Re: [ccp4bb] small crystals

[CHAVAS Leonard]

Dear Jens, Careina

this is not really an *advantage*, but rather a *convenience*. You can still 
use big crystals if you'd like to, but as they usually never survive more than 
one shot (few femtosec), you'd need a lot of these bigger crystals to collect a 
full data.

And yes, I would also highly recommend XFELs!

Cheers, Leo

On Dec 10, 2013, at 4:36 AM, Jens Kaiser  wrote:

> Careina,
>  If your target is interesting enough, try to reproduce the small
> crystals in batch and apply for FELS time. Small crystals are actually
> an advantage there.
> 
> Cheers,
> 
> Jens
> 
> 
> On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote:
>> Hi all
>> 
>> 
>> Any advice on how to get bigger crystals from conditions that give
>> showers of tiny crystals? I am getting small pretty looking individual
>> crystals but they are too small and they don't seem to grow. In fact,
>> in some instances if left for a couple of days they actually dissolve.
>> I have fiddled around with mother liquor volume, protein concentration
>> as well as drop volume (I am using hanging drop method) but none seem
>> to make any difference and I always get the same tiny crystals. I
>> think I might try microseeding but I haven't tried that yet. 
>> 
>> 
>> Any suggestions or tricks would be welcome 
>> Careina.

[ccp4bb] Rfree problem

[Nazia Nasir Phd2009,ProteinCrystall.Lab]

Hi,

I am facing a problem in refining one of my structures. With every
refinement the Rwork is going down. However, the Rfree is stuck and is
not going down with refinement. I have already solved the structure
without the co-factor bound. This structure is with the co-factor
bound. Can anyone suggest the reason for the Rfree getting stuck?

Thanks

-- 
Nazia Nasir
PhD Scholar
Protein Crystallography Lab
National Institute of Immunology
New Delhi

[ccp4bb] The cell parameter of the 2-D crystal

[Wei Feng]

Dear all,
Does anyone know how to calculate the cell parameter of the 2-D protein crystal?
The map of the crystal can be found in this blog:
http://dingding830106.blog.163.com/blog/static/351191702013111014448546/
Thanks for your time!
Best!
Wei

[ccp4bb] scala discrepancy between versions

[Jens Kaiser]

Dear developers,
  We have been doing the "local" scaling with scala of multiple
wavelengths for some time. We scaled one dataset collected remotely from
the absorption edge, and input this to scala as a reference as one batch
without any problems. With the latest release of CCP4, though, we get
the following error (reference dataset is assigned to batch 9):

  Missing phi limits for batch   9



  No valid orientation data for batch  9: this is not allowed
with
TAILS, SECONDARY, ABSORPTION or BEAMS options

 Scala:   Failed in SETSCL 
Times: User:   0.0s System:0.0s Elapsed: 0:00

Our current workaround is to use the last, not the latest, release of
CCP4.

Any help is appreciated,

Jens

Re: [ccp4bb] small crystals

[Jens Kaiser]

Careina,
  If your target is interesting enough, try to reproduce the small
crystals in batch and apply for FELS time. Small crystals are actually
an advantage there.

Cheers,

Jens


On Wed, 2013-12-04 at 21:41 -0800, Careina Edgooms wrote:
> Hi all
> 
> 
> Any advice on how to get bigger crystals from conditions that give
> showers of tiny crystals? I am getting small pretty looking individual
> crystals but they are too small and they don't seem to grow. In fact,
> in some instances if left for a couple of days they actually dissolve.
> I have fiddled around with mother liquor volume, protein concentration
> as well as drop volume (I am using hanging drop method) but none seem
> to make any difference and I always get the same tiny crystals. I
> think I might try microseeding but I haven't tried that yet. 
> 
> 
> Any suggestions or tricks would be welcome 
> Careina.

[ccp4bb] Postdoctoral position in Membrane Proteins in Australia

[Julia Archbold]

Hello,

We have a postdoctoral position available in 2014 studying membrane proteins 
involved in the innate immune system. This position is based in Professor 
Jennifer Martin's laboratory at the Institute for Molecular Bioscience in 
Brisbane, Australia. The applicant should hold a PhD in structural biology and 
previous experience with membrane proteins is highly desirable.

