Re: [ccp4bb] crystal orientation during data collection

[Frank von Delft]

I believe you achieve completeness more quickly (fewer crystals) if you 
just take random orientations.  At least, that's what I learnt from Dave 
Stuart.

phx



On 18/11/2011 04:20, Frank Murphy wrote:

Yanwu,

I surmise from your question that you are inquiring how to go about 
collecting from many crystals optimally. Merging data ex post facto is 
a totally different kettle of fish.


In my opinion, the most robust way to go about this is to use a kappa 
goniometer as Jim suggested (I am most familiar with the MK3). Since 
you intend to collect from many crystals, align the first and all 
subsequent crystals to the same easily attainable (or seemingly so) 
orientation, and then collect the sweep suggested by your data 
collection strategy program of choice.


To achieve this at NE-CAT, we have a GUI-based system that used STAC 
for orientation determination and BEST for strategy generation. As Jim 
suggested, more options than STAC exist.


If anyone is unable to get to a kappa goniometer, they can employ 
Mosflm or XDS (Xplan) to generate strategies for data collection from 
a crystal taking into account previously collected data. This is not 
nearly as robust a solution, but is a workable substitute (and also 
automated at NE-CAT).


I know there are other ways to achieve similar results, but I have 
suggested the methods I am most familiar with...



Yours,
Frank Murphy


Begin forwarded message:


*From: *yanwu huo mailto:applehu...@gmail.com>>
*Date: *November 17, 2011 4:00:06 PM CST
*To: *CCP4BB@JISCMAIL.AC.UK 
*Subject: **[ccp4bb] crystal orientation during data collection*
*Reply-To: *yanwu huo >


Hi,
I worked on a crystal sensitive to radiation damage, So I need to 
merge many crystal to obtain complete dataset, Does anyone know such 
program that can tell crystal orientation after first frame exposure.

Thank you in advance.


--
Thank you very much and all the best,

Yanwu Huo
Postdoctoral Associate
Department of Molecular Biology and Genetics
Cornell University
Ithaca, NY, 14853
Email:yh...@cornell.edu 

Re: [ccp4bb] crystal orientation during data collection

[Mark J van Raaij]

I don't think one can generalise that much.
In the Stuart case of BTV, only 1-3 useful images per crystal could be obtained 
if I remember correctly - so there the random strategy orientation makes sense. 
I.e. if you collect one image and then wait until the orientation and strategy 
is calculated, the crystal is probably already dead.
For projects where more images can be collected from a single crystal, I would 
think it makes more sense to use the strategy options described (like was done 
for F1-ATPase).
Another thing to keep in mind is how to achieve this random orientation, one 
can not always count on the crystal orienting randomly in a loop, i.e. many 
crystals have preferred orientations when mounted...and one tends to mount the 
loop on the goniometer the same way each time. So one would have to use 
"random" starting phis.

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/content/research/macromolecular/mvraaij





On 18 Nov 2011, at 09:26, Frank von Delft wrote:

> I believe you achieve completeness more quickly (fewer crystals) if you just 
> take random orientations.  At least, that's what I learnt from Dave Stuart.
> phx
> 
> 
> 
> On 18/11/2011 04:20, Frank Murphy wrote:
>> Yanwu,
>> 
>> I surmise from your question that you are inquiring how to go about 
>> collecting from many crystals optimally. Merging data ex post facto is a 
>> totally different kettle of fish.
>> 
>> In my opinion, the most robust way to go about this is to use a kappa 
>> goniometer as Jim suggested (I am most familiar with the MK3). Since you 
>> intend to collect from many crystals, align the first and all subsequent 
>> crystals to the same easily attainable (or seemingly so) orientation, and 
>> then collect the sweep suggested by your data collection strategy program of 
>> choice.
>> 
>> To achieve this at NE-CAT, we have a GUI-based system that used STAC for 
>> orientation determination and BEST for strategy generation. As Jim 
>> suggested, more options than STAC exist.
>> 
>> If anyone is unable to get to a kappa goniometer, they can employ Mosflm or 
>> XDS (Xplan) to generate strategies for data collection from a crystal taking 
>> into account previously collected data. This is not nearly as robust a 
>> solution, but is a workable substitute (and also automated at NE-CAT).
>> 
>> I know there are other ways to achieve similar results, but I have suggested 
>> the methods I am most familiar with...
>> 
>> 
>> Yours,
>> Frank Murphy
>> 
>> 
>> Begin forwarded message:
>> 
>>> From: yanwu huo 
>>> Date: November 17, 2011 4:00:06 PM CST
>>> To: CCP4BB@JISCMAIL.AC.UK
>>> Subject: [ccp4bb] crystal orientation during data collection
>>> Reply-To: yanwu huo 
>>> 
>>> Hi, 
>>> I worked on a crystal sensitive to radiation damage, So I need to merge 
>>> many crystal to obtain complete dataset, Does anyone know such 
>>> program that can tell crystal orientation after first frame exposure.
>>> Thank you in advance.
>>> 
>>> 
>>> -- 
>>> Thank you very much and all the best,
>>> 
>>> Yanwu Huo 
>>> Postdoctoral Associate
>>> Department of Molecular Biology and Genetics
>>> Cornell University
>>> Ithaca, NY, 14853
>>> Email:yh...@cornell.edu
>>> 
>> 
>> 
>> 
>> 

Re: [ccp4bb] crystal orientation during data collection

[Harry Powell]

Hi Frank

I believe (at least part of) the key to the 30S ribosome structure was being 
able to orient the crystals carefully and collect the data accordingly - see 
Brodersen et al, Acta Cryst. (2003). D59, 2044-2050 

Completeness, while important, ain't everything.

On 18 Nov 2011, at 08:26, Frank von Delft wrote:

> I believe you achieve completeness more quickly (fewer crystals) if you just 
> take random orientations.  At least, that's what I learnt from Dave Stuart.
> phx
> 
> 
> 
> On 18/11/2011 04:20, Frank Murphy wrote:
>> Yanwu,
>> 
>> I surmise from your question that you are inquiring how to go about 
>> collecting from many crystals optimally. Merging data ex post facto is a 
>> totally different kettle of fish.
>> 
>> In my opinion, the most robust way to go about this is to use a kappa 
>> goniometer as Jim suggested (I am most familiar with the MK3). Since you 
>> intend to collect from many crystals, align the first and all subsequent 
>> crystals to the same easily attainable (or seemingly so) orientation, and 
>> then collect the sweep suggested by your data collection strategy program of 
>> choice.
>> 
>> To achieve this at NE-CAT, we have a GUI-based system that used STAC for 
>> orientation determination and BEST for strategy generation. As Jim 
>> suggested, more options than STAC exist.
>> 
>> If anyone is unable to get to a kappa goniometer, they can employ Mosflm or 
>> XDS (Xplan) to generate strategies for data collection from a crystal taking 
>> into account previously collected data. This is not nearly as robust a 
>> solution, but is a workable substitute (and also automated at NE-CAT).
>> 
>> I know there are other ways to achieve similar results, but I have suggested 
>> the methods I am most familiar with...
>> 
>> 
>> Yours,
>> Frank Murphy
>> 
>> 
>> Begin forwarded message:
>> 
>>> From: yanwu huo 
>>> Date: November 17, 2011 4:00:06 PM CST
>>> To: CCP4BB@JISCMAIL.AC.UK
>>> Subject: [ccp4bb] crystal orientation during data collection
>>> Reply-To: yanwu huo 
>>> 
>>> Hi, 
>>> I worked on a crystal sensitive to radiation damage, So I need to merge 
>>> many crystal to obtain complete dataset, Does anyone know such program that 
>>> can tell crystal orientation after first frame exposure.
>>> Thank you in advance.
>>> 
>>> 
>>> -- 
>>> Thank you very much and all the best,
>>> 
>>> Yanwu Huo 
>>> Postdoctoral Associate
>>> Department of Molecular Biology and Genetics
>>> Cornell University
>>> Ithaca, NY, 14853
>>> Email:yh...@cornell.edu
>>> 
>> 
>> 
>> 
>> 

Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, 
Cambridge, CB2 0QH

[ccp4bb] La Caixa fellowships

[Mark J van Raaij]

Dear prospective PhD students,

for those considering coming to Spain, it may be worth applying for a La Caixa 
fellowship. 
The website is:
http://www.cnb.csic.es/content/about/lacaixa/index.php?l=0
You do not have to present a project, in fact you only choose which lab to go 
to after being selected.

greetings,

Mark

Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoleculas
Centro Nacional de Biotecnologia - CSIC
c/Darwin 3
E-28049 Madrid, Spain
tel. (+34) 91 585 4616
http://www.cnb.csic.es/content/research/macromolecular/mvraaij

Re: [ccp4bb] pointless problem

[Phil Evans]

Pointless will do the best it can with whatever data you give it, but if it is 
already merged then it can only check for under-merging, or for reindexing

The normal use is for unmerged data as output by an integration program such as 
Mosflm or XDS, as a prelude for scaling with Aimless or Scala. It that case 
Friedel pairs will be present.

Phil

On 17 Nov 2011, at 09:32, Tim Gruene wrote:

> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Dear all, dear Phil,
> 
> how can a dataset be merged if the space group is unknown? Does
> pointless require the Friedel pairs present in the file, too?
> 
> Tim
> 
> On 11/17/2011 08:34 AM, Graeme Winter wrote:
>> Dear Ping,
>> 
>> You need the unmerged data for this - for instance the output from
>> Mosflm or the XDS INTEGRATE step. Pointless works by comparing
>> potentially symmetry related measurements.
>> 
>> Best wishes,
>> 
>> Graeme
>> 
>> 2011/11/17 Ping Wang :
>>> Dear all,
>>> I have a dataset with spacegroup undetermined. When I use POINTLESS to
>>> determine laue group, the program gave the fatal error: HKLIN is merged and
>>> no HKLREF is defined, SPACEGROUP or REINDEX.
>>> In the protocol of POINTLESS for the determination of LAUE group, HKLREF can
>>> be not specified. So I have no idea where the problem is. Can anyone give me
>>> some suggestions?
>>> Kind regrads.
>>> Ping
>>> 
>>> 
>>> 
>> 
> 
> - -- 
> - --
> Dr Tim Gruene
> Institut fuer anorganische Chemie
> Tammannstr. 4
> D-37077 Goettingen
> 
> GPG Key ID = A46BEE1A
> 
> -BEGIN PGP SIGNATURE-
> Version: GnuPG v1.4.10 (GNU/Linux)
> Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/
> 
> iD8DBQFOxNSaUxlJ7aRr7hoRAiFQAKDuNVeWbieYvqwMZNIOnRLS7NaA1QCeNQ+3
> Sw5bBEwMUWWDWYunEJwWqsU=
> =Gzpp
> -END PGP SIGNATURE-

[ccp4bb] Manually setting 96 wells plates with lower volume samples!

[xaravich ivan]

Hi,
I apologize in advance as it is not a ccp4 related question, but over the
years, CCP4bb is synonymous with protein crystallographers virtual
university, at least for me.

Ok, now I do not have an easy access to crystallization robot, so I was
hoping if someone here have ever used the 96 well plates for manually
setting drops with much lower solution/sample volumes (0.1-0.2micro litres).

I heard about a clicker syringe that can be used to manually add lesser
volumes but I am not sure how to go about it.
Please let me know if you are aware of or  are routinely setting 96 well
trays with much lower volumes of crystallization solutions as well as
samples, manually.

thanks in advance,
ivan

[ccp4bb] FreeR in the case of few reflections

[Aaron Alt]

Hi all,

I have data indexed in I23 with ~3000 unique reflections. Having set
aside 10% of these my refinements still go berserk. The maps do look
fine though. The same happens when reindexed in lower symmetries.
Phenix autobuild finishes for example with 28/40 and I get similiar
results (although it took me longer) tracing manually and refining
with refmac. Does it make sense to set aside 500 reflections in my
case, which would be ~17% of the data? What is the correct way to
deal with data of this type? Ignoring the Rfree completely?
A nice weekend to all,
Aaron

Re: [ccp4bb] Manually setting 96 wells plates with lower volume samples!

[Shya Biswas]

Hi,
I am routinely using the gryphon robot from Art Robbins.This instrument can
dispense 0.02microlitre at minimum. Your drops can dry out if you use such
low volumes you have to be really fast. You can set up 0.2 to 0.1 micro
litre drops using this for 96 well plates however the instrument needs to
be calibrated well sometimes with low volume drop the robot dispenses the
protein at one corner of the well and the precipitant at another end so
eventually the outcome is the mixing of the precipitant with your protein
is not happening. However with 0.2micro litre drop or higher this is less
of a problem.
HTH,
shya

On Fri, Nov 18, 2011 at 9:34 AM, xaravich ivan wrote:

> Hi,
> I apologize in advance as it is not a ccp4 related question, but over the
> years, CCP4bb is synonymous with protein crystallographers virtual
> university, at least for me.
>
> Ok, now I do not have an easy access to crystallization robot, so I was
> hoping if someone here have ever used the 96 well plates for manually
> setting drops with much lower solution/sample volumes (0.1-0.2micro litres).
>
> I heard about a clicker syringe that can be used to manually add lesser
> volumes but I am not sure how to go about it.
> Please let me know if you are aware of or  are routinely setting 96 well
> trays with much lower volumes of crystallization solutions as well as
> samples, manually.
>
> thanks in advance,
> ivan
>

[ccp4bb] Combining big MTZs

[Jacob Keller]

Dear Crystallographers,

I am getting an error when I try to merge two mtz's from mosflm, one
with 180 and one with 360 frames, each from different but similar
crystals--see below. I can't imagine this really exceeds the max
number of records, so what am I doing wrong? Additionally but related,
what is the optimal procedure in CCP4 for combining data from two
similar crystals?

JPK



#CCP4I VERSION CCP4Interface 2.1.0
#CCP4I SCRIPT LOG sortmtz
#CCP4I DATE 18 Nov 2011  09:26:28
#CCP4I USER 'UNKNOWN'
#CCP4I PROJECT NatVSulP
#CCP4I JOB_ID 26
#CCP4I SCRATCH C:/Ccp4Temp
#CCP4I HOSTNAME chloe
#CCP4I PID 5396

 


SORTMTZ


 ###
 ###
 ###
 ### CCP4 6.2: SORTMTZ  version 6.2 : 06/09/05##
 ###
 User: Jacob  Run date: 18/11/2011 Run time: 09:26:28


 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.



Contents

Command Input
Input File Details
Output File Details
Header Information for Output MTZ File



Command Input
ASCEND/DESCEND
SORT KEYS

 Data line--- ASCEND
 Data line--- H K L M/ISYM BATCH
 Data line--- 
"C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz"



Input File Details


 OPENED INPUT MTZ FILE
 Logical Name: 
C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz
  Filename: 
C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz

 * Title:

 Untitled

 * Base dataset:

0 HKL_base
  HKL_base
  HKL_base

 * Number of Datasets = 1

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

1 New
  New
  New
 82.0600   82.0600  159.2500   90.   90.   90.
 0.97856

 * Number of Columns = 18

 * Number of Reflections = 1526614

 * Missing value set to NaN in input mtz file

 * Number of Batches = 360

 * Column Labels :

 H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH
LP MPART FLAG BGPKRATIOS

 * Column Types :

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

 * Associated datasets :

 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

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

   82.0600   82.0600  159.2500   90.   90.   90.

