Re: [ccp4bb] off topic

[Zhang, Yuzhu]

Hi Anamika,

 Proteins with high sequence identity can behave very differently when 
expressed in bacteria. Sumo (or other tags) may or may not help. If it does, 
you can express your SH2 with a tag and remove the tag after purification if 
the tag is a problem for functional studies. The vectors described in this 
article  may 
work for you if you cannot figure out why you get inconsistent expression.

YZ.


From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anamika 
Singh
Sent: Thursday, July 06, 2017 7:12 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off topic

Hi,

Is anyone has worked with STAT1 proteins?

I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was 
no expression so far or rather say inconsistent expression. Sometimes the 
expression was in inclusion bodies.  I have tried different methods to pull out 
the protein from inclusion bodies using urea, guanidium chloride tween20 but 
none of them worked well. The yield was very low (very faint band on SDS-PAGE ) 
from 3-liter culture. I changed the host cells from BL21 to Rosetta DE3 cells 
but no success so far.

We thought to use some other vector system like with SUMO tag but did not 
proceed because the aim of the project to design inhibitor and tag will 
interfere.

Please suggest me something so that I can complete my project in lesser time.


Looking forward to valuable suggestions.

Thanks
Anamika




This electronic message contains information generated by the USDA solely for 
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Re: [ccp4bb] off topic

[Vivoli, Mirella]

Hi Anamika,
I guess you tried several growth conditions.
You might try to do a heat shock growth.
Grow at 37C until OD 0.6, then move the bacteria to 42C for half hour, then to 
30C and grow them for 20 hours.
Another strategy is to add just before induction 1,2 or 3% Ethanol.
Not sure these will work with your protein but you might give a go.
Other strategies I could suggest is to clone STAT1 protein with other tags 
(MBP, GST, and so on) or in othe expression system, like ArticExpress.
If none of them works, then there is the painful strategy of refolding.
Feel free to contact me, I would be happy to help you.
Cheers,

Mirella

Get Outlook for Android



From: David Blum
Sent: Thursday, 6 July, 22:10
Subject: Re: [ccp4bb] off topic
To: ccp4bb@jiscmail.ac.uk


Hi Anamika,


I did a search and it looks like researchers are using either mammalian cells 
or baculovirus to express this protein.  I don't have experience with this 
particular construct so could you tell me why you choose E. coli?  I run a 
protein expression facility and we typically use HEK or CHO cells and get mg 
amounts from cell culture of mammalian derived proteins like STAT1.  Happy to 
talk offline if that would be easier.


Best,

David

--

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

(706) 542-1035 (Office)



On Thu, Jul 6, 2017 at 10:11 AM, Anamika Singh 
> wrote:

Hi,


Is anyone has worked with STAT1 proteins?


I have cloned the SH2 domain of STAT1 protein into pet28a vector but there was 
no expression so far or rather say inconsistent expression. Sometimes the 
expression was in inclusion bodies.  I have tried different methods to pull out 
the protein from inclusion bodies using urea, guanidium chloride tween20 but 
none of them worked well. The yield was very low (very faint band on SDS-PAGE ) 
from 3-liter culture. I changed the host cells from BL21 to Rosetta DE3 cells 
but no success so far.


We thought to use some other vector system like with SUMO tag but did not 
proceed because the aim of the project to design inhibitor and tag will 
interfere.


Please suggest me something so that I can complete my project in lesser time.



Looking forward to valuable suggestions.


Thanks

Anamika

Re: [ccp4bb] off topic

[David Blum]

Hi Anamika,

I did a search and it looks like researchers are using either mammalian
cells or baculovirus to express this protein.  I don't have experience with
this particular construct so could you tell me why you choose *E. coli*?  I
run a protein expression facility and we typically use HEK or CHO cells and
get mg amounts from cell culture of mammalian derived proteins like STAT1.
Happy to talk offline if that would be easier.

Best,

David

-- 

David L. Blum, Ph.D.

Director, Bioexpression and Fermentation Facility

Department of Biochemistry and Molecular Biology

University of Georgia

120 Green Street room A414A

Athens, GA 30602

http://bff.uga.edu/

(706) 542-1035 (Office)



On Thu, Jul 6, 2017 at 10:11 AM, Anamika Singh 
wrote:

