (-AUTONCS)   
REMARK   3TARGET RESTRAINTS.
REMARK   3 TARGET REPRESENTATION : NONE 
REMARK   3 TARGET STRUCTURE : NULL  
REMARK   3  
REMARK   3  TLS DETAILS.
REMARK   3   NUMBER OF TLS GROUPS  :2   
REMARK   3  
REMARK   3   TLS GROUP :1   
REMARK   3SET : { A|* } 
REMARK   3ORIGIN FOR THE GROUP (A):  -40.7570   45.89640.0654   
REMARK   3T TENSOR  
REMARK   3 T11:0.6834 T22:0.5325
REMARK   3 T33:   -0.6079 T12:   -0.0436
REMARK   3 T13:   -0.1033 T23:0.0313
REMARK   3L TENSOR  
REMARK   3 L11:1.6212 L22:0.8001
REMARK   3 L33:6.4728 L12:0.0460
REMARK   3 L13:0.0051 L23:0.7670
REMARK   3S TENSOR  
REMARK   3 S11:   -0.2854 S12:0.5434 S13:0.0788 
REMARK   3 S21:   -0.5270 S22:0.3608 S23:0.0731 
REMARK   3 S31:   -0.7141 S32:0.2459 S33:   -0.0754 
REMARK   3  
REMARK   3   TLS GROUP :2   
REMARK   3SET : { B|* } 
REMARK   3ORIGIN FOR THE GROUP (A):  -29.9645   52.6711   27.6973   
REMARK   3T TENSOR  
REMARK   3 T11:0.5441 T22:0.5948
REMARK   3 T33:   -0.6079 T12:   -0.0298
REMARK   3 T13:   -0.0992 T23:   -0.0084
REMARK   3L TENSOR  
REMARK   3 L11:1.7807 L22:1.1381
REMARK   3 L33:4.5990 L12:0.7938
REMARK   3 L13:0.4959 L23:1.3513
REMARK   3S TENSOR  
REMARK   3 S11:   -0.2911 S12:   -0.0941 S13:0.1302 
REMARK   3 S21:   -0.3180 S22:0.1719 S23:   -0.0989 
REMARK   3 S31:   -1.2554 S32:0.5321 S33:0.1192
I deleted Lot of waters on Ethan's suggestion and now have only 12 of them. I 
am refining now and it takes a while for a buster run in our computer and I 
will let you know the results.
So, I would appreciate if you could suggest what NCS restrains are needed and 
where they were needed and why,  if the above method is not a systematic 
approach use NCS after obtaining a solution. And I have same question for the 
TLS. I guess this would benefit many people like me who are dealing with their 
first low resolution data.
Many thanks to Francis for pointing good reference.Let me know if I need to 
give more information.I appreciate all the suggestions and your valuable time.
Thanks,RajDate: Fri, 2 Mar 2012 16:45:04 +
From: twom...@globalphasing.com
Subject: Re: [ccp4bb] sudden drop in R/Rfree
To: CCP4BB@JISCMAIL.AC.UK




On 2 Mar 2012, at 16:02, Regina Kettering wrote:Rajesh;
I am not sure that you have a high enough data:refinement parameters ratio to 
refine TLS.  It just adds more parameters to refine that can lead to 
over-refinement of your model, especially at the 3.3 A. 
TLS on

[ccp4bb] Na acetate buffer, purification of a high pI protein...wash H6 collumn with 1+M NaCl

[Paul Kraft]

I have purified dozens of very high pI viral proteins, and I can't stress 
enough the requirement to wash your protein with at least 1M NaCl after binding 
it to the histidine collumn. DNA fragments often require a 2M NaCl wash. High 
pI proteins are very soluble.


Dr. Paul Kraft
Structural Biologist
cell 586-596-2770
email: haresea...@yahoo.com
email: kraft_proteome_resea...@yahoo.com




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Re: [ccp4bb] Na acetate as purification buffer

[Kevin Jin]

Maybe you can this way.

Use Na acetate to elute your protein, then use EDTA to remove the Ni
from your protein, then  buffer exchange or dialysis to remove EDTA.



