[ccp4bb] vacancy for Java Web Application Developer at Diamond

2012-06-12 Thread Martin Walsh
Dear all, this new vacancy at Diamond may be of interest for some readers of 
this BB which is focused on further development of ISPyB at Diamond. Full 
details of the post can be found here:

http://www.diamond.ac.uk/Home/Jobs/Current/DIA0737_TH.html

Informal inquiries can be made to myself (martin.wa...@diamond.ac.uk ) or Alun 
Ashton (alun.ash...@diamond.ac.ukmailto:alun.ash...@diamond.ac.uk)

Thanks
Martin




Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

2012-06-12 Thread Eleanor Dodson
Why would anyone ignore the anomalous data they had collected? It will always 
help the phasing, and decide the hand for you..
  Eleanor 
On 6 Jun 2012, at 03:55, Stefan Gajewski wrote:

 Hey!
 
 I was just wondering, do you know of any recent (~10y) publication that 
 presented a structure solution solely based on MIR? Without the use of any 
 anomalous signal of some sort?  
 
 When was the last time you saw a structure that was solved without the use of 
 anomalous signal or homology model? Is there a way to look up the answer 
 (e.g. filter settings in the RCSB) I am not aware of?
 
 Thanks,
 S.
 
 (Disclaimer: I am aware that isomorpous data is a valuable source of 
 information)


[ccp4bb] how to interpret DALI search results

2012-06-12 Thread Jerry McCully

Dear ALL;

After we solved our structure by anomalous scattering, we did a DALI 
search. Here are the results but it is not easy to draw meaningful conclusions 
whether our structure represents a novel fold or is homologous to others.

   Basically the Z-score is between 2 and 6.4 since our structure only 
contains 130 residues. Sequence identity is between 5 to 15%.

   The RMSD of structural alignment is between 2.5 to 6 angstrom.  

   Any suggests to interpret the DALI results? Many thanks,

Jerry McCully


  

Re: [ccp4bb] how to interpret DALI search results

2012-06-12 Thread VAN RAAIJ , MARK JOHAN
if this is the first (or second, or third) time you do a DALI search,  
take the list output from DALI, start from the top and superpose each  
structure with yours and look at the superpositions with your  
favourite superposition software.
This is very educational and the only way you get a feeling for what  
numbers mean.



Quoting Jerry McCully:



Dear ALL;

After we solved our structure by anomalous scattering, we  
did a DALI search. Here are the results but it is not easy to draw  
meaningful conclusions whether our structure represents a novel fold  
or is homologous to others.


   Basically the Z-score is between 2 and 6.4 since our  
structure only contains 130 residues. Sequence identity is between 5  
to 15%.


   The RMSD of structural alignment is between 2.5 to 6 angstrom.

   Any suggests to interpret the DALI results? Many thanks,

Jerry McCully







Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es


Re: [ccp4bb] how to interpret DALI search results

2012-06-12 Thread VAN RAAIJ , MARK JOHAN

...I meant visualisation software of course...

Quoting VAN RAAIJ , MARK JOHAN:

if this is the first (or second, or third) time you do a DALI  
search, take the list output from DALI, start from the top and  
superpose each structure with yours and look at the superpositions  
with your favourite superposition software.
This is very educational and the only way you get a feeling for what  
numbers mean.



Quoting Jerry McCully:



Dear ALL;

   After we solved our structure by anomalous scattering, we  
did a DALI search. Here are the results but it is not easy to draw  
meaningful conclusions whether our structure represents a novel  
fold or is homologous to others.


  Basically the Z-score is between 2 and 6.4 since our  
structure only contains 130 residues. Sequence identity is between  
5 to 15%.


  The RMSD of structural alignment is between 2.5 to 6 angstrom.

  Any suggests to interpret the DALI results? Many thanks,

Jerry McCully







Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es





Mark J van Raaij
Laboratorio M-4
Dpto de Estructura de Macromoléculas
Centro Nacional de Biotecnología - CSIC
c/Darwin 3, Campus Cantoblanco
28049 Madrid
tel. 91 585 4616
email: mjvanra...@cnb.csic.es


Re: [ccp4bb] how to interpret DALI search results

2012-06-12 Thread Ethan Merritt
On Tuesday, June 12, 2012 02:29:13 pm Jerry McCully wrote:
 
 Dear ALL;
 
 After we solved our structure by anomalous scattering, we did a DALI 
 search. Here are the results but it is not easy to draw meaningful 
 conclusions whether our structure represents a novel fold or is homologous to 
 others.

