Re: [ccp4bb] New restraints, same name

[Ingo P. Korndoerfer]

... and why don't we move on from pdb format to something a bit more
modern ?

i think it would only require a handful of programmers to agree on this
and the rest would (have to) follow.

i.e. if coot and refmac could both read and write something more modern
we'd have a huge part of ccp4 world
covered.

are we thinking of ourselves at working at the frontiers of science or
not ... (... o.k. ... different topic, but you get the idea ...)

ingo

On 25.01.2012 03:53, Paul Emsley wrote:
> Thanks to all contributors, I have been informed, educated and
> entertained.
>
> A bit of background perhaps... (it seems that I have been living in
> the 0.7 world long enough to forget that not everyone else is here).
> "[T]he viewer programs don't care about the restraint dictionaries" 
> says Seth Harris - haha - in olden Coots that was the case... :)  It
> is my hope that Coot will be used for comparison, evaluation,
> validation and manipulation of ligands in protein-ligand complexes and
> their electron density.
>
> My current obsession is with chemical structure diagrams - here's a
> recent screenshot:
> http://lmb.bioch.ox.ac.uk/coot/screenshots/Screenshot-example-2010-01-02.png
>
>
> ... and here's one I made earlier today, illustrating the sorts of
> problems I am trying to handle (PI3 Kinase ligand, 4a55):
> http://lmb.bioch.ox.ac.uk/coot/screenshots/Screenshot-Coot-prodrg-valence-problem.png
>
> amusing, eh?
>
> Anyway, to make the chemical diagram and the 3D bonding representation
> I need to construct a description of the ligand that contains bond
> orders.  Hence restraints.  So yes, let me emphasize that this is
> mostly for drawing pictures and I don't see the use case of refinement
> of multiple different ligand complexes as very useful.
>
> I do like Dale's idea - the use of HETNAM and synonyms - so, as I
> understand it, the PDB file has a residue called LIG and the
> dictionary has a comp-id of
> "2-(N-methylmethanesulfonamido)-6-(propan-2-yl)pyrimidine" (or
> XYZ0123456 or whatever) and a HETNAM record in the PDB file provides
> the mapping.  Is this a solution?   It is attractive because it
> requires very little work from me.
>
> I did consider Judit's idea, i.e. check the atom names in the
> coordinates against the dictionary before bonding.  I thought that
> there may be (too many?) pathological cases where the names did match
> (at least for ligand fragments) but the chemistry did not.  Let me
> know if you think that I need not worry so much about that.  This is
> relatively easy to do.  However, this only solves the "tangle" problem
> - and I think that that for practical purposes that may be covered now
> as I have recently turned off restraints auto-loading for several
> "special" three-letter codes - one can (at least) see "noddy" bonding
> instead of a tangle.
>
> To answer Garib's point: yes, in Coot there is indeed a single
> table/dictionary of restraints, with the key/index being the
> comp-id/residue-name.  It applies to all molecules.  I had not before
> considered the option of embedding monomer restraints inside a Coot
> molecule - that might be a cleaner solution. I will ponder on that. 
> It does mean that you will have to read restraints after reading
> coordinates though.
>
> And yes, I do occasionally wonder how computational chemistry software
> (Maestro, Vida for example?) solves this problem.  Presumably such
> software is used to show several overlaying ligand structures (all
> called "LIG"?).  And computational chemists like to see chemistry, and
> not just coloured sticks, right?
>
> Thanks,
>
> Paul.
>

Re: [ccp4bb] New restraints, same name

[Garib N Murshudov]

That is the plan and hopefully with help of people from pdb coot, buster, 
phenix, refmac, ccp4 programs will all use mmcif with ligand descriptions 
inside. 
Good will is there, a little bit work is needed.

Regards
Garib

On 25 Jan 2012, at 08:15, Ingo P. Korndoerfer wrote:

> ... and why don't we move on from pdb format to something a bit more
> modern ?
> 
> i think it would only require a handful of programmers to agree on this
> and the rest would (have to) follow.
> 
> i.e. if coot and refmac could both read and write something more modern
> we'd have a huge part of ccp4 world
> covered.
> 
> are we thinking of ourselves at working at the frontiers of science or
> not ... (... o.k. ... different topic, but you get the idea ...)
> 
> ingo
> 
> On 25.01.2012 03:53, Paul Emsley wrote:
>> Thanks to all contributors, I have been informed, educated and
>> entertained.
>> 
>> A bit of background perhaps... (it seems that I have been living in
>> the 0.7 world long enough to forget that not everyone else is here).
>> "[T]he viewer programs don't care about the restraint dictionaries" 
>> says Seth Harris - haha - in olden Coots that was the case... :)  It
>> is my hope that Coot will be used for comparison, evaluation,
>> validation and manipulation of ligands in protein-ligand complexes and
>> their electron density.
>> 
>> My current obsession is with chemical structure diagrams - here's a
>> recent screenshot:
>> http://lmb.bioch.ox.ac.uk/coot/screenshots/Screenshot-example-2010-01-02.png
>> 
>> 
>> ... and here's one I made earlier today, illustrating the sorts of
>> problems I am trying to handle (PI3 Kinase ligand, 4a55):
>> http://lmb.bioch.ox.ac.uk/coot/screenshots/Screenshot-Coot-prodrg-valence-problem.png
>> 
>> amusing, eh?
>> 
>> Anyway, to make the chemical diagram and the 3D bonding representation
>> I need to construct a description of the ligand that contains bond
>> orders.  Hence restraints.  So yes, let me emphasize that this is
>> mostly for drawing pictures and I don't see the use case of refinement
>> of multiple different ligand complexes as very useful.
>> 
>> I do like Dale's idea - the use of HETNAM and synonyms - so, as I
>> understand it, the PDB file has a residue called LIG and the
>> dictionary has a comp-id of
>> "2-(N-methylmethanesulfonamido)-6-(propan-2-yl)pyrimidine" (or
>> XYZ0123456 or whatever) and a HETNAM record in the PDB file provides
>> the mapping.  Is this a solution?   It is attractive because it
>> requires very little work from me.
>> 
>> I did consider Judit's idea, i.e. check the atom names in the
>> coordinates against the dictionary before bonding.  I thought that
>> there may be (too many?) pathological cases where the names did match
>> (at least for ligand fragments) but the chemistry did not.  Let me
>> know if you think that I need not worry so much about that.  This is
>> relatively easy to do.  However, this only solves the "tangle" problem
>> - and I think that that for practical purposes that may be covered now
>> as I have recently turned off restraints auto-loading for several
>> "special" three-letter codes - one can (at least) see "noddy" bonding
>> instead of a tangle.
>> 
>> To answer Garib's point: yes, in Coot there is indeed a single
>> table/dictionary of restraints, with the key/index being the
>> comp-id/residue-name.  It applies to all molecules.  I had not before
>> considered the option of embedding monomer restraints inside a Coot
>> molecule - that might be a cleaner solution. I will ponder on that. 
>> It does mean that you will have to read restraints after reading
>> coordinates though.
>> 
>> And yes, I do occasionally wonder how computational chemistry software
>> (Maestro, Vida for example?) solves this problem.  Presumably such
>> software is used to show several overlaying ligand structures (all
>> called "LIG"?).  And computational chemists like to see chemistry, and
>> not just coloured sticks, right?
>> 
>> Thanks,
>> 
>> Paul.
>> 

Garib N Murshudov 
Structural Studies Division
MRC Laboratory of Molecular Biology
Hills Road 
Cambridge 
CB2 0QH UK
Email: ga...@mrc-lmb.cam.ac.uk 
Web http://www.mrc-lmb.cam.ac.uk

Re: [ccp4bb] Problem with getting Rfree and Rf down

[Miguel Ortiz Lombardía]

Le 24/01/12 21:18, Dale Tronrud a écrit :
> On 01/24/12 11:52, Miguel Ortiz Lombardia wrote:
>> El 24/01/12 18:56, Greg Costakes escribió:
>>> Whoops, I misspoke... I meant Rsym and Rmerge increase with higher
>>> redundancies. 
>>>
>>
>> But then suppose that one merges data from a crystal that is degrading
>> while exposed, sp the data gets degraded. This is not at all unusual. In
>> the absence of a deep understanding of refinement, intuition suggests
>> that degraded data should produce degraded models. If Rwork and Rfree
>> are measuring anything useful they should go up redundancy in those
>> not-so-unusual cases. Or intuition is misguiding me again.
>>
> 
>Yes, if one has a poorer quality data set one expects the Rw and Rf to
> be higher, but this is not necessarily a correlation to high redundancy.
> Surely if you have high redundancy and know the crystal is decaying you
> have to flexibility to not use the decayed data in the merge.  I would
> expect that decayed data would only be merged with the early data if
> the redundancy was so low that you had to just to get a full data set.
> 
> Dale Tronrud
> 

I agree. I would also expect so... unless the user simply runs the data
reduction software and does not check the log files to see, among other
important issues, at what point the data starts degrading due to a
decaying crystal. If the software is clever enough to decide by itself,
it will be all right or sort of, which is, I suppose, a good point for
automation. Unfortunately, there are many users of black boxes, which
is, I presume, a danger of automation. My answer was kind of a caveat
for such type of users.


-- 
Miguel

Architecture et Fonction des Macromolécules Biologiques (UMR7257)
CNRS, Aix-Marseille Université
Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
Tel: +33(0) 491 82 55 93
Fax: +33(0) 491 26 67 20
mailto:miguel.ortiz-lombar...@afmb.univ-mrs.fr
http://www.afmb.univ-mrs.fr/Miguel-Ortiz-Lombardia

Re: [ccp4bb] quasispecies

[Saul Hazledine]

On Jan 24, 2012, at 10:31 PM, Bart Hazes wrote:

> For some RNA viruses the rate of mutation is so high that they basically 
> sample a flat region of the fitness-landscape. If you could take two 
> individual viruses out of this sample to establish two independent infections 
> than over time each will start to re-sample the same flat landscape. In other 
> words, there is not a single unique, or predominant, sequence that represents 
> the species but a pool of "near-equal" fitness variants.
> 
> To some extend I feel that this is always the case but for "normal" organisms 
> the sampling rate of fitness space is slow and genetic differences between 
> individuals are dominated by mutations passed down by vertical descend. In 
> contrast, if you sequence two viruses from the above two infections there 
> genetic distance will be similar to the genetic distances between individuals 
> within a single infection.


