[ccp4bb] eBIC centre coordinator vacancy

[Martin Walsh]

Dear all, reminder that we have a vacancy at eBIC for a centre coordinator

Full details here:
http://www.diamond.ac.uk/Careers/Vacancies/All/061-17-CH.html

Please do contact Peijun Zhang for informal discussions

Best,
Martin


--
Martin A. Walsh,
Diamond Light Source Ltd
Diamond House,
Harwell Science & Innovation Campus
Didcot OX11 0DE
UK
T:+44 1235 778518
E: martin.wa...@diamond.ac.uk


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Re: [ccp4bb] Total occupancy of two conformations of one nucleotide is over 1.0? Need help!

[Pavel Afonine]

Hi Wei,

thanks for sharing the data (off-list). I did some detective work and yes,
Clemens is correct: what you see is the effect of bulk-solvent. After
adjusting the solvent contribution (mask) in regions occupied by altlocs A
and B the positive density mostly disappears.

Pavel

On Fri, Jul 21, 2017 at 12:26 AM, Clemens Vonrhein <
vonrh...@globalphasing.com> wrote:

> Dear Wei,
>
> remember that you might have "something else" in the location occupied
> by each alternate conformation when it is not occupied by that
> particular conformation. If you only model two alternate conformations
> you are saying something like
>
>  that space A is occupied 50% of the time by this A conformation and
>  50% it is vacuum
>
>  that space B is occupied 50% of the time by this B conformation and
>  50% it is vacuum
>
> It is more likely that you will have space A occupied N% by altConf A
> and (100-N)% e.g. by water/solvent. And for B you have (100-N)%
> altConf B and N% e.g. water/solvent. So you will need to model
> everything marked as N% as altConf A and everything marked (100-N)% as
> altConf B - including water/solvent.
>
> Because this is not yet done, the occupancy refinement might try to
> explain the non-vacuum density (water/solvent) by increasing the
> occupancy of each conformation so they sum up to larger than 1.
>
> Does that make sense?
>
> Cheers
>
> Clemens
>
> PS: in BUSTER (http://www.globalphasing.com/buster/) you can restrain
> the summed occupancy to one, which can help clarifying how to
> model the water/solvent region underneath each conformation
> ... depending on data quality/resolution of course. I'm sure other
> refinement programs have a similar feature.
>
> PPS: it can be sometimes useful to start refinement once from
> A=0.1/B=0.9 and a second run with A=0.9/B=0.1. Refinement should
> reach very similar final values in both cases - which is also a
> good way of providing confidence in the final results, I think.
>
>
> On Fri, Jul 21, 2017 at 01:10:15AM +, Wang, Wei wrote:
> > Hi Everyone,
> >
> >
> > One quick question:
> >
> >
> > When I do phenix refinement, I see continuous omitted map at one RNA
> chain 3'-end (Fig1).
> >
> > There is no possibility of next nucleotide because I have built
> full-length RNA sequence, which is well fitted into density.
> >
> > Hence two conformations is most possible for the last nucleotide. And
> our biochemical data also supported the result of two conformations.
> >
> > However, I found the occupancy is always > 1.0 (fig2 and fig3), when I
> did refinement of occupancy using phenix refinement,.
> >
> > I also think about the possibility of small molecules in the crystal
> buffer. But in most structures of this protein, there is always no small
> molecule here.
> >
> >
> > Any suggestions for occupancy refinement? I will appreciate your great
> help.
> >
> >
> > Best,
> >
> > Wei
> >
> >
> >
> > [cid:2b30f1a8-2a3a-4483-ac12-310f4bbed70b]
> >
> >
> >
> >
> >
> >
>
>
>
> --
>
> *--
> * Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
> * Global Phasing Ltd., Sheraton House, Castle Park
> * Cambridge CB3 0AX, UK   www.globalphasing.com
> *--
>

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Phoebe A. Rice]

You might also be getting aggregation.
If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the 
well?


++

Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago

pr...@uchicago.edu


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton 
[eilise.mil...@gmail.com]
Sent: Friday, July 21, 2017 9:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Completely agree, you need a higher DNA concentration. We have had luck with 10 
nM DNA.

Also, bubbles have a HUGE impact on how the fluorescent signal is measured. 
Make sure you spin your plates down (assuming you are using them) to remove any 
bubbles. We just had an undergrad read his anisotropy assay and the data looked 
horrible. He then realized he had not spun his plate down, after doing so the 
data was much more consistent.