> Hi,
>
> Is anyone has worked with STAT1 proteins?
>
> I have cloned the SH2 domain of STAT1 protein into pet28a vector but there
> was no expression so far or rather say inconsistent expression. Sometimes
> the expression was in inclusion bodies.  I have tried different methods to
> pull out the protein from inclusion bodies using urea, guanidium chloride
> tween20 but none of them worked well. The yield was very low (very faint
> band on SDS-PAGE ) from 3-liter culture. I changed the host cells from BL21
> to Rosetta DE3 cells but no success so far.
>
> We thought to use some other vector system like with SUMO tag but did not
> proceed because the aim of the project to design inhibitor and tag will
> interfere.
>
> Please suggest me something so that I can complete my project in lesser
> time.
>
>
> Looking forward to valuable suggestions.
>
> Thanks
> Anamika
>

Re: [ccp4bb] off topic

[zaigham mahmood khan]

Hey Anamika

I know SH2 domain is very much well-studied domain, and i am not sure why
are you facing troubles in the expression of this protein. Just go through
the literature and read about different protocol of expression of SH2
domain from several different proteins.. Well, reading that you have
"inconsistent" expression suggested me something else. Do verify the IPTG
stock. Also, confirm the expression via western using anti-His antibody.
You may also check the prevalence of rare codon in the proteins. If
present, then Rosetta would be a better option than DE3 cells. Eventually,
the best would be to follow the established protocol from the
closest-homology SH2 domain, and move from there onward.

Best wishes

-Z


Zaigham M Khan, PhD

Icahn School of Medicine at Mount Sinai
Department of Oncological Sciences
1470 Madison Avenue
New York

On Thu, Jul 6, 2017 at 10:11 AM, Anamika Singh 
wrote:

> Hi,
>
> Is anyone has worked with STAT1 proteins?
>
> I have cloned the SH2 domain of STAT1 protein into pet28a vector but there
> was no expression so far or rather say inconsistent expression. Sometimes
> the expression was in inclusion bodies.  I have tried different methods to
> pull out the protein from inclusion bodies using urea, guanidium chloride
> tween20 but none of them worked well. The yield was very low (very faint
> band on SDS-PAGE ) from 3-liter culture. I changed the host cells from BL21
> to Rosetta DE3 cells but no success so far.
>
> We thought to use some other vector system like with SUMO tag but did not
> proceed because the aim of the project to design inhibitor and tag will
> interfere.
>
> Please suggest me something so that I can complete my project in lesser
> time.
>
>
> Looking forward to valuable suggestions.
>
> Thanks
> Anamika
>

Re: [ccp4bb] ccp4i2 crashes on start

[Tom Burnley]

One possible source of the problem that we've come across before... ensure
the follow environment variable isn't set:

QT_PLUGIN_PATH

Best wishes,

Tom

On 4 July 2017 at 14:55, Stuart McNicholas 
wrote:

> Dear Johannes,
>Many apologies, this behaviour should certainly not be expected. The
> cause could be quite difficult to determine, I shall start by installing a
> Ubuntu virtualbox myself.
>
> Best wishes,
> Stuart
>
> On 4 July 2017 at 13:46, Johannes Cramer 
> wrote:
>
>> Dear CCP4 bb,
>>
>> I would like to use ccp4i2, but every time I try to start it via command
>> line, I get the following error message:
>>
>> Running CCP4i2 browser from: /home/cramejo/programs/ccp4-7.0/share/ccp4i2
>>> Python 2.7.10 (default, Aug 28 2015, 12:10:46)
>>> [GCC 4.8.2 20140120 (Red Hat 4.8.2-15)]
>>> Qt version 4.8.7
>>>
>>> 440 367
>>> Segmentation fault (core dumped)
>>
>>
>> Has anyone experienced this behavior? How (where) can I get a more
>> detailed log file? ccp4i (old GUI) works perfectly fine, but the session
>> log that is accessible via the interface is only valid for the current
>> session.
>> I am running 64 bit Kubuntu 10.16 on a windows host virtualbox.
>>
>> Cheers,
>> Johannes
>>
>
>


-- 
Dr Tom Burnley, PhD
CCP-EM | @ccp_em * | *www.ccpem.ac.uk

Science and Technology Facilities Council (STFC)
The Research Complex At Harwell
Rutherford Appleton Laboratory, R92
OX11 0FA
01235 56 7871

[ccp4bb] ARP/wARP webservice

[Victor Lamzin]

Dear Colleagues,

We are happy to announce a redesigned and extended webservice for remote 
ARP/wARP execution. In addition to the crystallographic protein chain 
tracing, many other functionalities of ARP/wARP are now also available 
online. These include:


- classic protein model building starting from phases or from existing model
- secondary structure building
- nucleotide building
- solvent modelling
- ligand modelling and identification

The webservice offers a registration with an email address, which 
provides more options to the users. For example, all jobs a user has 
previously submitted are now in one place, can be easily evaluated, 
compared or run again with different parameters.


