Re: [ccp4bb] Refinement at 4A resolution

[Appu kumar]

Dear All,
Sorry for the wrong pointless file. With this mail i have attached the
pointless run file from the unmerged data.  This file also suggests the
C2221 spacegroup.
Appu

On 22 April 2015 at 17:40, Christian Roth  wrote:

> Hi Appu,
>
> you start already with a fixed spacegroup (scaled merged data) according
> to your pointless log. So you can't get another possible solution from
> pointless.
>
> Cheers
>
>
>
> Am 22.04.2015 um 22:28 schrieb Appu kumar:
>
>> Dear CCP4 Member,
>> I seek your advice on the refinement issues at the low resolution 4A. I
>> am trying to refine a membrane protein structure after getting the
>> phases from MR using the PHASER. The soluble domain structure which
>> comprises of 40% of protein has been used as template (sequence identity
>> 80%) in MR search . The PHASER gave  a good solution having TFZ value of
>> about 14.3. I have then created the polyA model for the transmembrane
>> domain from distant homolog which share 30% sequence identity for TM
>> region and try to find the phases for whole TM domain keeping the
>> soluble domain fixed. I got lucky in getting the phases for the whole
>> protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork
>> and Rfree got stalled at the 41 and 44 respectively after several cycle
>> of the refinement in both refmac and phenix. I checked the spacegroup
>> with pointless and it suggests C2221. I have attached the pointless and
>> phenix.xtriage run file with this mail for your evaluation.
>> Phenix.xtriage suggests no major pathologies with the data except the
>> mild psuedomerohedral twining. There are two molecules of protein in
>> ASU. Evaluation of the density maps, suggest reasonable map for the most
>> of protein part. I am wondering why Rwork and Rfree are not coming down
>> despite of the good MR solution and what i am doing wrong with
>> refinement and if there is some pathologies associated with the data
>> which needs to be answered before heading to refinement.
>>
>> Thanks for your help in advance.
>>
>> Appu
>>
>>
#CCP4I VERSION CCP4Interface 2.2.1
#CCP4I SCRIPT LOG pointless
#CCP4I DATE 22 Apr 2015  17:39:30
#CCP4I USER appu
#CCP4I PROJECT AS015crv6ctd2nq
#CCP4I JOB_ID 119
#CCP4I SCRATCH /tmp/appu
#CCP4I HOSTNAME sasha
#CCP4I PID 47982

 
 ###
 ###
 ###
 ### CCP4 6.4: POINTLESS version 1.9.16 : 21/08/14##
 ###
 User: appu  Run date: 22/ 4/2015 Run time: 17:39:30 


 Please reference: Collaborative Computational Project, Number 4. 1994.
 "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 760-763.
 as well as any specific reference in the program write-up.

> Input command lines <

HKLIN /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz
HKLOUT /home/appu/xtal/2015_04_17_APS_24IDC/AS015c/refine/AS115c_1_0001_pointless1.mtz
## This script run with the command   ##
# /home/appu/Downloads/ccp4-6.4.0/bin/pointless


> End of input<

OS type:  linux
Release Date: 21st August2014


**
**
* POINTLESS  *
*   1.9.16   *
**
*   Determine Laue group from unmerged intensities   *
* Phil Evans MRC LMB, Cambridge  *
* Uses cctbx routines by Ralf Grosse-Kunstleve et al.*
**
**


 Spacegroup information obtained from library file: 
 Logical Name: SYMINFO   Filename: /home/appu/Downloads/ccp4-6.4.0/lib/data/syminfo.lib


Reflection list generated from file:
/home/appu/xtal/2015_04_17_APS_24IDC/AS015c/imosflm/AS115c_1_0001.mtz

Title: Untitled

   Space group from HKLIN file : C 2 2 21
   Cell:  130.42 213.36 160.80  90.00  90.00  90.00
   Resolution range in file: 65.213.75

Time for reading file(s):2.190 secs

===

>*> Summary of test data read in:
   Resolution range accepted:65.213.75

   Number of reflections  = 23371
   Number of observations =150865
   Number of parts=619082
   Number of batches in file  =   600
   Number of datasets = 1
  Project: New Crystal: New Dataset: New
  Run number:   1 consists of batches  1 to600
 Resolution range for run:65.213.75
 Phi range: 0.00 to   180.00   Time range: 0.0

Re: [ccp4bb] Refinement at 4A resolution

[Christian Roth]

Hi Appu,

you start already with a fixed spacegroup (scaled merged data) according 
to your pointless log. So you can't get another possible solution from 
pointless.