 *  Resolution Range :

0.000300.15998 ( 58.026 -  2.500 A )

 * Sort Order :

  0 0 0 0 0

 * Space group = 'P43212' (number 96)


 Spacegroup information obtained from library file:
 Logical Name: SYMINFO   Filename:
C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib

5 sort keys, in columns1   2   3   4   5



Output File Details

1526614 records read from file1
 Data line--- 
"C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz"



Input File Details


 OPENED INPUT MTZ FILE
 Logical Name: 
C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz
  Filename: 
C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz

 * Title:

 [No title given]

 * Base dataset:

0 HKL_base
  HKL_base
  HKL_base

 * Number of Datasets = 1

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

1 New
  New
  New
 82.3400   82.3400  161.4500   90.   90.   90.
 0.97940

 * Number of Columns = 18

 * Number of Reflections = 536433

 * Missing value set to NaN in input mtz file

 * Number of Batches = 180

 * Column Labels :

 H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH
LP MPART FLAG BGPKRATIOS

 * Column Types :

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

 * Associated datasets :

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

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

   82.3400   82.3400  161.4500   90.   90.   90.

 *  Resolution Range :

0.000150.16000 ( 82.340 -  2.500 A )

 * Sort Order :

  1 2 3 4 5

 * Space group = 'P43212' (number 96)

 Too many records
 SORTMTZ failed to release record to sort procedure, status = 1

 SORTMTZ:  Sorting failed
Times: User:   0.0s System:0.0s Elapsed: 0:05


***
* Information from CCP4Interface script
***
The program run with command: sortmtz HKLOUT
"C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_comb_raw.mtz"
has failed with error message
 SORTMTZ:  Sorting

Re: [ccp4bb] crystal orientation during data collection

[Sanishvili, Ruslan]

Depending on the crystal shape, "random orientation" is not always
random. Many crystals have tendencies of sitting themselves in one
predominant posture in the mount. Compounding this, many experimenters
have tendencies of rotating the mount into a specific orientation when
centering. Then crystal orientation ends up being not random at all, so
understanding it's true orientation as my neighbor Frank suggests can be
highly beneficial.

Cheers,

N.

 

Ruslan Sanishvili (Nukri), Ph.D.

GM/CA-CAT
Biosciences Division, ANL
9700 S. Cass Ave.
Argonne, IL 60439

Tel: (630)252-0665
Fax: (630)252-0667
rsanishv...@anl.gov 



From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Frank von Delft
Sent: Friday, November 18, 2011 2:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystal orientation during data collection

 

I believe you achieve completeness more quickly (fewer crystals) if you
just take random orientations.  At least, that's what I learnt from Dave
Stuart.
phx



On 18/11/2011 04:20, Frank Murphy wrote: 

Yanwu, 

 

I surmise from your question that you are inquiring how to go about
collecting from many crystals optimally. Merging data ex post facto is a
totally different kettle of fish.

 

In my opinion, the most robust way to go about this is to use a kappa
goniometer as Jim suggested (I am most familiar with the MK3). Since you
intend to collect from many crystals, align the first and all subsequent
crystals to the same easily attainable (or seemingly so) orientation,
and then collect the sweep suggested by your data collection strategy
program of choice.

 

To achieve this at NE-CAT, we have a GUI-based system that used STAC for
orientation determination and BEST for strategy generation. As Jim
suggested, more options than STAC exist.

 

If anyone is unable to get to a kappa goniometer, they can employ Mosflm
or XDS (Xplan) to generate strategies for data collection from a crystal
taking into account previously collected data. This is not nearly as
robust a solution, but is a workable substitute (and also automated at
NE-CAT).

 

I know there are other ways to achieve similar results, but I have
suggested the methods I am most familiar with...

 

 

Yours,

Frank Murphy

 

 

Begin forwarded message:





From: yanwu huo 

Date: November 17, 2011 4:00:06 PM CST

To: CCP4BB@JISCMAIL.AC.UK

Subject: [ccp4bb] crystal orientation during data collection

Reply-To: yanwu huo 


Hi, 
I worked on a crystal sensitive to radiation damage, So I need to merge
many crystal to obtain complete dataset, Does anyone know such program
that can tell crystal orientation after first frame exposure.
Thank you in advance.


-- 
Thank you very much and all the best,

Yanwu Huo 
Postdoctoral Associate
Department of Molecular Biology and Genetics
Cornell University
Ithaca, NY, 14853
Email:yh...@cornell.edu  


 

 

 

 

Re: [ccp4bb] crystal orientation during data collection

[Jacob Keller]

Generally speaking, don't we agree that "planned" or "rational" is
better than "random?" (Having trouble understanding the argument for
randomness here...)

Jacob

On Fri, Nov 18, 2011 at 9:40 AM, Sanishvili, Ruslan  wrote:
> Depending on the crystal shape, “random orientation” is not always random.
> Many crystals have tendencies of sitting themselves in one predominant
> posture in the mount. Compounding this, many experimenters have tendencies
> of rotating the mount into a specific orientation when centering. Then
> crystal orientation ends up being not random at all, so understanding it’s
> true orientation as my neighbor Frank suggests can be highly beneficial.
>
> Cheers,
>
> N.
>
>
>
> Ruslan Sanishvili (Nukri), Ph.D.
>
> GM/CA-CAT
> Biosciences Division, ANL
> 9700 S. Cass Ave.
> Argonne, IL 60439
>
> Tel: (630)252-0665
> Fax: (630)252-0667
> rsanishv...@anl.gov
>
> 
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank
> von Delft
> Sent: Friday, November 18, 2011 2:27 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] crystal orientation during data collection
>
>
>
> I believe you achieve completeness more quickly (fewer crystals) if you just
> take random orientations.  At least, that's what I learnt from Dave Stuart.
> phx
>
>
>
> On 18/11/2011 04:20, Frank Murphy wrote:
>
> Yanwu,
>
>
>
> I surmise from your question that you are inquiring how to go about
> collecting from many crystals optimally. Merging data ex post facto is a
> totally different kettle of fish.
>
>
>
> In my opinion, the most robust way to go about this is to use a kappa
> goniometer as Jim suggested (I am most familiar with the MK3). Since you
> intend to collect from many crystals, align the first and all subsequent
> crystals to the same easily attainable (or seemingly so) orientation, and
> then collect the sweep suggested by your data collection strategy program of
> choice.
>
>
>
> To achieve this at NE-CAT, we have a GUI-based system that used STAC for
> orientation determination and BEST for strategy generation. As Jim
> suggested, more options than STAC exist.
>
>
>
> If anyone is unable to get to a kappa goniometer, they can employ Mosflm or
> XDS (Xplan) to generate strategies for data collection from a crystal taking
> into account previously collected data. This is not nearly as robust a
> solution, but is a workable substitute (and also automated at NE-CAT).
>
>
>
> I know there are other ways to achieve similar results, but I have suggested
> the methods I am most familiar with...
>
>
>
>
>
> Yours,
>
> Frank Murphy
>
>
>
>
>
> Begin forwarded message:
>
> From:
>
> yanwu huo 
>
> Date:
>
> November 17, 2011 4:00:06 PM CST
>
> To:
>
> CCP4BB@JISCMAIL.AC.UK
>
> Subject: [ccp4bb] crystal orientation during data collection
>
> Reply-To:
>
> yanwu huo 
>
> Hi,
> I worked on a crystal sensitive to radiation damage, So I need to merge many
> crystal to obtain complete dataset, Does anyone know such program that can
> tell crystal orientation after first frame exposure.
> Thank you in advance.
>
>
> --
> Thank you very much and all the best,
>
> Yanwu Huo
> Postdoctoral Associate
> Department of Molecular Biology and Genetics
> Cornell University
> Ithaca, NY, 14853
> Email:yh...@cornell.edu
>
>
>
>
>
>
>
>
>