Kevin

On Fri, Mar 2, 2012 at 5:50 AM, Santosh  wrote:
> Hi Anita,
> As Artem noted, use of Histrap column at lower pH would not be a great idea
> and unless you want to elute your protein using pH Step gradient
> http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/A960DBAAE1C0C945C1257628001D29BB/$file/28404480AA.pdf
> You may also consider using CM (Carboxy Methyl weak cation exchanger) column
> at pH6.8 and load the peak fractions of your protein directly on Histrap in
> 20mM Hepes pH6.8 in higher salt ( match the conductivity using conductivity
> meter, high salt will rescue your protein from falling out of solution to
> some extent, you can always optimize salt up to 500mM)
> At this point you can concentrate your peak fractions in buffer of your
> choice before loading on SEC using your buffer of choice. Check for
> aggregates on peak fractions using DLS you may also be able to screen
> buffers using DLS that has 384 well plate set up. It is important to know
> the range of different buffers and their long term shelf life and stability.
> http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html
> I hope it helps you figure out the optimum pH conditions for you target
> protein.
> Best,
> Santosh Hodawadekar, PhD
> http://www.linkedin.com/in/shodawadekar/
>
>
> On Fri, Mar 2, 2012 at 8:14 AM, Artem Evdokimov 
> wrote:
>>
>> Proteins with high apparent pi value are often tricky because they tend to
>> bind anionic substances such as nucleic acids, other proteins, glass, etc.
>> Conversely given that most proteins have acidic-ish apparent pi its often
>> worth looking into why a particular protein is basic as it may be a fact of
>> biological relevance! Basic proteins can be purified using acidic resins
>> which is often a boon due to the same considerations (less competition, easy
>> separation). Of course the actual pi of a protein can deviate substantially
>> from its theoretical pi however it is fairly safe to assume that for a
>> theoretical pi of 9 the apprent pi will be at least above 8 provided there
>> is sufficient *number* of charged groups (I.e. this prediction is not based
>> on a lonely lysine and protein nterm quietly crying in its corner). The more
>> charges there are, the more likely theoretical and apparent pi are at least
>> in the same ballpark...
>>
>> His-based affinity chromatogaphy on immobilized metal works progressively
>> less effectively with ph decrease downwards from 7, pretty much regardless
>> of protein pi since his tag is not often involved in local interactions (and
>> if it is then it typically works badly as affinity tag due to steric
>> problems anyway). Ph 4 - 5 typically would elute bound protein off imac and
>> is in fact an optional elution metho if for some reason imidazole or
>> histidine are undesirable.
>>
>> Your mileage will vary.
>>
>> Artem
>>
>> On Mar 2, 2012 6:17 AM, "Carlos Kikuti"  wrote:
>>>
>>> You might be giving too much importance to the THEORETICAL pI of the
>>> protein. If it's supposed to be well charged at pH 7,4 (only a titration
>>> curve, and not simply knowing the pI will tell you this)  and it's still
>>> precipitating, the problem might be due to a bad fold, for instance, or to
>>> the lack of salt ...
>>>
>>> I've heard people saying that proteins with high pIs are more difficult
>>> to work with, but to be honest I don't know where that fear comes from.
>>>
>>> Carlos
>>>
>>> Em 02/03/2012, às 05:55, Artem Evdokimov escreveu:
>>>
>>> > This pH is generally incompatible with Ni IMAC, sorry :) If you have a
>>> > high pI your best bet is to employ ion exchange as primary capture,
>>> > specifically SP resin or if you're really lucky - CM resin. There are
>>> > only relatively few proteins in E. coli that bind to CM resin at pH 5
>>> > and virtually none (one-three) that will bind at pH 8. If your protein
>>> > still binds, then you're good to go.
>>> >
>>> > Artem
>>> >
>>> > On Thu, Mar 1, 2012 at 10:49 PM, anita p  wrote:
>>> >> Hi all,
>>> >> Has anyone used sodium acetate buffer pH (4-5) for purifying histag
>>> >> protein
>>> >> on Histrap column (AKTA) followed by SEC?
>>> >> My protein has a pI of 9. I tried pH7.4 but it has precipitation
>>> >> problems.
>>> >> While doing buffer screening using 24 well hanging drop I found that
>>> >> lower
>>> >> pI onces are clear, so just thinking can I use Na acetate at pH 5 for
>>> >> whole
>>> >> purification???
>>> >>
>>> >>
>>> >>  Thanks in advance
>>> >>  Anita
>
>

Re: [ccp4bb] sudden drop in R/Rfree

[Francis E Reyes]

I've found the following article  to be useful in defining suitable refinement 
strategies at a particular resolution. 

1.  Mueller, M., Jenni, S. & Ban, N. Strategies for crystallization and 
structure determination of very large macromolecular assemblies. Curr Opin 
Struct Biol 17, 572–579 (2007).

It can  be used as a rough guide line at the start of your refinement 
(incomplete model, fresh off of molecular replacement) with slow increase of 
refinement parameters the better your model becomes (and as Rfree permits). 


F
 





On Mar 2, 2012, at 9:39 AM, Ethan Merritt wrote:

> On Friday, 02 March 2012, Regina Kettering wrote:
>> Rajesh;
>> 
>> I am not sure that you have a high enough data:refinement parameters ratio 
>> to refine TLS. 
>> It just adds more parameters to refine that can lead to over-refinement of 
>> your model, 
>> especially at the 3.3 A. 
> 
> I'm afraid you've got this completely backwards.
> TLS uses very few parameters, and is especially useful at low resolution.
> At 3.3A I would recommend trying a TLS model _instead_ of refining
> individual B factors.
> 
> NCS restraints also help a lot at low resolution.
> 
> So the drop is believable, but...
> 
> You should first worry about "lots of waters were placed".
> It's there that many extra parameters have been added, perhaps leading
> to over-fitting.  I would not expect 3.3A data to justify placement of
> more than a handful of waters at most.
> 
> If you're parameter counting, you might note that 5 water molecules add 
> more parameters than 1 TLS model.  But the TLS model may improve the model
> everywhere, whereas the waters will only suppress a few local difference
> density peaks.
> 
>   cheers,
> 
>   Ethan
> 
> 
>> 
>> HTH,
>> 
>> Regina
>> 
>> 
>> 
>> 
>> From: Rajesh kumar 
>> To: CCP4BB@JISCMAIL.AC.UK 
>> Sent: Friday, March 2, 2012 10:54 AM
>> Subject: [ccp4bb] sudden drop in R/Rfree
>> 
>> 
>> 
>> 
>> Dear All, 
>> 
>> I have a 3.3 A data for a protein whose SG is P6522. Model used was wild 
>> type structure of same protein at 2.3 A.
>> 
>> After molecular replacement, first three rounds of refinement the R/Rf was  
>> 26/32.8,  27.1/31.72 % and 7.35/30.88 % respectively.
>> In the fourth round I refined with TLS and NCS abd added water and the R/Rf 
>> dropped to 19.34/26.46. It has almost 7% difference. I also see lot of 
>> unanswerable density in the map where lot of waters were placed. Model fits 
>> to the map like a low resolution data with most of side chains don't have 
>> best density.
>> 
>> I was not expecting such a sudden drop in the R/Rfree and a difference is 
>> 7.2%. 
>> I am wondering if I am in right direction. I am not sure if this usual for 
>> 3.3A data or in general any data if we consider the difference.
>> I appreciate your valuable  suggestions.
>> 
>> Thanks
>> Raj



-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] sudden drop in R/Rfree

[Thomas Womack]

On 2 Mar 2012, at 16:02, Regina Kettering wrote:

> Rajesh;
> 
> I am not sure that you have a high enough data:refinement parameters ratio to 
> refine TLS.  It just adds more parameters to refine that can lead to 
> over-refinement of your model, especially at the 3.3 A. 

TLS only adds twenty parameters per chain; so it's a really parsimonious thing 
to do at low resolution.

I'd say that adding lots of waters at 3.3A (at four parameters per added water) 
was much more likely to be the cause of a very wide R/Rfree gap.