I don't think the question is it homologous can ever be answered by 
a DALI score, whether it is high or low.  For that you need to think
about sequence families, inferred evolutionary history, shared function,
etc.

If your structure has a biological function related to that of your DALI
hit, I'd be inclined to consider seriously whether they could be distant
homologs.  If not, I doubt you will convince anyone based only on a 
vaguely similar fold.

A sequence identity of 15% is really low, but that is presumeably only
a pairwise comparison.  You should try PSIBLAST or similar to see if
both your protein and the DALI hit are recognizably members of the
same sequence family or superfamily.

Ethan



 
Basically the Z-score is between 2 and 6.4 since our structure only 
 contains 130 residues. Sequence identity is between 5 to 15%.
 
The RMSD of structural alignment is between 2.5 to 6 angstrom.  
 
Any suggests to interpret the DALI results? Many thanks,
 
 Jerry McCully
 
 
 

-- 
Ethan A Merritt
Biomolecular Structure Center,  K-428 Health Sciences Bldg
University of Washington, Seattle 98195-7742


[ccp4bb] how to get phase of huge complex

2012-06-12 Thread LISA
Hi all,

My work is to solve huge complex containing 4 different proteins and total
molecular weight is about 300 KD. I can purify the complex by co-expression
them in E.coli.  This complex contains 8 protein A, 2 protein B and 1
protein C and D. protein B and protein C  have homology structures
deposited in PDB database. No homology structure available for protein A
and D, which contribute 60% of the whole molecular weight for the complex.

  Now I am trying to find a way to solve the phase of this complex. I
am thinking of use sad or mad with se-Met.   There total 111 Met
residues in this complex. Is it possible to solve this complex by se-Met?
Does someone have experience to solve huge complex structure with se-met?
It is also very welcome for all the suggestion. Thank you.

All the best,

Lisa


Re: [ccp4bb] how to get phase of huge complex

2012-06-12 Thread Francis E Reyes
Do you have crystals? 

Do they diffract? If so, to what resolution?

What resolution do you require to answer your biological question?

F
On Jun 12, 2012, at 7:46 PM, LISA science...@gmail.com wrote:

 Hi all,
  
 My work is to solve huge complex containing 4 different proteins and total 
 molecular weight is about 300 KD. I can purify the complex by co-expression 
 them in E.coli.  This complex contains 8 protein A, 2 protein B and 1 protein 
 C and D. protein B and protein C  have homology structures deposited in PDB 
 database. No homology structure available for protein A and D, which 
 contribute 60% of the whole molecular weight for the complex. 
  
   Now I am trying to find a way to solve the phase of this complex. I am 
 thinking of use sad or mad with se-Met.   There total 111 Met residues in 
 this complex. Is it possible to solve this complex by se-Met? Does someone 
 have experience to solve huge complex structure with se-met? It is also very 
 welcome for all the suggestion. Thank you.
  
 All the best,
  
 Lisa


[ccp4bb] Where to get a thermo-cycling controlled chamber

2012-06-12 Thread Jun Ma
Hi everybody,

My experiment need me to try a thermo-cycling method for protein
crystallization. So I need a kind of temperature chamber, which can changes
temperature quickly, precisely and steadily. Furthermore, it should be
relatively quiet when running, because I'm afraid vibration will affect the
crystallization of my protein. I just need a small one, which can control
temperature from 0 C to 50 C.

I'm looking for this for some time, and still don't know where to get it.
If anyone can provide information about where to purchase, or how to solve
the thermo-cycling control if there's no commercial merchandise, I will
appreciate that very much.

Any other suggestions are welcome.
Jun Ma

2012-06-12


Re: [ccp4bb] how to get phase of huge complex

2012-06-12 Thread James Stroud
If you want to use se-Met, you might want to start by labeling only one protein 
at a time. For example, if you have A,B,C,D, grow crystals like this:

se-A, B, C, D
A, se-B, C, D,

etc.