This way of looking at species is described in a paper I once presented in a 
journal club. The work is computer based and describes putting an evolutionary 
algorithm in control of its own mutation rate. Its not my field, but I found 
the paper very approachable and extremely interesting. 

Emergence of species in evolutionary “simulated annealing”:
http://www.pnas.org/content/106/6/1869.full

Saul Hazledine

[ccp4bb] Introducing an ELN

[Chris Morris]

Tassos reports:

> 1. None of the twenty test-users was satisfied with any of the two
> solutions - and each was annoyed for a different reason.

This suggests that the choice of ELN is not the most difficult part of the 
adoption process. Maybe the test users at the NKI were annoyed by the idea of 
using an ELN at all. 

In my experience, the hardest part is ensuring that it provides benefits to the 
people who have to enter the data, and provides them early. The fact that it 
will make information retrieval easier in three years is not enough.

I suggest focussing on electronic support for housekeeping: booking time on an 
instrument, finding the files the instrument created, ordering oligos, 
recording when you use the last of a reagent. Scientists work very 
independently in most respects, but they do have certain obligations that flow 
from sharing the lab space. You can make use of these to encourage compliance 
with the ELN. If you do, then most of the science will get recorded in passing.

I suggest also ensuring that it includes electronic tools that actually help. 
Two examples from PiMS are primer design, and automatically uploading and 
interpreting results from the Caliper GX instrument.

It must allow round trips with spreadsheets, i.e. dump ELN data as a 
spreadsheet, edit it, upload it again. Despite their substantial disadvantages, 
some scientists will not give them up. It should also allow crossreferencing 
with paper note books. Some will continue to use a lab notebook. When they 
discover that the ELN serves as a searchable index to it, they will warm to the 
ELN.

I suggest aiming for "no paper" at your lab progress meetings within say 12 
months. When you reach that point, everything important is in the ELN. Before 
then, the ELN is not giving real value.

You will need someone who is keen on the introduction of the ELN, to customise 
it, provide first line user support, and act as a single point of contact with 
the supplier. This might be a scientist or an IT person. I have also seen this 
done well by a technician, Delphine Chesnel when she was at the EMBL Hamburg. 
If you can't find such a "champion", then introduction will not be successful.

Some of the problem here is an "own goal" by the community: scientists are 
trained to use paper during their degrees, so ELNs are a controversial change 
of practice. One person who, unusually, began with an ELN told me how 
inconvenient it is now she works in a paper-based lab.

PepTalk 2012 had a workshop on this topic. The recording and notes are here:

http://www.structuralbiology.eu/support/forums/networks/pims/why-dont-scientists-use-limselns

regards,
Chris

Chris Morris  
chris.mor...@stfc.ac.uk   
Tel: +44 (0)1925 603689  Fax: +44 (0)1925 603634
Mobile: 07921-717915
Skype: chrishgmorris
http://pims.structuralbiology.eu/
http://www.citeulike.org/blog/chrishmorris
Daresbury Lab,  Daresbury,  Warrington,  UK,  WA4 4AD
  

[ccp4bb] Postdoctoral position in Oxford Biochemistry

[John Vakonakis]

Dear all,

A postdoctoral position is available in Oxford Biochemistry for combined
crystallographic and NMR studies of centriolar proteins. The advert is
below and applications can be made online until Feb. 20th.

Regards,

John Vakonakis


-- 
Dr. Ioannis (John) Vakonakis

Wellcome Trust Research Fellow

Department of Biochemistry
University of Oxford
South Parks Road
Oxford, OX1 3QU
United Kingdom



  UNIVERSITY OF OXFORD


   DEPARTMENT OF BIOCHEMISTRY


 Postdoctoral Research Associate in Structural Biology


We are seeking to appoint a highly motivated Postdoctoral Research
Associate in the laboratory of Dr. Ioannis Vakonakis for studies of
proteins involved in centriole assembly. The successful candidate will
join a multidisciplinary collaboration between Oxford and the groups of
Prof. Pierre Gönczy (EPFL) and Dr. Michel Steinmetz (Paul Scherrer
Institute) in Switzerland. Recently, our joint efforts elucidated the
origins of centriolar 9-fold symmetry (Kitagawa, Vakonakis et
al.,Cell144, 364-375, 2011), a critical step in the centrosomal
duplication process that ensures genome stability in animals. We now
seek to build upon this breakthrough by studying the structure/function
relationship of centriolar components such as SAS-6, SAS-5, SAS-4,
ZYG-1, SPD-2 and others. The successful candidate will contribute to the
analysis of these targets using structural (crystallography, solution
NMR and/or small angle scattering) and biophysical methods.