Are you doing technical replicates? We do at least triplicates per plate.

All the best,
Morgan


Morgan E. Milton, PhD


On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi 
mailto:opher.gile...@sgc.ox.ac.uk>> wrote:
FP is the ratio between two fluorescence measurements; if the fluorescence 
signal is too low, you will still get a ratio but it will be essentially noise. 
Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in 
the low nM range, you may have to use other methods to measure KD.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Ruud Hovius]

if one has multiple species ,  e.g. bound and free, with each a 
different relative brightness and anisotropy/polarisation, then the use 
( and interpretation) of anisotropy is straight forward; just a sum 
weighted by molecular fraction and the relative brightness.

For polarisation is far more complicated.

See e.g. Lakowicz - Principles of fluorescence spectroscopy.

However, as long as one treats & interprets the data correctly there is 
no principle reason to forbid the use of polarisation.


Personally, I prefer anisotropy.

Ruud


On 21/7/17 17:57, Keller, Jacob wrote:


*>*Ideally you should convert polarization to anisotropy. Simple 
enough – but some referees can get picky…


What is the argument for anisotropy being better?


JPK



--

Ruud Hovius
EPFL SB ISIC LIP
BCH 4209
CH-1015 Lausanne
+41-21-693-9442
http://lip.epfl.ch

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Keller, Jacob]

>Ideally you should convert polarization to anisotropy. Simple enough – but 
>some referees can get picky…

What is the argument for anisotropy being better?

JPK

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Toth, Eric]

With this type of behavior, one suspicion is that you didn’t let the reaction 
come to equilibrium prior to taking the measurement and the variability between 
time of mixing and measuring between individual replicates is introducing extra 
variability. Take a concentration at which you know there is a significant 
signal change and measure the polarization every few minutes for an hour. You 
might find that you need to pre-incubate the samples for an extended time prior 
to taking measurements.

With that said, my first suspicion is that you have lousy binding.

Eric


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Mohammad 
Khan
Sent: Friday, July 21, 2017 9:33 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear all,

I am trying to measure the difference in polarization upon the binding of the 
DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions 
of my protein to it (100 microM to 1 nM). I do get a decrease in difference of 
polarization with decrease in protein concentration. However, the results are 
difficult to reproduce and also vary greatly within triplicates of an 
experiment.

Similar observations have been observed by my colleagues with their proteins.

Are there any tips or precautions to keep in mind while setting up these 
reactions?

Looking forward for suggestions.

Thank you.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Morgan Milton]

Completely agree, you need a higher DNA concentration. We have had luck
with 10 nM DNA.

Also, bubbles have a HUGE impact on how the fluorescent signal is measured.
Make sure you spin your plates down (assuming you are using them) to remove
any bubbles. We just had an undergrad read his anisotropy assay and the
data looked horrible. He then realized he had not spun his plate down,
after doing so the data was much more consistent.

Are you doing technical replicates? We do at least triplicates per plate.

All the best,
Morgan


Morgan E. Milton, PhD


On Fri, Jul 21, 2017 at 9:48 AM, Opher Gileadi 
wrote:

> FP is the ratio between two fluorescence measurements; if the fluorescence
> signal is too low, you will still get a ratio but it will be essentially
> noise. Try to perform the measurements at 10-50 nM DNA. If your binding
> affinity is in the low nM range, you may have to use other methods to
> measure KD.
>

[ccp4bb] postdoc position in protein MS

[Michael Weyand]

On behalf of Clemens Steegborn please note this job offer:

Dear colleagues,

Sorry for the slightly off-topic posting, but since almost everyone here
does use MS occasionally I consider it not too far off:

We have a postdoc / senior postdoc position in protein MS requiring a
rather experienced and independent researcher. The job ad is attached, I
would appreciate if you would bring it to the attention of potential
candidates.

Best

CSt



PostdocPosition_BC_MS_2017.pdf
Description: Adobe PDF document

[ccp4bb] PhD student position available in CBB Poznan, Poland

[Mirek Gilski]

Dear All,

I'd like to draw your attention to the opening for PhD student position 
at Center for Biocrystallographic Research (Poznan, Poland),
to work on a combination of protein crystallography and supramolecular 
chemistry (project PI prof. Mariusz Jaskolski)


More information at:

https://euraxess.ec.europa.eu/jobs/233708

The project overview can be found at:

http://www.man.poznan.pl/CBB/JOBS/POF.pdf

The deadline for applications is  31st August 2017.

