Re: [ccp4bb] problem in transformation of pqe 30 clone

- Protein crystallography

Main steps:

- Protein purification
- Crystallisation

Special:

- Programs for crystallography
- X-ray detectors

Basic tutorials:

   - Chemistry
   - Protein
   - Peptide
   - Amino Acids

Xtal community:

- CCP4BB

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: problem in transformation of pqe 30 clone
From: atul kumar atul {- dot -} kumar {- at -} IGIB {- dot -} RES {- dot -} IN
Date: 2009-05-04

Next message:
Subject: Re: problem in transformation of pqe 30 clone
From: Mensur Dlakic mdlakic {- at -} MONTANA {- dot -} EDU
Date: 2009-05-04

Subject: Re: problem in transformation of pqe 30 clone
From: Engin Ozkan eozkan {- at -} STANFORD {- dot -} EDU
Date: 2009-05-04

Have you tried M15[pREP4], which are the cells Qiagen would like you to
use? You can at least use pREP4 + your expression host, and have
repressor expression to prevent leaky expression. That can help you get
colonies of transformants.

Engin

On 5/4/09 7:04 AM, atul kumar wrote:
> xl1-blue is not an expression host,since i have cloned it
> successfully,i need to transform into expression host, i am able to
> transform it into dh5 alfa,but not in any of expression host
>
> ------------------------------------------------------------------------
> *From:* artem@xtals.org [mailto:artem@xtals.org]
> *Sent:* Mon 5/4/2009 6:32 PM
> *To:* atul kumar
> *Cc:* ccp4bb@jiscmail.ac.uk
> *Subject:* Re: [ccp4bb] problem in transformation of pqe 30 clone
>
> Hi,
>
> You are using a pQE vector which has a T5 promoter. T5 is a native-like
> promoter, recognized by the E. coli RNA polymerase - and this means that
> even with lac operatorsupstream there is a huge amount of leakage in this
> system. If your protein is even moderately toxic then you have issues.
>
> Your solutions are
> 1) to use cells with higher levels of lac repressor (XL1-blue for example)
> 2) to re-clone this ORF under some tightly controlled promoter
>
> Artem
>
> >
> > i have cloned my gene successfully into qiagen vector into pqe30 but
> i do
> > transformation of this into BL21,pLys,Rosseta,C41, i dont get any
> > colonies,comp cells are good other clones give good no of colonies upon
> > transformation.
> > can someone help???
> > thanks
> > atul
> >
> > -----Original Message-----
> > From: CCP4 bulletin board on behalf of
> Herman.Schreuder@SANOFI-AVENTIS.COM
> > Sent: Mon 5/4/2009 3:49 PM
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG
> molecule
> > crossing a two-fold crystallographic symmetry axis
> >
> > Dear Lidefeng,
> >
> > One "Tassos" afterthought; you are sure you traced the chain
> correctly? It
> > is not that both change make a crystal contact and then each go
> their own
> > (disordered) way?
> >
> > Best regards,
> > Herman
> >
> > -----Original Message-----
> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> > lidefeng
> > Sent: Friday, May 01, 2009 2:48 PM
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG
> molecule
> > crossing a two-fold crystallographic symmetry axis
> >
> > Dear Herman.Schreuder,
> >
> > Perhaps there is some misunderstanding about my question.
> There is
> > one molecule in the asymmetric unit (showed as symbos A). After the
> > 2nd crystallographic symmetric operation, another molecule appears
> > (symbol B). However, the density show that there is one peptide chain
> > cross the 2nd axis (symbol C). If we give chain C occupancy of 0.5,
> > only 50% of chain C belongs to molecules A and B, respectively. In
> > another word, , it looks like one half of chain C belongs to molecule
> > A and the other belongs to B. It means that one half of chain C
> > belongs to molecule A disappear, so do that belongs to molecule B.
> >
> > 2nd axis
> >

