Re: [ccp4bb] crystallizing fusion proteins

2021-03-15 Thread Pascal Egea
Hi Herman,
I will add a few points to all the excellent advice already provided to you.
If you decide to try this option of crystallizing a fusion protein of your
target, I would consider using N-terminal but also C-terminal fusions. We
have had success using MBP in N-ter and the superfolder GFP in C-terminal
for a few 'vexing' proteins from *Plasmodium falciparum. *It helped us
either to obtain crystals/structures (as you seek) but also provided a way
to circumvent a tendency for twinning from our target in some specific
cases.
GFP has the added benefit that your fusion crystals should be bright yellow
so that speeds up the screening process a bit.

I hope this helps. Good luck
Best regards,

Pascal Egea, PhD
UCLA School of Medicine


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Re: [ccp4bb] Data 3 A

2021-01-12 Thread Pascal Egea
Hi,
Have you looked at the packing of your crystal with that single molecule in
the ASU. Is it enough to build a crystal as in symmetry related mates
contact each other  (with probably quite a bit a solvent) but still a
crystal.
That would not be shocking since your resolution is average (on offense
intended).

Best,
Pascal Egea

On Tue, Jan 12, 2021 at 3:22 PM rohit kumar  wrote:

> Dear all,
>
> I am trying to solve a data with 3 A resolution, however data quality is
> very bad and mathews coffi. suggest  two molecules per ASU but It always
> gives one molecule in AU after phaser with the TFZ and RFZ score are 4.5
> and 3.5 respectively with LLG gain 121.
> And when I used that model for Refmac the final R/Rfee is 23/30 and with
> satisfactory Ramachandran statistics as well as electron density and model
> in agreement with each other  in coot. Does It mean that I have
> correct solution?
> Please suggest,
>
> --
> Regards
> Dr. Rohit Kumar Singh
> Postdoctoral fellow
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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-- 
Pascal F. Egea, PhD
Associate Project Scientist
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu



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Re: [ccp4bb] Cell disruption

2020-08-15 Thread Pascal Egea
Dear Bernhard ,
I would recommend the emulsified from Avestin. It is great . Depending on
your budget you can ask for stainless  steel (probe  to slow tear ) or
ceramic (longer lasting but a bit more expensive).
We have the ceramic but I have worked with the stainless steel one and it
is really good with E. coli cells. Not the best for
Yeast despite pressurizing more. Yeast are better dealt with bead beaters
or cryo-mill grinders.
3 passes are usually sufficient to process up to 250 ml of extract in a
decent time (15-20) minutes .

We operate ours at room temperature just cool the serpentine tubing coming
out of the chamber in ice . There is an option to add a cooling plate but
we have never considered it. Overheating comes from overpressurizing but
for E Coli at 15000 psi max  3 passes will do the trick without overhea tu
bf the sample. I have processEs successfully many membrane proteins And
protein complexes with it.

I have never seen one in a cold room to be honest but it should be fine
although I don’t think it would be necessary . I would Ask avestin about
that .
Maintenance and cleaning is simple . Once in a while I will replace the
seal, it s not very difficult to do. The whole assembly is metal so you can
sterilize the whole thing After complete disassembly in extreme cases .
This is a bit more involved but not overwhelming.
The people at Avestin are super nice and responsive ( Canadians) so they
will guide you if necessary . And we have had their engineers show up
sometimes to just check the instrument for free . I have had this one for
10 years in my lab now. And before that was using one for 7 years during my
post doc.  I would not lyse bacteria by any other method now.

I hope this helps.
All the best.
Pascal

On Sat, Aug 15, 2020 at 9:08 PM Bernhard Lechtenberg <
lechtenber...@wehi.edu.au> wrote:

> Dear colleagues,
>
>
>
> We are currently looking to purchase a cell disruptor/homogeniser mainly
> for routinely processing a few 100 mls of E. coli suspensions. With the
> current COVID-19 restrictions it is very difficult for us to test any
> equipment. I thus hope that some of you can share their experiences with
> the different models. I found a similar thread on the CCP4BB from 2013 but
> wondered if anybody had had some more up to date information.
>
>
>
> We are mainly looking at the Avestin Emulsiflex C3 homogeniser and the
> Microfluidics LM20 Microfluidizer. In particular we are interested to know
> more about ease of use, maintenance, reliability and if anybody operates
> these in a cold room (4°C).
>
>
>
> Thanks in advance,
>
>
>
> Bernhard
>
>
>
> 
>
>
>
> 
>
> --
>
> Bernhard C. Lechtenberg, Ph.D.
>
> Laboratory Head
>
> Ubiquitin Signalling Division
>
> The Walter and Eliza Hall Institute
>
> 1G Royal Parade
> 
>
> Parkville VIC 3052
> 
>
> Australia
> 
>
> Phone: +61 3 9345 2217
>
> Email: lechtenber...@wehi.edu.au
>
>
>
> ___
>
> The information in this email is confidential and intended solely for the
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> You must not disclose, forward, print or use it without the permission of
> the sender.
>
> The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of
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> Nation as the traditional owners of the land where our campuses are
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-- 
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Associate Project Scientist
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
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Re: [ccp4bb] Structural Importance of Methionine