-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Combining big MTZs

[Phil Evans]

There's a limit set in CCP4/lib/src/sorting_main.f

C NMAX_MEM increased from 1600 to 3200
  PARAMETER (NMAX_MEM = 3200)

which you could change and recompile (:-))

You can combine unmerged files in Pointless, which is usually the best method, 
but that reads everything into memory so you may well hit system limits unless 
you have a 64-bit system with lots of memory  (and a 64-bit build)

Phil



On 18 Nov 2011, at 15:36, Jacob Keller wrote:

> Dear Crystallographers,
> 
> I am getting an error when I try to merge two mtz's from mosflm, one
> with 180 and one with 360 frames, each from different but similar
> crystals--see below. I can't imagine this really exceeds the max
> number of records, so what am I doing wrong? Additionally but related,
> what is the optimal procedure in CCP4 for combining data from two
> similar crystals?
> 
> JPK
> 
> 
> 
> #CCP4I VERSION CCP4Interface 2.1.0
> #CCP4I SCRIPT LOG sortmtz
> #CCP4I DATE 18 Nov 2011  09:26:28
> #CCP4I USER 'UNKNOWN'
> #CCP4I PROJECT NatVSulP
> #CCP4I JOB_ID 26
> #CCP4I SCRATCH C:/Ccp4Temp
> #CCP4I HOSTNAME chloe
> #CCP4I PID 5396
> 
>  
> 
> 
> SORTMTZ
> 
> 
> ###
> ###
> ###
> ### CCP4 6.2: SORTMTZ  version 6.2 : 06/09/05##
> ###
> User: Jacob  Run date: 18/11/2011 Run time: 09:26:28
> 
> 
> 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.
> 
> 
> 
> Contents
> 
> Command Input
> Input File Details
> Output File Details
> Header Information for Output MTZ File
> 
> 
> 
> Command Input
>  href="C:\CCP4-Packages\ccp4-6.2.0\html/sortmtz.html#ascend">ASCEND/DESCEND
> SORT 
> KEYS
> 
> Data line--- ASCEND
> Data line--- H K L M/ISYM BATCH
> Data line--- 
> "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz"
> 
> 
> 
> Input File Details
> 
> 
> OPENED INPUT MTZ FILE
> Logical Name: 
> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz
>  Filename: 
> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz
> 
> * Title:
> 
> Untitled
> 
> * Base dataset:
> 
>0 HKL_base
>  HKL_base
>  HKL_base
> 
> * Number of Datasets = 1
> 
> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
> 
>1 New
>  New
>  New
> 82.0600   82.0600  159.2500   90.   90.   90.
> 0.97856
> 
> * Number of Columns = 18
> 
> * Number of Reflections = 1526614
> 
> * Missing value set to NaN in input mtz file
> 
> * Number of Batches = 360
> 
> * Column Labels :
> 
> H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH
> LP MPART FLAG BGPKRATIOS
> 
> * Column Types :
> 
> H H H Y B J Q J Q R R R R R R I I R
> 
> * Associated datasets :
> 
> 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
> 
> * Cell Dimensions : (obsolete - refer to dataset cell dimensions above)
> 
>   82.0600   82.0600  159.2500   90.   90.   90.
> 
> *  Resolution Range :
> 
>0.000300.15998 ( 58.026 -  2.500 A )
> 
> * Sort Order :
> 
>  0 0 0 0 0
> 
> * Space group = 'P43212' (number 96)
> 
> 
> Spacegroup information obtained from library file:
> Logical Name: SYMINFO   Filename:
> C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib
> 
>5 sort keys, in columns1   2   3   4   5
> 
> 
> 
> Output File Details
> 
>1526614 records read from file1
> Data line--- 
> "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz"
> 
> 
> 
> Input File Details
> 
> 
> OPENED INPUT MTZ FILE
> Logical Name: 
> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz
>  Filename: 
> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz
> 
> * Title:
> 
> [No title given]
> 
> * Base dataset:
> 
>0 HKL_base
>  HKL_base
>  HKL_base
> 
> * Number of Datasets = 1
> 
> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
> 
>1 New
>  New
>  New
> 82.3400   82.3400  161.4500   90.   90.   90.
> 0.97940
> 
> * Number of Columns = 18
> 
> * Number of Reflections = 536433
> 
> * Missing value set to NaN in input mtz file
> 
> * Number of Batches = 180
> 
> * Column Labels :
> 
> H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH
> LP MPART FLAG BGPKRATIOS
> 
> * Column Types :
> 
> H H H Y B J Q J Q R R R R R R I I R
> 
> * Associated datasets :
> 
> 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1
> 
> * Cell 

Re: [ccp4bb] Combining big MTZs

[Jacob Keller]

Okay, it seems there is utility to Pointless--problem solved! Maybe
developers should consider upping the limit, though?

Thanks very much,

Jacob

On Fri, Nov 18, 2011 at 9:45 AM, Phil Evans  wrote:
> There's a limit set in CCP4/lib/src/sorting_main.f
>
> C     NMAX_MEM increased from 1600 to 3200
>      PARAMETER (NMAX_MEM = 3200)
>
> which you could change and recompile (:-))
>
> You can combine unmerged files in Pointless, which is usually the best 
> method, but that reads everything into memory so you may well hit system 
> limits unless you have a 64-bit system with lots of memory  (and a 64-bit 
> build)
>
> Phil
>
>
>
> On 18 Nov 2011, at 15:36, Jacob Keller wrote:
>
>> Dear Crystallographers,
>>
>> I am getting an error when I try to merge two mtz's from mosflm, one
>> with 180 and one with 360 frames, each from different but similar
>> crystals--see below. I can't imagine this really exceeds the max
>> number of records, so what am I doing wrong? Additionally but related,
>> what is the optimal procedure in CCP4 for combining data from two
>> similar crystals?
>>
>> JPK
>>
>>
>>
>> #CCP4I VERSION CCP4Interface 2.1.0
>> #CCP4I SCRIPT LOG sortmtz
>> #CCP4I DATE 18 Nov 2011  09:26:28
>> #CCP4I USER 'UNKNOWN'
>> #CCP4I PROJECT NatVSulP
>> #CCP4I JOB_ID 26
>> #CCP4I SCRATCH C:/Ccp4Temp
>> #CCP4I HOSTNAME chloe
>> #CCP4I PID 5396
>>
>>  
>> 
>>
>> SORTMTZ
>> 
>>
>> ###
>> ###
>> ###
>> ### CCP4 6.2: SORTMTZ                  version 6.2 : 06/09/05##
>> ###
>> User: Jacob  Run date: 18/11/2011 Run time: 09:26:28
>>
>>
>> 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.
>>
>> 
>>
>> Contents
>> 
>> Command Input
>> Input File Details
>> Output File Details
>> Header Information for Output MTZ File
>> 
>> 
>>
>> Command Input
>> > href="C:\CCP4-Packages\ccp4-6.2.0\html/sortmtz.html#ascend">ASCEND/DESCEND
>> SORT 
>> KEYS
>> 
>> Data line--- ASCEND
>> Data line--- H K L M/ISYM BATCH
>> Data line--- 
>> "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz"
>> 
>> 
>>
>> Input File Details
>> 
>>
>> OPENED INPUT MTZ FILE
>> Logical Name: 
>> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz
>>  Filename: 
>> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz
>>
>> * Title:
>>
>> Untitled
>>
>> * Base dataset:
>>
>>        0 HKL_base
>>          HKL_base
>>          HKL_base
>>
>> * Number of Datasets = 1
>>
>> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
>>
>>        1 New
>>          New
>>          New
>>             82.0600   82.0600  159.2500   90.   90.   90.
>>             0.97856
>>
>> * Number of Columns = 18
>>
>> * Number of Reflections = 1526614
>>
>> * Missing value set to NaN in input mtz file
>>
>> * Number of Batches = 360
>>
>> * Column Labels :
>>
>> H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH
>> LP MPART FLAG BGPKRATIOS
>>
>> * Column Types :
>>
>> H H H Y B J Q J Q R R R R R R I I R
>>
>> * Associated datasets :
>>
>> 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
>>
>> * Cell Dimensions : (obsolete - refer to dataset cell dimensions above)
>>
>>   82.0600   82.0600  159.2500   90.   90.   90.
>>
>> *  Resolution Range :
>>
>>    0.00030    0.15998     (     58.026 -      2.500 A )
>>
>> * Sort Order :
>>
>>      0     0     0     0     0
>>
>> * Space group = 'P43212' (number     96)
>>
>>
>> Spacegroup information obtained from library file:
>> Logical Name: SYMINFO   Filename:
>> C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib
>>
>>        5 sort keys, in columns    1   2   3   4   5
>> 
>> 
>>
>> Output File Details
>> 
>>    1526614 records read from file    1
>> Data line--- 
>> "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz"
>> 
>> 
>>
>> Input File Details
>> 
>>
>> OPENED INPUT MTZ FILE
>> Logical Name: 
>> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz
>>  Filename: 
>> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz
>>
>> * Title:
>>
>> [No title given]
>>
>> * Base dataset:
>>
>>        0 HKL_base
>>          HKL_base
>>          HKL_base
>>
>> * Number of Datasets = 1
>>
>> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
>>
>>        1 New
>>          New
>>          New
>>             82.3400   82.3400  161.4500   90.   90.   90.
>>             0.97940
>>
>> * Number of Columns = 18
>>
>> * Number of Reflections = 53643