Cheers


Am 22.04.2015 um 22:28 schrieb Appu kumar:

Dear CCP4 Member,
I seek your advice on the refinement issues at the low resolution 4A. I
am trying to refine a membrane protein structure after getting the
phases from MR using the PHASER. The soluble domain structure which
comprises of 40% of protein has been used as template (sequence identity
80%) in MR search . The PHASER gave  a good solution having TFZ value of
about 14.3. I have then created the polyA model for the transmembrane
domain from distant homolog which share 30% sequence identity for TM
region and try to find the phases for whole TM domain keeping the
soluble domain fixed. I got lucky in getting the phases for the whole
protein using the PHASER (TFZ=17.6) but the during the refinement, Rwork
and Rfree got stalled at the 41 and 44 respectively after several cycle
of the refinement in both refmac and phenix. I checked the spacegroup
with pointless and it suggests C2221. I have attached the pointless and
phenix.xtriage run file with this mail for your evaluation.
Phenix.xtriage suggests no major pathologies with the data except the
mild psuedomerohedral twining. There are two molecules of protein in
ASU. Evaluation of the density maps, suggest reasonable map for the most
of protein part. I am wondering why Rwork and Rfree are not coming down
despite of the good MR solution and what i am doing wrong with
refinement and if there is some pathologies associated with the data
which needs to be answered before heading to refinement.

Thanks for your help in advance.

Appu

Re: [ccp4bb] Refinement at 4A resolution

[Phil Evans]

The Xtriage & Pointless logs don't show definitively that the space group is 
C2221 as they have been run on the merged data. You may need to check them with 
the unmerged data, and perhaps run molecular replacement in a lower symmetry 
such as C2

Phil

On 22 Apr 2015, at 22:28, Appu kumar  wrote:

> Dear CCP4 Member,
> I seek your advice on the refinement issues at the low resolution 4A. I am 
> trying to refine a membrane protein structure after getting the phases from 
> MR using the PHASER. The soluble domain structure which comprises of 40% of 
> protein has been used as template (sequence identity 80%) in MR search . The 
> PHASER gave  a good solution having TFZ value of about 14.3. I have then 
> created the polyA model for the transmembrane domain from distant homolog 
> which share 30% sequence identity for TM region and try to find the phases 
> for whole TM domain keeping the soluble domain fixed. I got lucky in getting 
> the phases for the whole protein using the PHASER (TFZ=17.6) but the during 
> the refinement, Rwork and Rfree got stalled at the 41 and 44 respectively 
> after several cycle of the refinement in both refmac and phenix. I checked 
> the spacegroup with pointless and it suggests C2221. I have attached the 
> pointless and phenix.xtriage run file with this mail for your evaluation. 
> Phenix.xtriage suggests no major pathologies with the data except the mild 
> psuedomerohedral twining. There are two molecules of protein in ASU. 
> Evaluation of the density maps, suggest reasonable map for the most of 
> protein part. I am wondering why Rwork and Rfree are not coming down despite 
> of the good MR solution and what i am doing wrong with refinement and if 
> there is some pathologies associated with the data which needs to be answered 
> before heading to refinement.  
> 
> Thanks for your help in advance. 
> 
> Appu
> 
> <93_pointless.log>

[ccp4bb] Refinement at 4A resolution

[Appu kumar]

Dear CCP4 Member,
I seek your advice on the refinement issues at the low resolution 4A. I am
trying to refine a membrane protein structure after getting the phases from
MR using the PHASER. The soluble domain structure which comprises of 40% of
protein has been used as template (sequence identity 80%) in MR search .
The PHASER gave  a good solution having TFZ value of about 14.3. I have
then created the polyA model for the transmembrane domain from distant
homolog which share 30% sequence identity for TM region and try to find the
phases for whole TM domain keeping the soluble domain fixed. I got lucky in
getting the phases for the whole protein using the PHASER (TFZ=17.6) but
the during the refinement, Rwork and Rfree got stalled at the 41 and 44
respectively after several cycle of the refinement in both refmac and
phenix. I checked the spacegroup with pointless and it suggests C2221. I
have attached the pointless and phenix.xtriage run file with this mail for
your evaluation. Phenix.xtriage suggests no major pathologies with the data
except the mild psuedomerohedral twining. There are two molecules of
protein in ASU. Evaluation of the density maps, suggest reasonable map for
the most of protein part. I am wondering why Rwork and Rfree are not coming
down despite of the good MR solution and what i am doing wrong with
refinement and if there is some pathologies associated with the data which
needs to be answered before heading to refinement.

Thanks for your help in advance.

Appu


93_pointless.log
Description: Binary data


xtriage_50.log
Description: Binary data

Re: [ccp4bb] Cleaved peptide density!

[Roger Rowlett]

If your protease depends on an oxyanion hole for stabilizing the 
transition state, fluoride ions are known to be a potent inhibitor of 
these proteases. (It is a quasi-diagnostic test for serine-type 
proteases, and related cysteine proteases.) This might allow you to get 
reactant bound without cleavage. It's worth a shot.


Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

On 4/22/2015 3:40 PM, Radisky, Evette S., Ph.D. wrote:


We had a similar situation with a catalytically dead serine protease.  
Initially I was excited to think we might be seeing residual catalytic 
activity of the mutant enzyme on a highly specific substrate; however, 
the activity turned out to result from contamination with a very small 
amount of wt enzyme, likely as a result of using the same affinity 
column to purify both.  When we incubated the enzyme preparation with 
PMSF (an inhibitor that covalently modifies the catalytic serine and 
would not have affected the mutant), we eliminated the residual 
activity of the enzyme preparation. However, in our case we never were 
able to get crystals with the intact substrate, which apparently was 
not compatible with our crystal form.


Is there a covalent inhibitor of your cysteine protease that you could 
use to pre-treat your enzyme, to see if this eliminates the activity?  
If so this might help distinguish between residual activity of the 
mutant vs. contamination with wt enzyme.


