Re: [ccp4bb] symmetry and pdb remark

[Smith Lee]

Dear All,
I got the each of 6 mates coordination for each pdb by Coot or by Chimera. Then 
viewed the arrangement of the 6 mates for 2zan or 2zao by Pymol or by Chimera. 
You will find, for the 6 mates of 2zan, they were separated, but for 2zao they 
were connected.
Then by pymol I align 2zan onto 2zam, and I get the PDB for 2zan fitted to 2zam 
(no any remark information left after pymol saving). Then I add all the remark 
information (in fact all text information except the coordination information) 
from 2zan to the PDB for 2zan fitted to 2zam, and then I view the mates for the 
remark （all text except coordination） added PDB for 2zan fitted to 2zam by 
Chimera or Pymol, I find the 6 mates of  the remark （all text except 
coordination）added PDB for 2zan fitted to 2zam were connected, rather than 
separated.
Based on what advised by Jared, by increasing the radius to 20 A by pymol, I am 
not sure they have the same molecule arrangement in the crystal.
I hope I can get more advice on my question.
Best regards.
Smith

 

On Thursday, February 2, 2017 12:03 PM, "Sampson, Jared" 
 wrote:
 

 Hi Smith - 
It seems that you may need to increase the radius of symmetry mate generation.  
Looking at these two PDBs after superimposition in PyMOL with symmetry mates 
generated within 20Å shows what appear to me as essentially identical lattice 
structures.
Also, the symmetry information is not stored in REMARK records, but rather in 
the CRYST1, ORIGXn, and SCALEn records 
(seehttp://deposit.rcsb.org/adit/docs/pdb_atom_format.html, for example).
Hope that helps.
Cheers,Jared
 

On Feb 1, 2017, at 10:00 PM, Smith Lee 
<0459ef8548d5-dmarc-requ...@jiscmail.ac.uk> wrote:
Dear All,

I have a symmetry problem, which I hope I can get your help.

For both PDB 2zan and 2zam, they are for the same protein, they conformation 
were similar except that 2zam was apo and 2zam was ATP binding. 2zaz was got by 
soaking the 2zam crystal with ATP. Both were P65 space group
However by getting the mates of 2zam and 2zan, you will find their 6 mates 
arrange differently. Can you explain to me why  their 6 mates arrange 
differently?
What is more, by pymol I align 2zan onto 2zam, and I get the PDB for 2zan 
fitted to 2zam (no any remark information left after pymol saving). Then I add 
all the remark information from 2zan to the PDB for 2zan fitted to 2zam, and 
then I view the mates for the remark added PDB for 2zan fitted to 2zam, I find 
the 6 mates arrange like 2zam, rather like 2zan.
Thus, will you please explain whi ch remark information decide the mate 
arrangement, as in the  remark added PDB for 2zan fitted to 2zam? Why after 
pymol alignment, the same remark information leads to different mates 
arrangement?
I am looking forward to getting a reply from you.
Smith



   

[ccp4bb] symmetry and pdb remark

[Smith Lee]

Dear All,

I have a symmetry problem, which I hope I can get your help.

For both PDB 2zan and 2zam, they are for the same protein, they conformation 
were similar except that 2zam was apo and 2zam was ATP binding. 2zaz was got by 
soaking the 2zam crystal with ATP. Both were P65 space group
However by getting the mates of 2zam and 2zan, you will find their 6 mates 
arrange differently. Can you explain to me why  their 6 mates arrange 
differently?
What is more, by pymol I align 2zan onto 2zam, and I get the PDB for 2zan 
fitted to 2zam (no any remark information left after pymol saving). Then I add 
all the remark information from 2zan to the PDB for 2zan fitted to 2zam, and 
then I view the mates for the remark added PDB for 2zan fitted to 2zam, I find 
the 6 mates arrange like 2zam, rather like 2zan.
Thus, will you please explain whi ch remark information decide the mate 
arrangement, as in the  remark added PDB for 2zan fitted to 2zam? Why after 
pymol alignment, the same remark information leads to different mates 
arrangement?
I am looking forward to getting a reply from you.
Smith

[ccp4bb] 10th CCP4/APS Crystallographic School in the USA

[Sanishvili, Ruslan]

Dear Colleagues,

We are pleased to announce the 10th annual CCP4 crystallographic school “>From 
data collection to structure refinement and beyond”, held at Advanced Photon 
Source (APS), Argonne National Laboratory (ANL). All details can be found at 
http://www.ccp4.ac.uk/schools/APS-2017/index.php