> > AAAAAA
BBBBBB
> > AAAAAA
BBBBBB
> > AA
BB
> >
/
> > CCCCCCC
CCCCCCC

> >

> >

> > Your sincerely
> > ????????De-Feng Li
> > ????????lidefeng@moon.ibp.ac.cn
> > ??????????2009-05-01
> >
> > Defeng Li, Dr.,
> > Email: lidefeng@moon.ibp.ac.cn
> > National Laboratory of Biomacromolecules, Institute of Biophysics,
> Chinese
> > Academy of Sciences,
> > 15 Datun Road, Chaoyang District,
> > Beijing 100101, China
> >
> >
> > ======= 2009-04-30 14:52:00 You writed in your letter:=======
> >
> >>Dear Feng-Li,
> >>
> >>The other half occupancy peptide is generated by crystallographic
> >> symmetry (the twofold), you need only to build one. To check that
> >> everything fits properly, you should switch on the crystallographic
> >> symmetry in coot. (Draw -> Cell & Symmetry).
> >>
> >>Good luck!
> >>Herman
> >>
> >>
> >>-----Original Message-----
> >>From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> >>lidefeng
> >>Sent: Thursday, April 30, 2009 3:44 AM
> >>To: CCP4BB@JISCMAIL.AC.UK
> >>Subject: Re: [ccp4bb] Peptide on two-fold axis - was:[ccp4bb] PEG
> >>molecule crossing a two-fold crystallographic symmetry axis
> >>
> >>Dear Tim Gruene,
> >>
> >> But how to illustrate the other one half occupancy of peptide?
> >> Disorder ?
> >>
> >>
> >> Your sincerely
> >>????????De-Feng Li
> >>????????lidefeng@moon.ibp.ac.cn
> >>??????????2009-04-30
> >>
> >>Defeng Li, Dr.,
> >>Email: lidefeng@moon.ibp.ac.cn
> >>National Laboratory of Biomacromolecules, Institute of Biophysics,
> >>Chinese Academy of Sciences,
> >>15 Datun Road, Chaoyang District,
> >>Beijing 100101, China
> >>
> >>
> >>======= 2009-04-29 10:55:00 You writed in your letter:=======
> >>
> >>>Hello De-Feng Li,
> >>>
> >>>first of all sorry for changing the subject: I think starting a new
> >>>thread from an existing one may hamper people who are going to search
> >>>the archives in the future, therefore it is good practice to give it
> >>>its separate subject line, even though it certainly is be very closely
> >>>related.
> >>>
> >>>In your case you can refine two peptides each with an occupancy of
> >>>0.5, one for each direction.
> >>>
> >>>Tim
> >>>--
> >>>Tim Gruene
> >>>Institut fuer anorganische Chemie
> >>>Tammannstr. 4
> >>>D-37077 Goettingen
> >>>
> >>>GPG Key ID = A46BEE1A
> >>>
> >>>
> >>>On Wed, 29 Apr 2009, lidefeng wrote:
> >>>
> >>>> Hi everyone,
> >>>>
> >>>> Following Chandrika's question, what should I do if one peptide
> >>>> chain crosses a two-fold crystallographic symmetry axis?
> >>>> The peptide is not symmetric and the sidechain of one Se-Met (two
> >>>> after CS operation) is determined and conformed by MAD.
> >>>>
> >>>> Your sincerely
> >>>> ????????De-Feng Li
> >>>> ????????lidefeng@moon.ibp.ac.cn
> >>>> ??????????2009-04-29
> >>>>
> >>>> Defeng Li, Dr.,
> >>>> Email: lidefeng@moon.ibp.ac.cn
> >>>> National Laboratory of Biomacromolecules, Institute of Biophysics,
> >>>> Chinese Academy of Sciences,
> >>>> 15 Datun Road, Chaoyang District,
> >>>> Beijing 100101, China
> >>>>
> >>>>
> >>>> ======= 2009-04-29 17:02:00 You writed in your letter?=======
> >>>>
> >>>>> Hello everyone,
> >>>>>
> >>>>> My protein crystallised in the spacegroup P6522 with one protein
> >>>>> molecule in the asymmetric unit. I have a PEG molecule from the
> >>>>> crystallization condition which crosses a two-fold crystallographic
> >>>>> symmetry axis. PEG is symmetric hence this does not violate the
> >>>>> crystal symmetry. However, this situation causes two problems
> which I
> >>>>> need to solve :
> >>>>>
> >>>>> First, How can I refine this structure ? I am using Phenix. Is there
> >>>>> a way to remove van der Waals repulsion between one half occupancy
> >>>>> PEG and its crystallographic symmetry mate ?
> >>>>>
> >>>>> Second, how do I submit this structure to PDB ? Do I include a full
> >>>>> PEG molecule at half occupancy even though one half is related
> to the
> >>>>> other via crystallographic symmetry ?
> >>>>>
> >>>>> Thanks,
> >>>>> Chandrika
> >>>>
> >>>> ========================================================
> >>>>
> >>>>
> >>
> >>========================================================
> >>
> >
> > ========================================================
> >
> >
> >
>

CCP4bb navigation

CCP4bb <-- 1999 <-- November 1999 <-- 30 November 1999

Previous message:
Subject: Re: problem in transformation of pqe 30 clone
From: atul kumar atul {- dot -} kumar {- at -} IGIB {- dot -} RES {- dot -} IN
Date: 2009-05-04

Next message:
Subject: Re: problem in transformation of pqe 30 clone
From: Mensur Dlakic mdlakic {- at -} MONTANA {- dot -} EDU
Date: 2009-05-04