Good luck!

Evette

Evette S. Radisky, Ph.D.
Associate Professor and Senior Associate Consultant

Department of Cancer Biology
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372

http://www.mayo.edu/research/labs/proteases-cancer/

*From:*CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf 
Of *Dipankar Manna

*Sent:* Monday, April 20, 2015 2:42 PM
*To:* CCP4BB@JISCMAIL.AC.UK
*Subject:* [ccp4bb] Cleaved peptide density!

Dear Crystallographers,

I am working with a cysteine protease. I co-crystallized the protease 
with some small chemically synthesised peptides of 7 amino acid 
residues. I mutated the active Cysteine residue with Alanine to avoid 
the peptide cleavage so that I can get the whole peptide bound with my 
protein. But interesting I got the density for a cleaved peptide with 
4 amino acids instead of the whole peptide. The resolution is 1.4 A, I 
can see the clear cleavage and the cleavage occurred exactly at the 
same peptide bond where it should. But I do not know how!


Now my question is why I am getting the cleaved peptide as I already 
mutated the active residue Cysteine with Alanine (this mutant did no 
show any activity when I checked with SDS-PAGE).


If anybody has the same kind of experience please advice me.

Thanks in advance.

Best,

Dipankar

--

Dipankar Manna

Research Scholar

Department of Chemistry

University of Oslo

Oslo, Norway

Re: [ccp4bb] Cleaved peptide density!

[Reza Khayat]

Hi Dipkar,

My understanding is that the majority of a protease's activity comes from the 
oxyanion hole activating the scissile bond. Mutating the nucleophile reduces 
activity, sometimes appreciably, but does not kill the enzyme. Were your 
SDS-PAGE experiments on the same time scale and buffer conditions as your 
crystallography experiments? Perhaps something in the crystallization buffer 
increases the activity of the enzyme, or all/some of the peptide is digested in 
the time it takes to attain crystals -or only crystals of the digested peptide 
complex can be attained.

Best wishes,
Reza

Reza Khayat, PhD
Assistant Professor
Department of Chemistry
City College of New York
New York, NY 10031
http://www.khayatlab.org/
212-650-6070

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Radisky, 
Evette S., Ph.D.
Sent: Wednesday, April 22, 2015 3:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Cleaved peptide density!

We had a similar situation with a catalytically dead serine protease.  
Initially I was excited to think we might be seeing residual catalytic activity 
of the mutant enzyme on a highly specific substrate; however, the activity 
turned out to result from contamination with a very small amount of wt enzyme, 
likely as a result of using the same affinity column to purify both.  When we 
incubated the enzyme preparation with PMSF (an inhibitor that covalently 
modifies the catalytic serine and would not have affected the mutant), we 
eliminated the residual activity of the enzyme preparation.  However, in our 
case we never were able to get crystals with the intact substrate, which 
apparently was not compatible with our crystal form.

Is there a covalent inhibitor of your cysteine protease that you could use to 
pre-treat your enzyme, to see if this eliminates the activity?  If so this 
might help distinguish between residual activity of the mutant vs. 
contamination with wt enzyme.

Good luck!
Evette

Evette S. Radisky, Ph.D.
Associate Professor and Senior Associate Consultant
Department of Cancer Biology
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372
http://www.mayo.edu/research/labs/proteases-cancer/

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar 
Manna
Sent: Monday, April 20, 2015 2:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cleaved peptide density!

Dear Crystallographers,

I am working with a cysteine protease. I co-crystallized the protease with some 
small chemically synthesised peptides of 7 amino acid residues. I mutated the 
active Cysteine residue with Alanine to avoid the peptide cleavage so that I 
can get the whole peptide bound with my protein. But interesting I got the 
density for a cleaved peptide with 4 amino acids instead of the whole peptide. 
The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred 
exactly at the same peptide bond where it should. But I do not know how!

Now my question is why I am getting the cleaved peptide as I already mutated 
the active residue Cysteine with Alanine (this mutant did no show any activity 
when I checked with SDS-PAGE).

If anybody has the same kind of experience please advice me.

Thanks in advance.

Best,

Dipankar

--
Dipankar Manna
Research Scholar
Department of Chemistry
University of Oslo
Oslo, Norway

Re: [ccp4bb] Cleaved peptide density!

[Radisky, Evette S., Ph.D.]

We had a similar situation with a catalytically dead serine protease.  
Initially I was excited to think we might be seeing residual catalytic activity 
of the mutant enzyme on a highly specific substrate; however, the activity 
turned out to result from contamination with a very small amount of wt enzyme, 
likely as a result of using the same affinity column to purify both.  When we 
incubated the enzyme preparation with PMSF (an inhibitor that covalently 
modifies the catalytic serine and would not have affected the mutant), we 
eliminated the residual activity of the enzyme preparation.  However, in our 
case we never were able to get crystals with the intact substrate, which 
apparently was not compatible with our crystal form.

Is there a covalent inhibitor of your cysteine protease that you could use to 
pre-treat your enzyme, to see if this eliminates the activity?  If so this 
might help distinguish between residual activity of the mutant vs. 
contamination with wt enzyme.