For a more detailed description of the project and facilities and information 
on how to apply, please head to:

http://uqjobs.uq.edu.au/jobDetails.asp?sJobIDs=495401&sReferrer=home&lApplicationSubSourceID=&lWorkTypeID=&lLocationID=&sJobNo=495401&lCategoryID=&lBrandID=&sKeywords=495401&stp=AW&sLanguage=en

Sincerely,

Julia Archbold

Dr Julia Archbold
NHMRC Postdoctoral Training Fellow
Institute for Molecular Bioscience
University of Queensland
Brisbane, Australia

Re: [ccp4bb] Experience with Biomek 2000 liquid-handling system

[Pius Padayatti]

Phil,
How about Alchemist from Rigaku automation
Please consider Alchemist for your next purchase
http://www.rigaku.com/products/automation/alchemistht

Padayatti PS


On Mon, Dec 9, 2013 at 10:46 AM, Phil Bourne  wrote:

> The Biomek 2000 is capable of setting up screens. It can handle viscous
> liquids if the liquid level sensing tools are combined with very slow
> pipetting. My experience with Biomek 2000 systems is that it is not quicker
> to prepare anything with them. Their advantage comes in reproducibility and
> consistency. The Interface is quite outdated but fairly easy to use once
> you
> get the hang of it. As with any system it will only do what it has been
> programmed to do, so you need to be very careful making sure every step of
> the method is correct. This can be time consuming for a long method but
> once
> it is done, it is done.
>
> Maintenance is a lottery. I have worked with four of these systems. Some
> required a lot of maintenance, some hardly any. It seems to be a matter of
> luck. We did not do maintenance ourselves, we had a service contract so I
> cannot say how difficult maintenance is.
>
> Good luck,
>
> Phil Bourne
>
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Anton
> Meinhart
> Sent: Monday, December 09, 2013 8:25 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] Experience with Biomek 2000 liquid-handling system
>
> Dear all,
>
> does anyone have experience in using a Biomek 2000 liquid-handling system
> for preparing screens? How does it perform with viscous solutions
> - e.g. PEGs? How difficult / time consuming are programming and
> maintenance?
>
> Your help is highly appreciated.
>
> Toni Meinhart
>



-- 
P

Re: [ccp4bb] Experience with Biomek 2000 liquid-handling system

[Phil Bourne]

The Biomek 2000 is capable of setting up screens. It can handle viscous
liquids if the liquid level sensing tools are combined with very slow
pipetting. My experience with Biomek 2000 systems is that it is not quicker
to prepare anything with them. Their advantage comes in reproducibility and
consistency. The Interface is quite outdated but fairly easy to use once you
get the hang of it. As with any system it will only do what it has been
programmed to do, so you need to be very careful making sure every step of
the method is correct. This can be time consuming for a long method but once
it is done, it is done.

Maintenance is a lottery. I have worked with four of these systems. Some
required a lot of maintenance, some hardly any. It seems to be a matter of
luck. We did not do maintenance ourselves, we had a service contract so I
cannot say how difficult maintenance is.

Good luck,

Phil Bourne

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Anton
Meinhart
Sent: Monday, December 09, 2013 8:25 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Experience with Biomek 2000 liquid-handling system

Dear all,

does anyone have experience in using a Biomek 2000 liquid-handling system
for preparing screens? How does it perform with viscous solutions
- e.g. PEGs? How difficult / time consuming are programming and maintenance?

Your help is highly appreciated.

Toni Meinhart

[ccp4bb] MrBUMP not running after update

[jie liu]

Dear All

I just installed CCP4-6.4 and all the subsequent updates. Now MrBump failed
with the following error message:

The program run with command: /usr/local/ccp4-6.4.0/bin/ccp4-python -u
/usr/local/ccp4-6.4.0/bin/mrbump HKLIN
/home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/high_x4c.mtz SEQIN
/home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/seq.seq HKLOUT
/home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/high_x4c_mrbump_soln1.mtz
XYZOUT
/home/jie/Structures/local/se-p26c8newn6/ccp4_6.3/high_x4c_mrbump_soln1.pdb
has failed with error message
Traceback (most recent call last):
 File "/usr/local/ccp4-6.4.0/bin/mrbump", line 107, in 
   queue = master.fast_search_MR(init, target_info, mstat)
 File
"/usr/local/ccp4-6.4.0/share/mrbump/include/initialisation/MRBUMP_master.py",
line 482, in fast_search_MR
   ps.get_top_hit(mstat.sorted_MR_list, init, mstat)
 File "/usr/local/ccp4-6.4.0/share/mrbump/include/structures/Matches.py",
line 1431, in get_top_hit
   CHAIN=mstat.chain_list[chain]
KeyError: '1e6j_p2'

It was running fine with CCP4-6.3. Could someone help please?