I'm a bit worried that a user working at low resolution on a protein with more 
than one chain per ASU is not using NCS from the very beginning; that's another 
good way of adding more restraints and effectively getting the 
parametersto-data ratio down (because the 'parameters' in that ratio is really 
'parameters minus K * number of restraints'; there is scope for a lot of debate 
as to the right value of K, it clearly depends on the strength of the 
restraints)

If he's using the Global Phasing refinement software, I would strongly suggest 
that Rajesh use targetting to the initial molecular replacement result 
throughout the refinement, as yet a third way of adding more restraints.

Tom Womack (Global Phasing)

> 
> HTH,
> Regina
> 
> From: Rajesh kumar 
> To: CCP4BB@JISCMAIL.AC.UK 
> Sent: Friday, March 2, 2012 10:54 AM
> Subject: [ccp4bb] sudden drop in R/Rfree
> 
> 
> Dear All, 
> 
> I have a 3.3 A data for a protein whose SG is P6522. Model used was wild type 
> structure of same protein at 2.3 A.
>  
> After molecular replacement, first three rounds of refinement the R/Rf was  
> 26/32.8,  27.1/31.72 % and 7.35/30.88 % respectively.
> In the fourth round I refined with TLS and NCS abd added water and the R/Rf 
> dropped to 19.34/26.46. It has almost 7% difference. I also see lot of 
> unanswerable density in the map where lot of waters were placed. Model fits 
> to the map like a low resolution data with most of side chains don't have 
> best density.
> 
> I was not expecting such a sudden drop in the R/Rfree and a difference is 
> 7.2%. 
> I am wondering if I am in right direction. I am not sure if this usual for 
> 3.3A data or in general any data if we consider the difference.
>  I appreciate your valuable  suggestions.
> 
> Thanks
> Raj
> 
> 
> 
> 

Re: [ccp4bb] sudden drop in R/Rfree

[Ethan Merritt]

On Friday, 02 March 2012, Regina Kettering wrote:
> Rajesh;
> 
> I am not sure that you have a high enough data:refinement parameters ratio to 
> refine TLS. 
> It just adds more parameters to refine that can lead to over-refinement of 
> your model, 
> especially at the 3.3 A. 

I'm afraid you've got this completely backwards.
TLS uses very few parameters, and is especially useful at low resolution.
At 3.3A I would recommend trying a TLS model _instead_ of refining
individual B factors.

NCS restraints also help a lot at low resolution.

So the drop is believable, but...

You should first worry about "lots of waters were placed".
It's there that many extra parameters have been added, perhaps leading
to over-fitting.  I would not expect 3.3A data to justify placement of
more than a handful of waters at most.

If you're parameter counting, you might note that 5 water molecules add 
more parameters than 1 TLS model.  But the TLS model may improve the model
everywhere, whereas the waters will only suppress a few local difference
density peaks.

cheers,

Ethan


> 
> HTH,
> 
> Regina
> 
> 
> 
> 
>  From: Rajesh kumar 
> To: CCP4BB@JISCMAIL.AC.UK 
> Sent: Friday, March 2, 2012 10:54 AM
> Subject: [ccp4bb] sudden drop in R/Rfree
>  
> 
>  
> 
> Dear All, 
> 
> I have a 3.3 A data for a protein whose SG is P6522. Model used was wild type 
> structure of same protein at 2.3 A.
>  
> After molecular replacement, first three rounds of refinement the R/Rf was  
> 26/32.8,  27.1/31.72 % and 7.35/30.88 % respectively.
> In the fourth round I refined with TLS and NCS abd added water and the R/Rf 
> dropped to 19.34/26.46. It has almost 7% difference. I also see lot of 
> unanswerable density in the map where lot of waters were placed. Model fits 
> to the map like a low resolution data with most of side chains don't have 
> best density.
> 
> I was not expecting such a sudden drop in the R/Rfree and a difference is 
> 7.2%. 
> I am wondering if I am in right direction. I am not sure if this usual for 
> 3.3A data or in general any data if we consider the difference.
>  I appreciate your valuable  suggestions.
> 
> Thanks
> Raj

Re: [ccp4bb] sudden drop in R/Rfree

[Regina Kettering]

Rajesh;

I am not sure that you have a high enough data:refinement parameters ratio to 
refine TLS.  It just adds more parameters to refine that can lead to 
over-refinement of your model, especially at the 3.3 A. 

HTH,

Regina




 From: Rajesh kumar 
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Friday, March 2, 2012 10:54 AM
Subject: [ccp4bb] sudden drop in R/Rfree
 

 

Dear All, 

I have a 3.3 A data for a protein whose SG is P6522. Model used was wild type 
structure of same protein at 2.3 A.
 
After molecular replacement, first three rounds of refinement the R/Rf was  
26/32.8,  27.1/31.72 % and 7.35/30.88 % respectively.
In the fourth round I refined with TLS and NCS abd added water and the R/Rf 
dropped to 19.34/26.46. It has almost 7% difference. I also see lot of 
unanswerable density in the map where lot of waters were placed. Model fits to 
the map like a low resolution data with most of side chains don't have best 
density.

I was not expecting such a sudden drop in the R/Rfree and a difference is 7.2%. 
I am wondering if I am in right direction. I am not sure if this usual for 3.3A 
data or in general any data if we consider the difference.
 I appreciate your valuable  suggestions.