The link for the new ARP/wARP webservice is 
https://arpwarp.embl-hamburg.de. The former link 
http://cluster.embl-hamburg.de/ARPwARP/remote-http.html continues to 
function too. The webservice is using the current ARP/wARP version 
7.6.1, which has been jointly released with the CCP4 software.


The website contains links to related webservices: Auto-Rickshaw for 
structure solution, ViCi for ligand scaffold hopping and DipCheck for 
protein model validation.


Your feedback, comments and suggestions are welcome.

Happy model building!
The ARP/wARP team at EMBL Hamburg

Re: [ccp4bb] REBATCH error mystery

[Phil Evans]

REBATCH is old and I can’t remember how it works :-(

> On 6 Jul 2017, at 15:18, Graeme Winter  wrote:
> 
> HI Phil,
> 
> Yes, probably. However it’s still a mystery I would like to understand. Also, 
> I use REBATCH for lots of things not just trimming data sets (i.e. manually 
> updating BATCH numbers) and - this is the real fun one - if I rerun the 
> processing with 900 images REBATCH works fine!
> 
> … following this train of thought …
> 
> Could this be as simple as MAXBAT needing to be reassigned / code recompiled? 
> In this happy world of 100,000 image + data sets guess 5,000 images may not 
> be enough (though the program does not check whether #batches > MAXBAT)
> 
> Anyhow, I suspect that the kernel of your message, that after 2.3 decades 
> there are better tools available, is almost certainly correct. Will look 
> elsewhere.
> 
> Thanks & best wishes Graeme
> 
> 
>> On 6 Jul 2017, at 15:10, Phil Evans  wrote:
>> 
>> REBATCH should generally be replaceable by Pointless I think
>> 
>> Latest latest Pointless (prerelease update 043, version 1.11.3) removes 
>> batch headers  for leading or trailing batch numbers which are not present 
>> in the reflection list (for you, Graeme)
>> 
>> 
>> 
>> 
>>> On 6 Jul 2017, at 15:04, Graeme Winter  wrote:
>>> 
>>> Afternoon all,
>>> 
>>> Technical harking back to the 90’s FORTRAN CCP4 question if I may
>>> 
>>> I have an MTZ file which has no spots on first couple of images, so the 
>>> batch headers at the start exist but there are no BATCH values which 
>>> correspond to these in the actual reflection data
>>> 
>>> I have been bashing my head against the wall trying to get away from this 
>>> error:
>>> 
>>> 
>>> 
>>> 
>>> 
>>> Output File
>>> 
>>> 
>>> *** Wrong batch! batch number in file is   3, batch number from 
>>> internal lookup1
>>> 
>>> 
>>> REBATCH:   *** Crazy batch number ***
>>> REBATCH:   *** Crazy batch number ***
>>> Times: User:   0.6s System:0.0s Elapsed: 0:01
>>> 
>>> 
>>> 
>>> 
>>> Does anyone out there know how one should “fix” this?
>>> 
>>> MTZDUMP output follows, which all looks sensible to me…
>>> 
>>> I also find the following at the top of the source code ;o)
>>> 
>>> C REBATCH
>>> C Copyright (C) 1994 Phil Evans
>>> 
>>> However I am certain other experts out there will also be able to help
>>> 
>>> I’ve worked my way through the source code but can’t really work out what 
>>> is happening here...
>>> 
>>> Cheers Graeme
>>> 
>>> 
>>>  
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> 
>>> ###
>>> ###
>>> ###
>>> ### CCP4 7.0.042: MTZDUMP  version 1.1 : ##
>>> ###
>>> User: graeme  Run date:  6/ 7/2017 Run time: 14:59:21
>>> 
>>> 
>>> Please reference: Collaborative Computational Project, Number 4. 2011.
>>> "Overview of the CCP4 suite and current developments". Acta Cryst. D67, 
>>> 235-242.
>>> as well as any specific reference in the program write-up.
>>> 
>>> 
>>> 
>>> OPENED INPUT MTZ FILE
>>> Logical Name: HKLIN   Filename: integrated.mtz
>>> 
>>> * Title:
>>> 
>>> from dials.export_mtz
>>> 
>>> * Base dataset:
>>> 
>>>  0 HKL_base
>>>HKL_base
>>>HKL_base
>>> 
>>> * Number of Datasets = 1
>>> 
>>> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
>>> 
>>>  1 DIALS
>>>XTAL
>>>FROMDIALS
>>>   54.2171   57.9174   66.5815   90.   90.   90.
>>>   0.96770
>>> 
>>> * Number of Columns = 17
>>> 
>>> * Number of Reflections = 2004565
>>> 
>>> * Missing value set to NaN in input mtz file
>>> 
>>> * Number of Batches = 12000
>>> 
>>> * HISTORY for current MTZ file :
>>> 
>>> From DIALS 1.dev.1541-ge526b95f, run on 06/07/2017 at 13:55:27
>>> 
>>> * Column Labels :
>>> 
>>> H K L M/ISYM BATCH IPR SIGIPR I SIGI BG SIGBG FRACTIONCALC XDET YDET ROT LP 
>>> DQE
>>> 
>>> * Column Types :
>>> 
>>> H H H Y B J Q J Q R R R R R R R R
>>> 
>>> * Associated datasets :
>>> 
>>> 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1
>>> 
>>> * Cell Dimensions : (obsolete - refer to dataset cell dimensions above)
>>> 
>>> 54.2171   57.9174   66.5815   90.   90.   90.
>>> 
>>> *  Resolution Range :
>>> 
>>>  0.000520.68412 ( 43.698 -  1.209 A )
>>> 
>>> * Sort Order :
>>> 
>>>0 0 0 0 0
>>> 
>>> * Space group = 'P222' (number 16)
>>> 
>>> 
>>> 
>>> 
>>> Batch number:
>>>1Batch 1
>>> Batch number:
>>>2Batch 2
>>> Batch number:
>>>3Batch 3
>>> Batch number:
>>>4Batch 4
>>> Batch number:
>>>5Batch 5
>>> Batch number:
>>>6Batch 6
>>> Batch number:
>>>7Batch 7
>>> Batch number:
>>>8Batch 8
>>> Batch number:
>>>9