Applicants should possess/be expected to obtain a PhD in natural
sciences and should have experience in aspects of the structural biology
pipeline, for example protein production and purification, NMR
spectroscopy or X-ray crystallography. Past experience with
complementary biophysical techniques (ITC, AUC, CD etc) and biomolecular
interactions is desirable. Previous familiarity with centriolar or
relevant biological systems would be an advantage but is not essential. 


This full-time post is funded by the Biotechnology and Biological
Sciences Research Council (BBSRC)for up to three years in the first
instance and is based in the New Biochemistry building, South Parks
Road, Oxford. More information about our group can be found by clicking
on the link below:


http://www2.bioch.ox.ac.uk/~rrnmr


This position is graded on the University’s grade 7 scale, for which the
salary range is £29,099 - £35,788 per annum. The actual starting salary
offered will be based on qualifications and relevant skills acquired and
will also be determined by the funding available.


For further general information, phone (01865) 613204, quoting reference
number BR/493.


Applications for this vacancy are to be made online.


To apply for this role and for further details, including the job
description and selection criteria,

please click on the link below:


https://www.recruit.ox.ac.uk/pls/hrisliverecruit/erq_jobspec_version_4.jobspec?p_id=101979


The closing date for applications is Monday, 20thFebruary 2012. It is
envisioned that interviews for shortlisted candidates will take place
within approximately 10-14 days. 


The University of Oxford is committed to equality and values diversity.

[ccp4bb] Fwd: [ccp4bb] quasispecies

[Carter, Charlie]

Begin forwarded message:

Date: January 24, 2012 7:16:42 PM EST
To: Bart Hazes mailto:bha...@ualberta.ca>>
Subject: Re: [ccp4bb] quasispecies

This remark brings to mind a paper published recently by Ariel Fernandez and 
Mike Lynch:

Nature 474:502, 2011:  Non-adaptive Origins of Interactome Complexity

The point, if I understand it correctly, is that when population sizes are 
small, as they are in multicellular eukaryotes, the effect of "drift" becomes 
more significant, and mutations to the surfaces of proteins that impair the 
protein surface - solvent interaction are established more easily in the 
population. A consequence suggested by the authors is that these "surface 
defects" provide an enhanced manifold from which to recruit novel 
surface-surface contacts that lead to an increase in the interactome".

Charlie

On Jan 24, 2012, at 4:31 PM, Bart Hazes wrote:

To some extend I feel that this is always the case but for "normal" organisms 
the sampling rate of fitness space is slow and genetic differences between 
individuals are dominated by mutations passed down by vertical descend. In 
contrast, if you sequence two viruses from the above two infections there 
genetic distance will be similar to the genetic distances between individuals 
within a single infection.

Re: [ccp4bb] writing scripts-off topic

[Mark Brooks]

Hi Yuri,
  If you don't like Python, like myself (and I'm not alone, it
would seem), you could try Ruby (http://www.ruby-lang.org/en/). Some
examples of PDB file manipulation are below (taken from [1]).

The language is a great improvement in Perl and Python in my opinion, but
the downside is that there aren't as many crystallographic features as
CCTBX et al, I don't think.

Yours,

Mark

[1] http://bioruby.open-bio.org/wiki/SampleCodes
 Structure I/O and Manipulation How do I read a structure file?

The current implementation only reads PDB format files, there will
hopefully be code for parsing mmCIF and PDB XML files in the future. Adding
these new formats will probably change the API for reading and writing
structure files.

Given the above caveat, the current way to read a PDB file is to slurp the
file into a string which is fed to the constructor of the Bio::PDB class:

require 'bio/db/pdb'string = IO.read('pdb1tim.ent')
structure = Bio::PDB.new(string)

The new Bio::PDB object now contains all the annotations and coordinate data.

How do I write a structure file?

As with reading, the current implementation can only write in PDB format.

All the coordinate classes in the Bio::PDB heirarchy have .to_s methods
which return the object in PDB format. This makes output as simple as:

require 'bio/db/pdb'
file = File.new('pdb1tim.ent').gets(nil)
structure = Bio::PDB.new(file)puts structure.to_s # Writes whole
structureputs structure[nil]['A'].to_s # Writes chain A only

How do I access annotations?

The annotations from the PDB file are stored in Bio::PDB::Record objects.
You can retrieve a specific annotation by calling the '.record' method of a
Bio::PDB object with the name of the annotation as the argument:

structure.record("HEADER")

In fact '.record' returns an array of all Bio::PDB::Records of the given
type, so you'll need to call '.first' to get to the actual
Bio::PDB::Record. Bio::PDB::Record provides methods to get to the
individual data in each record. E.g. The 'HEADER' record provides
classification, depDate and idCode methods. You can look at the PDB format
to see what fields are available in each record, or just look in the pdb.rb
file which has a hash of all the definitions.