Good luck!
Evette

Evette S. Radisky, Ph.D.
Associate Professor and Senior Associate Consultant
Department of Cancer Biology
Mayo Clinic Cancer Center
Griffin Cancer Research Building, Rm 310
4500 San Pablo Road
Jacksonville, FL 32224
(904) 953-6372
http://www.mayo.edu/research/labs/proteases-cancer/

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Dipankar 
Manna
Sent: Monday, April 20, 2015 2:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cleaved peptide density!

Dear Crystallographers,

I am working with a cysteine protease. I co-crystallized the protease with some 
small chemically synthesised peptides of 7 amino acid residues. I mutated the 
active Cysteine residue with Alanine to avoid the peptide cleavage so that I 
can get the whole peptide bound with my protein. But interesting I got the 
density for a cleaved peptide with 4 amino acids instead of the whole peptide. 
The resolution is 1.4 A, I can see the clear cleavage and the cleavage occurred 
exactly at the same peptide bond where it should. But I do not know how!

Now my question is why I am getting the cleaved peptide as I already mutated 
the active residue Cysteine with Alanine (this mutant did no show any activity 
when I checked with SDS-PAGE).

If anybody has the same kind of experience please advice me.

Thanks in advance.

Best,

Dipankar

--
Dipankar Manna
Research Scholar
Department of Chemistry
University of Oslo
Oslo, Norway

Re: [ccp4bb] Cleaved peptide density!

[Michael James]

hello Dipankar,
Your queries have generated much interest. It would be helpful if you told
us which clan the cysteine
peptidase you are working with is from. Also it would be very helpful if
you could show us the
electron density of the "cleaved" peptide in the active site. One presumes
that you have a product
complex with your peptidase and as has been pointed out this is very
interesting. Serine peptidases
often have products bound in the S1 to S4 subsites of the enzyme. They
remain bound throughout
the isolation, purification and crystallization steps of the structure
determination.

Regards,
Michael James

On Mon, Apr 20, 2015 at 2:47 PM, Dipankar Manna 
wrote:

> Thank you Rajesh,
>
> That could be a possibility though. I am planning to do some MS analysis
> from the extra gel bands, if I get any by running SDS-PAGE on the purified
> protein.
>
> Best,
>
> Dipankar
>
>
> On Mon, Apr 20, 2015 at 10:42 PM, rajesh ponnusamy <
> ponnusam...@googlemail.com> wrote:
>
>> just to add up: Thought about any E.Coli protease as an impurity.. very
>> small quantity?
>>
>> Cheers,
>> Rajesh.
>>
>>
>> On Mon, Apr 20, 2015 at 9:14 PM, Dipankar Manna <
>> dipankar.biot...@gmail.com> wrote:
>>
>>> Dear Barbel,
>>>
>>> Thank you!
>>>
>>> Yes you are right that I did the SDS-PAGE with bigger substrate.
>>> Regarding peptide, we did check the MS and HPLC profile for the peptides
>>> which clearly shows that there should not be any cleaved peptides!
>>>
>>> Best,
>>>
>>> Dipankar
>>>
>>> On Mon, Apr 20, 2015 at 9:46 PM, Bärbel Blaum <
>>> baerbel.bl...@uni-tuebingen.de> wrote:
>>>
 I suppose you do the SDS PAGE test not with the peptide but some bigger
 substrate. Are you sure your peptide is intact *before* soaking? I.e. have
 you checked the batch yourself with MS or NMR? We regularly get small
 compounds (sugars) that turn out not to be what the label says.

 Bärbel


 Zitat von Dipankar Manna :


  Dear Bonsor,
>
> Thanks for your suggestions!
>
> It need 2-3 weeks to get the fully grown crystals. And harvest, freeze
> and
> data collection just take usually next 3-5 days. I usually incubated
> substrate overnight. Initially I was purifying with the same column as
> the
> WT but in the next batch I used new beads to purify the mutant as you
> categorically pointed out. But results are the same, I mean I got the
> same
> cleaved peptide density! I tried soaking with different time frames and
> with different peptide concentrations as well but in this case I can't
> see
> any peptide density at all.
>
> Best,
>
> Dipankar
>
> On Mon, Apr 20, 2015 at 9:06 PM, D Bonsor 
> wrote:
>
>  First of all, you don't say how long it took to first set up
>> crystals, for
>> them to grow, harvest, freeze and collect data on. Secondly how long
>> did
>> leave the peptide/substrate for your SDS PAGE experiment? If they are
>> of a
>> different time scale e.g. 6 hours v.s. 30 days, it may be that your
>> enzyme
>> is not totally dead.
>>
>> Also how did you purify the alanine mutant? If you purified it on the
>> same
>> columns/beads as the WT protein you may have a residual amount of
>> active
>> protein which could cleave your peptide over the course of
>> crystallization.
>> You may want to use fresh beads, or treat columns with pepsin or
>> sodium
>> hydroxide.
>>
>> Not real answers I am afraid, more like suggestions.
>>
>>
>
>
> --
> Dipankar Manna
> Research Scholar
> Department of Chemistry
> University of Oslo
> Oslo, Norway
>
>