Thanks!

Jie

[ccp4bb] Off-Topic: NITPIC ITC software available

[Chad Brautigam]

Dear All,

Apologies for the off-topic post, but I think there might be a number of people 
subscribed to this list who also use ITC and would find this useful:

 
I am pleased to announce the immediate availability of the
software program NITPIC.  You may download it from 
http://biophysics.swmed.edu/MBR/software.html.
 
What does NITPIC do, you ask?  The software is designed
to read and integrate thermograms generated by GE, MicroCal, MCS, and TA
isothermal titration calorimeters.  It calculates the baseline in an
unbiased way, integrates the power peaks, generates error estimates for the
integrations, and outputs the data for analysis.  It interfaces seamlessly
with Peter Schuck’s SEDPHAT analysis program, which features a large variety of
interaction models.  When used with SEDPHAT, NITPIC also outputs data that
are compatible with the high-quality graphics renderer GUSSI.
 
We find that NITPIC generally outperforms the manufacturers’
analysis programs, and it removes the tedious step of manual baseline
adjustment.
 
The program is documented in a pdf file that accompanies the
downloaded executable.  It runs only under Windows operating systems; it
has been tested under Windows XP, 7, and 8.
 
The algorithms underlying the NITPIC program can be found in
Keller et al. (2012), Anal. Chem., Vol. 84, pp. 5066-5073.
 
If you find any bugs, please report them to me:  
chad.brauti...@utsouthwestern.edu
 
Enjoy,
Chad

[ccp4bb] Technical and Distributor Support Rôle at Molecular Dimensions Ltd.

[Jeanette Hobbs]

Hello,

Molecular Dimensions Ltd (Newmarket, England) is seeking an enthusiastic 
sales-oriented individual to help drive our marketing effort and support our 
distributors.

The rôle will be based in Newmarket and includes providing training and support 
for our distributor network and assisting at conferences and exhibitions, and 
will include some international travelling. The position will also involve 
preparing promotional material and newsletters, and updating our web site.
You should have a strong background in protein crystallography with laboratory 
experience in protein chemistry. Excellent interpersonal and presentation 
skills are essential in this role. Good written and verbal English is required.

Information on Molecular Dimensions and its products can be found here: 
http://www.moleculardimensions.com/

To find out more about this exciting position in this rapidly growing company 
please contact t...@moleculardimensions.com

Thanks,

Jeanette

=
Jeanette Hobbs Ph.D.
Business Development Manager
Molecular Dimensions Ltd.
Unit 6, Goodwin Business Park,
Willie Snaith Road
Newmarket
Suffolk
CB8 7SQ
Tel:+44(0)1638561051
Fax:+44(0)1638660674

[ccp4bb] Experience with Biomek 2000 liquid-handling system

[Anton Meinhart]

Dear all,

does anyone have experience in using a Biomek 2000 liquid-handling 
system for preparing screens? How does it perform with viscous solutions 
- e.g. PEGs? How difficult / time consuming are programming and maintenance?


Your help is highly appreciated.

Toni Meinhart

Re: [ccp4bb] How degenerate keyword in Phaser can be used?

[Randy Read]

Hi,

We used to allow degenerate solutions (i.e. ones in which only the orientation 
is known but not the position, or in which the orientation and position within 
a plane perpendicular to a rotation axes are known), because these can be 
scored by the same likelihood target used for the rotation search.  However, we 
never found these possibilities useful in practice compared to just doing a 
full translation search and they complicated the code tremendously, so these 
features were removed a couple of years ago.  It seems we had missed a couple 
of places in the documentation of script files where they were still mentioned, 
but the documentation has just now been updated.