Re: [ccp4bb] REBATCH error mystery

[Graeme Winter]

HI Phil,

Yes, probably. However it’s still a mystery I would like to understand. Also, I 
use REBATCH for lots of things not just trimming data sets (i.e. manually 
updating BATCH numbers) and - this is the real fun one - if I rerun the 
processing with 900 images REBATCH works fine!

… following this train of thought …

Could this be as simple as MAXBAT needing to be reassigned / code recompiled? 
In this happy world of 100,000 image + data sets guess 5,000 images may not be 
enough (though the program does not check whether #batches > MAXBAT)

Anyhow, I suspect that the kernel of your message, that after 2.3 decades there 
are better tools available, is almost certainly correct. Will look elsewhere.

Thanks & best wishes Graeme


> On 6 Jul 2017, at 15:10, Phil Evans  wrote:
> 
> REBATCH should generally be replaceable by Pointless I think
> 
> Latest latest Pointless (prerelease update 043, version 1.11.3) removes batch 
> headers  for leading or trailing batch numbers which are not present in the 
> reflection list (for you, Graeme)
> 
> 
> 
> 
>> On 6 Jul 2017, at 15:04, Graeme Winter  wrote:
>> 
>> Afternoon all,
>> 
>> Technical harking back to the 90’s FORTRAN CCP4 question if I may
>> 
>> I have an MTZ file which has no spots on first couple of images, so the 
>> batch headers at the start exist but there are no BATCH values which 
>> correspond to these in the actual reflection data
>> 
>> I have been bashing my head against the wall trying to get away from this 
>> error:
>> 
>> 
>> 
>> 
>> 
>> Output File
>> 
>> 
>> *** Wrong batch! batch number in file is   3, batch number from internal 
>> lookup1
>> 
>> 
>> REBATCH:   *** Crazy batch number ***
>> REBATCH:   *** Crazy batch number ***
>> Times: User:   0.6s System:0.0s Elapsed: 0:01
>> 
>> 
>> 
>> 
>> Does anyone out there know how one should “fix” this?
>> 
>> MTZDUMP output follows, which all looks sensible to me…
>> 
>> I also find the following at the top of the source code ;o)
>> 
>> C REBATCH
>> C Copyright (C) 1994 Phil Evans
>> 
>> However I am certain other experts out there will also be able to help
>> 
>> I’ve worked my way through the source code but can’t really work out what is 
>> happening here...
>> 
>> Cheers Graeme
>> 
>> 
>>  
>> 
>> 
>> 
>> 
>> 
>> 
>> 
>> ###
>> ###
>> ###
>> ### CCP4 7.0.042: MTZDUMP  version 1.1 : ##
>> ###
>> User: graeme  Run date:  6/ 7/2017 Run time: 14:59:21
>> 
>> 
>> Please reference: Collaborative Computational Project, Number 4. 2011.
>> "Overview of the CCP4 suite and current developments". Acta Cryst. D67, 
>> 235-242.
>> as well as any specific reference in the program write-up.
>> 
>> 
>> 
>> OPENED INPUT MTZ FILE
>> Logical Name: HKLIN   Filename: integrated.mtz
>> 
>> * Title:
>> 
>> from dials.export_mtz