I would be grateful if you could forward this opportunity to potential 
applicants.


Kind regards and all best wishes,
Mirek Gilski

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Corbin Black]

In addition to everyone else's suggestions (especially trying greater [probe]), 
I would look at optimizing the following:

- Sufficient rxn volume. In the 384 well plates, I found that consistency 
between replicates improved with greater volume
- Read height
- Scaling

Corbin Black, BSc
MSc Biochemistry Candidate
University of Northern British Columbia
Department of Chemistry
bla...@unbc.ca
blackcor...@gmail.com
250-552-5046

Sent from my iPhone

On Jul 21, 2017, at 7:11 AM, Thomas Edwards 
mailto:t.a.edwa...@leeds.ac.uk>> wrote:

A few tips, some/all of which may be relevant. Or not...

If you are in 96 or 384 well plates, they vary. We tried quite a few batches 
and brands before settling on one.

Keep your salt concentration as low as possible.

You should be able to measure Kd in a range of about 1nM to low uM. Outside 
that range is harder, or possibly impossible.

Buffers matter.
RNA binding proteins have preferred phosphates, PPIs Tris.
Some triton and/or some BSA can help.

All proteins are different, and each likes some or all or none of the above.

Ideally you should convert polarization to anisotropy. Simple enough - but some 
referees can get picky...

Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT ?Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka


From: CCP4 bulletin board mailto:CCP4BB@JISCMAIL.AC.UK>> 
on behalf of Mohammad Khan 
mailto:mohdkhan0...@gmail.com>>
Reply-To: Mohammad Khan mailto:mohdkhan0...@gmail.com>>
Date: Friday, 21 July 2017 at 14:32
To: "CCP4BB@JISCMAIL.AC.UK" 
mailto:CCP4BB@JISCMAIL.AC.UK>>
Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear all,

I am trying to measure the difference in polarization upon the binding of the 
DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions 
of my protein to it (100 microM to 1 nM). I do get a decrease in difference of 
polarization with decrease in protein concentration. However, the results are 
difficult to reproduce and also vary greatly within triplicates of an 
experiment.

Similar observations have been observed by my colleagues with their proteins.

Are there any tips or precautions to keep in mind while setting up these 
reactions?

Looking forward for suggestions.

Thank you.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Thomas Edwards]

A few tips, some/all of which may be relevant. Or not…

If you are in 96 or 384 well plates, they vary. We tried quite a few batches 
and brands before settling on one.

Keep your salt concentration as low as possible.

You should be able to measure Kd in a range of about 1nM to low uM. Outside 
that range is harder, or possibly impossible.

Buffers matter.
RNA binding proteins have preferred phosphates, PPIs Tris.
Some triton and/or some BSA can help.

All proteins are different, and each likes some or all or none of the above…..

Ideally you should convert polarization to anisotropy. Simple enough – but some 
referees can get picky…

Ed

T.A.Edwards Ph.D.
Deputy Director Astbury Centre for Structural Molecular Biology
Ass. Professor, School of Molecular and Cellular Biology
Garstang 8.53d
University of Leeds, Leeds, LS2 9JT 
Telephone: 0113 343 3031
http://www.astbury.leeds.ac.uk/people/staff/staffpage.php?StaffID=TE
Invention, my dear friends, is 93% perspiration, 6% electricity, 4% 
evaporation, and 2% butterscotch ripple. ~Willy Wonka
[cid:image001.png@01D30233.9026B5C0]

From: CCP4 bulletin board  on behalf of Mohammad Khan 

Reply-To: Mohammad Khan 
Date: Friday, 21 July 2017 at 14:32
To: "CCP4BB@JISCMAIL.AC.UK" 
Subject: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear all,

I am trying to measure the difference in polarization upon the binding of the 
DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions 
of my protein to it (100 microM to 1 nM). I do get a decrease in difference of 
polarization with decrease in protein concentration. However, the results are 
difficult to reproduce and also vary greatly within triplicates of an 
experiment.

Similar observations have been observed by my colleagues with their proteins.

Are there any tips or precautions to keep in mind while setting up these 
reactions?

Looking forward for suggestions.

Thank you.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Didier Spittler]

Hello,

What is your difference between the maximum and minimum value ? Have you
try to change the probe concentration?

