Re: [ccp4bb] circular peptide structure refinement

[Matthew Snee]

Hi All

To prevent Phenix removing link records, you can use Readyset to create a 
parameters file based on LINK records, and feed it to the refinement program, 
otherwhise they are deleted.

Best wishes

Matthew.

From: CCP4 bulletin board  on behalf of Nicholas Clark 

Sent: 14 August 2022 16:01
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] circular peptide structure refinement

Phenix in the past, and I’m assuming still, does not recognize “link records”. 
You’ll likely need to generate the peptide in AceDRG, making sure to link the 
carboxy- and amino-termini, and include the cif during refinement.

This was the only way I was able to get Phenix to properly refine an inhibitor 
covalently linked to an active site Cys. Thus, the same procedure may be 
required for your circular peptide.

Best,

Nick Clark

On Sun, Aug 14, 2022 at 1:06 AM Jiang Xu 
mailto:foxj...@gmail.com>> wrote:
Hi Joel,
 Thank you for your reply. I just got time to refine the circular peptide 
structure 1 month later. I use MR to solve the structure. I made the 
link(Calculate-->Modeling-->Make Link) as the guy who replied to my question 
suggested. The link generated is a dashed line but disappeared after refinement 
with Phenix.  It seemed that the program didn't consider the link made in coot 
as a valid bond and intentionally avoided forming a bond between the C atom and 
the N atom. I still don't know how to fix the problem.
Thank you,
Best regards,
Jiang
Lin Chen Lab
University of Southern California

P.S.
coot manually made link between the C and N terminal
[unnamed.jpg]
After refinement
[unnamed (1).jpg]



On Wed, Jul 6, 2022 at 3:29 PM Joel Tyndall 
mailto:joel.tynd...@otago.ac.nz>> wrote:

You will need to add the “link” line to the PDB file so the software recognises 
the covalent bond.

See the pdb file for 6U6K



Hope this helps



J



From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
On Behalf Of Jiang Xu
Sent: Thursday, 7 July 2022 10:15 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] circular peptide structure refinement



Hello everyone,

   I have a peptide that forms a peptide bond between the N terminal and C 
terminal.  I used X-ray crystallography to solve the structure and found the N 
and C terminals are pretty close to each other with extra electron densities 
clearly showing that they form a peptide bond. However in Coot I could not make 
the peptide bond, the two terminals seem to repel each other when I do real 
space refinement in coot and, couldn't form the peptide bond. Any suggestions 
on how to do it?

Thank you,

Best,

Jiang Xu

Lin Chen Research Group

Molecular and Computational Biology

Department of Biological Sciences

University of Southern California





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--
Nicholas D. Clark
PhD Candidate
Malkowski Lab
University at Buffalo
Department of Structural Biology
Jacob's School of Medicine & Biomedical Sciences
955 Main Street, RM 5130
Buffalo, NY 14203

Cell: 716-830-1908



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[ccp4bb] COOT RSR

[Matthew Snee]

Hi all

I had a bit of trouble getting used to the new COOT real space refine... it 
took me an embarrassingly long time to work out that there is nothing wrong 
with the fitting/restraints.. it just doesn't seem to allow clashes with parts 
of the model that aren't in the selected RSR zone even when the fit would be 
perfect.

Is there a quick way to toggle this feature on/off?
It seems like a nice addition, but it can cause problems where one incorrect 
part of a partially built model prevents me building another, or for cases 
where two different residues/ligands occupy the same space at partial occupancy.

Best wishes

Matthew.

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Re: [ccp4bb] convert loop into a sheet

[Matthew Snee]

Is it not just a case of adding "beta sheet restraints" in the real-space 
refine options menu?

If it goes back to a loop during refinement then that may mean that the atomic 
positions don't meet the requirements?

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From: CCP4 bulletin board  on behalf of rohit kumar 

Sent: Wednesday, August 11, 2021 5:41:55 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] convert loop into a sheet

Hello Everyone,

I was trying to build my peptide (8 AA long) as ligand in coot. Peptide should 
be in the form of Sheet, after multiple cycles of refinement peptide still in 
the loop form. If anyone could help me, how to convert this loop structure into 
a sheet in coot , would be very helpful?
I am using ccp4-Refmac5 for the refinement.


Thank you
Rohit




--
Regards
Dr. Rohit Kumar Singh






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Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Matthew Snee]

It seems immensely powerful, but my impression is it shows just how much 
information can be extrapolated from the PDB if a technique that can make use 
of "deep similarity" can be employed.

Obviously alphafold2 can make use of relationships that arent limited to direct 
homology, but if there is a fundamental "cellular context-free" relationship 
between sequence and structure (I'm sceptical about this) then it must be via 
the sidechains.

If the sidechains predictions are worse than the backbone, and loops are also 
imperfect, then it strongly suggests that the process is still inferring the 
structure (albeit in a very clever way that can determine and weight 
similarities that go far beyond those implied by direct homology) rather than  
"building" it de novo.