>> 
>> * Base dataset:
>> 
>>   0 HKL_base
>> HKL_base
>> HKL_base
>> 
>> * Number of Datasets = 1
>> 
>> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
>> 
>>   1 DIALS
>> XTAL
>> FROMDIALS
>>54.2171   57.9174   66.5815   90.   90.   90.
>>0.96770
>> 
>> * Number of Columns = 17
>> 
>> * Number of Reflections = 2004565
>> 
>> * Missing value set to NaN in input mtz file
>> 
>> * Number of Batches = 12000
>> 
>> * HISTORY for current MTZ file :
>> 
>> From DIALS 1.dev.1541-ge526b95f, run on 06/07/2017 at 13:55:27
>> 
>> * Column Labels :
>> 
>> H K L M/ISYM BATCH IPR SIGIPR I SIGI BG SIGBG FRACTIONCALC XDET YDET ROT LP 
>> DQE
>> 
>> * Column Types :
>> 
>> H H H Y B J Q J Q R R R R R R R R
>> 
>> * Associated datasets :
>> 
>> 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1
>> 
>> * Cell Dimensions : (obsolete - refer to dataset cell dimensions above)
>> 
>>  54.2171   57.9174   66.5815   90.   90.   90.
>> 
>> *  Resolution Range :
>> 
>>   0.000520.68412 ( 43.698 -  1.209 A )
>> 
>> * Sort Order :
>> 
>> 0 0 0 0 0
>> 
>> * Space group = 'P222' (number 16)
>> 
>> 
>> 
>> 
>> Batch number:
>> 1Batch 1
>> Batch number:
>> 2Batch 2
>> Batch number:
>> 3Batch 3
>> Batch number:
>> 4Batch 4
>> Batch number:
>> 5Batch 5
>> Batch number:
>> 6Batch 6
>> Batch number:
>> 7Batch 7
>> Batch number:
>> 8Batch 8
>> Batch number:
>> 9Batch 9
>> 
>> ——8<——
>> 
>> Batch number:
>> 11997Batch 11997
>> Batch number:
>> 11998Batch 11998
>> Batch number:
>> 11999Batch 11999
>> Batch number:
>> 12000Batch 12000
>> 
>> M/ISYM located at column4
>> 
>> BATCH located at column5
>> 
>> 
>> OVERALL FILE STATISTICS for resolution 

[ccp4bb] off topic

[Anamika Singh]

Hi,

Is anyone has worked with STAT1 proteins?

I have cloned the SH2 domain of STAT1 protein into pet28a vector but there
was no expression so far or rather say inconsistent expression. Sometimes
the expression was in inclusion bodies.  I have tried different methods to
pull out the protein from inclusion bodies using urea, guanidium chloride
tween20 but none of them worked well. The yield was very low (very faint
band on SDS-PAGE ) from 3-liter culture. I changed the host cells from BL21
to Rosetta DE3 cells but no success so far.

We thought to use some other vector system like with SUMO tag but did not
proceed because the aim of the project to design inhibitor and tag will
interfere.

Please suggest me something so that I can complete my project in lesser
time.


Looking forward to valuable suggestions.

Thanks
Anamika

Re: [ccp4bb] REBATCH error mystery

[Phil Evans]

REBATCH should generally be replaceable by Pointless I think

Latest latest Pointless (prerelease update 043, version 1.11.3) removes batch 
headers  for leading or trailing batch numbers which are not present in the 
reflection list (for you, Graeme)