Best,

Didier

Le 21 juil. 2017 15:34, "Mohammad Khan"  a écrit :

Dear all,

I am trying to measure the difference in polarization upon the binding of
the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
difference of polarization with decrease in protein concentration. However,
the results are difficult to reproduce and also vary greatly within
triplicates of an experiment.

Similar observations have been observed by my colleagues with their
proteins.

Are there any tips or precautions to keep in mind while setting up these
reactions?

Looking forward for suggestions.

Thank you.

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

[Opher Gileadi]

FP is the ratio between two fluorescence measurements; if the fluorescence 
signal is too low, you will still get a ratio but it will be essentially noise. 
Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in 
the low nM range, you may have to use other methods to measure KD.

[ccp4bb] Off topic: Flourescence anisotropy measurement

[Mohammad Khan]

Dear all,

I am trying to measure the difference in polarization upon the binding of
the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying
dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in
difference of polarization with decrease in protein concentration. However,
the results are difficult to reproduce and also vary greatly within
triplicates of an experiment.

Similar observations have been observed by my colleagues with their
proteins.

Are there any tips or precautions to keep in mind while setting up these
reactions?

Looking forward for suggestions.

Thank you.

[ccp4bb] AW: AW: [ccp4bb] high Rfree

[Herman . Schreuder]

Hi ???,

I would first try AMPLE, as was suggested by Daniel.

If separate helices still give you an anti-parallel coiled coil, your structure 
may in fact be anti-parallel. Your problem is not that the structure has 
another parallelity than you would like, but that you cannot solve the MR 
problem. If your structure turns out to be anti-parallel, that’s what it is, as 
long as the structure has been solved correctly.

How do your electron density maps look like: do you see any difference density 
for mutated side chains? Do you see any hints how to extend your shorter model? 
Did you do any refinement? If your MR solution is basically correct, the 
Rfactor might drop quite quickly during refinement, if not, the Rfactor may 
stay stuck or explode.

Best,
Herman


Von: 张士军 [mailto:21620150150...@stu.xmu.edu.cn]
Gesendet: Freitag, 21. Juli 2017 12:56
An: Schreuder, Herman /DE
Betreff: Re: AW: [ccp4bb] high Rfree


Hi

The resolution of my data is 2.8A, and the Rfree/Rwork are 0.55/0.49 which are 
very high. And I was separate them when I do MR, the solution is still  
anti-parallel coiled-coil after MR. Does it mean I MUST DO anomolous phasing ?
-原始邮件-
发件人:herman.schreu...@sanofi.com
发送时间:2017-07-21 15:37:33 (星期五)
收件人: 21620150150...@stu.xmu.edu.cn, 
CCP4BB@JISCMAIL.AC.UK
抄送:
主题: AW: [ccp4bb] high Rfree
Hi ???,

Coiled coil structures can be very tricky with MR, so your solution may not be 
correct. You could try to split your search model and run MR with the separate 
coils. If you have high enough resolution (> ~2.3 Å?) and your MR solution is 
basically correct, you may be able to solve your problem by rebuilding and 
refinement, but this may be a lot of work. Without any details (resolution of 
the data, current Rwork/Rfree etc.) it is difficult to give any more specific 
advice.

Best,
Herman

Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Freitag, 21. Juli 2017 03:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] high Rfree

Hi everyone

   I have got a anti-parallel coiled-coil structure in a short fragment 
recently, then I want to solve a longer fragment structure with phenix-MR using 
this short fragment structure as a model.The result is not good because of the 
Rwork and Rfree is 
high.So
 I think the longer fragment will be parallel coiled-coil which is different 
with the shorter one. I am wondering whether there are any other methods to 
handle this phenomenon besides heavy atom phasing? Thanks a lot!!!

Re: [ccp4bb] ample

[Randy Read]

Dear Patrick,

Apologies for a partly non-CCP4 answer!  You can use the HHPRED alignments in 
some of the software available in Phenix.  One option is to provide the .hhr 
file to MRage, which will fetch the PDB entries, run Sculptor to modify the 
templates with one or more protocols, based on that alignment, and then test 
all the models that have been generated.  That's fully automated and will often 
work.  If it doesn't, an alternative is to run phenix.mr_model_preparation, 
which again will fetch the PDB entries and run the single best Sculptor 
protocol on all of them.  I tend to prefer this option myself, because then you 
can look at whether it would be sensible to combine various alternative models 
as ensembles.  One of the most powerful approaches is to run Ensembler 
(available in both CCP4 and Phenix, as is Sculptor) to make an ensemble, with 
the "trim=True" option turned on to trim any bits that don't agree among the 
structures in the ensemble.  Cutting back to a conserved core has allowed a 
number of very recalcitrant structures to be solved with collections of models 
around the 20% sequence identity range.

