[ccp4bb] BBSRC-funded PDRA in CryoEM at University of Leeds

[Chi Trinh]

On behalf of Professor Neil Ranson (Director of EM, Astbury BioStructure 
Laboratory)


Dear All,

A new 3-year BBSRC-funded position in my lab is now available. The project is 
to work on high resolution structures of new plant viruses using the state of 
the art cryo-EM facilities with the Astbury Centre for Structural Molecular 
Biology at the University of Leeds.

We hope that the project will generate a number of novel, near-atomic 
resolution structures, and it would therefore benefit from extensive experience 
of model building. As such, while I'd ideally like cryo-EM experience, the 
position would also be suitable for a good X-ray crystallographer with the 
drive and commitment to learn cryo-EM.

For more info, including a candidate brief and info on the Astbury Centre:
https://jobs.leeds.ac.uk/Vacancy.aspx?id=9465=1

Please feel free to get in touch to discuss the position informally.

KR,
Neil Ranson


Professor Neil Ranson | Director of EM, Astbury BioStructure Laboratory
Astbury Centre for Structural Molecular Biology
School of Molecular & Cellular Biology
University of Leeds | LEEDS | LS2 9JT |  UK.
Astbury Building: Room 8.108
T: +44 (0) 113 343 7065| E: 
n.a.ran...@leeds.ac.uk | W: 
http://bit.ly/2voOQbI | Tw: @naranson

Re: [ccp4bb] AW: [ccp4bb] doubt regarding MR search model

[Eleanor Dodson]

Satvik - if you attach your data processing logs I can tell you what to
look out for - too abstract to do without a concrete example to discuss.
Eleanor

On 20 September 2017 at 14:00,  wrote:

> Dear Satvik,
>
>
>
> An R/Rfree of 0.29/0.35 after one round of automatic model building
> indicates that your solution is correct. You can proceed with refinement
> and rebuilding. You can take either the monomer-based or dimer-based
> solution, it does not matter. Personally I would take the monomer-based
> solution since it allowed for some small difference in orientation of the
> monomers within the dimer.
>
>
>
> Best,
>
> Herman
>
>
>
> *Von:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag von
> *Satvik Kumar
> *Gesendet:* Mittwoch, 20. September 2017 14:40
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* Re: [ccp4bb] doubt regarding MR search model
>
>
>
> Hello,
>
> Thanks all for your valuable suggestions. Two things that stuck me were "one
> gets to know if the space group is correct only after solving the
> structure" and "to try all possible space groups until one lands up with
> the correct solution".
>
> I ran Phaser with "all alternative space groups". The solution is still in
> the same space group P212121. This holds true with both the monomer as well
> as dimer search models. I hope I have checked all orthorhombic space groups
> by doing this as I am unsure what "all alternative space groups" means.
>
> I also ran Zanuda which tells me that the space group P212121 is correct
> in both cases.
>
> Pointless output indicates that "Systematic absence probability: 0.818".
> Can I take this to be convincing or do I need to run other tests?
>
>
>
> I dont know how to check for non-crystallographic symmetry? Please tell me
> how I can do so.
>
>
>
> The model is 60 percent identical to my protein. Also, one round of
> automated model building has brought down the R values. The Rwork,Rfree
> stand at 0.29,0.35 (monomer search model) and 0.30, 0.35 (dimer search
> model). There is good scope for building residues in both cases. But which
> one do I go ahead with is my question.
>
>
>
> Thanks,
>
> Satvik
>
>
>
>
>
> On Tue, Sep 19, 2017 at 11:40 PM, Eleanor Dodson <
> eleanor.dod...@york.ac.uk> wrote:
>
> Well - you haven't said what the sequence identity between model and your
> protein is, nor if you have a non-crystallographic translation.
>
>
>
> With low homology that R factor drop is acceptable and rebuilding can fix
> it, However if there is high homology you might expect better.
>
>
>
> But this sort of conjecture is pretty pointless - check in all
> orthorhombic  space groups as Mark suggests.
>
>
>
> Eleanor
>
>
>
> On 19 September 2017 at 15:16, Mark J van Raaij 
> wrote:
>
> With Rs of 43/48% I don't think you can be sure that your spacegroup is
> right.
>
> You should always try all the spacegroup possibilities until you get a
> solution you are sure is right, i.e. that refines to Rs of around 35% or
> preferably even lower.
>
> More so in the case of screw axes, so try P222, P2122, P2212, P2221,
> P21212, P21221, P22121 and P212121. Phaser can do this automatically for
> you by clicking the right box.
>
> If necessary, then try lower symmetry like P21 and perhaps P1.
>
> Programs like Xanuda can help.
>
>
>
>
>
> 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 <+34%20915%2085%2046%2016>
> http://wwwuser.cnb.csic.es/~mjvanraaij
> 
>
> Editor of Acta Crystallographica F, Structural Biology Communications
>
> http://journals.iucr.org/f/
> 
>
>
>
> On 19 Sep 2017, at 16:01, Satvik Kumar  wrote:
>
>
>
> Hello,
>
> Thanks everyone for your explanations.
>
> I have pasted the pointless output to provide more information.
>
> Best Solution:  space group P 21 21 21
>
> Laue group probability:   0.959
>
> Systematic absence probability: 0.818
>
> Total probability: 0.785
>
> Space group confidence: 