So, to get the title of a PDB file you would do it like this:

structure.record('TITLE').first.title

Some records have their own special methods:

structure.remark(num)  #Returns hash of the REMARK records based on 'num'
structure.jrnl #Returns a hash of the JRNL records
structure.jrnl('TITL') #Returns an array of all the TITL subfields from
   #the JRNL records
structure.helix(id)#Returns the HELIX records based on 'id'
   #Returns an array if no 'id' is given
structure.turn #Same interface as '.helix'
structure.sheet#Same interface as '.sheet'
structure.seqres(id)   #Returns the sequence given in the SEQRES record
   #as a Bio::Sequence::AA object
structure.keywords #returns a flattened lsit of keywords

And some methods are included for Bio::DB compatability:

structure.entry_id #Returns idCode
structure.accession#Same as entry_id
structure.definition   #Title
structure.version  #REVDAT

How do I access the coordinate data?

Coordinate data is stroed in a heirarchy of classes with Bio::PDB at the
top. A Bio::PDB object contains one or more Bio::PDB::Model objects which
contain one or more Bio::PDB::Chain objects which contain one or more
Bio::PDB::Residue objects which contain Bio::PDB::Atom objects.

There are two ways to access the coordinate data in a Bio::PDB object:
keyed access and iterators.

Keyed access provides the simplest way to get to data if you know which
residues or atoms you're interested in. For instance if you wanted to get
hold of chain 'A' you can do it like this:

chain = structure[nil]['A']

The nil refers to the model, which will be nil in crystal structures but
may have a number in NMR structures. Every level in the heirarchy can be
accessed this way. E.g. to get hold of the CA atom of residue 100 in chain
'A':

structure[nil]['A']['100']['CA']

or if you still have your chain object:

chain['100']['CA']

The residue identifier is not just the residue number. PDB residues can
have insertion codes and even negative numbers.

chain['100A']['CA']

could be valid.

Iterators are also provided so you can easily go through all objects in the
heirarchy. E.g:

structure.each{ |model|
  model.each{ |chain|
chain.each{ |residue|
  residue.each{ |atom|
puts atom
  }
}
  }}

Goes through every atom in the structure and outputs it. All the classes in
the heirarchy implement the standard Enumerable and Comparable mixins as
well.

Each class has a number of accessors to allow access to the data, most is
in the Bio::PDB::Atom class:

   - Bio::PDB::Model has a 'model_serial' accessor only.
   - Bio::PDB::Chain has an 'id' accessor for getting and setting the chain
   id.
 

[ccp4bb] Beamline Scientist position in SAXS@EMBL Hamburg

[Margret Fischer]

Dear all,

A beamline scientist position is available at the EMBL Hamburg Unit in Small 
Angle Scattering in the
research group of Dr. Dmitri Svergun.
I attach the Vacancy Notice below. Deadline for application is 4th March 2012.
Applications should be directed to:www.embl.org/jobs

regards
Margret
-
Vacancy Notice/Job Description

The European Molecular Biology Laboratory (EMBL) is one of the highest
ranked scientific research organisations in the world. The Headquarters
Laboratory is located in Heidelberg (Germany) and the outstations are in 
Grenoble (France),
Hamburg (Germany), Hinxton (UK) and Monterotondo (Italy).

Applications are invited for a Staff Scientist position at the Structural
Biology Unit of the EMBL in Hamburg, Germany. The position is within the
biological SAXS group, which runs a high brilliance user-oriented
synchrotron radiation P12 beamline on the storage ring PETRA 3. This
beamline is part of the integrated facilityofEMBL@PETRA3. The  successful
candidate will play a major role in scientific maintenance (development of
the beamline facilities, methods and technology), external user support and
collaborative projects at the beamline. The post is expected to be involved
in novel biological SAXS developments in collaboration with the other groups
of the EMBL@PETRA 3. There will be opportunities to link related
research activities with the operation of the beamline.

Qualifications and Experience

Applicants should have a PhD in a relevant field, proven record of
independent research, significant scientific accomplishments and expertise
in structure analysis of biological macromolecules using small-angle X-ray
scattering. Previous experience in hardware and/or software development for
synchrotron radiation beamlines is required.

Experience in work at a large-scale user-oriented facility, excellent
communication skills and ability to supervise and collaborate in
international team environment will be essential.

Please apply online throughwww.embl.org/jobs

Re: [ccp4bb] writing scripts-off topic

[Miguel Ortiz Lombardía]

Hi Yuri,

To add something nobody else has mentioned yet.
If you are interested in statistics and analysis you may find
interesting the R package. There is an excelent module, called Bio3D for
the analysis of protein structure and sequence data. Have a look here:

http://bio3d.pbworks.com/w/page/7824467/FrontPage

I use R for a number of things. The learning curve is not that steep.
But may not be what you're looking for.

Best regards,


-- 
Miguel

Architecture et Fonction des Macromolécules Biologiques (UMR7257)
CNRS, Aix-Marseille Université
Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
Tel: +33(0) 491 82 55 93
Fax: +33(0) 491 26 67 20
mailto:miguel.ortiz-lombar...@afmb.univ-mrs.fr
http://www.afmb.univ-mrs.fr/Miguel-Ortiz-Lombardia

Re: [ccp4bb] Expression of Viral proteins for crystallography

[Rubén Sánchez Eugenia]

Dear Gregory and Darren,

Thank you for your answers. They have been very useful.