2020-08-07 Thread Pascal Egea
Hi Samer,
In addition to what has been already mentioned to you, there are also the
two well illustrated cases of methionine rich domains.
- in the case of the M (for Met rich) domain of the Signal Recognition
Particle protein SRP54/Ffh involved in promiscuous binding of the
N-terminal hydrophobic signal sequences of nascent membrane proteins as
they exit the ribosome en route to the translocon and the membrane for
insertion or secretion.
- in the case of the ATP-ase Get3 (Guided Entry of Tail anchored proteins)
2 that also has an hydrophobic cleft enriched in Met residues also designed
to promiscuously bind the hydrophobic C-terminal transmembrane helices of
so-called Tailed Anchored proteins.
In both cases the substrates are hydrophobic helices and the greasy and
flexible side chains of Methionines of the receptor protein (SRP54/Ffh or
Get3) can accommodate a variety of hydrophobic substrates in the protein
binding clefts.

I hope this helps you.

All the best,

Pascal

On Fri, Aug 7, 2020 at 5:14 AM samer halabi <
30c2162795b2-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear All,
> I am working on structures where Methionine is important in binding of
> peptides to the MHC protein complex.
> Would anyone kindly like to share their knowledge about anything they find
> it important about this particular amino acid structurally? Sharing a paper
> or just few comments will be greatly appreciated.
>
> I know my question may sound very general (and kind of superficial) but
> there is definitely a reason, that I don't know and might be already known,
> why certain peptides (like CLIP) are rich in Methionine, and that lowers
> their affinity of binding.
> Thank you and sorry to disturb you all.
> Best regards,
> Samer
>
> --
>
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-- 
Pascal F. Egea, PhD
Associate Project Scientist
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu



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[ccp4bb] Post Doctoral Position at UCLA

2018-12-18 Thread Pascal Egea
Dear All,



A NIH-funded post-doctoral position is available in my group at UCLA to
study the structure, mechanisms, and functions of membrane protein
complexes involved in protein or phospholipid trafficking in eukaryotic
pathogens (such as fungi and parasitic protozoa) as described in:

AhYoung, Jiang et* al.* in *PNAS* 2015 (doi: 10.1073/pnas.1422363112).

Ho, Beck et* al.* in *Nature* 2018 (doi:
10.1038/s41586-018-0469-4).

Motivated and qualified candidates should hold a PhD in the field of
structural biology and be proficient in cloning, protein purification, and
structure solving by either crystallography or cryo-electron microscopy.
Experience with expression in insect cells and/or HEK293 cells is strongly
preferred.



Applicants should address their CV, a cover letter stating their
accomplishments, interests, and career plans together with the addresses of
three potential references to *pe...@mednet.ucla.edu
.*



Thanks.



All the best,
Pascal Egea

-- 
Pascal F. Egea, PhD
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu



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Re: [ccp4bb] Should I optimize these crystals?

2018-10-24 Thread Pascal Egea
Hi Marta,

1-- There is always a possibility of forming detergent/lipid (cholesterol
in your case) -containing crystals combined with other small molecule(s)
(organic or not).
Although you did not specify the width of the oscillation used to collect
the frame displayed in your mail (was it 45 degrees or 0.5 degrees?) I see
signs of a lattice at least on the vertical direction (3 rows can be
distinguished it seems), can you derive a "cell-dimension" along that
direction?
2- If you have access to a fluorescence-coupled light microscope (like a a
PRS-1000 Korima instrument) maybe you could see if your crystals glow
(provided your proteins contain tryptophans). Crystals from drop 1 seem
better candidates (they look 'thicker') for a fluorescence scan, the ones
from drop 2 are really thin and curvy, so it might be difficult to see in
that case. This could help you characterizing and ruling things out more
easily.
3- You mentioned that you analyzed the content of washed/dissolved crystals
by silver-stained SDS PAGE. Forgive me for asking if you also analyzed the
last wash step on this sivler-stained SDS PAGE gel to rule out that the
protein signal you observed was not  cross-contamination by a carry-over
effect.

Good luck.


Good luck



On Wed, Oct 24, 2018 at 1:31 PM marta borowska 
wrote:

> Dear all,
>
> I have grown crystals of a membrane protein complex, that I initially
> verified on SDS-PAGE. These crystals grew only if membrane protein
> component is there. The condition is polypropylene glycol 400,
> cryoprotected with ethylene glycol, sample buffer has 150mM NaCl on top of
> detergent with cholesterol. These long thin needles are quite sturdy and
> either didn't diffract or diffracted like small molecule (I cannot exclude
> the possibility of contamination stuck around crystal). After the
> synchrotron trip harvested crystals were washed (most did not dissolve in
> Urea or NaOH!), dissolved in 10% mild detergent and still showed protein
> from 4-6 crystals when analyzed on Silver Stain SDS-PAGE.
>
> I would appreciate your input on whether some of you encountered similar
> patterns and if you think I should proceed with more optimization on these
> crystals.
>
> Thank you,
> Marta
>
>
> --
>
> Marta T. Borowska
>
> Graduate Student
>
> Adams Lab
>
> Department of Biochemistry and Molecular Biology
>
> The University of Chicago
>
> 929 E. 57th Street
>
> Chicago, IL 60637
>
> Lab: GCIS W229
>
> Lab phone: 773-834-0660
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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-- 
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Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu



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Re: [ccp4bb] suggestions for cryoprotectant

2018-10-19 Thread Pascal Egea
Hi Firdous,

I noticed that conditions 2,4 and 5 are overall harsh agents (chaotropic
agents such as KSCN (probably not KCN) and Nitrate ions ), in my own
personal experience I have never been very lucky with crystals grown in
nitrate ions.
Condition 1 however looks a bit more soft with 'gentle' ions.
May I suggest trying a sodium malonate screen or seeding in such a
crystallization agent and maybe trying to cryprotect those acetate crystals
in malonate which is a cryo-friendly so-called 'magic salt'. crystals grown
in carboxylates such as citrate and malonate are usually easier to
cryo-protect.

Hope this helps

Pascal Egea

On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique 
wrote:

> Dear members
>
> I have got beautiful crystal hits in SaltRx screens which are not
> diffracting to a good resoultion. All of them are salt based condition and
> I am not able to formulate a good cryoprotectant for these crystals. I also
> think that in my case the poor resolution is due to a poor cryoprotectant
> selection.
>
> The conditions are as follows:
>
> 1> 4M Ammonium Acetate 100mM Bis Tris Propane pH 7.0
> 2>0.5M KCN 100mM Tris pH8.5
> 3>1.5M LiSo4 100mM Bris Tris Propane pH 7.0
> 4>4M Sodium Nitrate 100mM Tris pH8.5
> 5>1.5M Sodium Nitrate 100mM Sodium acetate pH 4.6
>
> There are few more conditions but so far not able to see good diffraction
> with using lower peg and glycerol based cryoprotectants.
>
> Can anybody suggest me good cryos conditions for salt based
> crystallization conditions or anything good for SaltRx crystallization hits.
>
> Thanks
>
> Firdous
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu



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Re: [ccp4bb] Protein toxicity or low expression

2017-12-09 Thread Pascal Egea
Hi Firdous,,

As you mentioned they are many things to change and try.
before changing vector it is more efficient in my opinion to change
expression conditions (temperature, amount of IPTG, length of culture),
culture medium (regular ones against M9 or  auto induction media) and
expression strains BL21, BL21gold C43 and and BL21 Lemo cells.

Then you can change vector (arabinose or rhamnose controlled promoters) and
fusion (MBP or GST maybe).

Leaky expression can have deleterious effects on expression levels and
culture stability/degenerescence (especially dependent on the type of
antibiotic you are using). Tightening up the expression control maybe
important in the case of a toxic target). BL21 Lemo cells can be very
useful in those cases.

If your gene has an odd codon usage (compared to your expression host) you
may want to think about recoding it or use rare tRNA plasmids to try to
compensate for that.

The fact that you mentioned that you see the Sumo-his but not the following
target protein is worrisome. Since you mentioned the vector is fine I would
think that there is a problem of translation/folding of the target
sequence, maybe check the presence of rare codons that could promote
pausing for example. I don t know how easy it is to check for sequences
that would affect the local structure of the mRNA but codon optimization
programs should take this into account.

Good luck

On Sat, Dec 9, 2017 at 5:27 PM, Firdous Tarique 
wrote:

> Hello everyone.
>
> I am struggling with the solubility or expression of my proteins in
> BL21DE3 E.coli expression cells. In one of my construct I have a Sumo-His
> fusion at the n-terminal of my protein. After induction what I see is the
> expression of the Sumo tag only. My sequencing results are fine and there
> is no mistake in cloning. I wonder why I am seeing only the expression of
> the Sumo tag although my fusion protein is in frame with this tag.
> My second question is related with the very low expression of one of my
> gene. A brief literature search suggests so many things to improve the
> expression like changing the host, vector, media etc. It is a nuclease and
> cloned in vector with a Sumo-His tag on it. I am able to purify the little
> amount from E coli BL21DE3 host. The problem is related with low expression
> which is clearly observed in difference in the pre and post induction
> lysate on SDS PAGE. Out of so many option available in the literature I am
> confused what to  try first. Any general idea?
>
> Your suggestions can help a lot.
>
> Kahkashan
> Ph.D student
> Delhi University
> India
>



-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu


Re: [ccp4bb] the purification process of protein, used for crystallization

2017-11-17 Thread Pascal Egea
Hi liuqing,

It is more usual to finish with a sec because you control the composition
of the final conditioning buffer of your sample and remove any aggregates.
 We personally favor the following sequence
iMac - desalt - iex if necessary - sec

Desalt meaning one of those small Pd10 like column that enable the quick
removal of imidazole and conditioning of your protein for either cleavage
with a protease of you wish to remove tags and/or lowering of salt
concentration to bind on an iex column if u wish to do one.

That said . In some cases
Doing a real sec straight after IMac has the advantage to remove some large
molecular weight contaminants ( usually DNA) and some aggregates that are
annoying.

These choices depend on your target of course and the level of abundance
too.