[ccp4bb] AW: [ccp4bb] doubt regarding MR search model

[Herman . Schreuder]

Dear Satvik,

An R/Rfree of 0.29/0.35 after one round of automatic model building indicates 
that your solution is correct. You can proceed with refinement and rebuilding. 
You can take either the monomer-based or dimer-based solution, it does not 
matter. Personally I would take the monomer-based solution since it allowed for 
some small difference in orientation of the monomers within the dimer.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Satvik 
Kumar
Gesendet: Mittwoch, 20. September 2017 14:40
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] doubt regarding MR search model

Hello,
Thanks all for your valuable suggestions. Two things that stuck me were "one 
gets to know if the space group is correct only after solving the structure" 
and "to try all possible space groups until one lands up with the correct 
solution".
I ran Phaser with "all alternative space groups". The solution is still in the 
same space group P212121. This holds true with both the monomer as well as 
dimer search models. I hope I have checked all orthorhombic space groups by 
doing this as I am unsure what "all alternative space groups" means.
I also ran Zanuda which tells me that the space group P212121 is correct in 
both cases.
Pointless output indicates that "Systematic absence probability: 0.818". Can I 
take this to be convincing or do I need to run other tests?

I dont know how to check for non-crystallographic symmetry? Please tell me how 
I can do so.

The model is 60 percent identical to my protein. Also, one round of automated 
model building has brought down the R values. The Rwork,Rfree stand at 
0.29,0.35 (monomer search model) and 0.30, 0.35 (dimer search model). There is 
good scope for building residues in both cases. But which one do I go ahead 
with is my question.


Thanks,
Satvik


On Tue, Sep 19, 2017 at 11:40 PM, Eleanor Dodson 
> wrote:
Well - you haven't said what the sequence identity between model and your 
protein is, nor if you have a non-crystallographic translation.

With low homology that R factor drop is acceptable and rebuilding can fix it, 
However if there is high homology you might expect better.

But this sort of conjecture is pretty pointless - check in all orthorhombic  
space groups as Mark suggests.

Eleanor

On 19 September 2017 at 15:16, Mark J van Raaij 
> wrote:
With Rs of 43/48% I don't think you can be sure that your spacegroup is right.
You should always try all the spacegroup possibilities until you get a solution 
you are sure is right, i.e. that refines to Rs of around 35% or preferably even 
lower.
More so in the case of screw axes, so try P222, P2122, P2212, P2221, P21212, 
P21221, P22121 and P212121. Phaser can do this automatically for you by 
clicking the right box.
If necessary, then try lower symmetry like P21 and perhaps P1.
Programs like Xanuda can help.


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
Editor of Acta Crystallographica F, Structural Biology Communications
http://journals.iucr.org/f/

On 19 Sep 2017, at 16:01, Satvik Kumar 
> wrote:

Hello,
Thanks everyone for your explanations.
I have pasted the pointless output to provide more information.

Best Solution:  space group P 21 21 21

Laue group probability:   0.959

Systematic absence probability: 0.818

Total probability: 0.785

Space group confidence:0.751

Laue group confidence   0.951


Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00

Also based on L-test, pointless says data does not suggest twinning.

Yes, the R values go down when I refine in both cases. After 20 rounds of 
restrained refinement using the coordinates generated by monomer as search 
model, the Rwork and Rfree are 

Re: [ccp4bb] doubt regarding MR search model

[Satvik Kumar]

Hello,

Thanks all for your valuable suggestions. Two things that stuck me were "one
gets to know if the space group is correct only after solving the
structure" and "to try all possible space groups until one lands up with
the correct solution".