2012/1/24 Gregory Verdon 
>
> it possible that the protein is toxic (even when slightly expressed from
> your possibly leaky pET vector), so that e.coli select for mutations that
> kill expression of your recombinant gene ...
>
>
> Gregory, the protein may be toxic, but I do not think that could be the
problem. BSJ cells are not meant to be for expression, but for cloning, so
they are not DE3 lysogenic. That is, they do not have the T7 RNApol
necessary for the expression of the proteins inserted into pET plasmids.

In any case, thank you very much for you help.


>
> 2012/1/24 Darren Hart :
>  > I think the explanation is this:
> > The source is natural viral RNA which is a mixture of naturally mutated
> > sequences (e.g. flu forms such a quasispecies)
> > See:
> > http://www.virology.ws/2009/05/11/the-quasispecies-concept/
> >
> > The pooled RNA has an average sequence that you see when you sequence the
> > pooled cDNA (individual mutations are hidden by the averaging effect of
> > having many sequences present).
> >
> > But when you clonally separate DNA molecules by transformation (1 plasmid
> > enters 1 cell to yield 1 colony), you see each individual molecule
> > represented 100% in the sequencing chromatogram from the plasmid DNA that
> > you have isolated from colonies.
> >
> > This is effect is commonly observed when sequencing influenza virus
> isolates
> > from patients. It will have nothing to do with the E. coli strain. You
> can
> > avoid it completely by using gene synthesis.
> >
> > Darren
>


Darren, thanks to your information about the quasi-species, now we are
convinced that this may be the problem. We have been thinking about this
and we have conclude that the average sequence may not even exist and if it
was, it might not be the active one. That is to say, maybe we can not
synthesize our gene because we don't know whether the mutations are needed
or in any case what mutations would be needed. Do you agree? And if I am
right, is it the only solution to try different mutants to get the active
one?

Anyway, I think I would not need to synthesize the gene because I can
select one monoclonal colony and delete the mutation by site-directed PCR
mutagenesis.

Best regards,

[ccp4bb] BioSAXS Essentials III Workshop

[David Schuller]

MacCHESS is pleased to announce that the BioSAXS Essentials III Workshop 
will once again be offered at Cornell University.




*BioSAXS Essentials III: Getting Started in Biological Small-Angle X-ray 
Solution Scattering*


*

*Date:*February  24-26, 2012

*Location:*Cornell High Energy Synchrotron Source, Ithaca, NY

** to be held in the new CHESS eXploration Station educational Facility

*Cost: *$150

*Deadline for Registration: February 10, 2012*

MacCHESS is offering an intensive HOWTO course in BioSAXS. A day of 
lectures on basics and software tutorials by experienced practitioners 
in the field will be followed by actual data collection on two MacCHESS 
beamlines (F2 and G1 stations).


While the primary purpose of the course is educational, students may 
bring a limited number of their own research samples. Prepared standard 
protein samples will be available to all students for practice.


*Course topics*:

·Basic principles and processing methods

·Critical sample preparation tips

·Evaluating data quality

·Computing envelopes and data modeling

·Overview of advanced and emerging methods

·What you need to know for publication

*Speakers:*

R. Gillilan (MacCHESS)

S. Nielsen (Danish Technical University)

M. Moller (University of Copenhagen)

K.  Gupta (UPENN Medical school)

T. Grant (Hauptman-Woodward Institute)

N. Ando (MIT)

L. Pollack (Cornell University)

*SPACE IS LIMITED*

*REGISTRATION:*http://www.chess.cornell.edu/BioSAXS%20course/Registration/register02242012.html 



*REQUIRED:*

·Once registration is complete you must submit a short express-mode 
proposal form http://express.chess.cornell.edu/EM_form.php.


·If you are bringing your own samples please notate in Section 9 of the 
proposal form the solution you will be using for your samples to 
determine hazards, if any.


·Additionally please notate "BioSAXS Essentials Course" in Section 9 of 
the form.


·You are also required to complete an on-line safety training at 
http://www.chess.cornell.edu/onlnequiz/index.htm (this safety training 
is necessary in order to use our facility).


*HOTEL:*We have a block of rooms set up at the Best Western 
University Inn located just one block from CHESS at 1020 Ellis Hollow 
Road, Ithaca, NY. The rate of the room is *$89/night*.  To make a 
reservation call the hotel direct at *(607) 272-6100* and ask for the 
BioSAXS workshop room block OR the CHESS rate.  The Best Western offers 
a free shuttle service to and from the Ithaca airport and to Cornell Campus.


Hope you can join us.  Should you have additional questions, please call 
(607) 255-7163.