> On 6 Jul 2017, at 15:04, Graeme Winter  wrote:
> 
> Afternoon all,
> 
> Technical harking back to the 90’s FORTRAN CCP4 question if I may
> 
> I have an MTZ file which has no spots on first couple of images, so the batch 
> headers at the start exist but there are no BATCH values which correspond to 
> these in the actual reflection data
> 
> I have been bashing my head against the wall trying to get away from this 
> error:
> 
> 
> 
> 
> 
> Output File
> 
> 
> *** Wrong batch! batch number in file is   3, batch number from internal 
> lookup1
> 
> 
> REBATCH:   *** Crazy batch number ***
> REBATCH:   *** Crazy batch number ***
> Times: User:   0.6s System:0.0s Elapsed: 0:01
> 
> 
> 
> 
> Does anyone out there know how one should “fix” this?
> 
> MTZDUMP output follows, which all looks sensible to me…
> 
> I also find the following at the top of the source code ;o)
> 
> C REBATCH
> C Copyright (C) 1994 Phil Evans
> 
> However I am certain other experts out there will also be able to help
> 
> I’ve worked my way through the source code but can’t really work out what is 
> happening here...
> 
> Cheers Graeme
> 
> 
>  
> 
> 
> 
> 
> 
> 
> 
> ###
> ###
> ###
> ### CCP4 7.0.042: MTZDUMP  version 1.1 : ##
> ###
> User: graeme  Run date:  6/ 7/2017 Run time: 14:59:21
> 
> 
> Please reference: Collaborative Computational Project, Number 4. 2011.
> "Overview of the CCP4 suite and current developments". Acta Cryst. D67, 
> 235-242.
> as well as any specific reference in the program write-up.
> 
> 
> 
> OPENED INPUT MTZ FILE
> Logical Name: HKLIN   Filename: integrated.mtz
> 
> * Title:
> 
> from dials.export_mtz
> 
> * Base dataset:
> 
>0 HKL_base
>  HKL_base
>  HKL_base
> 
> * Number of Datasets = 1
> 
> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
> 
>1 DIALS
>  XTAL
>  FROMDIALS
> 54.2171   57.9174   66.5815   90.   90.   90.
> 0.96770
> 
> * Number of Columns = 17
> 
> * Number of Reflections = 2004565
> 
> * Missing value set to NaN in input mtz file
> 
> * Number of Batches = 12000
> 
> * HISTORY for current MTZ file :
> 
> From DIALS 1.dev.1541-ge526b95f, run on 06/07/2017 at 13:55:27
> 
> * Column Labels :
> 
> H K L M/ISYM BATCH IPR SIGIPR I SIGI BG SIGBG FRACTIONCALC XDET YDET ROT LP 
> DQE
> 
> * Column Types :
> 
> H H H Y B J Q J Q R R R R R R R R
> 
> * Associated datasets :
> 
> 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1
> 
> * Cell Dimensions : (obsolete - refer to dataset cell dimensions above)
> 
>   54.2171   57.9174   66.5815   90.   90.   90.
> 
> *  Resolution Range :
> 
>0.000520.68412 ( 43.698 -  1.209 A )
> 
> * Sort Order :
> 
>  0 0 0 0 0
> 
> * Space group = 'P222' (number 16)
> 
> 
> 
> 
> Batch number:
>  1Batch 1
> Batch number:
>  2Batch 2
> Batch number:
>  3Batch 3
> Batch number:
>  4Batch 4
> Batch number:
>  5Batch 5
> Batch number:
>  6Batch 6
> Batch number:
>  7Batch 7
> Batch number:
>  8Batch 8
> Batch number:
>  9Batch 9
> 
> ——8<——
> 
> Batch number:
>  11997Batch 11997
> Batch number:
>  11998Batch 11998
> Batch number:
>  11999Batch 11999
> Batch number:
>  12000Batch 12000
> 
> M/ISYM located at column4
> 
> BATCH located at column5
> 
> 
> OVERALL FILE STATISTICS for resolution range   0.001 -   0.684
> ===
> 
> 
> Col SortMinMaxNum  % Mean Mean   Resolution   Type 
> Column
> num order   Missing complete  abs.   LowHigh   
> label
> 
>   1 NONE 0  36  0  100.00 15.0 15.0  43.70   1.21   H  H
>   2 NONE 0  39  0  100.00 16.6 16.6  43.70   1.21   H  K
>   3 NONE 0  49  0  100.00 18.2 18.2  43.70   1.21   H  L
>   4 NONE 1   8  0  100.00  4.4  4.4  43.70   1.21   Y  
> M/ISYM
>   5 ASC  3   11998  0  100.00   5997.2   5997.2  43.70   1.21   B  
> BATCH
>   6 NONE   -1.6   894.4 0  100.0014.3014.31  43.70   1.21   J  IPR
>   7 NONE0.016.5 0  100.00 1.92 1.92  43.70   1.21   Q  
> SIGIPR
>   8 NONE   -5.8   982.8 0  100.0015.9516.09  43.70   1.21   J  I
>   9 NONE0.017.4 0  100.00 2.38 2.38  43.70   1.21   Q  
> SIGI
>  10 

[ccp4bb] Scientific Software Developer position at RCSB Protein Data Bank (RCSB.org) at the University of California San Diego (UCSD)

[Christine Zardecki]

For contact information and other positions, please see 
http://www.rcsb.org/pdb/static.do?p=general_information/about_pdb/contact/job_listings.html
 


Scientific Software Developer

Develop, implement, and maintain complex scientific and web-based software 
systems for the RCSB Protein Data Bank (RCSB PDB; http://www.rcsb.org 
) at the University of California San Diego (UCSD).