Dates: June 19 through 26, 2017

Site: Advanced Photon Source, Argonne National Laboratory, Argonne (Near 
Chicago), Illinois, USA

The school comprises
Data collection workshop: beamline training; data collection on GM/CA@APS 
beamlines 23ID-D and 23ID-B equipped with Pilatus3 6M and Eiger 16M detectors, 
respectively; and data processing. For data collection, only the participants' 
crystals will be used.
Crystallographic computation workshop: will feature many modern 
crystallographic software packages taught by authors and other experts. The 
daily schedule will be organized in three sections – lectures, tutorials, and 
hands-on, interactive trouble-shooting of the technical difficulties the 
participants face in their projects. We have had considerable success resolving 
these problems in past years, attested by resulting publications 
http://www.ccp4.ac.uk/schools/APS-school/publications.php

Applicants: Graduate students, postdoctoral researchers and young scientists at 
the assistant professor level, along with commercial/industrial researchers 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 
and/or computation workshops.

Application: Application deadline is April 19. The application form, the 
program, contact info and other details can be found at 
http://www.ccp4.ac.uk/schools/APS-2017/index.php

Registration fees: The registration for application is free but there is $500 
participation fee for the selected academic students and $950 for the 
industrial researchers. The link for the on-line payments and instructions will 
be provided once the selection process is completed. The students will be 
responsible for their transportation, lodging and breakfast. The workshop 
organizers can assist in making the reservations at economical lodging at the 
Argonne Guest House. The workshop will cover all other expenses.

We hope to see you at the school.
Garib, Ronan and Nukri


Ruslan Sanishvili (Nukri), Ph.D.
Macromolecular Crystallographer
GM/CA@APS
X-ray Science Division, ANL
9700 S. Cass Ave.
Lemont, IL 60439

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

Re: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures

[Gerard DVD Kleywegt]

The main issue is that carboxyls seem to be invisible and Coot tries to fit 
them as though the map had them there


Well, if the atoms are part of the model, then Coot will try to fit them to 
the "data." I routinely chop off GLU and ASP side-chain when modelling into 
cryo-EM maps (that's what the K key is for).


That seems unnecessarily harsh. Remember that EM maps do not show electron 
density but electric potential, so undamaged negatively charged moieties 
should occur at negative density levels. There is an "Aha-Erlebnis" paper (at 
least, that's the effect it had on me :-) ) by Jimin Wang and Peter Moore 
(entitled "On the interpretation of electron microscopic maps of biological 
macromolecules") on this very topic in last month's EM-special of Protein 
Science, Vol 26, pp 122-129.


--Gerard

**
   Gerard J. Kleywegt

  http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
**
   The opinions in this message are fictional.  Any similarity
   to actual opinions, living or dead, is purely coincidental.
**
   Little known gastromathematical curiosity: let "z" be the
   radius and "a" the thickness of a pizza. Then the volume
of that pizza is equal to pi*z*z*a !
**

Re: [ccp4bb] intermolecular dissulphides

[Tom Peat]

Hello Eleanor,


We found some intermolecular vicinal disulfides recently that we think are 
'real'. This class of proteins forms tetramers and in one version we find these 
intermolecular disulfides across molecules. We did some tests and found that 
oxidation or reduction has an effect on the stability of the protein. ?We also 
saw this was consistent across multiple space groups. If you would like to have 
a look, they were just released: 5HY0, 5HY2, 5HY4.

As a comparison to another protein in this fold class that doesn't have the 
disulfide is 5HWE.


cheers, tom


Tom Peat
Proteins Group
Biomedical Program, CSIRO
343 Royal Parade
Parkville, VIC, 3052
+613 9662 7304
+614 57 539 419
tom.p...@csiro.au

From: CCP4 bulletin board  on behalf of Eleanor Dodson 

Sent: Thursday, February 2, 2017 2:17 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] intermolecular dissulphides

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556  2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556  2.15

We seem to have one but it would have to form after crystalisation?

Eleanor

Re: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures

[Paul Emsley]

On 01/02/2017 17:11, Trevor Sewell wrote:

I have a 3.4 A (enzyme – protein only) map that I have fitted manually using 
Coot and
automatically with Rapper. It all looks very nice – I can fit all but 13 of the 
330
residues. I have the following questions:



Is there a way of having Coot know that it is an electron map and not an x-ray 
map?


Yes, and/but it's not under user control.