Richard Gillilan

Sr Scientist

MacCHESS

Ithaca, NY

[ccp4bb] British Crystallographic Association

[Elspeth Garman]

Dear Crystallographer
Following my pitch on behalf of the British Crystallographic Association (BCA) 
at the 2012 CCP4 Study Weekend in Warwick, I wanted to encourage any interested 
macromolecular crystallographers to become members of the Association.
The UK will host the European Crystallographic  Meeting in Warwick in 2013 
(August 25th-29th) and 2014 is designated as the 'International Year of 
Crystallography'.  For these events and for the health of the Association, it 
would be really good to have strong involvement of the UK MX community in the 
BCA.
Membership of the BCA offers:
*   Reduced registration for all BCA meetings
*   Four copies of Crystallography News per year to keep up to date with 
news and happenings in the field
*   The opportunity to actively contribute to the development of 
crystallography
*   Networking opportunities
*   Eligibility for students to apply to the Arnold Beevers Bursary Fund to 
obtain grants for meetings.
*   Opportunity to interact and learn from scientists outside your own area 
of crystallography
Membership registration is on-line at
www.crystallography.org.uk
We look forward to welcoming you as a member!
Best wishes
Elspeth

  Professor Elspeth F. Garman,
  President, British Crystallographic Association
  Director of Systems Biology Programme, Doctoral Training Centre.
Postal address:
  Laboratory of Molecular Biophysics,
 Department of Biochemistry,
  University of Oxford,  Tel: (44)-1865-613297
  South Parks Road,  FAX: (44)-1865-613201
  OXFORD, OX1 3QU, U.K. E-mail: 
elspeth.gar...@bioch.ox.ac.uk
 http://lmb.bioch.ox.ac.uk/www/garman/gindex.html
 -
The University of Oxford's Doctoral Training Centre has three available 
programmes for October 2012 entry: Life Sciences Interface Doctoral Training 
Centre; Systems Biology Doctoral Training Centre; and Systems Approaches to 
Biomedical Science Industrial Doctorate Centre.

To find out more, visit: http://www.dtc.ox.ac.uk/

Re: [ccp4bb] Expression of Viral proteins for crystallography

[Chun Luo]

Toxicity of target proteins in pET vectors can manifest itself without DE3.
Some people suggest E. coli polymerases causes low level of expression. The
observed mutation rate is thousands of fold higher than what the textbooks
say. It’s easy to tell toxicity from other causes of heterogeneity. Less
than expected numbers of colonies from transformation. The growth rates of
cultures from different colonies are quite different. Most mutations are
single nucleotide change (and single amino acid change) that reduces certain
functionalities of target proteins. However the mutations can be outside
those functional domains. In my experience with a few toxic proteins, the
mutations are evenly distributed, no over representation of any mutation.
Isolated wild type clones generate mutants after transformation.

Chun

 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Rubén
Sánchez Eugenia
Sent: Wednesday, January 25, 2012 8:52 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Expression of Viral proteins for crystallography

 

Dear Gregory and Darren,

Thank you for your answers. They have been very useful. 

2012/1/24 Gregory Verdon 

it possible that the protein is toxic (even when slightly expressed from
your possibly leaky pET vector), so that e.coli select for mutations that
kill expression of your recombinant gene ...

 

 

Gregory, the protein may be toxic, but I do not think that could be the
problem. BSJ cells are not meant to be for expression, but for cloning, so
they are not DE3 lysogenic. That is, they do not have the T7 RNApol
necessary for the expression of the proteins inserted into pET plasmids. 

In any case, thank you very much for you help.
 


2012/1/24 Darren Hart :

> I think the explanation is this:
> The source is natural viral RNA which is a mixture of naturally mutated
> sequences (e.g. flu forms such a quasispecies)
> See:
> http://www.virology.ws/2009/05/11/the-quasispecies-concept/
>
> The pooled RNA has an average sequence that you see when you sequence the
> pooled cDNA (individual mutations are hidden by the averaging effect of
> having many sequences present).
>
> But when you clonally separate DNA molecules by transformation (1 plasmid
> enters 1 cell to yield 1 colony), you see each individual molecule
> represented 100% in the sequencing chromatogram from the plasmid DNA that
> you have isolated from colonies.
>
> This is effect is commonly observed when sequencing influenza virus
isolates
> from patients. It will have nothing to do with the E. coli strain. You can
> avoid it completely by using gene synthesis.
>
> Darren




Darren, thanks to your information about the quasi-species, now we are
convinced that this may be the problem. We have been thinking about this and
we have conclude that the average sequence may not even exist and if it was,
it might not be the active one. That is to say, maybe we can not synthesize
our gene because we don't know whether the mutations are needed or in any
case what mutations would be needed. Do you agree? And if I am right, is it
the only solution to try different mutants to get the active one?

Anyway, I think I would not need to synthesize the gene because I can select
one monoclonal colony and delete the mutation by site-directed PCR
mutagenesis.

Best regards,

Re: [ccp4bb] Expression of Viral proteins for crystallography

[Phoebe Rice]

We've seen nice in vivo activity (on purpose) from proteins cloned under T7 
promoters but transformed into non-DE3 cells.  In fact, friends working with 
more zesty enzymes who wanted a more "tunable" in vivo assay have had to mutate 
the ribosome binding sites for proteins under T7 promotors to knock down 
expression in non-DE3 cells.   