Then try combinations of 2, then 3, then if you haven't got the phases you 
need, try all 4.

And remember, if 2 different combinations diffract and they are isomorphous, 
then you can try MIR too.

Also, if your complex can stay intact on a gel shift, look at this paper: 
http://www.ncbi.nlm.nih.gov/pubmed/10903954

James


On Jun 12, 2012, at 8:46 PM, LISA wrote:

 Hi all,
  
 My work is to solve huge complex containing 4 different proteins and total 
 molecular weight is about 300 KD. I can purify the complex by co-expression 
 them in E.coli.  This complex contains 8 protein A, 2 protein B and 1 protein 
 C and D. protein B and protein C  have homology structures deposited in PDB 
 database. No homology structure available for protein A and D, which 
 contribute 60% of the whole molecular weight for the complex. 
  
   Now I am trying to find a way to solve the phase of this complex. I am 
 thinking of use sad or mad with se-Met.   There total 111 Met residues in 
 this complex. Is it possible to solve this complex by se-Met? Does someone 
 have experience to solve huge complex structure with se-met? It is also very 
 welcome for all the suggestion. Thank you.
  
 All the best,
  
 Lisa


Re: [ccp4bb] Where to get a thermo-cycling controlled chamber

2012-06-12 Thread Roger Rowlett
Why wouldn't a peltier-cooled/heated (PCR) thermal cycler be adaptable?

Roger Rowlett
On Jun 12, 2012 11:15 PM, Jun Ma mrmar2...@gmail.com wrote:

 Hi everybody,

 My experiment need me to try a thermo-cycling method for protein
 crystallization. So I need a kind of temperature chamber, which can changes
 temperature quickly, precisely and steadily. Furthermore, it should be
 relatively quiet when running, because I'm afraid vibration will affect the
 crystallization of my protein. I just need a small one, which can control
 temperature from 0 C to 50 C.

 I'm looking for this for some time, and still don't know where to get it.
 If anyone can provide information about where to purchase, or how to solve
 the thermo-cycling control if there's no commercial merchandise, I will
 appreciate that very much.

 Any other suggestions are welcome.
 Jun Ma

 2012-06-12



Re: [ccp4bb] how to get phase of huge complex

2012-06-12 Thread Frank von Delft
If you don't have crystals yet, you'll find getting it to crystallize is 
your main problem.


Finding 111 sites should be feasible without other tricks than very 
careful data collection (see below);  if you have two or more copies in 
the ASU, you may find you need to do what the ribosome guys did, namely 
use other derivatives (e.g TaBr clusters) to locate your seleniums, and 
then phase.


If your resolution is low, you definitely want to do 2- or 3-wavelength 
MAD.


So yes, it's definitely feasible.  If you have crystals...


von Delft, Inoue, et al.  'Structure of E. coli ketopantoate 
hydroxymethyl transferase complexed with ketopantoate and Mg2+, solved 
by locating 160 selenomethionine sites'. /Structure /11, no. 8 (2003): 
985--996.

http://www.cell.com/structure/retrieve/pii/S0969212603001588



On 13/06/2012 03:55, Francis E Reyes wrote:

Do you have crystals?

Do they diffract? If so, to what resolution?

What resolution do you require to answer your biological question?

F
On Jun 12, 2012, at 7:46 PM, LISAscience...@gmail.com  wrote:


Hi all,

My work is to solve huge complex containing 4 different proteins and total 
molecular weight is about 300 KD. I can purify the complex by co-expression 
them in E.coli.  This complex contains 8 protein A, 2 protein B and 1 protein C 
and D. protein B and protein C  have homology structures deposited in PDB 
database. No homology structure available for protein A and D, which contribute 
60% of the whole molecular weight for the complex.

   Now I am trying to find a way to solve the phase of this complex. I am 
thinking of use sad or mad with se-Met.   There total 111 Met residues in this 
complex. Is it possible to solve this complex by se-Met? Does someone have 
experience to solve huge complex structure with se-met? It is also very welcome 
for all the suggestion. Thank you.