> The main

issue is that carboxyls seem to be invisible and Coot tries to fit them as 
though the map
had them there


Well, if the atoms are part of the model, then Coot will try to fit them to the "data." I 
routinely chop off GLU and ASP side-chain when modelling into cryo-EM maps (that's what the 
K key is for).


Coot refines differently to Refmac.


What is a reasonable starting weight for vdwrestraints ? Is there a rule of 
thumb for
choosing this?


I don't typically adjust this.  What make you think that you need to do so?

Paul.

Re: [ccp4bb] intermolecular dissulphides

[Edward A. Berry]

Does this count as an example?
grep SSBOND /a/pdb/pdb4e9m.ent
SSBOND   1 CYS A   39CYS B   39  1555   1555  2.05
SSBOND   2 CYS C   39CYS D   39  1555   1555  2.04
SSBOND   3 CYS E   39CYS F   39  1555   1555  2.03

The A.U. contains three domain-swapped dimers. The cys are not on the swapped 
helix
but the swapping fortuitously brings the same cys in the two molecules into 
proximity
to make a disulfide. There are two or three other x-ray structures that show 
the same
domain-swapped, disulfide-clinched dimer in different packing. However an NMR 
structure
shows it to be monomeric in solution, based on estimated tumbling speed since
nmr restraints might not distinguish inter- from intramolecular contacts in a
domain-swapped dimer.
The cys is not conserved, and although this protein is expected to oligomerize,
the putative oligomerization domain is not included in this construct.

On 02/01/2017 10:17 AM, Eleanor Dodson wrote:

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556  2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556  2.15

We seem to have one but it would have to form after crystalisation?

Eleanor

Re: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures

[Fislage, Marcus]

Dear Trevor,

I do not know if you are aware of it but there is a different version of refmac 
which is just made for Cryo-EM refinements.
(http://www2.mrc-lmb.cam.ac.uk/groups/murshudov/content/software.html)

In there, Refmac reports an average FSC value which is way more useful than the 
R-factor.
You want to have this value as high as possible (based on your resolution it 
might be around 0.79)

But you always have to make sure to not do overfitting. You will have to test 
for overfitting as it is described and done for example in the most recent 
publications of Venki Ramakrishnan.

Cheers
Marcus

Marcus Fislage, PhD

Howard Hughes Medical Institute (HHMI)
Columbia University
Department of Biochemistry and Biophysics
Lab of Joachim Frank
New York, NY

email address: mf2...@cumc.columbia.edu
Phone: 212.305.9524
Fax: 212.305.9500

Marcus Fislage, PhD

Howard Hughes Medical Institute (HHMI)
Columbia University
Department of Biochemistry and Biophysics
Lab of Joachim Frank
New York, NY

email address: mf2...@cumc.columbia.edu
Phone: 212.305.9524
Fax: 212.305.9500


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Trevor Sewell 
[trevor.sew...@uct.ac.za]
Sent: Wednesday, February 01, 2017 12:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Refmac5 and Coot for refining cryo-EM structures

I have a 3.4 A (enzyme – protein only) map that I have fitted manually using 
Coot and automatically with Rapper. It all looks very nice – I can fit all but 
13 of the 330 residues. I have the following questions:

Does refmac5 have a way of refining the scale of the map? – I did my best to 
calibrate the microscope – but it would be nice to have the scale as a 
refinable parameter.

Is there a way of having Coot know that it is an electron map and not an x-ray 
map? The main issue is that carboxyls seem to be invisible and Coot tries to 
fit them as though the map had them there – which results in some pretty dopey 
stuff. The problem doesn’t seem to apply in refmac5 if you use the source EM as 
described in the instructions.

What is a reasonable starting weight for vdwrestraints ? Is there a rule of 
thumb for choosing this?

What are acceptable values for the Rfactor produced by Refmac5? The fit looks 
pretty good with an Rfactor of .39. What is the experience of the community in 
this regard?

Many thanks

Trevor Sewell


Disclaimer - University of Cape Town This e-mail is subject to UCT policies and 
e-mail disclaimer published on our website at 
http://www.uct.ac.za/about/policies/emaildisclaimer/ or obtainable from +27 21 
650 9111. If this e-mail is not related to the business of UCT, it is sent by 
the sender in an individual capacity. Please report security incidents or abuse 
via cs...@uct.ac.za

[ccp4bb] Refmac5 and Coot for refining cryo-EM structures

[Trevor Sewell]

I have a 3.4 A (enzyme – protein only) map that I have fitted manually using 
Coot and automatically with Rapper. It all looks very nice – I can fit all but 
13 of the 330 residues. I have the following questions:

Does refmac5 have a way of refining the scale of the map? – I did my best to 
calibrate the microscope – but it would be nice to have the scale as a 
refinable parameter.