 --
 Bärbel Blaum, Ph.D.
 Interfakultäres Institut für Biochemie (IFIB)
 Hoppe-Seyler-Strasse 4
 D-72076 Tübingen
 Germany
 +49 70 71 29 75 359
 http://www.ifib.uni-tuebingen.de/research/blaum.html

>>>
>>>
>>>
>>> --
>>> Dipankar Manna
>>> Research Scholar
>>> Department of Chemistry
>>> University of Oslo
>>> Oslo, Norway
>>>
>>
>>
>>
>> --
>> Dr. Rajesh Ponnusamy
>> Macromolecular Crystallography Unit
>> Instituto de Tecnologia Química e Biológica - ITQB-UNL
>> Oeiras - Portugal
>>
>> phone: (+351) 21 446 96 63
>> fax:   (+351) 21 443 36 44
>> email: raj...@itqb.unl.pt
>>
>
>
>
> --
> Dipankar Manna
> Research Scholar
> Department of Chemistry
> University of Oslo
> Oslo, Norway
>

Re: [ccp4bb] Phaser going into infinite loop in Ample

[Dale Tronrud]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

   Thanks for all the help!

   We will restart the job with the KILL option as Jens suggested.

   We will also send a copy of the Phaser log file to Randy.  This is
not a case of Phaser simply trying harder - it is doing the same
search over and over again.  After four days the log file is getting
pretty long.  I may have to use DropBox.

   We considered trying ARCIMBOLDO but the resolution of the data did
not reach the 2.1 A limit usually suggested for that program.  If we
get AMPLE running, and it fails, we will give ARCIMBOLDO a try.

Dale Tronrud
Sarah Clark

On 4/22/2015 2:06 AM, Thomas, Jens wrote:
> Dear Dale,
> 
> This is a known issue with AMPLE and will be fixed with the next
> release.
> 
> In the meantime you can tell AMPLE to pass the KILL option that
> Randy mentions to PHASER, by adding the following arguments to your
> script:
> 
> -mr_keys PKEY KILL TIME 360
> 
> this will kill PHASER after 360 minutes (6 hours), which we've
> found if normally enough, although pick whatever time works for
> you,
> 
> I should also point out that I think George is doing a disservice
> to SHELXE. In our last paper looking at coiled-coils, we saw
> successes at resolutions much lower than 2.1, in one case, AMPLE
> was able to solve a structure with a resolution of 2.9A:
> 
> http://dx.doi.org/10.1107/S2052252515002080
> 
> If you have any issues getting the KILL command to work, please
> feel free to contact me off-list.
> 
> Best wishes,
> 
> Jens
> 
>  From: CCP4 bulletin board
> [CCP4BB@JISCMAIL.AC.UK] on behalf of Randy Read [rj...@cam.ac.uk] 
> Sent: 22 April 2015 09:04 To: CCP4BB@JISCMAIL.AC.UK Subject: Re:
> [ccp4bb] Phaser going into infinite loop in Ample
> 
> Hi Dale,
> 
> It must actually be AMPLE deciding how many copies to search for.
> Phaser will give you some information about how consistent the
> specified composition is with the Matthews volume, but it just
> searches for the number of copies that it's instructed to look for.
> We haven't put the intelligence into it to correlate the model(s)
> with the composition information and try out different
> possibilities.  At the moment, we're leaving that level of analysis
> to pipelines like MRage.
> 
> We're well aware of the tension between looking hard enough to find
> a solution in a difficult case and not throwing good CPU cycles
> after bad when it's hopeless.  We're gradually introducing new
> features to make these decisions better, but we do tend to prefer
> wasting CPU time to missing solutions.  However, we've introduced a
> couple of ways to limit the amount of time that Phaser spends
> pursuing very difficult or hopeless solutions, partly for the
> benefit of people such as the developers of Arcimboldo, AMPLE and
> the wide-search molecular replacement pipeline.  One is the KILL
> command, which is a rather blunt instrument saying to give up if a
> solution isn't found in a certain length of time.  In AMPLE, if you
> set quick mode, then the KILL time is set to 15 minutes.  Another
> option (which I don't think AMPLE uses) is the PURGE command, where
> you can say (for instance) that Phaser should pursue a maximum of
> 25 partial solutions when adding the next component.
> 
> If you're seeing an infinite loop, it would be handy if you could
> send me a copy of the logfile showing what is going on.  There have
> been some bugs leading to such infinite loops under some
> circumstances, and if you're running into one of those there's a
> good chance that it has been fixed in a recent nightly build of
> Phaser available through Phenix.  You can instruct CCP4 to use the
> Phaser executable from Phenix, and I think this should work fine in
> AMPLE, though I haven't tested it — I don't think any relevant
> syntax has changed.  It's been a while since we've had a new stable
> release of Phaser in either CCP4 or Phenix, so we're aiming to get
> one out in the not too distant future.
> 
> All the best,
> 
> Randy
> 
>> On 22 Apr 2015, at 05:56, Dale Tronrud 
>> wrote:
>> 
> 
> We are having a problem with AMPLE and hope someone can help.
> 
> The protein is about 70 amino acids long and we suspect it forms a 
> coiled-coil.  Our previous attempts at molecular replacement have 
> failed so we hoped that AMPLE, with its ability to generate a
> variety of potential models, would do the trick.
> 
> Our problem is that all of our CPU cores are consumed by Phaser 
> jobs that are not making progress.  With this protein Phaser
> decides that it will look for 11 copies in the asymmetric unit.
> For a few of the possible ensembles it fails to find even one copy
> and gives up. That's fine with us.  For other ensembles it finds a
> handful of possible first positions, goes on to look for a second
> and fails, then goes back to try to place a second copy again.  We
> presume that the intent is to lower the acceptance criteria in the
> second pass, but in actuality 

