Re: [ccp4bb] analytical gel filtration columns

[David Blum]

Mohinder,

You could also try HPLC where you should get better results due to more
theoretical plates.  One option would from Phenomenex.
10E6A Phase Information
--

   - Molecular Weight Range: 60 K - 10,000 K

https://www.phenomenex.com/Products/HPLCDetail/phenogel

*David L. Blum, Ph.D.*

*Bioexpression and Fermentation Facility | Director*

*Master of Biomanufacturing and Bioprocessing | Director*

*120 Green Street | Life Sciences Bldg rm A414A | Athens, GA 30602 *

*706-542-1035* <706-542-1035>* | **b...@uga.edu* * | *
*bff.uga.edu* * | **biomanufacturing.uga.edu*





On Wed, Apr 17, 2019 at 11:11 AM Mohinder Pal 
wrote:

> Dear all,
>
> I would like to gel filter a multi protein complex (1.4MDa) as the final
> purification step. I have tried tandem Superose 6 columns and this complex
> elutes close to void volume as it is a very elongated molecule.
>
>
> I was wondering if someone could suggest different analytical columns for
> better resolution as I plan to add more components to this existing
> complex.
>
> Best wishes,
>
> Mohinder Pal
>
> --
> "Whatever you’re meant to do, do it now. The conditions are
> always impossible.”
> Doris Lessing
> --
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

Re: [ccp4bb] analytical gel filtration columns

[Roger Rowlett]

How about BioGel-A50m with an exclusion limit of 50 million Da? I've used
A15m for complexes as large as 500 kDa and it seemed to work well.

__
Roger Rowlett

On Wed, Apr 17, 2019, 11:11 AM Mohinder Pal 
wrote:

> Dear all,
>
> I would like to gel filter a multi protein complex (1.4MDa) as the final
> purification step. I have tried tandem Superose 6 columns and this complex
> elutes close to void volume as it is a very elongated molecule.
>
>
> I was wondering if someone could suggest different analytical columns for
> better resolution as I plan to add more components to this existing
> complex.
>
> Best wishes,
>
> Mohinder Pal
>
> --
> "Whatever you’re meant to do, do it now. The conditions are
> always impossible.”
> Doris Lessing
> --
>
>
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

Re: [ccp4bb] analytical gel filtration columns

[David Briggs]

Hi Mohinder,

Have you tried SEC columns like Sephacryl S-400 or CL-4B?

They have a higher fractionation range than Superose 6

HTH,

Dave

From: CCP4 bulletin board  on behalf of Mohinder Pal 

Sent: 17 April 2019 16:10
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] analytical gel filtration columns

Dear all,

I would like to gel filter a multi protein complex (1.4MDa) as the final 
purification step. I have tried tandem Superose 6 columns and this complex 
elutes close to void volume as it is a very elongated molecule.


I was wondering if someone could suggest different analytical columns for 
better resolution as I plan to add more components to this existing complex.

Best wishes,

Mohinder Pal

--
"Whatever you’re meant to do, do it now. The conditions are always impossible.”
Doris Lessing
--







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

The Francis Crick Institute Limited is a registered charity in England and 
Wales no. 1140062 and a company registered in England and Wales no. 06885462, 
with its registered office at 1 Midland Road London NW1 1AT



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

Re: [ccp4bb] A ccp4I2 question

[Jan Stransky]

I can comfirm, it is my favourite feature of i2.

I would like to see it automated (or -able using CLI) for flawless
backup recovery.

Cheers,

Jan

On 16/04/2019 14:22, Christian Roth wrote:
> yes one can
>
> Cheers
> Christian
>
> On Tue, Apr 16, 2019 at 1:19 PM Eleanor Dodson
> <176a9d5ebad7-dmarc-requ...@jiscmail.ac.uk
> > wrote:
>
> Can one export of project started under linux to a windows system?
>
> Eleanor
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

Re: [ccp4bb] analytical gel filtration columns

[Mohinder Pal]

Dear all,

I would like to gel filter a multi protein complex (1.4MDa) as the final 
purification step. I have tried tandem Superose 6 columns and this complex 
elutes close to void volume as it is a very elongated molecule.