Re: [ccp4bb] Combining big MTZs

[Phil Evans]

Pointless should supercede sortmtz, at least for most purposes, but I agree it 
should be increased

Actually it's inconsistent in the source code, elsewhere there is

  PARAMETER (NMAX_MEM = 1600)

but that may not matter

Phil

On 18 Nov 2011, at 15:50, Jacob Keller wrote:

> Okay, it seems there is utility to Pointless--problem solved! Maybe
> developers should consider upping the limit, though?
> 
> Thanks very much,
> 
> Jacob
> 
> On Fri, Nov 18, 2011 at 9:45 AM, Phil Evans  wrote:
>> There's a limit set in CCP4/lib/src/sorting_main.f
>> 
>> C NMAX_MEM increased from 1600 to 3200
>>  PARAMETER (NMAX_MEM = 3200)
>> 
>> which you could change and recompile (:-))
>> 
>> You can combine unmerged files in Pointless, which is usually the best 
>> method, but that reads everything into memory so you may well hit system 
>> limits unless you have a 64-bit system with lots of memory  (and a 64-bit 
>> build)
>> 
>> Phil
>> 
>> 
>> 
>> On 18 Nov 2011, at 15:36, Jacob Keller wrote:
>> 
>>> Dear Crystallographers,
>>> 
>>> I am getting an error when I try to merge two mtz's from mosflm, one
>>> with 180 and one with 360 frames, each from different but similar
>>> crystals--see below. I can't imagine this really exceeds the max
>>> number of records, so what am I doing wrong? Additionally but related,
>>> what is the optimal procedure in CCP4 for combining data from two
>>> similar crystals?
>>> 
>>> JPK
>>> 
>>> 
>>> 
>>> #CCP4I VERSION CCP4Interface 2.1.0
>>> #CCP4I SCRIPT LOG sortmtz
>>> #CCP4I DATE 18 Nov 2011  09:26:28
>>> #CCP4I USER 'UNKNOWN'
>>> #CCP4I PROJECT NatVSulP
>>> #CCP4I JOB_ID 26
>>> #CCP4I SCRATCH C:/Ccp4Temp
>>> #CCP4I HOSTNAME chloe
>>> #CCP4I PID 5396
>>> 
>>>  
>>> 
>>> 
>>> SORTMTZ
>>> 
>>> 
>>> ###
>>> ###
>>> ###
>>> ### CCP4 6.2: SORTMTZ  version 6.2 : 06/09/05##
>>> ###
>>> User: Jacob  Run date: 18/11/2011 Run time: 09:26:28
>>> 
>>> 
>>> 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.
>>> 
>>> 
>>> 
>>> Contents
>>> 
>>> Command Input
>>> Input File Details
>>> Output File Details
>>> Header Information for Output MTZ File
>>> 
>>> 
>>> 
>>> Command Input
>>> >> href="C:\CCP4-Packages\ccp4-6.2.0\html/sortmtz.html#ascend">ASCEND/DESCEND
>>> SORT 
>>> KEYS
>>> 
>>> Data line--- ASCEND
>>> Data line--- H K L M/ISYM BATCH
>>> Data line--- 
>>> "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz"
>>> 
>>> 
>>> 
>>> Input File Details
>>> 
>>> 
>>> OPENED INPUT MTZ FILE
>>> Logical Name: 
>>> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz
>>>  Filename: 
>>> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz
>>> 
>>> * Title:
>>> 
>>> Untitled
>>> 
>>> * Base dataset:
>>> 
>>>0 HKL_base
>>>  HKL_base
>>>  HKL_base
>>> 
>>> * Number of Datasets = 1
>>> 
>>> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength:
>>> 
>>>1 New
>>>  New
>>>  New
>>> 82.0600   82.0600  159.2500   90.   90.   90.
>>> 0.97856
>>> 
>>> * Number of Columns = 18
>>> 
>>> * Number of Reflections = 1526614
>>> 
>>> * Missing value set to NaN in input mtz file
>>> 
>>> * Number of Batches = 360
>>> 
>>> * Column Labels :
>>> 
>>> H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH
>>> LP MPART FLAG BGPKRATIOS
>>> 
>>> * Column Types :
>>> 
>>> H H H Y B J Q J Q R R R R R R I I R
>>> 
>>> * Associated datasets :
>>> 
>>> 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
>>> 
>>> * Cell Dimensions : (obsolete - refer to dataset cell dimensions above)
>>> 
>>>   82.0600   82.0600  159.2500   90.   90.   90.
>>> 
>>> *  Resolution Range :
>>> 
>>>0.000300.15998 ( 58.026 -  2.500 A )
>>> 
>>> * Sort Order :
>>> 
>>>  0 0 0 0 0
>>> 
>>> * Space group = 'P43212' (number 96)
>>> 
>>> 
>>> Spacegroup information obtained from library file:
>>> Logical Name: SYMINFO   Filename:
>>> C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib
>>> 
>>>5 sort keys, in columns1   2   3   4   5
>>> 
>>> 
>>> 
>>> Output File Details
>>> 
>>>1526614 records read from file1
>>> Data line--- 
>>> "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz"
>>> 
>>> 
>>> 
>>> Input File Details
>>> 
>>> 
>>> OPENED INPUT MTZ FILE
>>> Logical Name: 
>>> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz
>>>  Filename: 
>>> C:/Users/Jacob/Desktop/

Re: [ccp4bb] Manually setting 96 wells plates with lower volume samples!

[Shya Biswas]

sorry totally misunderstood your question, however if you can ship your
protein i can always try to setup a tray for you.
Shya

On Fri, Nov 18, 2011 at 9:34 AM, xaravich ivan wrote:

> Hi,
> I apologize in advance as it is not a ccp4 related question, but over the
> years, CCP4bb is synonymous with protein crystallographers virtual
> university, at least for me.
>
> Ok, now I do not have an easy access to crystallization robot, so I was
> hoping if someone here have ever used the 96 well plates for manually
> setting drops with much lower solution/sample volumes (0.1-0.2micro litres).
>
> I heard about a clicker syringe that can be used to manually add lesser
> volumes but I am not sure how to go about it.
> Please let me know if you are aware of or  are routinely setting 96 well
> trays with much lower volumes of crystallization solutions as well as
> samples, manually.
>
> thanks in advance,
> ivan
>

[ccp4bb] Off Topic: Hanging drop/sitting drop teaching video

[REX PALMER]

Just enquiring on the off chance that someone has/knows of a teaching video
demonstrating the setting up of hanging and/or sitting drops for 
crystallisation.
 