Is there a way of having Coot know that it is an electron map and not an x-ray 
map? The main issue is that carboxyls seem to be invisible and Coot tries to 
fit them as though the map had them there – which results in some pretty dopey 
stuff. The problem doesn’t seem to apply in refmac5 if you use the source EM as 
described in the instructions.

What is a reasonable starting weight for vdwrestraints ? Is there a rule of 
thumb for choosing this?

What are acceptable values for the Rfactor produced by Refmac5? The fit looks 
pretty good with an Rfactor of .39. What is the experience of the community in 
this regard?

Many thanks

Trevor Sewell


Disclaimer - University of Cape Town This e-mail is subject to UCT policies and 
e-mail disclaimer published on our website at 
http://www.uct.ac.za/about/policies/emaildisclaimer/ or obtainable from +27 21 
650 9111. If this e-mail is not related to the business of UCT, it is sent by 
the sender in an individual capacity. Please report security incidents or abuse 
via cs...@uct.ac.za

Re: [ccp4bb] comparison of different protein states

[Guillermo Montoya]

Dear all,

 thanks for your
input


cheers!!


G.



On 1 Feb 2017, at 17:17, Debanu 
> wrote:

Dear Xavier,
Great point and reminder!
Thanks,
Debanu

On Feb 1, 2017, at 6:44 AM, Boaz Shaanan 
> wrote:

Hi,
One possible (formal, I should say) way around this would be to use one of the 
homology modeling servers (my favourite recently is phyre2 but go for any 
server you prefer) and feed it with the sequence of the protein in your 
structure as if you're trying to get its structure in the''apo'' form (a wrong 
term, as was pointed out on the bb recently). I'm quite certain that with the 
degree of conservation you mentioned it'll superpose extremely well on the 
other ''apo'' structure. This should satisfy the referee I would think (it's 
not me though).
Cheers,
Boaz


 Original message 
From: Guillermo Montoya 
>
Date: 01/02/2017 07:40 (GMT+02:00)
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] comparison of different protein states


Dear all,

first of all sorry for this off-topic question. I am requesting your help to
find some papers  to convince one referee about the comparison
of two different protein states.

In our manuscript we show the crystal structure of an
enzyme. This structure represents the enzyme after catalysis in complex with 
the product.
 In the discussion we have superimposed the enzyme in the apo conformation
and the enzyme after catalysis in complex with the product
and we have  commented the conformational changes observed
between these 2 states to propose a model.

The point of the referee is that this comparison is not valid because the
enzymes that we used in the comparison belong to different species.
They are not the same protein.

However, and this is stated by us  in the figs and the manuscript, these two 
proteins are
40% identical and 60% conserved, the polypeptide length is the same, and
the key amino acids and the domain structure are fully conserved. They are
obviously orthologs.

I´d really appreciate if you can send me some literature/information
to support our approach

Thanks a lot for your input


best




Guillermo Montoya, Prof., Dr.
Research Director, Protein Structure and Function Programme
Novo Nordisk Foundation Center for Protein Research
Faculty of Health and Medical Sciences, University of Copenhagen,
Blegdamsvej 3A, DK-2200 Copenhagen, Denmark
web: www.cpr.ku.dk

Re: [ccp4bb] intermolecular dissulphides

[Eleanor Dodson]

Thank you for all this information, and especially Paul for having a good
search query - I had got nothing useful from any I tried at PDBe or RSCB.

No idea why it has been formed - the fold is identical to a high resolution
example (1.3A)  where the disulphide links the N & C termini within a
single molecule.