Best,
Pascal Egea, PHD
UCLA Geffen School of Medicine

On Fri, Nov 17, 2017 at 5:46 AM Liuqing Chen <519198...@163.com> wrote:

> Hello everyone!
> I have listened someone suggested that,  first use affinity chromatography
> (Ni-NTA),  then use SEC (superdex200 increase),  and finally used ion
> exchange (monoQ),   to purified protein,  which will be used to
> crystallization.
> My question  is why  the monoQ used in the finally step,  why not the SEC
> used at the finally step?
>
> sincerely
> Liuqing Chen
>
-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu


[ccp4bb] MR phasing using Negative Stain EM reconstruction

2016-10-24 Thread Pascal Egea
Dear All,

I would like to know if it is possible to use a low resolution EM
reconstruction of a complex obtained in negative stain EM (not cryo EM) to
help molecular replacement in a 4.5A resolution X-ray diffraction data set
of the same complex
I am aware of the possibility of using low resolution cryoEM maps for MR as
described in the review from Jackson et al in Nature Protocols but I was
wondering if there is an intrinsically impossibility for negative stain
reconstructions.

Any thoughts or advice will be greatly appreciated.

Best,

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pegea at mednet.ucla.edu


[ccp4bb] fluorescence question

2015-04-23 Thread Pascal Egea
Dear All,

This is more a biochemical/biophysics question but since we need to
complete our structural analysis with functional or biophysical data, I
thought I would ask. We crystallized a protein as an MBP fusion and solved
its structure. Based on previous knowledge we know that the small protein
binds peptides. So we are now trying to measure binding of the protein to
peptides labeled with a fluorophore using fluorescence anisotropy.
Unfortunately the protein has to be used  with its MBP still attached.

 We have a resilient problem with the MBP carrier. It has some ‘residual’
fluorescence that interferes severely with our measurements (we are using
tetramethyl rhodamine as fluorescent reporter).

I was curious to know if anyone else had encountered this problem and
figured out a solution. Any suggestion will be greatly appreciated.


Many thanks in advance.


Pascal Egea

Assistant Professor of Biological Chemistry

UCLA David Geffen School of Medicine


Re: [ccp4bb] Zinc binding protein expressed from insect cells

2014-08-15 Thread Pascal Egea
Hi Heng,

DTT can react with metal cations such as Zinc or Iron. This is why people
to tend to use little DTT or no DTT at all on metal affinity columns or
replace it b-mercaptoethanol or TCEP that do not interfere.
Regarding the incorporation of Zinc into the culture media.
I recall that Zinc finger domain expression in E coli (for nuclear
receptors dna binding domains) is classically described to be done in
presence of Zinc acetate added into the culture media (around 100
microMolar I think ) this is already a lot of zinc (more might be toxic to
the cells).

depending on what your process of purification is.
I would try to add zinc in the expression media (this has probably been
descrived also for insect cells)
if you use a Ni or Co chelating column I would NOT add any free zinc at
this stage, you can, if you wish it, add it to eluate of your affinity
column and to gel filtration buffers or ion exchange buffers.

That said, I would suspect that  having enough zinc around is mostly
beneficial at the expression stage as the protein folding machinery is
dealing with your target protein.zinc misloaded protein is likely to be
unsoluble.

for nuclear receptors DBDs there are protocols describing reincorporation
(or even exchange) of metal ions inside the domain (maybe even starting
from inclusion bodies) but the more cysteines you have the more likely it
is to be difficult to get the right folded protein back. so I would
rather favor a strategy trying to get as much folded protein as possible by
natural means (let the cells do what we biochemists still don t know to do
very well).

if you add free zinc I would be careful with the concentration and the pH
of your buffers. at basic pH zinc and other ions (such as Ca and Fe) form
insoluble hydroxydes.

if you have managed to purify some protein I would try to do some emission
spectroscopy to see what ions are bound (zinc and or iron ) you may be
surprised by what you will see.

sorry for the lengthy response but I hope this helps.
all the best,

Pascal Egea



On Fri, Aug 15, 2014 at 8:03 AM, Harvey Rodriguez 
h.rodriguez.x...@gmail.com wrote:

 Dear all,

 Sorry for the non-crystallographic question. Currently I am working on a
 zinc binding protein which is expressed in insect cells and may contain 4-6
 zinc ions. As we know, so many zinc binding proteins can absorb the iron
 ions from the culture medium and the protein looks from yellow to dark red
 when concentrated. But when I concentrate the protein, I didn’t see the red
 color even in the very high concentration. I am just wondering if a zinc
 binding protein is expressed from insect or mammalian cells, can the zinc
 binding sites grab the irons instead of zinc or the zinc binding site can
 be empty loaded if there is not enough zinc in the culture medium? If so,
 do I need to include some zinc salt into the culture medium when doing
 expression or I can add some zinc ions when purifying? Usually, how much
 zinc and at which step of purification can we add the zinc into the
 solution when doing purification?

 Another question is that we know DTT can react with the heavy atoms to
 form the insoluble sulfide precipitates and if the zinc binding protein is
 purified with DTT at a final concentration of 1-5 mM, can it strip the zinc
 ions from the protein?

 I am appreciated if someone has this kind of experimental experiences and
 thanks in advance!