Best wishes,

Randy

> On 20 Jul 2017, at 23:13, Patrick Loll  wrote:
> 
> I’m intrigued by the prospect of using AMPLE to test multiple distant 
> homologs in a MR problem. I’ve used HHPRED to identify about 20 
> high-probability homologs of known structure, each of which has about 20-25% 
> identity with the unknown protein. However, it’s not clear to me from the 
> documentation whether the program will use the alignments from HHPRED, and, 
> if so, how I should provide that information. 
> 
> Or does AMPLE perform its own alignment? I.e., do I simply point the program 
> to a directory containing 20 different PDB files and stand back?
> 
> Thanks for any insights.
> 
> Cheers,
> 
> Pat 
> ---
> Patrick J. Loll, Ph. D.  
> Professor of Biochemistry & Molecular Biology
> Drexel University College of Medicine
> Room 10-102 New College Building
> 245 N. 15th St., Mailstop 497
> Philadelphia, PA  19102-1192  USA
> 
> (215) 762-7706
> pjl...@gmail.com
> pj...@drexel.edu

--
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] ample

[Daniel Rigden]

Dear Pat

Yes, AMPLE does it's own, purely structure-based, alignment of the 
homologs provided using GESAMT. You provide a directory of structures to 
work with and, crucially, give AMPLE the


-homologs True

flag so that it knows the inputs have different sequences (unlike in 
modelling mode), and need to be dealt with accordingly.


There's more explanation and example files here

http://ample.readthedocs.io/en/latest/examples/rst/homologs.html#example-dist-homologs

In this mode AMPLE first finds the common core shared by all inputs and 
then produces graded further truncations of that with different side 
chain treatments. This sampling is often necessary in the hardest cases. 
What this means, however, is if the input set are ultra-diverse, then 
the GESAMT shared common core can be quite small. It can therefore also 
be worth trying a smaller set of not quite so diverse structures which 
share a somewhat larger core structure.


Best wishes

Daniel

On 20/07/17 23:13, Patrick Loll wrote:

I’m intrigued by the prospect of using AMPLE to test multiple distant homologs 
in a MR problem. I’ve used HHPRED to identify about 20 high-probability 
homologs of known structure, each of which has about 20-25% identity with the 
unknown protein. However, it’s not clear to me from the documentation whether 
the program will use the alignments from HHPRED, and, if so, how I should 
provide that information.

Or does AMPLE perform its own alignment? I.e., do I simply point the program to 
a directory containing 20 different PDB files and stand back?

Thanks for any insights.

Cheers,

Pat
---
Patrick J. Loll, Ph. D.
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu


--
Dr Daniel John Rigden Tel:(+44) 151 795 4467
Institute of Integrative Biology  FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpool   http://pcwww.liverpool.ac.uk/~drigden/
Crown St.,
Liverpool L69 7ZB, U.K.

Re: [ccp4bb] AW: [ccp4bb] high Rfree

[Daniel Rigden]

Hi there

AMPLE works very well for coiled-coil structures, irrespective of 
parallel/anti-parallel etc


http://journals.iucr.org/m/issues/2015/02/00/lz5005/

Resolution permitting, SHELXE tracing and phase modification, built into 
the AMPLE pipeline, gives statistics that differentiate very clearly 
between success and failure.


I also understand that ARCIMBOLDO works very well for coiled-coils

Good luck

Daniel


On 21/07/17 08:37, herman.schreu...@sanofi.com wrote:


Hi ???,

Coiled coil structures can be very tricky with MR, so your solution 
may not be correct. You could try to split your search model and run 
MR with the separate coils. If you have high enough resolution (> ~2.3 
Å?) and your MR solution is basically correct, you may be able to 
solve your problem by rebuilding and refinement, but this may be a lot 
of work. Without any details (resolution of the data, current 
Rwork/Rfree etc.) it is difficult to give any more specific advice.


Best,

Herman

*Von:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *Im Auftrag 
von *???