Best wishes,

Randy Read

On 9 Dec 2013, at 09:20, Rojan Shrestha  wrote:

> Hello,
>  
> Can somebody tell me how the Phaser degenerate function can be used with 
> MR_BTF?
>  
> My script is as follows:
>  
> #my_phaser_run.com
>   phaser << eof
>   TITLe BTF_RUN
>   MODE MR_BTF
>   HKLIn a.mtz
>   LABIn F=Fobs SIGF=Sigma
>   ENSEmble mol1 PDB a.pdb IDENtity 100 # this is model structure not solution 
> after MR
>   ENSEmble mol2 PDB b.pdb IDENtity 100 # this is model structure not solution 
> after MR
>   COMPosition PROTein SEQuence a.fasta NUM 1 
>   COMPosition PROTein SEQuence b.fasta NUM 1 
>   SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74
>   SOLUtion TRIAl ENSEmble mol2 EULEr 22 25 40 DEGEnerate X FRACtional 0.05 
> 0.74 
> ROOT ab
>   eof 
>  
> …
>  
> When I run this script, I got an error:
> $TEXT:Script: $$ Baubles Markup $$
> TITLE GPCR_MR 
> JOBS 1
> ENSEmble mol1 PDB a.pdb IDENtity 100 # this is model structure not solution 
> after MR
> ENSEmble mol2 PDB b.pdb IDENtity 100 # this is model structure not solution 
> after MR
> COMPosition PROTein SEQuence a.fasta NUM 1 
> COMPosition PROTein SEQuence b.fasta NUM 1 
> SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74
> SOLUtion TRIAl ENSEmble mol2 EULEr 22 25 40 DEGEnerate
> $$
> 
> $TEXT:Warning: $$ Baubles Markup $$
> -
> SYNTAX ERROR: Use (optionally) ZSCORE RFZ or PTNCS
> -
> 
> Regards,
> 
>  
> 
> Rojan
> 

--
Randy J. Read
Department of Haematology, University of Cambridge
Cambridge Institute for Medical Research  Tel: + 44 1223 336500
Wellcome Trust/MRC Building   Fax: + 44 1223 336827
Hills RoadE-mail: rj...@cam.ac.uk
Cambridge CB2 0XY, U.K.   www-structmed.cimr.cam.ac.uk

Re: [ccp4bb] Off topic: Problem in reproducing crystal screen

[Bosch, Juergen]

another option might be the age of the screen perhaps ?
very silly suggestion/question: your pH meter is calibrated before you make 
your measurements right ?

If you have a nano-pH probe (anything that can be added to a 96well reservoir 
well) you could test what the final pH of your composition is in which you do 
obtain crystals and then pH the final mixture to that value with the same 
electrode.

Jürgen

On Dec 9, 2013, at 4:59 AM, Evgeny Osipov 
mailto:e.m.osi...@gmail.com>> wrote:

Hello, Nasir,
What concentration of your ammonium sulphate stock solution? Do your
solution remains immiscible without ammonium sulphate?May be addition of
concentrated solution of ammonium sulphate to PEG MME could cause phase
separation? Anyway, try to heat your solution a bit as recommended by
Tim or use ammonium sulphate stock solution with low ( probably 1 to 2
M) concetration and tell us if it works
06.12.2013 20:39, Tim Gruene пишет:
Dear Nazia Nasir,

did you warm up the solution a little? This makes working with PEG
solutions a lot easier. You may also note the Hampton do not maintain
the pH: they use the buffers as indicated but this does not mean that
the final solution still has pH 6.5. With your ingredients it probably
is, but e.g. with high concentrations of imidazole the final pH would
differ a lot.

Best,
Tim

On 12/06/2013 02:37 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab wrote:
I have obtained crystals in Hampton Crustal Screen 2, condition 26.The
condition has the following components:
1. 0.2 M ammonium sulphate
2. 0.1 M MES buffer pH 6.5
3. 30% PEG mononmethyl ether 5,000.

When I try to prepare the screen in-house, I use the Hampton stocks of 3.5
M ammonium sulphate and 50%  PEG mononmethyl ether 5,000. I make the MES
buffer using MES hydrate, SigmaUltra from Sigma and maintain the pH
accurately. The water I use is filtered MilliQ. However, the PEG MME and
the rest of the solution remains immiscible and thus, forms droplets of
separated PEG when I set up crystallization. The original condition from
Hampton has no such problems and gives good crystals.