 Heng




-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Difficult MR with MBP fusion protein

2014-05-18 Thread Pascal Egea
Dear Niu,

It is very unlikely that MBP will be disordered. We use that protein a
standard for SAXS calibration and gel filtration it is extremely well
behaved.
There is an excellent review about MBP enhanced crystallization (by Moon et
al)  and you can do a quick survey of the PDB to find the cornucopia of
structures obtained using this strategy. I would suggest maybe an ensemble
MR search in phaser using a sum of all conformations adopted by MBP in
these different structures. You have 4 A data and it is difficult sometimes
to pull out the solution at low resolution. Trimming the few disordered
loops in the MBP model could help you, so you avoid throwing away the good
solution because of a few clashes when it packs solutions after rotation
and translation searches.
We recently solved one structure MBP + target protein of about 10 kDal
(2.7A) and it worked extremely well. A linker of 30 aminoacids connected
the target to MBP and it ended up tightly packed against the carrier MBP
protein even if  we were still missing about 25 residues of linker that was
actually part of the natural sequence of the protein (we had to generate 5
constructs to get it right).
Another approach, may not be smart but it should get the job done, is to
selenolabel your fusion and crystallize it. MBP has 5 or 6 methionines ,
even if you target has none, this will be enough to phase (although you are
only at 4 A if I understood well). That should enable you to lock the MBP
in position and see what is going on in your crystals.

Good luck

Pascal Egea


On Fri, May 16, 2014 at 8:03 AM, Niu Tou niutou2...@gmail.com wrote:

 Dear All,

 Recently we collected some data of a MBP fusion protein, at around 4A
 resolution. The protein itself is about half of the MBP size. However when
 we tried to solve it with MR, it failed. We tried to use MBP alone,
 homology model of target protein alone, and MBP+model. It is very strange
 that MBP alone can not yield any reasonable solution at all, so does
 searching with MBP and model together. While searching with model alone
 could get some better results, but when fix it to search MBP, it failed.
 There are 1 molecule per ASU with solvent content 55%. The spacegroup
 should be right and we tried to search all possible alternatives in each
 run, we also tried to lower it down, but did not work either. When running
 Phenix.phaser, there is a warning at the beginning saying eLLG suggests
 placing of ensembles will be very difficult.

 I wonder if anybody has encountered similar situation before. Any
 suggestions will be greatly appreciated!

 Regards,
 Niu




-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] off-topic: bug busting

2014-02-04 Thread Pascal Egea
Hi Phoebe,

Another possibility is the Emulsiflex (from Avestin, in canada). Not a
cheap piece of equipment, but very sturdy and efficient to up to 200 mL of
extract (runs on house-air). Can deal with E coli (and even yeast if you
have the models with internal compressor). It comes in 3 sizes I think we
have the middle one (C-3) with a compressor. It is basically a french press
but without the inconvenient of being french (I am french myself). It is
quite gentle, and does not overheat samples as much as the sonicator. We
make a lot of membrane protein purifications and I have been working with
this since my post-doc
Hope this helps.

Best regards,

Pascal Egea


On Tue, Feb 4, 2014 at 8:49 AM, Phoebe A. Rice pr...@uchicago.edu wrote:

  Some time ago, there was a nice discussion of cost-effective, wimpy
 protein-friendly ways to break open E. coli.  We're thinking about
 replacing an aging sonicator.  If people have a favorite gizmo, could
 they repeat that advice?
 thank you,
   Phoebe Rice

  ++

 Phoebe A. Rice
 Dept. of Biochemistry  Molecular Biology
 The University of Chicago

 773 834 1723; pr...@uchicago.edu
 http://bmb.bsd.uchicago.edu/Faculty_and_Research/

 http://www.rsc.org/shop/books/2008/9780854042722.asp




-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


[ccp4bb] membrane protein and phase separation

2013-08-01 Thread Pascal Egea
Dear All,

I have a question tailored for the membrane protein and detergent folks. We
are purifying a membrane protein that associates into an homoligomeric pore
and we have been successfully preparing it in two detergents: FC-12 or a
mild lipid. The two Protein Detergent Complexes look very homogenous by SEC
and can be concentrated without protein loss on membranes with MW cutoffs
(100kDal) way larger that the expected their respective free detergent
micelles.
Everything looks good so far... until we get to the crystallization stage.
While the PDC in FC12 does not tend to form too much phase separation, the
PDC in the lipid does. This looks a bit odd to me since these lipid
micelles are supposed to be a bit smaller than the FC-12 micelles. We are
working at twice the CMC and besides lowering the detergent concentration,
I am a bit perplex about what I am observing. Intuitively I would have
expected to observe the reverse behavior: worst in FC-12 than the
lipid. This lipid is a very mild solubilizing/reconstituting agent that has
already been successfully used for structure determination. Any advice or
thoughts will be greatly appreciated. Is this something that some of you
have already observed?

Many thanks in advance,

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


[ccp4bb] post-doctoral in membrane protein structural biology

2013-07-18 Thread Pascal Egea
We are seeking a post-doctoral researcher to join my research group at
UCLA. The lab focuses on the structural studies of challenging targets:
membrane protein complexes and channels from eukaryotes (yeast and several
parasites) and seeks to characterize their architecture and molecular
mechanisms of action combining X-ray crystallography, SAXS and cryo-EM when
necessary and possible.