*Gesendet:* Freitag, 21. Juli 2017 03:17
*An:* CCP4BB@JISCMAIL.AC.UK
*Betreff:* [ccp4bb] high Rfree

Hi everyone

   I have got a anti-parallel coiled-coil structure in a short 
fragment recently, then I want to solve a longer fragment structure 
with phenix-MR using this short fragment structure as a model.The 
result is not good because of the Rwork and Rfree is high.So 
 
I think the longer fragment will be parallel coiled-coil which is 
different with the shorter one. I am wondering whether there are any 
other methods to handle this phenomenon besides heavy atom phasing? 
Thanks a lot!!!




--
Dr Daniel John Rigden Tel:(+44) 151 795 4467
Institute of Integrative Biology  FAX:(+44) 151 795 4406
Room 101, Biosciences Building
University of Liverpool   http://pcwww.liverpool.ac.uk/~drigden/
Crown St.,
Liverpool L69 7ZB, U.K.

[ccp4bb] AW: [ccp4bb] high Rfree

[Herman . Schreuder]

Hi ???,

Coiled coil structures can be very tricky with MR, so your solution may not be 
correct. You could try to split your search model and run MR with the separate 
coils. If you have high enough resolution (> ~2.3 Å?) and your MR solution is 
basically correct, you may be able to solve your problem by rebuilding and 
refinement, but this may be a lot of work. Without any details (resolution of 
the data, current Rwork/Rfree etc.) it is difficult to give any more specific 
advice.

Best,
Herman

Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von ???
Gesendet: Freitag, 21. Juli 2017 03:17
An: CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] high Rfree

Hi everyone

   I have got a anti-parallel coiled-coil structure in a short fragment 
recently, then I want to solve a longer fragment structure with phenix-MR using 
this short fragment structure as a model.The result is not good because of the 
Rwork and Rfree is 
high.So
 I think the longer fragment will be parallel coiled-coil which is different 
with the shorter one. I am wondering whether there are any other methods to 
handle this phenomenon besides heavy atom phasing? Thanks a lot!!!

Re: [ccp4bb] Total occupancy of two conformations of one nucleotide is over 1.0? Need help!

[Clemens Vonrhein]

Dear Wei,

remember that you might have "something else" in the location occupied
by each alternate conformation when it is not occupied by that
particular conformation. If you only model two alternate conformations
you are saying something like

 that space A is occupied 50% of the time by this A conformation and
 50% it is vacuum

 that space B is occupied 50% of the time by this B conformation and
 50% it is vacuum

It is more likely that you will have space A occupied N% by altConf A
and (100-N)% e.g. by water/solvent. And for B you have (100-N)%
altConf B and N% e.g. water/solvent. So you will need to model
everything marked as N% as altConf A and everything marked (100-N)% as
altConf B - including water/solvent.

Because this is not yet done, the occupancy refinement might try to
explain the non-vacuum density (water/solvent) by increasing the
occupancy of each conformation so they sum up to larger than 1.

Does that make sense?

Cheers

Clemens

PS: in BUSTER (http://www.globalphasing.com/buster/) you can restrain
the summed occupancy to one, which can help clarifying how to
model the water/solvent region underneath each conformation
... depending on data quality/resolution of course. I'm sure other
refinement programs have a similar feature.

PPS: it can be sometimes useful to start refinement once from
A=0.1/B=0.9 and a second run with A=0.9/B=0.1. Refinement should
reach very similar final values in both cases - which is also a
good way of providing confidence in the final results, I think.


On Fri, Jul 21, 2017 at 01:10:15AM +, Wang, Wei wrote:
> Hi Everyone,
> 
> 
> One quick question:
> 
> 
> When I do phenix refinement, I see continuous omitted map at one RNA chain 
> 3'-end (Fig1).
> 
> There is no possibility of next nucleotide because I have built full-length 
> RNA sequence, which is well fitted into density.
> 
> Hence two conformations is most possible for the last nucleotide. And our 
> biochemical data also supported the result of two conformations.
> 
> However, I found the occupancy is always > 1.0 (fig2 and fig3), when I did 
> refinement of occupancy using phenix refinement,.
> 
> I also think about the possibility of small molecules in the crystal buffer. 
> But in most structures of this protein, there is always no small molecule 
> here.
> 
> 
> Any suggestions for occupancy refinement? I will appreciate your great help.
> 
> 
> Best,
> 
> Wei
> 
> 
> 
> [cid:2b30f1a8-2a3a-4483-ac12-310f4bbed70b]
> 
> 
> 
> 
> 
> 



-- 

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* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
* Global Phasing Ltd., Sheraton House, Castle Park 
* Cambridge CB3 0AX, UK   www.globalphasing.com
*--