Except for 2-3 residues which cause the domain swap the CA rmsd is < 2. It
must be a crystallisation artefact I think,. or my mistake in
interpretation... This new data is lower resolution but after omitting the
hings region the density is pretty reliable..
Eleanor






On 1 February 2017 at 16:13, Eleanor Dodson 
wrote:

> Thank you VERY much - how did you generate that?
> E
>
> On 1 February 2017 at 16:10, Paul Emsley 
> wrote:
>
>> On 01/02/2017 15:17, Eleanor Dodson wrote:
>>
>>> Does anyone know of examples of these?
>>>
>>
>> Here's a rough list - not all of them are real.
>>
>> P.
>>
>>
>>
>>
>

Re: [ccp4bb] comparison of different protein states

[Debanu]

Dear Xavier,
Great point and reminder!
Thanks,
Debanu

> On Feb 1, 2017, at 6:44 AM, Boaz Shaanan  wrote:
> 
> Hi,
> One possible (formal, I should say) way around this would be to use one of 
> the homology modeling servers (my favourite recently is phyre2 but go for any 
> server you prefer) and feed it with the sequence of the protein in your 
> structure as if you're trying to get its structure in the''apo'' form (a 
> wrong term, as was pointed out on the bb recently). I'm quite certain that 
> with the degree of conservation you mentioned it'll superpose extremely well 
> on the other ''apo'' structure. This should satisfy the referee I would think 
> (it's not me though).
> Cheers,
> Boaz
> 
> 
>  Original message 
> From: Guillermo Montoya  
> Date: 01/02/2017 07:40 (GMT+02:00) 
> To: CCP4BB@JISCMAIL.AC.UK 
> Subject: [ccp4bb] comparison of different protein states 
> 
> 
> Dear all, 
> 
> first of all sorry for this off-topic question. I am requesting your help to 
> find some papers  to convince one referee about the comparison
> of two different protein states.
> 
> In our manuscript we show the crystal structure of an 
> enzyme. This structure represents the enzyme after catalysis in complex with 
> the product.
>  In the discussion we have superimposed the enzyme in the apo conformation 
> and the enzyme after catalysis in complex with the product 
> and we have  commented the conformational changes observed
> between these 2 states to propose a model.
> 
> The point of the referee is that this comparison is not valid because the 
> enzymes that we used in the comparison belong to different species.
> They are not the same protein.
> 
> However, and this is stated by us  in the figs and the manuscript, these two 
> proteins are 
> 40% identical and 60% conserved, the polypeptide length is the same, and
> the key amino acids and the domain structure are fully conserved. They are 
> obviously orthologs.
> 
> I´d really appreciate if you can send me some literature/information
> to support our approach 
> 
> Thanks a lot for your input
> 
> 
> best 
> 
> 
> 
> 
> Guillermo Montoya, Prof., Dr.
> Research Director, Protein Structure and Function Programme 
> Novo Nordisk Foundation Center for Protein Research
> Faculty of Health and Medical Sciences, University of Copenhagen,
> Blegdamsvej 3A, DK-2200 Copenhagen, Denmark
> web: www.cpr.ku.dk
> 
> 
> 
> 

[ccp4bb] Symmetry problems..

[Eleanor Dodson]

Oh dear - more symmetry Qs.

This has arisen from the SSBOND to a symmetry equivalent puzzle with
links 1_555 (X,Y,Z) to 9_565 (X,1+X-y,1/6-Z) IF you are using the $CLIB/
symlib.info
but links 1_555 to 12_565 if you are using the mmdb order of symmetry
operators..
.

Aaaah!!!

Eleanor

[ccp4bb] AW: [ccp4bb] intermolecular dissulphides

[Herman . Schreuder]

Dear Eleanor,

I did not check the pdb file you mentioned, but I have had a case like that. 
The protein formed a complex of 8 large and 8 small subunits with internal 422 
symmetry. There was a disulfide link across the internal twofolds and in one of 
the crystal forms we got, this internal twofold came on top of a 
crystallographic twofold. At the time, I did not know about these sophisticated 
SSBOND records (if they existed at the time), so I assigned a very small van 
der Waals radius to the SGs to solve the repulsion problem.

So, if you have a dimer with an existing disulfide link across the twofold 
axis, this twofold axis may become a crystallographic twofold and there is no 
need for the disulfide bond to form afterwards.

Best regards,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Eleanor 
Dodson
Gesendet: Mittwoch, 1. Februar 2017 16:18
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] intermolecular dissulphides

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556  2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556  2.15
We seem to have one but it would have to form after crystalisation?
Eleanor

Re: [ccp4bb] intermolecular dissulphides

[Keller, Jacob]

I solved a structure years ago (1L31 and a couple related entries) which had 
intermolecular oligomerizing disulfides in some of the crystals (reciprocal 
C23-C27’ and C23’-C27). I thought at the time that they seemed too “deliberate” 
to be just an artifact, but others thought that since the enzyme was 
intracellular, the disulfides would not be physiologically relevant. I still 
think they’re too perfect to be artefactual, and since the protein comes from 
Methanobacterium thermoautotrophicum, all usual bets might be off. I’ve always 
wondered about it, although I am not sure how profound the consequences are. I 
would love to hear peoples’ two cents on this.