 We will exclusively consider applicants with proven experience in
protein crystallography, cloning, protein expression and purification. Previous
experience with membrane proteins is highly desirable but not required.
Candidates should hold or expected to soon hold a PhD degree in a relevant
area (Biophysics, Structural Biology) and have excellent social and
communication skills in english (very important). Interested candidates
should send a CV and personal statement together with the name and
addresses of three references.

   I will be attending the ACA-2013 meeting in Hawaii this coming week and
will present a poster presented from 5:30-07:30pm on Sunday, July 21.
Motivated individuals are invited to directly contact with me.


Pascal F. Egea, PhD
-
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Membrane Protein Optimisation

2013-05-09 Thread Pascal Egea
Hi Rhys,

I suspect that what you call a gel might be phase separation (correct me if
this is wrong) like an oily phase enriched in protein and detergent. you
may have too much detergent in your drop.
may I ask what detergent you are using (low or high CMC) and at what
concentration of detergent do you think you are when you setup your drops.
you might have concentrated more detergent than you think along your
protein. the MW cutoff of the membrane you are using to concentrate is
important relative to the size of the free detergent micelle and of course
the protein-detergent micelle you are trying to concentrate.
too much detergent staying around is a major cause of trouble (i.e. poor
diffraction and phase separation competing with productive crystal growth)
besides many other parameters

Hope this helps

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER r.grinte...@research.gla.ac.uk
 wrote:

 Hi All,

 A quick question if you've ever worked on membrane proteins, I'm trying to
 optimize crystals for bacterial integral outer membrane protein I'm working
 on. I'm getting some fairly modest rod like crystals in a  0.1M Tris pH
 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of
 birefringent gel forming in the same condition, I get the feeling that this
 is Detergent/Protein complex and is robbing my crystals of material to grow
 bigger.
 These crystals diffract to 5 A so I'd quite like to make them bigger and
 better,

 Cheers,

 Rhys


[ccp4bb] gelification of a pure protein

2013-04-22 Thread Pascal Egea
Dear All,

I am presently faced with a peculiar case in the lab. We are expressing a
protein in E. coli and we are able to express it as a fusion
protein without problems . Fusion cleavage goes well and the final product
looks homogenous by size-exclusion chromatography with the expected
molecular weight. There are no signs of aggregation. However when we lower
the salt concentration by dialysis then the protein forms a gel.
transparent , optically clear, with no fluffy material (in the cold room).

Gelification seems to occur when we lower the concentration below 100 mM
NaCl. This protein has a fairly high pI (~9.0). Attempts to reverse the
process by gentle heating or salt addition have been so far unsuccessful.
It is not a thermophilic protein. We have not been able to obtain crystals
so far.

Has anyone already observed this kind of behavior and/or have any
suggestions?

Many thanks in advance .

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] gelification of a pure protein

2013-04-22 Thread Pascal Egea
Thanks to All for the diligent answers to my query,

The protein is not thermophilic and has only one cysteine. We are working
in presence of freshly added reducing agent and glycerol to promote
solubility (well kinda it seems).
This is not an RNA or DNA binding protein and it has no low-complexity
regions except at the N terminus, there maybe some left over from a cryptic
transit peptide (somehow basic) that supposedly targets the protein to a
specific organelle. We are probably going to truncate further to see if it
solves our problem

I appreciate all the comments and suggestions,
Cheers,

Pascal

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag

2013-02-20 Thread Pascal Egea
Hi Raji,

Thrombin is a rather good protease and behaves well in a large set of
different detergents ( there is a paper by Michael Wiener that describes
the relative efficiencies of several usual proteases, amongst those
thrombin is inlcuded, used routinely for cleavage of membrane protein
fusions in detergents. This is a rather extensive survey henceforth it is
very informative.

The variable detergent sensitivity of proteases that are utilized

for recombinant protein affinity tag removal

James M. Vergis 1, Michael C. Wiener


in

Protein Expression and Purification 78 (2011) 139–142


thrombin tends to be sensitive to reducing agents so I would stay away from
DTT and TCEP , b-mercapto is acceptable in reasonable amounts. We cut in
salt concentrations as high as 500 mM (NaCl) at pH 7.5-8.5 with 5-10%
glycerol around

no chelating agents should be present and imidazole in our hands tends to
be an inhibitors (probably because it has some chelating/complexing
activity).

we have good cleavage in DDM , OG and somehow more difficulties in
foscholines but it still cuts reasonnably well given the cost of the enzyme
and the targets,


the most crucial parameter is Protease/target ratio and incubation time and
temp. you can do trials on small scale digests in PCR tubes at different
temperatures. we usually cut at 4 or room temp.