I ran Phaser with "all alternative space groups". The solution is still in
the same space group P212121. This holds true with both the monomer as well
as dimer search models. I hope I have checked all orthorhombic space groups
by doing this as I am unsure what "all alternative space groups" means.

I also ran Zanuda which tells me that the space group P212121 is correct in
both cases.

Pointless output indicates that "Systematic absence probability: 0.818".
Can I take this to be convincing or do I need to run other tests?

I dont know how to check for non-crystallographic symmetry? Please tell me
how I can do so.

The model is 60 percent identical to my protein. Also, one round of
automated model building has brought down the R values. The Rwork,Rfree
stand at 0.29,0.35 (monomer search model) and 0.30, 0.35 (dimer search
model). There is good scope for building residues in both cases. But which
one do I go ahead with is my question.




Thanks,
Satvik


On Tue, Sep 19, 2017 at 11:40 PM, Eleanor Dodson 
wrote:

> Well - you haven't said what the sequence identity between model and your
> protein is, nor if you have a non-crystallographic translation.
>
> With low homology that R factor drop is acceptable and rebuilding can fix
> it, However if there is high homology you might expect better.
>
> But this sort of conjecture is pretty pointless - check in all
> orthorhombic  space groups as Mark suggests.
>
> Eleanor
>
> On 19 September 2017 at 15:16, Mark J van Raaij 
> wrote:
>
>> With Rs of 43/48% I don't think you can be sure that your spacegroup is
>> right.
>> You should always try all the spacegroup possibilities until you get a
>> solution you are sure is right, i.e. that refines to Rs of around 35% or
>> preferably even lower.
>> More so in the case of screw axes, so try P222, P2122, P2212, P2221,
>> P21212, P21221, P22121 and P212121. Phaser can do this automatically for
>> you by clicking the right box.
>> If necessary, then try lower symmetry like P21 and perhaps P1.
>> Programs like Xanuda can help.
>>
>>
>> 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 <+34%20915%2085%2046%2016>
>> http://wwwuser.cnb.csic.es/~mjvanraaij
>> Editor of Acta Crystallographica F, Structural Biology Communications
>> http://journals.iucr.org/f/
>>
>> On 19 Sep 2017, at 16:01, Satvik Kumar  wrote:
>>
>> Hello,
>>
>> Thanks everyone for your explanations.
>>
>> I have pasted the pointless output to provide more information.
>>
>> Best Solution:  space group P 21 21 21
>>
>> Laue group probability:   0.959
>>
>> Systematic absence probability: 0.818
>>
>> Total probability: 0.785
>>
>> Space group confidence:0.751
>>
>> Laue group confidence   0.951
>>
>>
>> Unit cell:   82.10 100.51 157.11 90.00  90.00  90.00
>>
>> Also based on L-test, pointless says data does not suggest twinning.
>>
>> Yes, the R values go down when I refine in both cases. After 20 rounds of 
>> restrained refinement using the coordinates generated by monomer as search 
>> model, the Rwork and Rfree are 0.43 and 0.48
>>
>> respectively. Refinement using the coordinates generated by using dimer
>> as search model also results in similar R values. I have attached the plots
>> to show that the R values indeed reduce in both cases.
>>
>> Is my space group correct? Do I need to reexamine the space group even
>> though the probability is high?
>>
>> If my space group is indeed correct, how do I decide whether to go ahead
>> with the results generated by the monomer search model or the dimer?
>>
>> Please share your thoughts.
>>
>>
>> Thanks,
>> Satvik
>>
>> On Mon, Sep 18, 2017 at 7:36 PM, Eleanor Dodson <
>> eleanor.dod...@york.ac.uk> wrote:
>>
>>> You need to provide a bit more information.
>>>
>>> First of all about the data processing..
>>>
>>> Is the space group correct?
>>> ways of being misled are:
>>> Non-crystallographic translations with a shift of ~0.5 along an axis -
>>> say a.  This will generate absences in the odd h 0 0 reflections and can
>>> make the space group appear to be P 21 21 21 whilst it is really P 2 21 21..
>>>
>>> Perfect twinning can have the same effect. In an orthorhombix space
>>> group this can usually only occur if two axes have approximately the same
>>> length, but the data processing stats can indicate if that is the case.
>>>
>>> Then - re PHASER. The packing rejection criteria may be set too severely
>>> - that 

Re: [ccp4bb] R free flag

[Robbie Joosten]

Hi Rashi,

Rather than use the mtz file from EDS you should use he mmCIF reflection file 
from the PDB directly and convert it. If the authors deposited the test set, 
you can keep it this way. Otherwise you need to select a new test set.
Note that if you want to optimize an existing PDB entry, the pdb-redo databank 
is a good starting point. It already did (part of) the work for you.