Re: [ccp4bb] Phaser going into infinite loop in Ample

[Claudia Millán Nebot]

Dear Dale,

as George points out, you may be interested in trying ARCIMBOLDO, as it has
been successfully applied to coiled coil proteins recently, as in the case
of
Franke et al (2014)  Open Biology, 4. p. 130172 or  Sammito et al (2013)
Nature Methods 10: 1099-1101 . Different models can be searched for,
starting from single helices with or without sidechains, going to
precomputed libraries of helices in parallel or antiparallel configurations.

If you need help or support to use it, please feel free to ask any question.
Best wishes,

Claudia



Claudia Millán

2015-04-22 6:56 GMT+02:00 Dale Tronrud :

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
>
>We are having a problem with AMPLE and hope someone can help.
>
>The protein is about 70 amino acids long and we suspect it forms a
> coiled-coil.  Our previous attempts at molecular replacement have
> failed so we hoped that AMPLE, with its ability to generate a variety
> of potential models, would do the trick.
>
>Our problem is that all of our CPU cores are consumed by Phaser
> jobs that are not making progress.  With this protein Phaser decides
> that it will look for 11 copies in the asymmetric unit.  For a few of
> the possible ensembles it fails to find even one copy and gives up.
> That's fine with us.  For other ensembles it finds a handful of
> possible first positions, goes on to look for a second and fails, then
> goes back to try to place a second copy again.  We presume that the
> intent is to lower the acceptance criteria in the second pass, but in
> actuality Phaser simply repeats the same search that failed before and
> fails again.  The leads to an infinite loop.
>
>Once all the cores are occupied in this futile endeavor AMPLE makes
> no further progress.
>
>How can we get Phaser to either try harder to place a molecule or
> to give up?
>
>We are using CCP4 6.5.008 and the copy of Phaser that came with it.
>  We used CCP4i to create a script which we modified slightly and ran
> using the "at" command.  The command is:
>
> /usr/local/ccp4-6.5/bin/ccp4-python -u /usr/local/ccp4-6.5/bin/ample
> - -mtz /user/sarah/xray/1Apr_Athena/SD6004_2_001_mergedunique14.mtz
> - -fasta /user/sarah/xray/1Apr_Athena/swaseq.fa -mr_sequence
> /user/sarah/xray/1Apr_Athena/swaseq.fa -nmodels 500 -name MVD0
> - -run_dir /home/sarah/AMPLE -nproc 6 -make_models True -rosetta_dir
> /usr/local/rosetta-3.5 -frags_3mers
> /user/sarah/xray/1Apr_Athena/aat000_03_05.200_v1_3 -frags_9mers
> /user/sarah/xray/1Apr_Athena/aat000_09_05.200_v1_3 -make_frags False
> - -F F -SIGF SIGF -FREE FreeR_flag  -early_terminate True   -use_shelxe
> True -shelx_cycles 15 -use_arpwarp False
>
> Any help is appreciated,
> Dale Tronrud
> Sarah Clark
> -BEGIN PGP SIGNATURE-
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>
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> p6UAnAmKkUZe46zbiXckdDTNgUQ0dgWq
> =A/Sj
> -END PGP SIGNATURE-
>

Re: [ccp4bb] Phaser going into infinite loop in Ample

[Thomas, Jens]

Dear Dale,

This is a known issue with AMPLE and will be fixed with the next release.

In the meantime you can tell AMPLE to pass the KILL option that Randy mentions 
to PHASER, by adding the following arguments to your script:

-mr_keys PKEY KILL TIME 360

this will kill PHASER after 360 minutes (6 hours), which we've found if 
normally enough, although pick whatever time works for you,

I should also point out that I think George is doing a disservice to SHELXE. In 
our last paper looking at coiled-coils, we saw successes at resolutions much 
lower than 2.1, in one case, AMPLE was able to solve a structure with a 
resolution of 2.9A:

http://dx.doi.org/10.1107/S2052252515002080

If you have any issues getting the KILL command to work, please feel free to 
contact me off-list.

Best wishes,

Jens


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Randy Read 
[rj...@cam.ac.uk]
Sent: 22 April 2015 09:04
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Phaser going into infinite loop in Ample

Hi Dale,

It must actually be AMPLE deciding how many copies to search for.  Phaser will 
give you some information about how consistent the specified composition is 
with the Matthews volume, but it just searches for the number of copies that 
it's instructed to look for.  We haven't put the intelligence into it to 
correlate the model(s) with the composition information and try out different 
possibilities.  At the moment, we're leaving that level of analysis to 
pipelines like MRage.