Thanks
Raj

[ccp4bb] sudden drop in R/Rfree

[Rajesh kumar]

Dear All, 
I have a 3.3 A data for a protein whose SG is P6522. Model used was wild type 
structure of same protein at 2.3 A. After molecular replacement, first three 
rounds of refinement the R/Rf was  26/32.8,  27.1/31.72 % and 7.35/30.88 % 
respectively.In the fourth round I refined with TLS and NCS abd added water and 
the R/Rf dropped to 19.34/26.46. It has almost 7% difference. I also see lot of 
unanswerable density in the map where lot of waters were placed. Model fits to 
the map like a low resolution data with most of side chains don't have best 
density.
I was not expecting such a sudden drop in the R/Rfree and a difference is 7.2%. 
I am wondering if I am in right direction. I am not sure if this usual for 3.3A 
data or in general any data if we consider the difference. I appreciate your 
valuable  suggestions.
ThanksRaj

  

Re: [ccp4bb] Na acetate as purification buffer

[Santosh]

Hi Anita,
As Artem noted, use of Histrap column at lower pH would not be a great idea
and unless you want to elute your protein using pH Step gradient
http://www.gelifesciences.com/aptrix/upp00919.nsf/Content/A960DBAAE1C0C945C1257628001D29BB/$file/28404480AA.pdf
You may also consider using CM (Carboxy Methyl weak cation exchanger)
column at pH6.8 and load the peak fractions of your protein directly on
Histrap in 20mM Hepes pH6.8 in higher salt ( match the conductivity using
conductivity meter, high salt will rescue your protein from falling out of
solution to some extent, you can always optimize salt up to 500mM)
At this point you can concentrate your peak fractions in buffer of your
choice before loading on SEC using your buffer of choice. Check for
aggregates on peak fractions using DLS you may also be able to screen
buffers using DLS that has 384 well plate set up. It is important to know
the range of different buffers and their long term shelf life and stability.
http://www.sigmaaldrich.com/life-science/core-bioreagents/biological-buffers/learning-center/buffer-reference-center.html
I hope it helps you figure out the optimum pH conditions for you target
protein.
Best,
Santosh Hodawadekar, PhD
http://www.linkedin.com/in/shodawadekar/

On Fri, Mar 2, 2012 at 8:14 AM, Artem Evdokimov
wrote:

> Proteins with high apparent pi value are often tricky because they tend to
> bind anionic substances such as nucleic acids, other proteins, glass, etc.
> Conversely given that most proteins have acidic-ish apparent pi its often
> worth looking into why a particular protein is basic as it may be a fact of
> biological relevance! Basic proteins can be purified using acidic resins
> which is often a boon due to the same considerations (less competition,
> easy separation). Of course the actual pi of a protein can deviate
> substantially from its theoretical pi however it is fairly safe to assume
> that for a theoretical pi of 9 the apprent pi will be at least above 8
> provided there is sufficient *number* of charged groups (I.e. this
> prediction is not based on a lonely lysine and protein nterm quietly crying
> in its corner). The more charges there are, the more likely theoretical and
> apparent pi are at least in the same ballpark...
>
> His-based affinity chromatogaphy on immobilized metal works progressively
> less effectively with ph decrease downwards from 7, pretty much regardless
> of protein pi since his tag is not often involved in local interactions
> (and if it is then it typically works badly as affinity tag due to steric
> problems anyway). Ph 4 - 5 typically would elute bound protein off imac and
> is in fact an optional elution metho if for some reason imidazole or
> histidine are undesirable.
>
> Your mileage will vary.
>
> Artem
> On Mar 2, 2012 6:17 AM, "Carlos Kikuti"  wrote:
>
>> You might be giving too much importance to the THEORETICAL pI of the
>> protein. If it's supposed to be well charged at pH 7,4 (only a titration
>> curve, and not simply knowing the pI will tell you this)  and it's still
>> precipitating, the problem might be due to a bad fold, for instance, or to
>> the lack of salt ...
>>
>> I've heard people saying that proteins with high pIs are more difficult
>> to work with, but to be honest I don't know where that fear comes from.
>>
>> Carlos
>>
>> Em 02/03/2012, às 05:55, Artem Evdokimov escreveu:
>>
>> > This pH is generally incompatible with Ni IMAC, sorry :) If you have a
>> > high pI your best bet is to employ ion exchange as primary capture,
>> > specifically SP resin or if you're really lucky - CM resin. There are
>> > only relatively few proteins in E. coli that bind to CM resin at pH 5
>> > and virtually none (one-three) that will bind at pH 8. If your protein
>> > still binds, then you're good to go.
>> >
>> > Artem
>> >
>> > On Thu, Mar 1, 2012 at 10:49 PM, anita p  wrote:
>> >> Hi all,
>> >> Has anyone used sodium acetate buffer pH (4-5) for purifying histag
>> protein
>> >> on Histrap column (AKTA) followed by SEC?
>> >> My protein has a pI of 9. I tried pH7.4 but it has precipitation
>> problems.
>> >> While doing buffer screening using 24 well hanging drop I found that
>> lower
>> >> pI onces are clear, so just thinking can I use Na acetate at pH 5 for
>> whole
>> >> purification???
>> >>
>> >>
>> >>  Thanks in advance
>> >>  Anita
>>
>

Re: [ccp4bb] Na acetate as purification buffer

[Roger Rowlett]

  
  
Classical protein purification by IEX, HIC,
  GEC, etc. is apparently a dying art. It is typically quite easy to
  purify proteins using non-affinity methods from overexpression
  mixtures using an AKTA system. (Gosh, in the old days we used to
  purify to homogeneity proteins with 0.1% abundance in a natural
  source--a 15% overexpression crude extract is trivial by
comparison.) It is fairly quick to scout good step gradient
conditions for partially purifying your protein by ion exchange by
using a 1 mL or 5 mL ion-exchange column, then scale up to a 1.6x10
cm or 2.6x10 cm column, depending on your crude extract sample
volume. (We maintain Q- and SP-sepharose columns in our lab for low-
and high-pI proteins, respectively.) A secondary purification via
hydrophobic interaction or even salt fractionation is typically
sufficient to clean up well-overexpressed proteins. Desalting and
polishing can be accomplished on a large (1.6x60 cm) gel exclusion
column. We purify our current crop of proteins we are studying this
way--no tags to remove later. Sometimes, the time saved by affinity
purification on the front end is eaten up on the back end with the
necessity for purification tag removal. Don't be afraid to give the
"old" methods a go. As Artem has pointed out, if your target protein
has an unusual pI, it may essentially purify in one step (IEX) and
you can then do a quick polish on GEC.

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 3/1/2012 11:55 PM, Artem Evdokimov wrote:

  This pH is generally incompatible with Ni IMAC, sorry :) If you have a
high pI your best bet is to employ ion exchange as primary capture,
specifically SP resin or if you're really lucky - CM resin. There are
only relatively few proteins in E. coli that bind to CM resin at pH 5
and virtually none (one-three) that will bind at pH 8. If your protein
still binds, then you're good to go.