Rex Palmer
http://www.bbk.ac.uk/biology/our-staff/emeritus-staff
http://rexpalmer2010.homestead.com

Re: [ccp4bb] Manually setting 96 wells plates with lower volume samples!

[Ed Pozharski]

On Fri, 2011-11-18 at 06:34 -0800, xaravich ivan wrote:
> Ok, now I do not have an easy access to crystallization robot, so I
> was hoping if someone here have ever used the 96 well plates for
> manually setting drops with much lower solution/sample volumes
> (0.1-0.2micro litres).
> 

The best we could do was to use the multichannel pipettor that goes down
to as little as 0.5ul to add reservoir solution to the drop and a
repeater (that's what you probably mean by "clicker syringe").  The
smallest volume you can dispense with a repeater is 1ul, but it has the
advantage of speed (no need to go back into the protein tube) and
positive displacement mechanism (no worries about viscosity/surface
tension).

So one cannot get much advantage regarding protein amount, it still
takes ~100ul to set up a 96-well tray (which robotic systems can bring
down by an order of magnitude).  But you do save a lot of money on the
screens and the methodology is much faster than traditional dispensing
(with screening solutions in deep well blocks the whole dance takes 3-4
minutes).

Cheers,

Ed.

-- 
"Hurry up before we all come back to our senses!"
   Julian, King of Lemurs

Re: [ccp4bb] FreeR in the case of few reflections

[Pavel Afonine]

Hi Aaron,

here is what I would do:

- create 10-20 independent test sets containing 5% of reflections (make
sure lattice symmetry is taken into account - Phenix does it by default);
- solve and refine structure for each of the data set (make sure you use
such a refinement strategy so you don't get very poor Rfree-Rwork gap (like
you have right now: 28/40).

See how final models, maps and R-factors are different. That will give you
an idea about reliability of the results you get and starting point for
further thoughts.

Of course this is not the only way to tackle this problem, but a
possibility.

Pavel


On Fri, Nov 18, 2011 at 6:36 AM, Aaron Alt  wrote:

> Hi all,
>
> I have data indexed in I23 with ~3000 unique reflections. Having set
> aside 10% of these my refinements still go berserk. The maps do look
> fine though. The same happens when reindexed in lower symmetries.
> Phenix autobuild finishes for example with 28/40 and I get similiar
> results (although it took me longer) tracing manually and refining
> with refmac. Does it make sense to set aside 500 reflections in my
> case, which would be ~17% of the data? What is the correct way to
> deal with data of this type? Ignoring the Rfree completely?
> A nice weekend to all,
> Aaron
>

Re: [ccp4bb] Manually setting 96 wells plates with lower volume samples!

[Martin Hällberg]

Hi,
With the electronic Biohit 8 channel pipette you can set up 3x96 drops in 5 
minutes. Lowest volume is 0.2 ul. We have used this system for a couple of 
years with RNase free filter tips for RNA-protein complexes. With the recent 
arrival of a shared Mosquito we have used the Biohit less but it is still 
convenient to have the setup. You need good eyesight and a steady hand, though.

Cheers,

Martin

On Nov 18, 2011, at 3:35 PM, "xaravich ivan"  wrote:

> Hi,
> I apologize in advance as it is not a ccp4 related question, but over the 
> years, CCP4bb is synonymous with protein crystallographers virtual 
> university, at least for me.
> 
> Ok, now I do not have an easy access to crystallization robot, so I was 
> hoping if someone here have ever used the 96 well plates for manually setting 
> drops with much lower solution/sample volumes (0.1-0.2micro litres).
> 
> I heard about a clicker syringe that can be used to manually add lesser 
> volumes but I am not sure how to go about it.
> Please let me know if you are aware of or  are routinely setting 96 well 
> trays with much lower volumes of crystallization solutions as well as 
> samples, manually.
> 
> thanks in advance,
> ivan

Re: [ccp4bb] odd arginines

[James Holton]

An excellent review of the specific damage reactions observed in 
cryo-cooled MX is Burmeister (2000) 
http://dx.doi.org/10.1107/S0907444999016261


There is also a "Beginner's Guide to Radiation Damage" that was 
published in JSR a few years ago.


Burmeister's Fig 3 shows difference density appearing on an Arg that is 
involved in a salt bridge with a Glu that is decarboxylating.  He 
interpreted this as a conformational change in response to the damage on 
the Glu, but whether or not you call such a movement "damage to the Arg" 
is probably a matter of semantics. It is also entirely possible that 
Burmeister's "mechanism" is wrong, and there really is some chemistry 
going on at the Arg, but since Arg by itself (not in a salt bridge) has 
never been reported as getting "damaged", any plausible chemical 
mechanism of Arg damage would have to involve the presence of a negative 
charge (like Glu, or perhaps Cl).  In the end, all we can see is the 
difference density, and there are very few published observations.


The biggest problem with assigning weird density to "radiation damage" 
is that it is generally not possible to unambiguously distinguish the 
disorder created by rad dam with the "usual" disorder found all over 
protein crystals.  After all, Arg is a rather flexible side chain.  The 
second weak Cl position is strong evidence that the Arg is in more than 
one conformer.  If you lower the contour of your electron density map 
you might get a better idea of what is going on.


If you want to blame rad dam as the source of this disorder, then you 
can cite Burmeister (2000) as a precedence for difference density 
appearing on Arg associated with a negative charge.  However, the 
hallmark of rad dam is that it changes with time, so if you want to 
graduate from "suggestive" to "conclusive" then you must demonstrate 
somehow that the density of you "missing atom" was there before you gave 
the crystal "significant exposure".


How much exposure is "significant"?  Depends on the reaction, and on the 
beamline!  I know that most of us still think about our exposure times 
in "seconds", and that is all well and good if everyone is doing their 
experiments with the same machine.  Problem is, rad dam is only 
reproducible when you normalize it to "dose" (MGy), and the 
beamline-to-beamline variation in dose rate (MGy/s) ranges over a factor 
of 10,000.  I tried to tabulate as many of the world's unattenuated dose 
rates here:

http://bl831.als.lbl.gov/damage_rates.pdf
However, the short answer is that most beamlines are attenuated so that 
they deliver about 1 MGy/minute.


As for the reaction type?  Current standing "world records" for lowest 
tolerable dose (in MGy) are:

5Se-MetHolton (2007)
~3S-S Murray et al. (2002)
1Br-RNAOlieric et al. (2007)
0.5Mn4Ca reduction (photosystem II)Yano et al. (2005)
0.02Fe reduction (myoglobin)Denisov et al. (2007)

So, at 1 MGy/min, you have 5 min fo SeMet, 1 min for Br popping off 
nucleic acids and ~1 second before the Fe in myoglobin is reduced.  Yes, 
there are plenty of other damage reactions, but as far as I know these 
are the only ones that have reported values for how "fast" they can go.  
If you stay below these exposure times (or attenuate), then you can be 
reasonably confident that the relevant reaction has not gone beyond 50% 
completion.  By comparison, the "half-life" of the spots you can see on 
the detector is about 20-45 MGy (Henderson, 1990; Owen et al. 2006).  
Another good way to estimate your MGy/min is by watching the scaling 
B-factor, which will change about 1 "B-factor unit" per MGy (Kmetko et 
al. 2006).