The Scientific Software Developer will work closely and collaboratively with 
other software developers and scientists at the San Diego Supercomputer Center 
(SDSC) and the RCSB PDB partner sites to expand RCSB.org 's 
functionality and reliability as a premier biological data and information 
resource.

* Develop new scalable algorithms for the mining and analysis of the rapidly 
growing PDB archive using leading edge Big Data technologies. Design and 
implement user interfaces for the query, analysis, reporting, and visualization 
of 3D structural information and associated annotations.
* Integrate external database resources with RCSB PDB to provide a structural 
view of biology. Help lead the design of databases and data warehouses to store 
and aid in the query of data.
* Recommend and implement changes in software development, maintenance and 
system standards for analysis algorithms, tools, and infrastructure.
* Serve as a recognized expert on relevant scientific and technical aspects of 
the various web, web services, and database components of the RCSB PDB. Stays 
abreast of the latest development in structural and computational biology and 
new technologies.
* Applies advanced bioinformatics concepts to design, develop, modify, debug, 
and evaluate highly complex software programs and web tools. Translates 
scientific problems into scalable and maintainable software solutions that meet 
end user needs.

Qualifications: Advanced understanding of programming in one or more languages 
is required (Java, Python or Javascript); In-depth understanding of 
Bioinformatics; understanding of Biology or Chemistry; and an advanced degree

--
Twitter: http://twitter.com/#!/buildmodels
Facebook:  http://www.facebook.com/RCSBPDB

[ccp4bb] REBATCH error mystery

[Graeme Winter]

Afternoon all,

Technical harking back to the 90’s FORTRAN CCP4 question if I may

I have an MTZ file which has no spots on first couple of images, so the batch 
headers at the start exist but there are no BATCH values which correspond to 
these in the actual reflection data

I have been bashing my head against the wall trying to get away from this error:





Output File


 *** Wrong batch! batch number in file is   3, batch number from internal 
lookup1


 REBATCH:   *** Crazy batch number ***
 REBATCH:   *** Crazy batch number ***
Times: User:   0.6s System:0.0s Elapsed: 0:01




Does anyone out there know how one should “fix” this?

MTZDUMP output follows, which all looks sensible to me…

I also find the following at the top of the source code ;o)

C REBATCH
C Copyright (C) 1994 Phil Evans

However I am certain other experts out there will also be able to help

I’ve worked my way through the source code but can’t really work out what is 
happening here...

Cheers Graeme


 







 ###
 ###
 ###
 ### CCP4 7.0.042: MTZDUMP  version 1.1 : ##
 ###
 User: graeme  Run date:  6/ 7/2017 Run time: 14:59:21


 Please reference: Collaborative Computational Project, Number 4. 2011.
 "Overview of the CCP4 suite and current developments". Acta Cryst. D67, 
235-242.
 as well as any specific reference in the program write-up.



 OPENED INPUT MTZ FILE
 Logical Name: HKLIN   Filename: integrated.mtz

 * Title:

 from dials.export_mtz

 * Base dataset:

0 HKL_base
  HKL_base
  HKL_base

 * Number of Datasets = 1

 * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:

1 DIALS
  XTAL
  FROMDIALS
 54.2171   57.9174   66.5815   90.   90.   90.
 0.96770

 * Number of Columns = 17

 * Number of Reflections = 2004565

 * Missing value set to NaN in input mtz file

 * Number of Batches = 12000

 * HISTORY for current MTZ file :

 From DIALS 1.dev.1541-ge526b95f, run on 06/07/2017 at 13:55:27

 * Column Labels :

 H K L M/ISYM BATCH IPR SIGIPR I SIGI BG SIGBG FRACTIONCALC XDET YDET ROT LP DQE

 * Column Types :

 H H H Y B J Q J Q R R R R R R R R

 * Associated datasets :

 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1

 * Cell Dimensions : (obsolete - refer to dataset cell dimensions above)

   54.2171   57.9174   66.5815   90.   90.   90.

 *  Resolution Range :

0.000520.68412 ( 43.698 -  1.209 A )

 * Sort Order :

  0 0 0 0 0

 * Space group = 'P222' (number 16)




 Batch number:
  1Batch 1
 Batch number:
  2Batch 2
 Batch number:
  3Batch 3
 Batch number:
  4Batch 4
 Batch number:
  5Batch 5
 Batch number:
  6Batch 6
 Batch number:
  7Batch 7
 Batch number:
  8Batch 8
 Batch number:
  9Batch 9

——8<——

 Batch number:
  11997Batch 11997
 Batch number:
  11998Batch 11998
 Batch number:
  11999Batch 11999
 Batch number:
  12000Batch 12000