Artem

On Thu, Mar 1, 2012 at 10:49 PM, anita p  wrote:

  
Hi all,
Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein
on Histrap column (AKTA) followed by SEC?
My protein has a pI of 9. I tried pH7.4 but it has precipitation problems.
While doing buffer screening using 24 well hanging drop I found that lower
pI onces are clear, so just thinking can I use Na acetate at pH 5 for whole
purification???


 Thanks in advance
 Anita

  

  

Re: [ccp4bb] Na acetate as purification buffer

[Artem Evdokimov]

Proteins with high apparent pi value are often tricky because they tend to
bind anionic substances such as nucleic acids, other proteins, glass, etc.
Conversely given that most proteins have acidic-ish apparent pi its often
worth looking into why a particular protein is basic as it may be a fact of
biological relevance! Basic proteins can be purified using acidic resins
which is often a boon due to the same considerations (less competition,
easy separation). Of course the actual pi of a protein can deviate
substantially from its theoretical pi however it is fairly safe to assume
that for a theoretical pi of 9 the apprent pi will be at least above 8
provided there is sufficient *number* of charged groups (I.e. this
prediction is not based on a lonely lysine and protein nterm quietly crying
in its corner). The more charges there are, the more likely theoretical and
apparent pi are at least in the same ballpark...

His-based affinity chromatogaphy on immobilized metal works progressively
less effectively with ph decrease downwards from 7, pretty much regardless
of protein pi since his tag is not often involved in local interactions
(and if it is then it typically works badly as affinity tag due to steric
problems anyway). Ph 4 - 5 typically would elute bound protein off imac and
is in fact an optional elution metho if for some reason imidazole or
histidine are undesirable.

Your mileage will vary.

Artem
On Mar 2, 2012 6:17 AM, "Carlos Kikuti"  wrote:

> You might be giving too much importance to the THEORETICAL pI of the
> protein. If it's supposed to be well charged at pH 7,4 (only a titration
> curve, and not simply knowing the pI will tell you this)  and it's still
> precipitating, the problem might be due to a bad fold, for instance, or to
> the lack of salt ...
>
> I've heard people saying that proteins with high pIs are more difficult to
> work with, but to be honest I don't know where that fear comes from.
>
> Carlos
>
> Em 02/03/2012, às 05:55, Artem Evdokimov escreveu:
>
> > This pH is generally incompatible with Ni IMAC, sorry :) If you have a
> > high pI your best bet is to employ ion exchange as primary capture,
> > specifically SP resin or if you're really lucky - CM resin. There are
> > only relatively few proteins in E. coli that bind to CM resin at pH 5
> > and virtually none (one-three) that will bind at pH 8. If your protein
> > still binds, then you're good to go.
> >
> > Artem
> >
> > On Thu, Mar 1, 2012 at 10:49 PM, anita p  wrote:
> >> Hi all,
> >> Has anyone used sodium acetate buffer pH (4-5) for purifying histag
> protein
> >> on Histrap column (AKTA) followed by SEC?
> >> My protein has a pI of 9. I tried pH7.4 but it has precipitation
> problems.
> >> While doing buffer screening using 24 well hanging drop I found that
> lower
> >> pI onces are clear, so just thinking can I use Na acetate at pH 5 for
> whole
> >> purification???
> >>
> >>
> >>  Thanks in advance
> >>  Anita
>

Re: [ccp4bb] Na acetate as purification buffer

[Carlos Kikuti]

You might be giving too much importance to the THEORETICAL pI of the protein. 
If it's supposed to be well charged at pH 7,4 (only a titration curve, and not 
simply knowing the pI will tell you this)  and it's still precipitating, the 
problem might be due to a bad fold, for instance, or to the lack of salt ...

I've heard people saying that proteins with high pIs are more difficult to work 
with, but to be honest I don't know where that fear comes from.

Carlos

Em 02/03/2012, às 05:55, Artem Evdokimov escreveu:

> This pH is generally incompatible with Ni IMAC, sorry :) If you have a
> high pI your best bet is to employ ion exchange as primary capture,
> specifically SP resin or if you're really lucky - CM resin. There are
> only relatively few proteins in E. coli that bind to CM resin at pH 5
> and virtually none (one-three) that will bind at pH 8. If your protein
> still binds, then you're good to go.
> 
> Artem
> 
> On Thu, Mar 1, 2012 at 10:49 PM, anita p  wrote:
>> Hi all,
>> Has anyone used sodium acetate buffer pH (4-5) for purifying histag protein
>> on Histrap column (AKTA) followed by SEC?
>> My protein has a pI of 9. I tried pH7.4 but it has precipitation problems.
>> While doing buffer screening using 24 well hanging drop I found that lower
>> pI onces are clear, so just thinking can I use Na acetate at pH 5 for whole
>> purification???
>> 
>> 
>>  Thanks in advance
>>  Anita

Re: [ccp4bb] twin refinement in refmac

[arka chakraborty]

Hi all,

Thanks for the express replies. Your insights along with the article by
Prof. Garib pointed to by Prof. Pavel completes the story for me.

Regards,

ARKO

On Fri, Mar 2, 2012 at 3:09 PM, Steiner, Roberto
wrote:

> On 2 Mar 2012, at 08:01, arka chakraborty wrote:
>
> Hi all,
>
> I will like to know, as a follow up of what Prof. Randy Read said, what
> should be done to do the refinement against the measured data and not the
> detwinned F( which refmac outputs in the mtz after twin refinement), during
> subsequent refinements.
>
>
> Operationally, don't put in the "MTZ in" field of the GUI something that
> Refmac generated for you in a previous run as "MTZ out" file. Always use as
> "MTZ in" your original data file.
>
> And also, I would like to know how to ensure that the free R generated
> takes twinning into account if I am not using phenix.
>
>
> Refmac does take in consideration the twin law(s) when handling free
> reflections . This is the case even if you have generated your free
> reflections randomly. Internally Refmac will modify your Free set in such a
> way that twin related reflections are in the same group (free or working)
> --> classes mentioned by Garib
>
> The good thing about this is that twin-related reflections are handled
> properly during refinement irrespective of your Free-set choice.
> The bad thing (Garib please correct me if I am wrong here) is that you
> might end up depositing a Free set which is not that actually used in
> refinement.
>
> Best wishes
> Roberto
>
>
>
> Thanks in advance,
>
> Regards,
>
> ARKO
>
> On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read  wrote:
>
>> I'm worried when you say that you use the initial job's output MTZ.
>> Refmac replaces F with a detwinned F in the output file so you wouldn't be
>> refining against your measured data in the subsequent round.
>>
>> Best wishes
>>
>> Randy Read
>>
>> 
>> Randy J. Read
>>
>> On 2 Mar 2012, at 02:00, wtempel  wrote:
>>
>> > Dear CCp4ers,
>> > A good morning to everyone.
>> > Today, I have a structure that I initially refined in space group
>> P6522, 1mol/asu.
>> > Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; / > 3
>> > 2.61-2.55A: Rsym=39.6%, / > 10
>> > 50.00-6.13: Rsym=6.4%
>> > Some mild anisotropy in the resolution limits is apparent on the
>> diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in
>> the other.
>> > Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like
>> 3.5A resolution, with some difference density for loops that cannot be
>> interpreted with reasonable geometry.
>> > Rsym is very similar for data scaled in P3, in all resolution shells.
>> Xtriage does not suggest merohedral twinning.
>> > Nevertheless, I extended my free flags in sftools from P6522 to P32 and
>> cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I
>> expanded my model to a homotetramer and ran Refmac with amplitude based
>> twinning. (Would this be a reasonable input to twin refinement?)
>> > From the output coordinates:
>> > REMARK   3  TWIN DETAILS
>> > REMARK   3   NUMBER OF TWIN DOMAINS  :4
>> > REMARK   3  TWIN DOMAIN   :1
>> > REMARK   3  TWIN OPERATOR :  H,  K,  L
>> > REMARK   3  TWIN FRACTION : 0.269
>> > REMARK   3  TWIN DOMAIN   :2
>> > REMARK   3  TWIN OPERATOR : -K, -H, -L
>> > REMARK   3  TWIN FRACTION : 0.171
>> > REMARK   3  TWIN DOMAIN   :3
>> > REMARK   3  TWIN OPERATOR :  K,  H, -L
>> > REMARK   3  TWIN FRACTION : 0.258
>> > REMARK   3  TWIN DOMAIN   :4
>> > REMARK   3  TWIN OPERATOR : -H, -K,  L
>> > REMARK   3  TWIN FRACTION : 0.302
>> > Does this establish twinning versus underestimated symmetry? And what
>> do I need to know about my free-R? Did refmac assign a new flag? Whereas
>> the output file's flags are all 1s and 0s, the input file had 0 ... 19.
>> During the first run, Rfree dropped to <28%. But on a subsequent run, Rfree
>> was stuck >30% when I used the initial job's output MTZ.
>> > Many thanks in advance for your helpful comments.
>> > Wolfram Tempel
>> >
>>
>
>
>
> --
>
> *ARKA CHAKRABORTY*
> *CAS in Crystallography and Biophysics*
> *University of Madras*
> *Chennai,India*
>
>
> Roberto Steiner, PhD
> Group Leader
> Randall Division of Cell and Molecular Biophysics
> King's College London
>
> Room 3.10A
> New Hunt's House
> Guy's Campus
> SE1 1UL, London, UK
> Tel 0044-20-78488216
> Fax 0044-20-78486435
> roberto.stei...@kcl.ac.uk
>
>
>
>
>


-- 

*ARKA CHAKRABORTY*
*CAS in Crystallography and Biophysics*
*University of Madras*
*Chennai,India*

Re: [ccp4bb] twin refinement in refmac

[Steiner, Roberto]

On 2 Mar 2012, at 08:01, arka chakraborty wrote:

Hi all,

I will like to know, as a follow up of what Prof. Randy Read said, what should 
be done to do the refinement against the measured data and not the detwinned F( 
which refmac outputs in the mtz after twin refinement), during subsequent 
refinements.

Operationally, don't put in the "MTZ in" field of the GUI something that Refmac 
generated for you in a previous run as "MTZ out" file. Always use as "MTZ in" 
your original data file.

And also, I would like to know how to ensure that the free R generated takes 
twinning into account if I am not using phenix.

Refmac does take in consideration the twin law(s) when handling free 
reflections . This is the case even if you have generated your free reflections 
randomly. Internally Refmac will modify your Free set in such a way that twin 
related reflections are in the same group (free or working) --> classes 
mentioned by Garib

The good thing about this is that twin-related reflections are handled properly 
during refinement irrespective of your Free-set choice.
The bad thing (Garib please correct me if I am wrong here) is that you might 
end up depositing a Free set which is not that actually used in refinement.

Best wishes
Roberto



Thanks in advance,

Regards,

ARKO

On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read 
mailto:rj...@cam.ac.uk>> wrote:
I'm worried when you say that you use the initial job's output MTZ. Refmac 
replaces F with a detwinned F in the output file so you wouldn't be refining 
against your measured data in the subsequent round.

Best wishes

Randy Read


Randy J. Read

On 2 Mar 2012, at 02:00, wtempel mailto:wtem...@gmail.com>> 
wrote:

> Dear CCp4ers,
> A good morning to everyone.
> Today, I have a structure that I initially refined in space group P6522, 
> 1mol/asu.
> Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; / > 3
> 2.61-2.55A: Rsym=39.6%, / > 10
> 50.00-6.13: Rsym=6.4%
> Some mild anisotropy in the resolution limits is apparent on the diffraction 
> images. Say, visible spots at 2.2A in one direction, 2.6A in the other.
> Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A 
> resolution, with some difference density for loops that cannot be interpreted 
> with reasonable geometry.
> Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage 
> does not suggest merohedral twinning.
> Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd 
> them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my 
> model to a homotetramer and ran Refmac with amplitude based twinning. (Would 
> this be a reasonable input to twin refinement?)
> From the output coordinates:
> REMARK   3  TWIN DETAILS
> REMARK   3   NUMBER OF TWIN DOMAINS  :4
> REMARK   3  TWIN DOMAIN   :1
> REMARK   3  TWIN OPERATOR :  H,  K,  L
> REMARK   3  TWIN FRACTION : 0.269
> REMARK   3  TWIN DOMAIN   :2
> REMARK   3  TWIN OPERATOR : -K, -H, -L
> REMARK   3  TWIN FRACTION : 0.171
> REMARK   3  TWIN DOMAIN   :3
> REMARK   3  TWIN OPERATOR :  K,  H, -L
> REMARK   3  TWIN FRACTION : 0.258
> REMARK   3  TWIN DOMAIN   :4
> REMARK   3  TWIN OPERATOR : -H, -K,  L
> REMARK   3  TWIN FRACTION : 0.302
> Does this establish twinning versus underestimated symmetry? And what do I 
> need to know about my free-R? Did refmac assign a new flag? Whereas the 
> output file's flags are all 1s and 0s, the input file had 0 ... 19. During 
> the first run, Rfree dropped to <28%. But on a subsequent run, Rfree was 
> stuck >30% when I used the initial job's output MTZ.
> Many thanks in advance for your helpful comments.
> Wolfram Tempel
>



--

ARKA CHAKRABORTY
CAS in Crystallography and Biophysics
University of Madras
Chennai,India


Roberto Steiner, PhD
Group Leader
Randall Division of Cell and Molecular Biophysics
King's College London

Room 3.10A
New Hunt's House
Guy's Campus
SE1 1UL, London, UK
Tel 0044-20-78488216
Fax 0044-20-78486435
roberto.stei...@kcl.ac.uk

Re: [ccp4bb] twin refinement in refmac

[Garib N Murshudov]

1) You should use measured data (after scala/aimless/truncate). In general 
there may not be one to one relationship between observed data and asymmetric 
unit (e.g. non-merohedral twinning) and it would not be possible to bring input 
data to output file. Use original data
2) Internally refmac groups reflection into classes. All twin related 
reflections belong to one class. In the example you give four reflections 
belong to one class. FreeR is property of the class not individual relfections. 
I.e. all twin related reflections belong either to free or working class. If 
your input data has this property then output should also have this. In the 
output file there will be 0 (for free) and 1 (working)
3) At early stages it is not easy to factorise twin and underestimated 
symmetry. It seems that L-test is the best way of making decision if you 
"crystals" are twinnined. If you collect data from many crystals and merge them 
then you artificially twin (or increase twinning). Using pointless to index all 
datasets consistently may sort some of the problems 
4) In this case it seems that you may need to reindex. Largest twin domain is 
not the first one

Regards
Garib


On 2 Mar 2012, at 02:00, wtempel wrote:

> Dear CCp4ers,
> A good morning to everyone.
> Today, I have a structure that I initially refined in space group P6522, 
> 1mol/asu.
> Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; / > 3
> 2.61-2.55A: Rsym=39.6%, / > 10
> 50.00-6.13: Rsym=6.4%
> Some mild anisotropy in the resolution limits is apparent on the diffraction 
> images. Say, visible spots at 2.2A in one direction, 2.6A in the other.
> Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A 
> resolution, with some difference density for loops that cannot be interpreted 
> with reasonable geometry.
> Rsym is very similar for data scaled in P3, in all resolution shells. Xtriage 
> does not suggest merohedral twinning.
> Nevertheless, I extended my free flags in sftools from P6522 to P32 and cad'd 
> them to amplitudes merged in spacegroup P32. Correspondingly, I expanded my 
> model to a homotetramer and ran Refmac with amplitude based twinning. (Would 
> this be a reasonable input to twin refinement?)
> From the output coordinates:
> REMARK   3  TWIN DETAILS
> REMARK   3   NUMBER OF TWIN DOMAINS  :4
> REMARK   3  TWIN DOMAIN   :1
> REMARK   3  TWIN OPERATOR :  H,  K,  L
> REMARK   3  TWIN FRACTION : 0.269
> REMARK   3  TWIN DOMAIN   :2
> REMARK   3  TWIN OPERATOR : -K, -H, -L
> REMARK   3  TWIN FRACTION : 0.171
> REMARK   3  TWIN DOMAIN   :3
> REMARK   3  TWIN OPERATOR :  K,  H, -L
> REMARK   3  TWIN FRACTION : 0.258
> REMARK   3  TWIN DOMAIN   :4
> REMARK   3  TWIN OPERATOR : -H, -K,  L
> REMARK   3  TWIN FRACTION : 0.302
> Does this establish twinning versus underestimated symmetry? And what do I 
> need to know about my free-R? Did refmac assign a new flag? Whereas the 
> output file's flags are all 1s and 0s, the input file had 0 ... 19. During 
> the first run, Rfree dropped to <28%. But on a subsequent run, Rfree was 
> stuck >30% when I used the initial job's output MTZ.
> Many thanks in advance for your helpful comments.
> Wolfram Tempel
> 

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] twin refinement in refmac

[Randy Read]

Garib may have more to say, but the first point would be to always include the 
original data file as your input MTZ file for any cycle of refinement, whether 
you're using Refmac in CCP4 or phenix.refine.  (In phenix.refine, if you assign 
the R-free data the first time you do refinement, it will produce a file 
containing all the information used in that cycle of refinement, including the 
new R-free flags and possibly any Hendrickson-Lattman coefficients, and you 
should use that file for all subsequent refinements.)

As for whether R-free takes twinning into account, I think it's fair to say 
that all the refinement programs that handle twinning will do this 
appropriately.