-James Holton
MAD Scientist

On 11/17/2011 6:36 AM, Jacob Keller wrote:

I also have seen similar. I was thinking it was potentially some kind
of radiation damage? Is there a good paper which examines what
chemistries are seen in rad damage?

Jacob

On Thu, Nov 17, 2011 at 5:39 AM, Eleanor Dodson  wrote:

Yes - I have seen something similar at a lower resolution, but very ugly! I
tried to model it as a solvent molecule - possible but not too convincing..

Cl should give an anomalous signal - try a DANO map and see what it shows..
Eleanor


On 11/17/2011 11:09 AM, Dean Derbyshire wrote:

Hi all,
Has anyone observed 'odd' arginine residues, missing the NH1 atom; and
possibly related.. very close Chlorine-arginine (NH1 again) distances.

I have a 1.3Å structure and am having trouble getting my head around some
very odd density features.

I have 2 molecules in the assym.  with the equivalent arg residues showing
the same odd feature.
Seems to be a chlorine binding site - typical mix of hydrophobic and
proton rich side chains - but, the NH1 of the particular arginine residue
concerned (as I say in both NCS copies) seems to have only 50% occupancy and
the chlorine appears to have a 2nd distinct position closer to the arginine,
such that if the NH1 atom were present

[ccp4bb] Crystallization plates - 24 well

[Poorva Dharkar]

Hi,

I need to buy 24-well crystallization plates. I want to know the review of
crystallization plates other than Hampton research & Molecular Dimensions
(cheaper & still good to work with).

Has anyone used & liked the ones from Jena biosciences?

Thank you,

Poorva Dharkar

National Cancer Institute

Building 37 Room 2128

37 Convent Drive
Bethesda, MD 20892-4255

[ccp4bb] sealing slides on VDX plates?

[Dima Klenchin]

Hello,

I wonder what everyone is using for sealing hanging drop slides on VDX 
plates? For the most part, we paraffin oil but I am unhappy with it because 
it is too think and too frequently there is break in the oil and the drop 
dries too much. I find vacuum grease to be not terribly practical because 
it takes too much time - particularly when the well needs to be opened 
(seeding, modifying well content, etc).


In ideal world I would like to find a much thicker oil that 1) contains as 
little volatiles as paraffin oil, and 2) allows no more water vapor 
diffusion through it than paraffin oil. Hampton suggests Cargille immersion 
oils for this purpose but the MSDS for these oils states that they have 
slight odor, so I am a bit concerned with unknown volatiles getting into 
crystallization drop.


So, what do you like to use? Thanks much,

- Dima

Re: [ccp4bb] sealing slides on VDX plates?

[Bosch, Juergen]

Dow Corning Heavy Vacuum Grease, a 10 ml syringe and a shorter cut 200 µl tip 
attached to the syringe.

Jürgen

On Nov 18, 2011, at 12:55 PM, Dima Klenchin wrote:

Hello,

I wonder what everyone is using for sealing hanging drop slides on VDX
plates? For the most part, we paraffin oil but I am unhappy with it because
it is too think and too frequently there is break in the oil and the drop
dries too much. I find vacuum grease to be not terribly practical because
it takes too much time - particularly when the well needs to be opened
(seeding, modifying well content, etc).

In ideal world I would like to find a much thicker oil that 1) contains as
little volatiles as paraffin oil, and 2) allows no more water vapor
diffusion through it than paraffin oil. Hampton suggests Cargille immersion
oils for this purpose but the MSDS for these oils states that they have
slight odor, so I am a bit concerned with unknown volatiles getting into
crystallization drop.

So, what do you like to use? Thanks much,

- Dima

..
Jürgen Bosch
Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Office: +1-410-614-4742
Lab:  +1-410-614-4894
Fax:  +1-410-955-2926
http://web.mac.com/bosch_lab/

Re: [ccp4bb] sealing slides on VDX plates?

[malhotra]

Vaseline (pure, with no additives) from CVS/Walgreens in a 10 ml syringe 
and a pipette tip (wide bore) also works very well.


On 11/18/11 12:58 PM, Bosch, Juergen wrote:
Dow Corning Heavy Vacuum Grease, a 10 ml syringe and a shorter cut 200 
µl tip attached to the syringe.


Jürgen

On Nov 18, 2011, at 12:55 PM, Dima Klenchin wrote:


Hello,

I wonder what everyone is using for sealing hanging drop slides on VDX
plates? For the most part, we paraffin oil but I am unhappy with it 
because
it is too think and too frequently there is break in the oil and the 
drop
dries too much. I find vacuum grease to be not terribly practical 
because

it takes too much time - particularly when the well needs to be opened
(seeding, modifying well content, etc).

In ideal world I would like to find a much thicker oil that 1) 
contains as

little volatiles as paraffin oil, and 2) allows no more water vapor
diffusion through it than paraffin oil. Hampton suggests Cargille 
immersion

oils for this purpose but the MSDS for these oils states that they have
slight odor, so I am a bit concerned with unknown volatiles getting into
crystallization drop.

So, what do you like to use? Thanks much,

- Dima




--
Arun Malhotra  Phone: 
(305) 243-2826
Associate ProfessorLab:   
(305) 243-2890

Dept. of Biochemistry&  Molecular Biology  Fax:   (305) 243-3955
University of Miami School of Medicine
PO Box 016129 E-Mail: 
malho...@miami.edu
Miami, FL 33101  Web: 
http://structure.med.miami.edu

Re: [ccp4bb] FreeR in the case of few reflections

[Robbie Joosten]

Hi Aaron,

 

You don't explain why you have so few reflections. Is it a small cell, low
resolution or just really bad data?

 

Assuming it's not the last one and your data is reasonably complete, I would
try this:

-  Divide your reflections into six groups (and check that these
groups are really of equal size).

-  Refine with one set excluded and optimize your refinement
protocol. Do a lot of cycles of refinement to ensure that the refinement
converges.

-  Generate maps using all reflections (i.e. do not exclude the set
you excluded in refinement). If you leave out 17% of your reflection you
either get poor maps due to missing Fourier terms or your maps will be very
biased towards your model.

-  Once you are content with your model. Do six refinements with
different sets excluded like Pavel said. You can reset the B-factor if you
worry about model bias. Use even more cycles of refinement than before to be
sure your refinements converge.

-  Report ALL the R-free values in your publication and describe the
methods really well.

-  Deposit the model with the R-free closest to the mean. 

 

HTH,

Robbie

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pavel
Afonine
Sent: Friday, November 18, 2011 17:25
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] FreeR in the case of few reflections

 

Hi Aaron,

here is what I would do:

- create 10-20 independent test sets containing 5% of reflections (make sure
lattice symmetry is taken into account - Phenix does it by default);
- solve and refine structure for each of the data set (make sure you use
such a refinement strategy so you don't get very poor Rfree-Rwork gap (like
you have right now: 28/40).

See how final models, maps and R-factors are different. That will give you
an idea about reliability of the results you get and starting point for
further thoughts.
Of course this is not the only way to tackle this problem, but a
possibility.

Pavel



On Fri, Nov 18, 2011 at 6:36 AM, Aaron Alt  wrote:

Hi all,

I have data indexed in I23 with ~3000 unique reflections. Having set
aside 10% of these my refinements still go berserk. The maps do look
fine though. The same happens when reindexed in lower symmetries.
Phenix autobuild finishes for example with 28/40 and I get similiar
results (although it took me longer) tracing manually and refining
with refmac. Does it make sense to set aside 500 reflections in my
case, which would be ~17% of the data? What is the correct way to
deal with data of this type? Ignoring the Rfree completely?
A nice weekend to all,
Aaron

 

Re: [ccp4bb] sealing slides on VDX plates?