Can anyone let me know how to overcome the problem insolubility of PEG MME
in the buffer mix? I have not faced such problems with other conditions
which I prepare in house and have PEG in them. Further, it's not possible
for me to use the original screen all the time.

Thanks a lot!


Regards



--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com

..
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://lupo.jhsph.edu

Re: [ccp4bb] Off topic: Problem in reproducing crystal screen

[Evgeny Osipov]

Hello, Nasir,
What concentration of your ammonium sulphate stock solution? Do your 
solution remains immiscible without ammonium sulphate?May be addition of 
concentrated solution of ammonium sulphate to PEG MME could cause phase 
separation? Anyway, try to heat your solution a bit as recommended by 
Tim or use ammonium sulphate stock solution with low ( probably 1 to 2 
M) concetration and tell us if it works

06.12.2013 20:39, Tim Gruene пишет:

Dear Nazia Nasir,

did you warm up the solution a little? This makes working with PEG
solutions a lot easier. You may also note the Hampton do not maintain
the pH: they use the buffers as indicated but this does not mean that
the final solution still has pH 6.5. With your ingredients it probably
is, but e.g. with high concentrations of imidazole the final pH would
differ a lot.

Best,
Tim

On 12/06/2013 02:37 PM, Nazia Nasir Phd2009,ProteinCrystall.Lab wrote:

I have obtained crystals in Hampton Crustal Screen 2, condition 26.The
condition has the following components:
1. 0.2 M ammonium sulphate
2. 0.1 M MES buffer pH 6.5
3. 30% PEG mononmethyl ether 5,000.

When I try to prepare the screen in-house, I use the Hampton stocks of 3.5
M ammonium sulphate and 50%  PEG mononmethyl ether 5,000. I make the MES
buffer using MES hydrate, SigmaUltra from Sigma and maintain the pH
accurately. The water I use is filtered MilliQ. However, the PEG MME and
the rest of the solution remains immiscible and thus, forms droplets of
separated PEG when I set up crystallization. The original condition from
Hampton has no such problems and gives good crystals.

Can anyone let me know how to overcome the problem insolubility of PEG MME
in the buffer mix? I have not faced such problems with other conditions
which I prepare in house and have PEG in them. Further, it's not possible
for me to use the original screen all the time.

Thanks a lot!


Regards




--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
A.N. Bach Institute of Biochemistry
Russian Academy of Sciences
Leninsky pr. 33, 119071 Moscow, Russia
e-mail: e.m.osi...@gmail.com

[ccp4bb] How degenerate keyword in Phaser can be used?

[Rojan Shrestha]

Hello,

 

Can somebody tell me how the Phaser degenerate function can be used with
MR_BTF?

 

My script is as follows:

 

#my_phaser_run.com
  phaser << eof
  TITLe BTF_RUN
  MODE MR_BTF
  HKLIn a.mtz
  LABIn F=Fobs SIGF=Sigma
  ENSEmble mol1 PDB a.pdb IDENtity 100 # this is model structure not
solution after MR
  ENSEmble mol2 PDB b.pdb IDENtity 100 # this is model structure not
solution after MR
  COMPosition PROTein SEQuence a.fasta NUM 1 
  COMPosition PROTein SEQuence b.fasta NUM 1 

  SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

  SOLUtion TRIAl ENSEmble mol2 EULEr 22 25 40 DEGEnerate X FRACtional 0.05
0.74 
ROOT ab
  eof 

 

.

 

When I run this script, I got an error: 

$TEXT:Script: $$ Baubles Markup $$
TITLE GPCR_MR 
JOBS 1
ENSEmble mol1 PDB a.pdb IDENtity 100 # this is model structure not solution
after MR
ENSEmble mol2 PDB b.pdb IDENtity 100 # this is model structure not solution
after MR
COMPosition PROTein SEQuence a.fasta NUM 1 
COMPosition PROTein SEQuence b.fasta NUM 1 

SOLUtion 6DIM ENSEmble mol1 EULEr 17 20 32 FRACtional 0.12 0.05 0.74

SOLUtion TRIAl ENSEmble mol2 EULEr 22 25 40 DEGEnerate
$$

$TEXT:Warning: $$ Baubles Markup $$

-
SYNTAX ERROR: Use (optionally) ZSCORE RFZ or PTNCS

-

Regards,

 

Rojan