I was wondering if someone could suggest different analytical columns for 
better resolution as I plan to add more components to this existing complex. 

Best wishes,

Mohinder Pal

--
"Whatever you’re meant to do, do it now. The conditions are always impossible.” 
Doris Lessing
--







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

Re: [ccp4bb] naive question how to add GTP as a residue to a nucleic acid chain and refine!

[Almudena Ponce Salvatierra]

Hi Phoebe,

that actually worked! thank you very much!

Best,

Almudena

El mar., 16 abr. 2019 a las 20:32, Phoebe A. Rice ()
escribió:

> At least last fall, the state of the art for getting your T7-transcribed
> RNA to start with a triphosphate in phenix was to add this file (edited for
> your chains, of course) through the gui (if you use the gui).
>
> Thanks to Deepak Koirala and Nigel Moriarty for help in working this out.
>
>- Phoebe
>
>
>
> refinement.geometry_restraints.edits {
>
> bond {
>
>   action = *add
>
>   atom_selection_1 = chain A and resname GTP and name O3'
>
>   atom_selection_2 = chain A and resid 594 and name P
>
>   symmetry_operation = None
>
>   distance_ideal = 1.6
>
>   sigma = 0.02
>
>   slack = None
>
> }
>
> angle {
>
>   action = *add
>
>   atom_selection_1 = chain A and resname GTP and name C3'
>
>   atom_selection_2 = chain A and resname GTP and name O3'
>
>   atom_selection_3 = chain A and resid 594 and name P
>
>   angle_ideal = 120
>
>   sigma = 1
>
> }
>
> bond {
>
>   action = *add
>
>   atom_selection_1 = chain B and resname GTP and name O3'
>
>   atom_selection_2 = chain B and resid 594 and name P
>
>   symmetry_operation = None
>
>   distance_ideal = 1.6
>
>   sigma = 0.02
>
>   slack = None
>
> }
>
> angle {
>
>   action = *add
>
>   atom_selection_1 = chain B and resname GTP and name C3'
>
>   atom_selection_2 = chain B and resname GTP and name O3'
>
>   atom_selection_3 = chain B and resid 594 and name P
>
>   angle_ideal = 120
>
>   sigma = 1
>
> }
>
> }
>
>
>
>
>
>
>
> *From: *CCP4 bulletin board  on behalf of Almudena
> Ponce Salvatierra 
> *Reply-To: *Almudena Ponce Salvatierra 
> *Date: *Friday, April 12, 2019 at 10:43 AM
> *To: *"CCP4BB@JISCMAIL.AC.UK" 
> *Subject: *[ccp4bb] naive question how to add GTP as a residue to a
> nucleic acid chain and refine!
>
>
>
> Dear all,
>
>
>
> I would like to add a GTP residue to a nucleic acid chain. For so I
> followed the following steps in coot: add monomer from library, GTP,
> changed chain ID and residue number accordingly, and then Extensions -->
> Modelling --> link 2 atoms. I got a dashed line between the 3'O of the GTP
> and the phosphate of the neighboring nucleotide, and then I used the real
> space refine tool. Up to here, it all looked promising.
>
>
>
> In phenix refine I run elbow and readyset and provided cif files, but
> after phenix.refine I find out that the link between GTP and it's neighbor
> is gone.
>
> How to preserve the bond?
>
>
>
> Thank you very much in advance!!
>
>
>
> Best,
>
>
>
> Almudena
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>



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

Re: [ccp4bb] High Rfree in last Shell

[Randy Read]

Hi,

No-one else seems to have said this, but just from the data in that table, if I 
were a referee I would almost certainly be asking why you seem to have 
discarded useful data at higher resolution than 3.1A!