Cheers,
Robbie

Sent from my Windows 10 phone

From: Rashi Aggarwal
Sent: woensdag 20 september 2017 08:49
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] R free flag

Hello,

I am trying to refine a structure that is deposited in PDB. I have downloaded 
the _sigmaa.mtz file from the electron density server.

Trying to run refmac using this mtz file gives me the popup free R label has 
not been set.

How can I set free R label in an existing mtz file so that after refinement I 
can get the R and Rfree?

-Rashi

Re: [ccp4bb] AW: [ccp4bb] question regarding sequence numbering

[John Berrisford]

Dear Tony

The only requirements we have for numbering is that every residue must 
be unique when using a combination of residue name (to handle 
microheterogeneity), residue number, insertion code and chain ID.


During curation we will try to map your protein sequence to UniProt - 
please see the following documentation on this process:


https://www.wwpdb.org/documentation/procedure#toc_1

The exact numbering scheme you choose is up to you (especially for 
expression tags), however users of your entry may find it difficult to 
use your entry is you decided to number your protein randomly or with 
decreasing residue numbers. We may suggest that you changed the 
numbering if you did this.


Our official wording from the above link is:

"The wwPDB encourages deposition of polymer chains with sequential 
residue numbering. For protein chains, the authors are encouraged to 
follow the UniProt residue numbering, wherever possible. The use of 
non-sequential residue numbering and insertion codes should be avoided 
as far as possible in order to make structures easily interpretable by 
the larger scientific community. If the coordinate residue numbers, as 
provided by the author, are unique and sequential within a particular 
chain ID, the residues will not be renumbered."


this is from the section "How are chain IDs related to residue numbering?"


I hope this helps

John
PDBe


On 19/09/2017 13:51, herman.schreu...@sanofi.com wrote:


Hi Dave and Tony,

Upon submission, the pdb checks the sequence and automatically 
generates comments about sequences derived from the expression vector. 
So you do not have to do anything. Given the issues many programs have 
with non-sequentially numbered residues, I would also number them 7,8,9.


Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag 
von *Briggs, David C

*Gesendet:* Dienstag, 19. September 2017 14:24
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* Re: [ccp4bb] question regarding sequence numbering

Hi Tony,

When I've had similar issues, I've numbered them sequentially (i.e.  
7,8,9) and remarked in the PDB header that they are vector-derived 
sequence.


I believe that is what the PDB ask you to do in situations like this 
(maybe they can comment?).


If they are not numbered sequentially, then often graphics and 
refinement software won't treat them as linked.


Dave

--

Dr David C Briggs

Hohenester Lab

Department of Life Sciences

Imperial College London

UK

http://about.me/david_briggs 





From: Antonio Ariza

Sent: Tuesday 19 September, 13:15

Subject: [ccp4bb] question regarding sequence numbering

To: ccp4bb@jiscmail.ac.uk 

Hi all,

Here's a problem I haven't come across before. I'm working on a 
structure whose expression plasmid was designed to remove the first 9 
amino acids from the protein of interest and to which an N-terminal 
tag was added. After cleaving the tag I am left with 3 amino 
acids (GPM) followed by the original sequence. Obviously the residues 
of interest should follow the numbering of the original sequence (i.e. 
10, 11, 12, ...). What numbers would you assign to the first 3 
residues (GPM)? 7, 8, 9? -2, -1, 0?


Cheers,

Tony

--

*Dr. Antonio Ariza*

*University of Oxford*

*Sir William Dunn School of Pathology*

*South Parks Road*

*Oxford*

*OX1 3RE*

*e-mail: *antonio.ar...@path.ox.ac.uk 

*Tel: 00 +44 1865 285655*

*Links to my public profiles:*

ResearchGate 



LinkedIn 



GoogleScholar 



Twitter 

[ccp4bb] R free flag

[Rashi Aggarwal]

Hello,

I am trying to refine a structure that is deposited in PDB. I have
downloaded the _sigmaa.mtz file from the electron density server.

Trying to run refmac using this mtz file gives me the popup free R label
has not been set.

How can I set free R label in an existing mtz file so that after refinement
I can get the R and Rfree?

-Rashi