Jacob

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Eleanor 
Dodson
Sent: Wednesday, February 01, 2017 10:18 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] intermolecular dissulphides

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556  2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556  2.15
We seem to have one but it would have to form after crystalisation?
Eleanor

Re: [ccp4bb] Manual His-Tag Purification

[David Blum]

Hi Sundaram,

The binding capacity of this column is 40 mg/mL of resin so a 5mL column
will hold a maximum of 200 mg of protein.  If you run your cleared lysate
on a gel you may be able to estimate how much protein there is.  Our
facility purifies a range of different proteins for investigators and our
rule of thumb is not to load more than 1/3 of the column capacity so if
your construct expresses more than 66.6 mg/L then you may want to batch
load the protein.  Without any knowledge of expression levels, I would
recommend loading 1/10 of your cleared lysate then estimating total protein
from your purified sample before loading the entire lysate.

Best,

David

-- 

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

Skype: dlblum11

(706) 542-1035 (Office)




On Wed, Feb 1, 2017 at 5:08 AM, Tim Gruene  wrote:

> Dear Sundaram S,
>
> during my PhD I used 4-7.5ml resin per l of culture, but I also had a large
> yield of 60-100mg protein per litre. Try to use as little as possible - at
> first trial check both flow-through and retentate by SDS-PAGE. (see. p. 40
> and
> 54 of my thesis).
>
> My constructs would also bind greatly to Ni-IDA, but not at all to the much
> more common Ni-NTA.
>
> When you expect low yields, and a protein that may be sensitive to the
> immidazole concentration, you can also try Co instead of Ni.
>
> Best regards,
> Tim
>
> On Wednesday 01 February 2017 02:54:09 PM Sundaram wrote:
> > Hello ,
> >
> > It's an off topic question.
> >
> > I'm planning to do manual purification a 6 his tagged protein of size
> > around 20kDa from 1L E.coli culture using
> > COHISC-RO Roche cOmplete™ His-Tag Purification Column.
> >
> >
> > Can I get some advice regarding the lysate loading volume and retention
> > time.
> > This is the first time I going to use this column and I have no idea
> about
> > my protein yield from 1L culture.
> >
> > Sorry if I spammed your inbox.
> >
> >
> > Thanks!
> >
> > Yours Sincerely,
> > Sundaram.S
> --
> --
> Paul Scherrer Institut
> Dr. Tim Gruene
> - persoenlich -
> Principal Investigator
> Biology and Chemistry
> OFLC/102
> CH-5232 Villigen PSI
>
> Phone: +41 (0)56 310 5297
>
> GPG Key ID = A46BEE1A
>
>

[ccp4bb] intermolecular dissulphides

[Eleanor Dodson]

Does anyone know of examples of these?
I have found one - 2WQW with these SSBOND records
2WQW
SSBOND   1 CYS A  206CYS A  227  1555   6556
2.07
SSBOND   2 CYS B  206CYS B  227  1555   5556
2.15

We seem to have one but it would have to form after crystalisation?

Eleanor

Re: [ccp4bb] RMSD plot

[Tanner, John J.]

CNS, if sequences are identical.


Sent from Jack's iPhone

> On Feb 1, 2017, at 4:26 AM, Madhuranayaki Thulasingam  
> wrote:
> 
> Dear all,
> 
> I would like to plot RMSD vs amino acids for two superimposed crystal 
> structures.
> 
> Can anyone suggest me a way to do it.
> 
> Thanks in advance,
> Madhu.

Re: [ccp4bb] RMSD plot

[Graeme Winter]

Dear Madhu

I think LSQKAB does what you want

http://www.ccp4.ac.uk/html/lsqkab.html

(at least, from the description you gave)

Best wishes Graeme

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Madhuranayaki Thulasingam
Sent: 01 February 2017 10:25
To: ccp4bb
Subject: [ccp4bb] RMSD plot

Dear all,

I would like to plot RMSD vs amino acids for two superimposed crystal 
structures.

Can anyone suggest me a way to do it.

Thanks in advance,
Madhu.

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[ccp4bb] RMSD plot

[Madhuranayaki Thulasingam]

Dear all,

I would like to plot RMSD vs amino acids for two superimposed crystal
structures.

Can anyone suggest me a way to do it.