 M/ISYM located at column4

 BATCH located at column5


 OVERALL FILE STATISTICS for resolution range   0.001 -   0.684
 ===


 Col SortMinMaxNum  % Mean Mean   Resolution   Type 
Column
 num order   Missing complete  abs.   LowHigh   
label

   1 NONE 0  36  0  100.00 15.0 15.0  43.70   1.21   H  H
   2 NONE 0  39  0  100.00 16.6 16.6  43.70   1.21   H  K
   3 NONE 0  49  0  100.00 18.2 18.2  43.70   1.21   H  L
   4 NONE 1   8  0  100.00  4.4  4.4  43.70   1.21   Y  
M/ISYM
   5 ASC  3   11998  0  100.00   5997.2   5997.2  43.70   1.21   B  
BATCH
   6 NONE   -1.6   894.4 0  100.0014.3014.31  43.70   1.21   J  IPR
   7 NONE0.016.5 0  100.00 1.92 1.92  43.70   1.21   Q  
SIGIPR
   8 NONE   -5.8   982.8 0  100.0015.9516.09  43.70   1.21   J  I
   9 NONE0.017.4 0  100.00 2.38 2.38  43.70   1.21   Q  SIGI
  10 NONE0.0   140.2 0  100.00 6.29 6.29  43.70   1.21   R  BG
  11 NONE0.013.4 0  100.00 2.59 2.59  43.70   1.21   R  
SIGBG
  12 BOTH1.0 1.0 0  100.00 1.00 1.00  43.70   1.21   R  
FRACTIONCALC
  13 NONE7.5  2062.3 0  100.00  1032.71  1032.71  43.70   1.21   R  XDET
  14 NONE6.9  2160.1 0  100.00  1084.27  1084.27  43.70   1.21   R  YDET
  15 NONE0.2  1199.8 0  100.00   599.47   599.47  43.70   1.21   R  ROT
  16 NONE0.0 0.7 0  100.00 0.44 0.44  43.70   1.21   R  LP
  17 NONE0.8 0.9 0  100.00 0.87 0.87  43.70   

[ccp4bb] RELEASE NOTE SIMPLE 2.5

[Hans Elmlund]

Dear All:

We announce the release of a new version of our program package SIMPLE
(v2.5) for single-particle cryo-EM *ab initio* 3D reconstruction (web:
simplecryoem.com)

New features include:

* A new DDD movie pre-processing program that implements motion correction
based the same principal strategy as Grigorieff's Unblur. There are two
important differences: automatic weighting of the frames using a
correlation-based M-estimator and stochastic continuous optimisation of the
shift parameters. This enables analysis of movies with severe pathologies
due to radiation damage or extreme drift.

* A new program for DDD movie pre-processing of tomographic tilt-series.

* Improved simultaneous 2D alignment and clustering with PRIME2D using a
hybrid extremal/stochastic hill-climbing search approach, Wiener
restoration-based CTF correction and acceleration of the search using
Hadamard projection matching. It is now possible to generate a
sub-nanometer resolution *ab initio* 3D reconstruction from class averages
obtained with PRIME2D in a about 10 minutes on a laptop (MacBook Pro mid
2015, 2.8 GHz Intel i7, four physical cores).

* Improved *ab initio* 3D reconstruction from class averages using
stochastic neighbourhood hill-climbing for initialisation of the 3D
orientation search, improving the success rate from around 40% to 90-100%
for more challenging starting model generation problems, executed with the
new program ini3D_from_cavgs.

* Serial CPU code optimisation through data re-organisation and pipelining.

* Improved parallel CPU performance through load balancing as well as data
and algorithm re-organisation. It is now possible to process data sets of
realistic size on laptops or lightweight workstations that cost less than
2,000 USD.

* High-level workflows for 2D analysis and initial 3D model generation that
automates initialisation, updates search parameters automatically and
dynamically down-scales the images for improved performance.

If you need help installing SIMPLE, optimising its execution for your
computer architecture or experience any problems or bugs please use the
contact form on the webpage (www.simplecryoem.com/contact.html) to file a
help ticket. We will endeavour to respond within two days.

Happy image processing!

The SIMPLE team:

Cyril F Reboul
Michael Eager
Dominika & Hans Elmlund

-- 
*HANS ELMLUND PhD *
Associate Professor

*ARC Centre of Excellence for Advanced Molecular Imaging*
*Dept. of Biochemistry and Molecular Biology*
Monash University
Room 228, Level 2, Building 77, Clayton Campus
23 Innovation Walk
Clayton VIC 3800
Australia

T: +61 399029310
M: +61 (0)45271213
E: hans.elml...@monash.edu
simplecryoem.com