Best wishes,

Randy Read

> On 16 Apr 2019, at 20:59, Jan van Agthoven  wrote:
> 
> Dear all,
> 
> The last resolution shell is 3.1-3.2 Å. I corrected high resolution Rmerge 
> and Rmeas which seemed truncated, and added cc1/2. Rsym is Rpim which was a 
> typo.
> 
> Resolution range (Å)   49.2-3.1   
>49.3-3.1
> Rfactor (%)24.0 (32.4)
>   23.4 (32.0)
> Rfree (%)  26.6 (29.2)
>   26.3 (31.6)
> 
> Data collection
> Completeness 100 (100)
> 100 (100)
> 
> Redundancy6.9 (7.0)   
>6.2 (6.3)
> 
> Molecules in asymmetric unit  1   
>1
> 
> Average I/σ 14.1 (1.7)
> 15.3 (2.0)
> 
> Rmerge (%)  14.9 (169.4)  
>12.7 (131.8)
> 
> Rmeas (%)16.2 (183.2) 
>  13.9 (143.9)
> 
> Rpim (%)   6.2 (68.6) 
>5.5 (57.1)
> Wilson B-factor 65.6  
>   62.7
> 
> cc1/20.997 (0.561)
>   0.998 (0.616)
> 
> 
>> On Apr 16, 2019, at 2:21 PM, Diana Tomchick 
>> > > wrote:
>> 
>> Why are the the values for R(free) in parentheses higher than R(factor) in 
>> parentheses? I would not expect that to happen, if the same resolution 
>> limits for the last shell are used for both values. And I echo the previous 
>> commentator, please provide the resolution limits for the values in 
>> parentheses.
>> 
>> What exactly is R(factor) in this case? The R(work), or the [R(work) + 
>> R(free)]? Please define.
>> 
>> Diana
>> 
>> **
>> Diana R. Tomchick
>> Professor
>> Departments of Biophysics and Biochemistry
>> UT Southwestern Medical Center
>> 5323 Harry Hines Blvd.
>> Rm. ND10.214A
>> Dallas, TX 75390-8816
>> diana.tomch...@utsouthwestern.edu 
>> (214) 645-6383 (phone)
>> (214) 645-6353 (fax)
>> 
>> On Apr 16, 2019, at 11:57 AM, Jan van Agthoven > > wrote:
>> 
>> Hi everyone,
>> I’m trying to publish two structures at 3.1Å resolution with the following 
>> refinement statistics:
>> 
>> Resolution range (Å)   49.2-3.1  
>> 49.3-3.1
>> Rfactor (%)24.0 (32.4)   
>>23.4 (32.0)
>> Rfree (%)  26.6 (29.2)   
>>26.3 (31.6)
>> 
>> Data collection
>> Completeness  100 (100)  
>>   100 (100)
>> 
>> Redundancy6.9 (7.0)  
>> 6.2 (6.3)
>> 
>> Molecules in asymmetric unit  1  
>> 1
>> 
>> Average I/σ 14.1 (1.7)   
>>  15.3 (2.0)
>> 
>> Rmerge (%)  14.9 (100)   
>> 12.7 (100)
>> 
>> Rmeas (%)16.2 (100)  
>>  13.9 (100)
>> 
>> Rsym (%)   6.2 (68.6)
>> 5.5 (57.1)
>> Wilson B-factor 65.6 
>>62.7
>> 
>> I’ve been told that the Rfree factor in the last shell are too high. Does 
>> anyone know how I can improve these Rfree factors other then cutting the 
>> resolution, which already is rather low?
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
>> 
>> 
>> UT Southwestern 
>> 
>> Medical Center
>> 
>> The future of medicine, today.
>> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 
--
Randy J. Read
Department of Haematology, University of Cambridge

Re: [ccp4bb] High Rfree in last Shell

[Mark J van Raaij]

Send your manuscripts to any of the IUCr journals! 
Might still get the occasional referee like that, but the editors should ignore 
those comments.
plus you'd be supporting a scientific society [sorry for the advert...]