Regards,

Randy Read

On 2 Mar 2012, at 08:01, arka chakraborty wrote:

> Hi all,
> 
> I will like to know, as a follow up of what Prof. Randy Read said, what 
> should be done to do the refinement against the measured data and not the 
> detwinned F( which refmac outputs in the mtz after twin refinement), during 
> subsequent refinements. And also, I would like to know how to ensure that the 
> free R generated takes twinning into account if I am not using phenix.
> 
> Thanks in advance,
> 
> Regards,
> 
> ARKO
> 
> On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read  wrote:
> I'm worried when you say that you use the initial job's output MTZ. Refmac 
> replaces F with a detwinned F in the output file so you wouldn't be refining 
> against your measured data in the subsequent round.
> 
> Best wishes
> 
> Randy Read
> 
> 
> Randy J. Read
> 
> On 2 Mar 2012, at 02:00, wtempel  wrote:
> 
> > Dear CCp4ers,
> > A good morning to everyone.
> > Today, I have a structure that I initially refined in space group P6522, 
> > 1mol/asu.
> > Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; / > 3
> > 2.61-2.55A: Rsym=39.6%, / > 10
> > 50.00-6.13: Rsym=6.4%
> > Some mild anisotropy in the resolution limits is apparent on the 
> > diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in 
> > the other.
> > Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like 3.5A 
> > resolution, with some difference density for loops that cannot be 
> > interpreted with reasonable geometry.
> > Rsym is very similar for data scaled in P3, in all resolution shells. 
> > Xtriage does not suggest merohedral twinning.
> > Nevertheless, I extended my free flags in sftools from P6522 to P32 and 
> > cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I 
> > expanded my model to a homotetramer and ran Refmac with amplitude based 
> > twinning. (Would this be a reasonable input to twin refinement?)
> > From the output coordinates:
> > REMARK   3  TWIN DETAILS
> > REMARK   3   NUMBER OF TWIN DOMAINS  :4
> > REMARK   3  TWIN DOMAIN   :1
> > REMARK   3  TWIN OPERATOR :  H,  K,  L
> > REMARK   3  TWIN FRACTION : 0.269
> > REMARK   3  TWIN DOMAIN   :2
> > REMARK   3  TWIN OPERATOR : -K, -H, -L
> > REMARK   3  TWIN FRACTION : 0.171
> > REMARK   3  TWIN DOMAIN   :3
> > REMARK   3  TWIN OPERATOR :  K,  H, -L
> > REMARK   3  TWIN FRACTION : 0.258
> > REMARK   3  TWIN DOMAIN   :4
> > REMARK   3  TWIN OPERATOR : -H, -K,  L
> > REMARK   3  TWIN FRACTION : 0.302
> > Does this establish twinning versus underestimated symmetry? And what do I 
> > need to know about my free-R? Did refmac assign a new flag? Whereas the 
> > output file's flags are all 1s and 0s, the input file had 0 ... 19. During 
> > the first run, Rfree dropped to <28%. But on a subsequent run, Rfree was 
> > stuck >30% when I used the initial job's output MTZ.
> > Many thanks in advance for your helpful comments.
> > Wolfram Tempel
> >
> 
> 
> 
> -- 
> 
> ARKA CHAKRABORTY
> CAS in Crystallography and Biophysics
> University of Madras
> Chennai,India
> 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] twin refinement in refmac

[arka chakraborty]

Hi all,

I will like to know, as a follow up of what Prof. Randy Read said, what
should be done to do the refinement against the measured data and not the
detwinned F( which refmac outputs in the mtz after twin refinement), during
subsequent refinements. And also, I would like to know how to ensure that
the free R generated takes twinning into account if I am not using phenix.

Thanks in advance,

Regards,

ARKO

On Fri, Mar 2, 2012 at 12:44 PM, Randy J. Read  wrote:

> I'm worried when you say that you use the initial job's output MTZ. Refmac
> replaces F with a detwinned F in the output file so you wouldn't be
> refining against your measured data in the subsequent round.
>
> Best wishes
>
> Randy Read
>
> 
> Randy J. Read
>
> On 2 Mar 2012, at 02:00, wtempel  wrote:
>
> > Dear CCp4ers,
> > A good morning to everyone.
> > Today, I have a structure that I initially refined in space group P6522,
> 1mol/asu.
> > Scaling stats (scalepack): 2.30-2.26A: Rsym=99.9%; / > 3
> > 2.61-2.55A: Rsym=39.6%, / > 10
> > 50.00-6.13: Rsym=6.4%
> > Some mild anisotropy in the resolution limits is apparent on the
> diffraction images. Say, visible spots at 2.2A in one direction, 2.6A in
> the other.
> > Rfree, using data to 2.3A, was stuck in mid-30%s. The map appears like
> 3.5A resolution, with some difference density for loops that cannot be
> interpreted with reasonable geometry.
> > Rsym is very similar for data scaled in P3, in all resolution shells.
> Xtriage does not suggest merohedral twinning.
> > Nevertheless, I extended my free flags in sftools from P6522 to P32 and
> cad'd them to amplitudes merged in spacegroup P32. Correspondingly, I
> expanded my model to a homotetramer and ran Refmac with amplitude based
> twinning. (Would this be a reasonable input to twin refinement?)
> > From the output coordinates:
> > REMARK   3  TWIN DETAILS
> > REMARK   3   NUMBER OF TWIN DOMAINS  :4
> > REMARK   3  TWIN DOMAIN   :1
> > REMARK   3  TWIN OPERATOR :  H,  K,  L
> > REMARK   3  TWIN FRACTION : 0.269
> > REMARK   3  TWIN DOMAIN   :2
> > REMARK   3  TWIN OPERATOR : -K, -H, -L
> > REMARK   3  TWIN FRACTION : 0.171
> > REMARK   3  TWIN DOMAIN   :3
> > REMARK   3  TWIN OPERATOR :  K,  H, -L
> > REMARK   3  TWIN FRACTION : 0.258
> > REMARK   3  TWIN DOMAIN   :4
> > REMARK   3  TWIN OPERATOR : -H, -K,  L
> > REMARK   3  TWIN FRACTION : 0.302
> > Does this establish twinning versus underestimated symmetry? And what do
> I need to know about my free-R? Did refmac assign a new flag? Whereas the
> output file's flags are all 1s and 0s, the input file had 0 ... 19. During
> the first run, Rfree dropped to <28%. But on a subsequent run, Rfree was
> stuck >30% when I used the initial job's output MTZ.
> > Many thanks in advance for your helpful comments.
> > Wolfram Tempel
> >
>



-- 

*ARKA CHAKRABORTY*
*CAS in Crystallography and Biophysics*
*University of Madras*
*Chennai,India*