All the best,

Lisa


Re: [ccp4bb] Where to get a thermo-cycling controlled chamber

2012-06-12 Thread Zhijie Li
Hi Jun,

In this following article an metallic adapter was used to mount a 192 well 
sitting drop plate on a conventional thermocycler.
http://journals.iucr.org/d/issues/2006/05/00/av5047/av5047bdy.html. 
However I guess there would be significant evaporation and condensation if you 
use vapor diffusion setup and cycle the temperatures.

On the other hand, if you do microbatch under oil, then you should be able to 
easily setup the experiment on a 96-tube PCR plate and mount it on a PCR 
machine. I guess the temperature can be more precisely controlled this way with 
no special equipment needed. Especially, the evaporation and condensation 
during thermo cycles would probably be less problematic.

Zhijie



From: Jun Ma 
Sent: Tuesday, June 12, 2012 11:15 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: [ccp4bb] Where to get a thermo-cycling controlled chamber


Hi everybody,

My experiment need me to try a thermo-cycling method for protein 
crystallization. So I need a kind of temperature chamber, which can changes 
temperature quickly, precisely and steadily. Furthermore, it should be 
relatively quiet when running, because I'm afraid vibration will affect the 
crystallization of my protein. I just need a small one, which can control 
temperature from 0 C to 50 C. 
I'm looking for this for some time, and still don't know where to get it. If 
anyone can provide information about where to purchase, or how to solve the 
thermo-cycling control if there's no commercial merchandise, I will appreciate 
that very much.

Any other suggestions are welcome.

Jun Ma

2012-06-12

Re: [ccp4bb] how to get phase of huge complex

2012-06-12 Thread Nat Echols
On Tue, Jun 12, 2012 at 8:53 PM, Frank von Delft
frank.vonde...@sgc.ox.ac.uk wrote:
 Finding 111 sites should be feasible without other tricks than very careful
 data collection (see below);  if you have two or more copies in the ASU, you
 may find you need to do what the ribosome guys did, namely use other
 derivatives (e.g TaBr clusters) to locate your seleniums, and then phase.

With 40% of the complex having homologues in the PDB, you may be able
to place those subunits by MR, then use the phases from the incomplete
model to locate the seleniums.

-Nat


Re: [ccp4bb] how to get phase of huge complex

2012-06-12 Thread Vellieux Frederic

Hi Lisa, hi all,

Please do not discard the alternative method(s) of conventional heavy 
atoms. Co-crystallisation or heavy-atom containing mother liquor soaks. 
You may remember that monster complexes have been solved in the past 
by such methods, and sometimes there are difficulties in crystallising 
the Se-Met version of a protein (the native protein gives crystals, 
the Se-Met version does not). And there is still some work going on 
regarding the development of novel (lanthanide-based) heavy-atom 
compounds that may provide both isomorphous and anomalous differences, 
the anomalous signal being extremely useful in the case of lanthanides 
and can be used on its own to solve 3D structures (one can then go 
back to the structure of the native macromolecule or complex if need be).


See e.g. Talon, R. et al. (2011), J. Sync. Rad. 18, 74-78 (PMID: 21169697)
and
http://www.natx-ray.com/products/catalogue_consum_CSM002.html

HTH,

Fred.

F.M.D. Vellieux (B.Sc., Ph.D., hdr)
Institut de Biologie Structurale J.-P. Ebel CEA CNRS UJF
LBM/ELMA
41 rue Jules Horowitz
38027 Grenoble Cedex 01
France
Tel: +33 (0) 438789605 (direct line), +33 (0) 663482891 (mobile phone)
Fax: +33 (0) 438785494
e-mail: frederic.velli...@ibs.fr

LISA wrote:

Hi all,
 
My work is to solve huge complex containing 4 different proteins and 
total molecular weight is about 300 KD. I can purify the complex by 
co-expression them in E.coli.  This complex contains 8 protein A, 2 
protein B and 1 protein C and D. protein B and protein C  have 
homology structures deposited in PDB database. No homology structure 
available for protein A and D, which contribute 60% of the whole 
molecular weight for the complex. 
 
  Now I am trying to find a way to solve the phase of this 
complex. I am thinking of use sad or mad with se-Met.   There total 
111 Met residues in this complex. Is it possible to solve this complex 
by se-Met? Does someone have experience to solve huge complex 
structure with se-met? It is also very welcome for all the suggestion. 
Thank you.
 
All the best,
 
Lisa