Thanks in advance,
Madhu.

Re: [ccp4bb] Manual His-Tag Purification

[Tim Gruene]

Dear Sundaram S,

during my PhD I used 4-7.5ml resin per l of culture, but I also had a large 
yield of 60-100mg protein per litre. Try to use as little as possible - at 
first trial check both flow-through and retentate by SDS-PAGE. (see. p. 40 and 
54 of my thesis).

My constructs would also bind greatly to Ni-IDA, but not at all to the much 
more common Ni-NTA.

When you expect low yields, and a protein that may be sensitive to the 
immidazole concentration, you can also try Co instead of Ni.

Best regards,
Tim

On Wednesday 01 February 2017 02:54:09 PM Sundaram wrote:
> Hello ,
> 
> It's an off topic question.
> 
> I'm planning to do manual purification a 6 his tagged protein of size
> around 20kDa from 1L E.coli culture using
> COHISC-RO Roche cOmplete™ His-Tag Purification Column.
> 
> 
> Can I get some advice regarding the lysate loading volume and retention
> time.
> This is the first time I going to use this column and I have no idea about
> my protein yield from 1L culture.
> 
> Sorry if I spammed your inbox.
> 
> 
> Thanks!
> 
> Yours Sincerely,
> Sundaram.S
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Dr. Tim Gruene
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Biology and Chemistry
OFLC/102
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Phone: +41 (0)56 310 5297

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Re: [ccp4bb] Manual His-Tag Purification

[Horrell, Sam]

This should give you everything you need to know about the column 
http://www.sigmaaldrich.com/technical-documents/protocols/biology/roche/complete-his-tag-purification-column.html



I'd definitely say load all of your lysate, possibly run it through twice if 
you really want to. If you're worried about it not binding run it at a slower 
flow rate. You would have to have a lot of expression from one litre of media 
to saturate the column. Not sure what exactly you mean by retention time but 
the protein should stay there indefinitely until you elute it.


Cheers,


Sam

[http://www.sigmaaldrich.com/etc/designs/sigma-aldrich/images/sial-logo-sharing.png]

cOmplete™ His-Tag Purification Column Protocol 
...
www.sigmaaldrich.com
Protocol. Purification Protocols. cOmplete His-Tag Purification Columns are 
compatible with automated chromatography systems such as ÄKTAexplorer.




From: CCP4 bulletin board  on behalf of Sundaram 

Sent: 01 February 2017 09:24:09
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Manual His-Tag Purification

Hello ,

It's an off topic question.

I'm planning to do manual purification a 6 his tagged protein of size around 
20kDa from 1L E.coli culture using
COHISC-RO Roche cOmplete™ His-Tag Purification Column.


Can I get some advice regarding the lysate loading volume and retention time.
This is the first time I going to use this column and I have no idea about my 
protein yield from 1L culture.

Sorry if I spammed your inbox.


Thanks!

Yours Sincerely,
Sundaram.S

[ccp4bb] Manual His-Tag Purification

[Sundaram]

Hello ,

It's an off topic question.

I'm planning to do manual purification a 6 his tagged protein of size
around 20kDa from 1L E.coli culture using
COHISC-RO Roche cOmplete™ His-Tag Purification Column.


Can I get some advice regarding the lysate loading volume and retention
time.
This is the first time I going to use this column and I have no idea about
my protein yield from 1L culture.

Sorry if I spammed your inbox.


Thanks!

Yours Sincerely,
Sundaram.S

[ccp4bb] Fwd: Re: [ccp4bb] comparison of different protein states

[F.Xavier Gomis-Rüth]

Dear all,

just a side comment to Guillermo's and Debanu´s discussion, which deals 
with the term "apo".


I got the text below from a reviewer, who—as an exception ;-)—was 
absolutely right:


In enzymology, enzymes that require the aid of cofactors such as metal 
ions or prosthetic groups (e.g. a heme group, etc.) are holo-enzymes. In 
the absence of these groups, the enzyme is non-functional and is termed 
apo-enzyme. A common mistake that can be detected lately in the 
literature is to call a holo-enzyme that lacks a bound substrate or 
product an apo-form. Please, replace apo form with unbound form 
throughout the text except when dealing with forms lacking the catalytic 
zinc ion.