We're well aware of the tension between looking hard enough to find a solution 
in a difficult case and not throwing good CPU cycles after bad when it's 
hopeless.  We're gradually introducing new features to make these decisions 
better, but we do tend to prefer wasting CPU time to missing solutions.  
However, we've introduced a couple of ways to limit the amount of time that 
Phaser spends pursuing very difficult or hopeless solutions, partly for the 
benefit of people such as the developers of Arcimboldo, AMPLE and the 
wide-search molecular replacement pipeline.  One is the KILL command, which is 
a rather blunt instrument saying to give up if a solution isn't found in a 
certain length of time.  In AMPLE, if you set quick mode, then the KILL time is 
set to 15 minutes.  Another option (which I don't think AMPLE uses) is the 
PURGE command, where you can say (for instance) that Phaser should pursue a 
maximum of 25 partial solutions when adding the next component.

If you're seeing an infinite loop, it would be handy if you could send me a 
copy of the logfile showing what is going on.  There have been some bugs 
leading to such infinite loops under some circumstances, and if you're running 
into one of those there's a good chance that it has been fixed in a recent 
nightly build of Phaser available through Phenix.  You can instruct CCP4 to use 
the Phaser executable from Phenix, and I think this should work fine in AMPLE, 
though I haven't tested it — I don't think any relevant syntax has changed.  
It's been a while since we've had a new stable release of Phaser in either CCP4 
or Phenix, so we're aiming to get one out in the not too distant future.

All the best,

Randy

> On 22 Apr 2015, at 05:56, Dale Tronrud  wrote:
>
> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
>
>
>   We are having a problem with AMPLE and hope someone can help.
>
>   The protein is about 70 amino acids long and we suspect it forms a
> coiled-coil.  Our previous attempts at molecular replacement have
> failed so we hoped that AMPLE, with its ability to generate a variety
> of potential models, would do the trick.
>
>   Our problem is that all of our CPU cores are consumed by Phaser
> jobs that are not making progress.  With this protein Phaser decides
> that it will look for 11 copies in the asymmetric unit.  For a few of
> the possible ensembles it fails to find even one copy and gives up.
> That's fine with us.  For other ensembles it finds a handful of
> possible first positions, goes on to look for a second and fails, then
> goes back to try to place a second copy again.  We presume that the
> intent is to lower the acceptance criteria in the second pass, but in
> actuality Phaser simply repeats the same search that failed before and
> fails again.  The leads to an infinite loop.
>
>   Once all the cores are occupied in this futile endeavor AMPLE makes
> no further progress.
>
>   How can we get Phaser to either try harder to place a molecule or
> to give up?
>
>   We are using CCP4 6.5.008 and the copy of Phaser that came with it.
> We used CCP4i to create a script which we modified slightly and ran
> using the "at" command.  The command is:
>
> /usr/local/ccp4-6.5/bin/ccp4-python -u /usr/local/ccp4-6.5/bin/ample
> - -mtz /user/sarah/xray/1Apr_Athena/SD6004_2_001_mergedunique14.mtz
> - -fasta /user/sarah/xray/1Apr_Athena/swaseq.fa -mr_sequence
> /user/sarah/xray/1Apr_Athena/swaseq.fa -nmodels 5

Re: [ccp4bb] Phaser going into infinite loop in Ample

[Randy Read]

Hi Dale,

It must actually be AMPLE deciding how many copies to search for.  Phaser will 
give you some information about how consistent the specified composition is 
with the Matthews volume, but it just searches for the number of copies that 
it's instructed to look for.  We haven't put the intelligence into it to 
correlate the model(s) with the composition information and try out different 
possibilities.  At the moment, we're leaving that level of analysis to 
pipelines like MRage.

We're well aware of the tension between looking hard enough to find a solution 
in a difficult case and not throwing good CPU cycles after bad when it's 
hopeless.  We're gradually introducing new features to make these decisions 
better, but we do tend to prefer wasting CPU time to missing solutions.  
However, we've introduced a couple of ways to limit the amount of time that 
Phaser spends pursuing very difficult or hopeless solutions, partly for the 
benefit of people such as the developers of Arcimboldo, AMPLE and the 
wide-search molecular replacement pipeline.  One is the KILL command, which is 
a rather blunt instrument saying to give up if a solution isn't found in a 
certain length of time.  In AMPLE, if you set quick mode, then the KILL time is 
set to 15 minutes.  Another option (which I don't think AMPLE uses) is the 
PURGE command, where you can say (for instance) that Phaser should pursue a 
maximum of 25 partial solutions when adding the next component.

If you're seeing an infinite loop, it would be handy if you could send me a 
copy of the logfile showing what is going on.  There have been some bugs 
leading to such infinite loops under some circumstances, and if you're running 
into one of those there's a good chance that it has been fixed in a recent 
nightly build of Phaser available through Phenix.  You can instruct CCP4 to use 
the Phaser executable from Phenix, and I think this should work fine in AMPLE, 
though I haven't tested it — I don't think any relevant syntax has changed.  
It's been a while since we've had a new stable release of Phaser in either CCP4 
or Phenix, so we're aiming to get one out in the not too distant future.