Obviously sidechain and loop positions are important when we think about the 
applications of macro molecular structures, but I'm not qualified to say 
whether there is actually enough data in the PDB to beat the law of diminishing 
returns and reliably get trustworthy "experimental quality" predictions, and 
how that will scale with complex proteins which may be very context dependent 
in their ability to fold.

We probably dont need a universal understanding of sequence/structure to get 
there, but the claim that this is just a matter of time only really follows on 
from the assumption of a true de-novo method.  Without it, the learning set may 
need to be bigger than all solved (or even solveable) structures.

This could have been framed as something really exciting and complementary to 
experimental structural biology (trivial MR, much better denovo EM etc..) at a 
time when multi-disciplinary approaches are producing incredible insights, but 
the press that has been generated, seems  misleading, and I fear this is what 
the public and funders will base their decisions upon.

Just my two cents.

Matthew.




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From: CCP4 bulletin board  on behalf of Cedric Govaerts 

Sent: Wednesday, December 9, 2020 9:37:17 AM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less 
pipetting (?)

Dear All

After about 10 (!) years of (very) hard work we solved the structures of our 
dearest membrane transporter.  Dataset at 2.9 And resolution, fairly 
anisotropic, experimental phasing, and many long nights with Coot and 
Buster to achieve model refinement.

The experimental structure had a well defined ligand nicely coordinated but 
also a lipid embedded inside the binding cavity (a complete surprise but 
biologically relevant) and two detergent molecules well defined 
(experimental/crystallisation artefact).

As our paper was accepted basically when CASP organisers were calling for 
targets I offered my baby to the computing Gods. However we only provided the 
sequence to CASP, no info regarding any ligand or lipid.

Less than a month after, the CASP team contacted us and send us the best model. 
 In fact it was 2 half models as the transporter is a pseudo dimer, with the 
N-lobe and C-lobe moving relative to each other during transport cycle, thus 
divided as two domains in CASP.

The results were breathtaking. 0.7 And RSMD on one half, 0.6 on the other. And 
yes, group 427 was the superpower (did not know at the time that it was 
AlphaFold).

We had long discussions with the CASP team, as -for us- this almost exact 
modelling was dream-like (or science fiction) and -at some point- we were even 
suspecting fraud, as our coordinates had travelled over the internet a few 
times around when interacting with colleagues.  The organisers reassured us 
that we were not the only target that had been “nailed” so no reason to suspect 
any wrongdoing.

To this day I am still baffled and I would be happy to hear from the community, 
maybe from some of the CASP participants.

The target is T024, the “perfect" models are domain-split version (T024-D1 and 
T024-D2), as AlphaFold2 did not perform so well on the complete assembly.
Deposited PDB is 6T1Z

Cedric

PS: I should also note that many other groups performed very well, much better 
than I would have dreamed, including on the full protein but just not as 
crazy-good.
—
Prof. Cedric Govaerts, Ph.D.
Universite Libre de Bruxelles
Campus Plaine. Phone :+32 2 650 53 77
Building BC, Room 1C4 203
Boulevard du Triomphe, Acces 2
1050 Brussels
Belgium
http://govaertslab.ulb.ac.be/




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[ccp4bb] Protein expression (codon bias)

[Matthew Snee]

Hi all

I'm designing genes for expression in M. smegmatis (safe host for Mtb 
proteins), but its not possible (or advisable due to the GC content) to 
optimise for
mycobacterial expression.

Would anyone with experience be able to tell me if its fine to stick with the 
E. coli codon optimisation, or if there is an advantage going with b. subtilis 
(or another G+ organism that is in Thermo's list).

Best wishes

Matthew.



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[ccp4bb] Ideas for improving refinement with anisotopic data.

[Matthew Snee]

Hi everyone.

I have an x ray structure that I am finishing up, and there are a few ambiguous 
regions where the peptide is poorly resolved.

The data is highly anisotrophic, and requires truncation to around 2.4A to 
achieve acceptable merging stats, although there is data in the "good" 
direction going as high as 1.8-1.9A (determined by merging stats when using 
elliptical cutoff).

I have tried feeding my integration outputs to STARANISO with both the 
elliptical and spherical cutoffs, but neither produce better results than Xia2 
Dials, as both need to be truncated further than 2.3A before they give the same 
refinement stats (i.e it's best to just let refmac/phenix.refine deal with the 
anistropy).

As I understand it, anisotrophy can lead to loss of high resolution detail 
because weak observations from the high res shells in the bad direction are 
down-weighted, So any tricks to improve map quality (or conversely refining 
data with poor completeness) would be appreciated.

I'm not holding out hope that I can deposit anything better than 2.3A, but 
Improving the maps might really help me with some of these troublesome loops :)

Cheers

Matthew.

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