Sometimes it doesn't take much of a protein for its activity to have an effect! 

=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.rsc.org/shop/books/2008/9780854042722.asp


 Original message 
>Date: Wed, 25 Jan 2012 09:48:55 -0800
>From: CCP4 bulletin board  (on behalf of Chun Luo 
>)
>Subject: Re: [ccp4bb] Expression of Viral proteins for crystallography  
>To: CCP4BB@JISCMAIL.AC.UK
>
>   Toxicity of target proteins in pET vectors can
>   manifest itself without DE3. Some people suggest E.
>   coli polymerases causes low level of expression. The
>   observed mutation rate is thousands of fold higher
>   than what the textbooks say. It's easy to tell
>   toxicity from other causes of heterogeneity. Less
>   than expected numbers of colonies from
>   transformation. The growth rates of cultures from
>   different colonies are quite different. Most
>   mutations are single nucleotide change (and single
>   amino acid change) that reduces certain
>   functionalities of target proteins. However the
>   mutations can be outside those functional domains.
>   In my experience with a few toxic proteins, the
>   mutations are evenly distributed, no over
>   representation of any mutation. Isolated wild type
>   clones generate mutants after transformation.
>
>   Chun
>
>
>
>   From: CCP4 bulletin board
>   [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of RubA(c)n
>   SA!nchez Eugenia
>   Sent: Wednesday, January 25, 2012 8:52 AM
>   To: CCP4BB@JISCMAIL.AC.UK
>   Subject: Re: [ccp4bb] Expression of Viral proteins
>   for crystallography
>
>
>
>   Dear Gregory and Darren,
>
>   Thank you for your answers. They have been very
>   useful.
>
>   2012/1/24 Gregory Verdon  
>
>   it possible that the protein is toxic (even when
>   slightly expressed from your possibly leaky pET
>   vector), so that e.coli select for mutations that
>   kill expression of your recombinant gene ...
>
>
>
>
>
>   Gregory, the protein may be toxic, but I do not
>   think that could be the problem. BSJ cells are not
>   meant to be for expression, but for cloning, so they
>   are not DE3 lysogenic. That is, they do not have the
>   T7 RNApol necessary for the expression of the
>   proteins inserted into pET plasmids.
>
>   In any case, thank you very much for you help.
>
>
> 2012/1/24 Darren Hart :
>
> > I think the explanation is this:
> > The source is natural viral RNA which is a
> mixture of naturally mutated
> > sequences (e.g. flu forms such a quasispecies)
> > See:
> >
> http://www.virology.ws/2009/05/11/the-quasispecies-concept/
> >
> > The pooled RNA has an average sequence that you
> see when you sequence the
> > pooled cDNA (individual mutations are hidden by
> the averaging effect of
> > having many sequences present).
> >
> > But when you clonally separate DNA molecules by
> transformation (1 plasmid
> > enters 1 cell to yield 1 colony), you see each
> individual molecule
> > represented 100% in the sequencing chromatogram
> from the plasmid DNA that
> > you have isolated from colonies.
> >
> > This is effect is commonly observed when
> sequencing influenza virus isolates
> > from patients. It will have nothing to do with
> the E. coli strain. You can
> > avoid it completely by using gene synthesis.
> >
> > Darren
>
>   Darren, thanks to your information about the
>   quasi-species, now we are convinced that this may be
>   the problem. We have been thinking about this and we
>   have conclude that the average sequence may not even
>   exist and if it was, it might not be the active one.
>   That is to say, maybe we can not synthesize our gene
>   because we don't know whether the mutations are
>   needed or in any case what mutations would be
>   needed. Do you agree? And if I am right, is it the
>   only solution to try different mutants to get the
>   active one?
>
>   Anyway, I think I would not need to synthesize the
>   gene because I can select one monoclonal colony and
>   delete the mutation by site-directed PCR
>   mutagenesis.
>
>   Best regards,

[ccp4bb] Position available: Research Technician, High Throughput Crystallisation

[Jose A. Marquez]

Dear All,

A Research Technician position is available at the High Throughput 
Crystallisation laboratory of the EMBL Grenoble Outstation.

You will find the details in the link below.

http://www.embl.de/aboutus/jobs/searchjobs/index.php?newlang=1&newms=sr&searchregion=670 

 



Best regards
Josan

--
_
Jose A. Marquez Ph.D.
The High Throughput Crystallization Laboratory
EMBL Grenoble Outstation
Postal address:   EMBL Grenoble Outstation, 6 Rue Jules Horowitz,  BP181
38042 Grenoble Cedex 9, France
Delivery address: EMBL Grenoble Outstation, Polygone Scientifique,  6 
Rue Jules Horowitz

38000 Grenoble, France
Phone +33 (0)476 20 74 25
Fax. +33 (0)476 20 71 99
https://htxlab.embl.fr
__