All the best,

Randy

> On 22 Apr 2015, at 05:56, Dale Tronrud  wrote:
> 
> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> 
>   We are having a problem with AMPLE and hope someone can help.
> 
>   The protein is about 70 amino acids long and we suspect it forms a
> coiled-coil.  Our previous attempts at molecular replacement have
> failed so we hoped that AMPLE, with its ability to generate a variety
> of potential models, would do the trick.
> 
>   Our problem is that all of our CPU cores are consumed by Phaser
> jobs that are not making progress.  With this protein Phaser decides
> that it will look for 11 copies in the asymmetric unit.  For a few of
> the possible ensembles it fails to find even one copy and gives up.
> That's fine with us.  For other ensembles it finds a handful of
> possible first positions, goes on to look for a second and fails, then
> goes back to try to place a second copy again.  We presume that the
> intent is to lower the acceptance criteria in the second pass, but in
> actuality Phaser simply repeats the same search that failed before and
> fails again.  The leads to an infinite loop.
> 
>   Once all the cores are occupied in this futile endeavor AMPLE makes
> no further progress.
> 
>   How can we get Phaser to either try harder to place a molecule or
> to give up?
> 
>   We are using CCP4 6.5.008 and the copy of Phaser that came with it.
> We used CCP4i to create a script which we modified slightly and ran
> using the "at" command.  The command is:
> 
> /usr/local/ccp4-6.5/bin/ccp4-python -u /usr/local/ccp4-6.5/bin/ample
> - -mtz /user/sarah/xray/1Apr_Athena/SD6004_2_001_mergedunique14.mtz
> - -fasta /user/sarah/xray/1Apr_Athena/swaseq.fa -mr_sequence
> /user/sarah/xray/1Apr_Athena/swaseq.fa -nmodels 500 -name MVD0
> - -run_dir /home/sarah/AMPLE -nproc 6 -make_models True -rosetta_dir
> /usr/local/rosetta-3.5 -frags_3mers
> /user/sarah/xray/1Apr_Athena/aat000_03_05.200_v1_3 -frags_9mers
> /user/sarah/xray/1Apr_Athena/aat000_09_05.200_v1_3 -make_frags False
> - -F F -SIGF SIGF -FREE FreeR_flag  -early_terminate True   -use_shelxe
> True -shelx_cycles 15 -use_arpwarp False
> 
> Any help is appreciated,
> Dale Tronrud
> Sarah Clark
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v2.0.22 (MingW32)
> 
> iEYEARECAAYFAlU3KhEACgkQU5C0gGfAG117JACfXahEX8z1k3ev043a7V2SzhNh
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> -END PGP SIGNATURE-

--
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] Phaser going into infinite loop in Ample

[George Sheldrick]

Dear Dale,

Isabel Uson's ARCIMBOLDO-LITE works well for coiled coils and has the 
same resolution
requirements (2.1A or better) as AMPLE because both use SHELXE to expand 
the solution.
It also employs PHASER to place a small fragment but it is often 
sufficient to let it search for

just two or three copies in the asymmetric unit.

Best wishes, George


On 04/22/2015 06:56 AM, Dale Tronrud wrote:

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1


We are having a problem with AMPLE and hope someone can help.

The protein is about 70 amino acids long and we suspect it forms a
coiled-coil.  Our previous attempts at molecular replacement have
failed so we hoped that AMPLE, with its ability to generate a variety
of potential models, would do the trick.

Our problem is that all of our CPU cores are consumed by Phaser
jobs that are not making progress.  With this protein Phaser decides
that it will look for 11 copies in the asymmetric unit.  For a few of
the possible ensembles it fails to find even one copy and gives up.
That's fine with us.  For other ensembles it finds a handful of
possible first positions, goes on to look for a second and fails, then
goes back to try to place a second copy again.  We presume that the
intent is to lower the acceptance criteria in the second pass, but in
actuality Phaser simply repeats the same search that failed before and
fails again.  The leads to an infinite loop.

Once all the cores are occupied in this futile endeavor AMPLE makes
no further progress.

How can we get Phaser to either try harder to place a molecule or
to give up?

We are using CCP4 6.5.008 and the copy of Phaser that came with it.
  We used CCP4i to create a script which we modified slightly and ran
using the "at" command.  The command is:

/usr/local/ccp4-6.5/bin/ccp4-python -u /usr/local/ccp4-6.5/bin/ample
- -mtz /user/sarah/xray/1Apr_Athena/SD6004_2_001_mergedunique14.mtz
- -fasta /user/sarah/xray/1Apr_Athena/swaseq.fa -mr_sequence
/user/sarah/xray/1Apr_Athena/swaseq.fa -nmodels 500 -name MVD0
- -run_dir /home/sarah/AMPLE -nproc 6 -make_models True -rosetta_dir
/usr/local/rosetta-3.5 -frags_3mers
/user/sarah/xray/1Apr_Athena/aat000_03_05.200_v1_3 -frags_9mers
/user/sarah/xray/1Apr_Athena/aat000_09_05.200_v1_3 -make_frags False
- -F F -SIGF SIGF -FREE FreeR_flag  -early_terminate True   -use_shelxe
True -shelx_cycles 15 -use_arpwarp False

Any help is appreciated,
Dale Tronrud
Sarah Clark
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Version: GnuPG v2.0.22 (MingW32)

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--
Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-33021 or -33068
Fax. +49-551-39-22582