I hope this helps,


Best regards,


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Off-topic: heterologous co-expression in yeast

2012-11-19 Thread Pascal Egea
Hi Andre,

There is set of plasmids allowing coexpression in yeast
they are described in the following article

Constructionand characterization of bidirectional expression

vectors in Saccharomyces cerevisiae

Aimin Li1,2, Zengshan Liu1, Qianxue Li2, Lu Yu1, Dacheng Wang2  Xuming Deng
1,2


in FEMS Yeast Res 8 (2008) 6–9 !c


Those plasmids are designed for coexpression and use two different
promoters a constitutive one (GPD) and a strong inducible one (GAL). You
have several selection markers and they are shuttle vectors for easy
manipulation and amplification in E. coli.


Hope this helps.


Best of luck



-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] 24 well screw-cap crystallization plates

2012-03-27 Thread Pascal Egea
Hi Brad,

I am afraid that there is no alternate source for these plates. The screw
cap system , I believe, was patented by the canadian company NEXTAL that
was then assimilated by Q...N and the patent is probably still holding.

Pascal


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


[ccp4bb] recombinant sources of N-glycanase

2012-03-11 Thread Pascal Egea
Hi All,

We are trying to deal with a eukaryotic membrane protein that crystallizes
but, as usual, diffracts poorly so far. As it is heavily glycosylated, we
are considering enzymatic deglycosylation.
Does anyone know about a recombinant source of N-glycanase ; we would like
to prepare it ourselves because the commercial sources are apparently too
expensive.

Thanks in advance.

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


[ccp4bb] Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues

2012-02-01 Thread Pascal Egea
Hi ,

To add to the previous comments,
crystallization of GTP or ATP (or their analogues) with their kinase/ A- or
G-tpases can depend on a lot of factors that were mentioned (such as
packing).

A simple common problem is that ATP solutions should be carefully buffered
prior to their use for soaking, people tend to forget about this. A 100 mM
ATP solution is pH 3 (probably not good for your protein and it has 3
acidic groups)

For some classes of ATP binding proteins, acidic pH have also been shown to
lower chances of successful soaking or co-crystallization.
The crystallization condition is also important. High concentrations of
sulphates or phosphates tend to complicate things . Same thing for high
concentrations of di or tri carboxylic acids (such as citrate, tartrate or
malonate). Sulfates tend to occupy the beta phosphate binding sites and at
high concentrations they can outcompete an analogue.
For first hand experience, I would not assume that all analogues behave the
same. Especially between AMPPNP, ATPgammaS and AMPPCP (or their Guanine
counterparts). the Cp analogues in our hands tend to have lower affinities.
You can always try ADP AlF4 combination or ADP BeF3, if you are not afraid
of beryllium .

Hope this helps

Good luck


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
Boyer Hall room 356
611 Charles E Young Drive East
Los Angeles CA 90095
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

2011-06-16 Thread Pascal Egea
I would like to add something about the NanoDrop versus NanoPearl,

I don't think that the path length is fixed on this instrument (the
NanoDrop) since if I recall well, the instruments sets the path length as it
scans through the droplet, hence the characteristic clicky noise that you
hear as the handle moves. This instrument requires recalibration every year
(or so according to the vendors, and this is of course not cheap) since
there is a moving part that can get out of alignment.
On the other hand, the NanoPear from Implen as a fixed geometry for the same
tiny amount of sample required. Thus you do not have to deal with moving
parts and recalibration.
We just bought a NanoPearl and I should also mention that this instrument
does both things: nano-drop size measurement like the NanoVue AND cuvette
size measurements (for OD600 or old school Bradford).

All the best,

On Thu, Jun 16, 2011 at 4:57 PM, Shaun Lott s.l...@auckland.ac.nz wrote:

 Just to add my 2c worth...

 The department here has a couple of nanodrops as a shared facility, one for
 DNA/RNA and one for protein. It has been noticeable that over time people
 has been getting decreased reliability of measurements on the latter machine
 cf cuvette measurements, presumably due to the build-up of protein deposits
 over time - so I would say that although it's easier to clean than a
 cuvette, the nanodrop is not immune to the problem. The biggest issue I see
 with the nanodrop is evaporation of sample. Even here in moist Auckland,
 where RH is very often 80%+, taking a series of measurements with the
 nanodrop over a period of just a minute or two shows increasing
 concentration in the sample. So, for consistent results, one has to be
 careful to measure quickly. It's probably fine for comparative measurements,
 but as has been observed above, not great for super-accurate values for
 biophysics, and I think rather operator dependent. But all our students are
 super-careful, right? ;) Worth to note also that ProtParam calculates
 extinction coefficients based on Gill  von Hippel, (Gill, S.C. and von
 Hippel, P.H. (1989) Calculation of protein extinction coefficients from
 amino acid sequence data. Anal. Biochem. 182:319-326) who claim accuracy of
 ~5% for 'normal globular' proteins without extra chromophores. Whilst on
 this subject, I would put in a plug for the good old BCA (aka Pierce) assay
 for protein concentration. It's a little slow, but gets away from sequence
 dependency somewhat as it is primarily dependent on the peptide backbone
 rather than sidechains  and works well in micro-titre plates etc. It is
 certainly very superior to Bradford. (Smith, P.K., et al. (1985).
 Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1):
 76–85. doi:10.1016/0003-2697(85)90442-7).

 cheers

 Shaun




-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] GTP Agarose Resin

2011-06-13 Thread Pascal Egea
Hi Matthew,

Most GTPases required Magnesium to hydrolyze so you maybe able to reduce
this by working in presence of EDTA and absence of magnesium. This may
promote removal of traces of GTP or GDP ( from previous experience with SRP
GTPases I would be more worried about residual GDP). Having EDTA and no
magnesium during purification helps.
If your GTPase has a very low basal GTPase activity ( and some do as they
require a cognate GAP to really get in the mood to hydrolyze) this might be
enough to minimize hydrolysis on this resin.