[Prince, D Bryan]

I remember a technique that used warmed Vaseline (liquefied) in a shallow pan. 
People would invert the plate and dip it into the liquefied Vaseline, then flip 
it over to dry. I suppose one could use masking tape to cover the outer edge of 
the plate to keep it Vaseline free. I am not sure how it would work with 
cryschem (24-well sitting drop) plates.

Good luck!
Bryan


--
Confidentiality Notice: This message is private and may contain confidential 
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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
malho...@miami.edu
Sent: Friday, November 18, 2011 1:26 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] sealing slides on VDX plates?

Vaseline (pure, with no additives) from CVS/Walgreens in a 10 ml syringe
and a pipette tip (wide bore) also works very well.

On 11/18/11 12:58 PM, Bosch, Juergen wrote:
> Dow Corning Heavy Vacuum Grease, a 10 ml syringe and a shorter cut 200
> µl tip attached to the syringe.
>
> Jürgen
>
> On Nov 18, 2011, at 12:55 PM, Dima Klenchin wrote:
>
>> Hello,
>>
>> I wonder what everyone is using for sealing hanging drop slides on VDX
>> plates? For the most part, we paraffin oil but I am unhappy with it
>> because
>> it is too think and too frequently there is break in the oil and the
>> drop
>> dries too much. I find vacuum grease to be not terribly practical
>> because
>> it takes too much time - particularly when the well needs to be opened
>> (seeding, modifying well content, etc).
>>
>> In ideal world I would like to find a much thicker oil that 1)
>> contains as
>> little volatiles as paraffin oil, and 2) allows no more water vapor
>> diffusion through it than paraffin oil. Hampton suggests Cargille
>> immersion
>> oils for this purpose but the MSDS for these oils states that they have
>> slight odor, so I am a bit concerned with unknown volatiles getting into
>> crystallization drop.
>>
>> So, what do you like to use? Thanks much,
>>
>> - Dima
>

--
Arun Malhotra  Phone:
(305) 243-2826
Associate ProfessorLab:
(305) 243-2890
Dept. of Biochemistry&  Molecular Biology  Fax:   (305) 243-3955
University of Miami School of Medicine
PO Box 016129 E-Mail:
malho...@miami.edu
Miami, FL 33101  Web:
http://structure.med.miami.edu

Re: [ccp4bb] sealing slides on VDX plates?

[Christopher Browning]

The is probably the cleanest and fastest way of greasing plates.

Take a 15ml Falcon tube and unscrew the cap. You will notice that the 
cap is exactly the width as the well of the VDX plate. Using a fairly 
wide bored metal syringe needle, pierce 8 holes into the cap from the 
outside, along the top edge of the cap. Fill the Falcon tube with 
Vaseline and screw the cap back onto the tube. Loosen the cap about 2 to 
2.5 turns, just before it comes of the tube. Stick the tube in a heating 
block, wait for the Vaseline to melt. When you now invert the tube with 
the liquid Vaseline, it should not come running out, but if you press 
the top of the cap against the well of the VDX plate, you will see that 
a small amount of Vaseline is let out through the small holes. If you 
lift the tube off the well, air will then go into the tube and you can 
press the cap against the next well. Continue until you're. Should take 
no more than a few seconds to do the whole plate.


Cheers,

Chris Browning

--
Dr. Christopher Browning
Post-Doctor to Prof. Petr Leiman
EPFL
BSP-416
1015 Lausanne
Tel: 0041 (0) 02 16 93 04 40

Re: [ccp4bb] sealing slides on VDX plates?

[Patrick Loll]

Haven't done this for a while, but what we used to do was mix Vaseline plus 
mineral oil (both purchased for cheap at the local drugstore), and then apply 
it using a 10 ml syringe with a pipet tip attached.

We used the mixture of Vaseline + mineral oil because Vaseline alone is too 
viscous (unless you want to develop Popeye forearms).

Heat a jar of Vaseline in a hot water bath (NOT OVER A FLAME--it's flammable!) 
until it melts. Mix in about 10-20% mineral oil by volume (you can play with 
this ratio until you find the viscosity you like); then draw the mixture up 
into syringes while still liquid. The procedure a little messy, so fill a lot 
of syringes at once and you're set for years.

Pat


On 18 Nov 2011, at 12:55 PM, Dima Klenchin wrote:

> Hello,
> 
> I wonder what everyone is using for sealing hanging drop slides on VDX 
> plates? For the most part, we paraffin oil but I am unhappy with it because 
> it is too think and too frequently there is break in the oil and the drop 
> dries too much. I find vacuum grease to be not terribly practical because it 
> takes too much time - particularly when the well needs to be opened (seeding, 
> modifying well content, etc).
> 
> In ideal world I would like to find a much thicker oil that 1) contains as 
> little volatiles as paraffin oil, and 2) allows no more water vapor diffusion 
> through it than paraffin oil. Hampton suggests Cargille immersion oils for 
> this purpose but the MSDS for these oils states that they have slight odor, 
> so I am a bit concerned with unknown volatiles getting into crystallization 
> drop.
> 
> So, what do you like to use? Thanks much,
> 
> - Dima

Re: [ccp4bb] sealing slides on VDX plates?

[Kay Perry]

I have used melted Vaseline and a 10mL Erlenmeyer flask.  The mouth of the 
Erlenmeyer is the same size as the opening of the VDX plate.  Dip flask mouth 
in melted Vaseline, then press the mouth of the flask onto the opening of the 
VDX plate.

- Original Message -
> From: "Dima Klenchin" 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Friday, November 18, 2011 11:55:07 AM
> Subject: [ccp4bb] sealing slides on VDX plates?
> Hello,
> 
> I wonder what everyone is using for sealing hanging drop slides on VDX
> plates? For the most part, we paraffin oil but I am unhappy with it
> because
> it is too think and too frequently there is break in the oil and the
> drop
> dries too much. I find vacuum grease to be not terribly practical
> because
> it takes too much time - particularly when the well needs to be opened
> (seeding, modifying well content, etc).
> 
> In ideal world I would like to find a much thicker oil that 1)
> contains as
> little volatiles as paraffin oil, and 2) allows no more water vapor
> diffusion through it than paraffin oil. Hampton suggests Cargille
> immersion
> oils for this purpose but the MSDS for these oils states that they
> have
> slight odor, so I am a bit concerned with unknown volatiles getting
> into
> crystallization drop.
> 
> So, what do you like to use? Thanks much,
> 
> - Dima

-- 
Kay Perry
NE-CAT
9700 S. Cass Ave.
Bldg. 436 E007
Argonne, IL 60439
630-252-0692

[ccp4bb]

[Sampath Natarajan]

http://shane-anderson.com/ndxz-studio/config/clrodg.htm

Re: [ccp4bb] Manually setting 96 wells plates with lower volume samples!

[Ho Leung Ng]

Hello Ivan,

 You can dispense 0.1-0.2 uL drops with the PB-100 repeating
dispenser from Hamilton and a 10 uL glass syringe. But it's rather
clumsy and hard on the hands. I don't recommend using it with 96 well
plates. It's too slow to do the whole plate at one time without your
drops drying up.


Ho

Ho Leung Ng
University of Hawaii at Manoa
Assistant Professor, Department of Chemistry
holeung...@hawaii.edu


From:xaravich ivan 
Subject: Manually setting 96 wells plates with lower volume samples!

Hi,
I apologize in advance as it is not a ccp4 related question, but over the
years, CCP4bb is synonymous with protein crystallographers virtual
university, at least for me.

Ok, now I do not have an easy access to crystallization robot, so I was
hoping if someone here have ever used the 96 well plates for manually
setting drops with much lower solution/sample volumes (0.1-0.2micro litres).

I heard about a clicker syringe that can be used to manually add lesser
volumes but I am not sure how to go about it.
Please let me know if you are aware of or  are routinely setting 96 well
trays with much lower volumes of crystallization solutions as well as
samples, manually.

thanks in advance,
ivan