Mark J van Raaij
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
calle Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://wwwuser.cnb.csic.es/~mjvanraaij
Section Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/


> On 16 Apr 2019, at 22:29, Diana Tomchick  
> wrote:
> 
> Ah, even though it seems a long time ago, approximately one out of every two 
> referee comments that I receive complain about our modern methods for 
> processing, scaling and reporting our data collection/processing/refinement 
> statistics. I’m afraid it’s an ongoing effort at education.
> 
> Diana
> 
> **
> Diana R. Tomchick
> Professor
> Departments of Biophysics and Biochemistry
> UT Southwestern Medical Center
> 5323 Harry Hines Blvd.
> Rm. ND10.214A
> Dallas, TX 75390-8816
> diana.tomch...@utsouthwestern.edu 
> (214) 645-6383 (phone)
> (214) 645-6353 (fax)
> 
> On Apr 16, 2019, at 3:09 PM, Frank von Delft  > wrote:
> 
> Jan, tell your reviewer to join us all in the 21st century.  
> 
> Diederichs and Karplus, Science, about 2 decades ago.  (Technically, 2012 
> ,
>  but it really is a long long time ago now.) 
> 
> 
> On 16/04/2019 18:06, Tim Gruene wrote:
>> Dear Jan,
>> 
>> You statistics look quite solid.
>> 
>> R-factors are not good criteria to judge the resolution cut-off. The 
>> weighting 
>> schemes in refinement programs have much improved since the late 1990s. A 
>> good 
>> starting point to learn more is 
>> Rupp's "Against Method: Table 1 -Cui Bono?", https://doi.org/10.1016/j.str 
>> .
>> 2018.04.013
>> 
>> Best regards,
>> Tim
>> 
>> On Tuesday, April 16, 2019 6:57:06 PM CEST Jan van Agthoven wrote:
>>> Hi everyone,
>>> I’m trying to publish two structures at 3.1Å resolution with the following
>>> refinement statistics:
>>> 
>>> Resolution range (Å)   49.2-3.1 
>>> 49.3-3.1 Rfactor (%)   
>>> 24.0 (32.4)  23.4 (32.0) Rfree (%) 
>>> 26.6 (29.2) 
>>> 26.3 (31.6)
>>> 
>>> Data collection
>>> Completeness  100 (100) 
>>>   100 (100)
>>> 
>>> Redundancy6.9 (7.0) 
>>> 6.2 (6.3)
>>> 
>>> Molecules in asymmetric unit  1 
>>> 1
>>> 
>>> Average I/σ 14.1 (1.7)  
>>>  15.3 (2.0)
>>> 
>>> Rmerge (%)  14.9 (100)  
>>> 12.7 (100)
>>> 
>>> Rmeas (%)16.2 (100) 
>>>  13.9 (100)
>>> 
>>> Rsym (%)   6.2 (68.6)   
>>> 5.5 (57.1) Wilson B-factor 
>>>65.662.7
>>> 
>>> I’ve been told that the Rfree factor in the last shell are too high. Does
>>> anyone know how I can improve these Rfree factors other then cutting the
>>> resolution, which already is rather low?
>>> 
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
>>> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 
> 
> UT Southwestern 
> 
> Medical Center
> 
> The future of medicine, today.
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1 
> 



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

Re: [ccp4bb] Big density blobs near Arg and His residues or Thr residues

[Robbie Joosten]

I would try chloride and potassium in those sites and see whether that refines 
well.

Cheers,
Robbie

On 17 Apr 2019 07:49, WENHE ZHONG  wrote:
Dear all,

We found a huge density blob close to residues Arg and His (see attached) and 
another big density blob near three Thr residues (see attached) in the 
structure with an resolution of 2.6A. They are too big to place water atoms. In 
the crystallization condition, we have PEG 8000, glycerol, triethanolamine-HCl 
(TEA) buffer pH 7.2, KCl, MgCl2 and ATP. Do you have any idea about what can be 
in the density? Covalent modification?

Thanks.

Kind regards,
Wenhe

[cid:48C74A30-E0A3-4EA8-BFE6-E0EB418751C2@local.local][cid:E8DDA617-5B85-4883-AF05-E8175CC33F51@local.local]
[cid:C3ADA69F-7A08-41E0-9B71-362BE360F09E@local.local][cid:5CFE7EF9-6FCD-4C69-941B-4A72B3776108@local.local]



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




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