Best regards,
Xavier


 Forwarded Message 
Subject:Re: [ccp4bb] comparison of different protein states
Date:   Tue, 31 Jan 2017 22:23:27 -0800
From:   Debanu Das 
Reply-To:   debanu@gmail.com
To: CCP4BB@JISCMAIL.AC.UK



Dear Guillermo,

I think the referee has a point because you are comparing two
different proteins to propose a model of 2 states even though the
enzymes are highly similar with conservation of key features. With 40%
id/60% similarity, even if core domain structure and length are
conserved, there are likely differences in loops, etc.? Are any such
structural variations in the vicinity of the functional site or in
regions that could impact approach or function of the active site? If
so, the apo and bound forms could also be due to intrinsic differences
between the two orthologs?

In the absence of additional supporting evidence, I suppose one way to
frame it would be to emphasize that you are proposing a model based on
available experimental evidence on closely related proteins and also
admit the limitations and unconfirmed/speculative nature of the
proposed model?

However, I think there may be something more interesting you can do
for supporting your theory in case there are other experimental
structures of closely related members of the same enzyme family. If
so, you could compare in detail additional apo forms of multiple
related enzymes and then point to the complex form as an outlier due
to its structural differences, which then may represent your proposed
model.

In addition, do you have assay/mutational analysis of your protein
compared to the other one? If so, and if they have similar activity
and full conservation of active sites, then that can also be used to
support your theory that the 2 enzymes are structurally and
functionally very similar and so the two structures are representative
of your proposed pathway/model.

I had a similar situation a few years ago in comparative structural
analysis of 2 nucleases. Active site residues were identical with the
same metal and overall architecture was similar despite other
structure/dimer differences. Referee insisted we mutate our active
site residues and compare, which we did.

Hope this helps.
Best,
Debanu
--
Debanu Das,
Accelero Biostructures

On Tue, Jan 31, 2017 at 9:40 PM, Guillermo Montoya
 wrote:


Dear all,

first of all sorry for this off-topic question. I am requesting your help to
find some papers  to convince one referee about the comparison
of two different protein states.

In our manuscript we show the crystal structure of an
enzyme. This structure represents the enzyme after catalysis in complex with
the product.
 In the discussion we have superimposed the enzyme in the apo conformation
and the enzyme after catalysis in complex with the product
and we have  commented the conformational changes observed
between these 2 states to propose a model.

The point of the referee is that this comparison is not valid because the
enzymes that we used in the comparison belong to different species.
They are not the same protein.

However, and this is stated by us  in the figs and the manuscript, these two
proteins are
40% identical and 60% conserved, the polypeptide length is the same, and
the key amino acids and the domain structure are fully conserved. They are
obviously orthologs.

I´d really appreciate if you can send me some literature/information
to support our approach

Thanks a lot for your input


best




Guillermo Montoya, Prof., Dr.
Research Director, Protein Structure and Function Programme
Novo Nordisk Foundation Center for Protein Research
Faculty of Health and Medical Sciences, University of Copenhagen,
Blegdamsvej 3A, DK-2200 Copenhagen, Denmark
web: www.cpr.ku.dk

Re: [ccp4bb] Coot and pymol 3D for Centos 7?

[mesters]

Hi,

indeed, very difficult nowadays to get your hands on a monitor with 
build-in IR-emitter in order to avoid needing to buy an expensive Nvidia 
card with a 3-pin connector.


On the other hand, on EBAY many older Nvidia cards are being offered 
that have a 3-pin connector and that are fast enough to run 
crystallographic programs such as coot.


Look for the Nvidia FX3700 (very good card with 512 MB of DDR3 memory, 
more than fast enough for Coot) or even an FX4800 (big card, make sure 
it fits into the housing), plenty of interesting offers for little 
money. With these older cards you can drive any modern 120-144 Hz 
Monitor (but obviously need the 3-pin emitter equipment to drive the 
glasses).


Good luck,

Jeroen

Am 31.01.17 um 20:44 schrieb Kay Diederichs:

Hi Jun,

on CentOS 7 it works as described at 
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Stereo#Nvidia_3D_Vision_2
 , with a Quadro K620 and BenQ XL2420TX . Unfortunately this monitor type seems 
difficult to find nowadays.

best,

Kay


On Tue, 31 Jan 2017 16:29:17 +, Jun Dong  wrote:


I have made coot and pymol 3D work with NVIDIA Quadro K5200 under Windows 7 but 
I could not make it work under Centos 7. WinCoot was not able to load big virus 
maps, it crashed all the time. I really want to make coot 3D work under Centos 
7. Has anybody succeeded at running coot 3D in Centos 7?
Jun



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