Good luck,


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu

On Mon, Jun 13, 2011 at 12:21 PM, Matthew Bratkowski mab...@cornell.edu
 wrote:

 Hi.



 I was considering using GTP Agarose Resin for the final clean up step of
 the purification of a GTPase and was wondering if anyone has had experience
 using this resin.  My main concerns are whether it actually has a decent
 binding capacity for GTP binding proteins, considering that endogenous
 GTP/GDP may remain tightly bound during purification, and if an active
 GTPase would bind without cleaving the GTP off of the resin itself.

 I found only a few companies that still carry the resin, but the price for
 each is very different.  The resin from Sigma is fairly cheap and is linked
 to the resin via ribose hydroxyls, while the resin from Innova Biosciences
 is more than four times as much but is linked to the resin via the gamma
 phosphate, which supposively prevents cleavage by contaminating
 phophatases.  Considering that my protein should be relatively pure during
 this purification step, I was wondering whether or not GTP cleavage of the
 resin by the GTPase and loss of binding to the column would be a problem if
 using the Sigma resin.

 If anyone has any other information about purification using this resin,
 such as resin binding capacity, an effective protocol with relevant buffers,
 and the lifetime of the resin after regeneration, I would be happy to hear
 it.

 Thanks,
 Matt



[ccp4bb] expression of membrane proteins as GST fusions

2011-05-27 Thread Pascal Egea
Dear All,

This is not strictly a crystallography related question.
We are trying to express several membrane proteins and were considering the
use of GST as a fusion partner instead of the HIS or FLAG purification tags.
I would like to know if anyone had any experience (positive or negative) to
share with us.

Many thanks in advance.

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
364 Boyer Hall
Molecular Biology Institute
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
364 Boyer Hall
Molecular Biology Institute
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Hydrophobic protein surface and SDS page

2011-05-26 Thread Pascal Egea
Hi Debajyoti,

Migration of proteins in SDS containing gels is dependent on hydrophobicity,
the amount of SDS that your protein binds (despite the fact that in theory
all proteins should behave the same way under these conditions) and charge.
Highly basic or acidic proteins will migrate anomalously, thermophilic
proteins (which are usually more highly charged and hydrophobic) also tend
to migrate anomalously..membrane proteins also because they usually tend to
interact differently with the detergent; it is not unusual to observe stable
non-covalent homo-oligomers of membrane proteins even in an SDS-PAGE gel.
Phosphorylation and glycosylation will also affect the apparent MW as
estimated from the SDS-PAGE experiment.

What you observe is not unusual at all.

If you want to be sure of your MW, Mass Spec will tell you what you want to
know.

Hope this helps, good luck

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] crystallization of a weird protein

2011-05-20 Thread Pascal Egea
Hi Wei,
this milk is precipitated/microcrystals of SDS probably with some phosphate
since you are in PBS buffer.
I would have two suggestions.
1-Can't you find a better detergent than SDS for your membrane protein? Have
you run a detergent screen for this protein to find a milder and more
crystallization friendly detergent for the reconstiution/purification and
handling of your sample. SDS is very very rarely used for membrane protein
crystallization.
2- I would try to avoid preparing a protein for crystallization in a
phosphate containing buffer (unless you have no choice). Phosphates tend to
yield more salt crystals at the screening stage.

Hope this helps.


 --
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
356 Boyer Hall
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] highly glycosylated protein

2011-05-13 Thread Pascal Egea
Hi Wei,

Glycosylation usually stabilize proteins although it is a source of
structural heterogeneity for us crystallographers.Since you are expressing
in HEK293 cells, there is a strain of cells that is deficient for
glycosylation (it was designed by Gobind Khorana at the MIT I believe). You
may want to try this. This is particularly useful when you express membrane
proteins, it avoids hyperglycosylation. You may want to try a lightly
glycosylated version of your protein and see if it behaves correctly,
The other extreme solution is to identify all occupied sequons in your
protein and eventually inactivate them by mutagenesis to have a completely
deglycosylated protein. This solution is probably not the best since
glycosylation usually stabilize proteins and may be essential to their
biological function and activity. So it is to be considered with a lot of
caution.

Hope this helps.


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
356 Boyer Hall
office (310)-983-3515
lab  (310)-983-3516
email   pe...@mednet.ucla.edu


Re: [ccp4bb] reproducibility of protein crystals

2011-05-02 Thread Pascal Egea
Anita,

Proteolysis and oxydation are the most common alteration affecting proteins
during the course of crystallization.
If you have drops of the trays that yielded crystals I would run a gel on
those drops and look at the aspect of protein still around in the drop. That
would give you some clues. If there was no reducing agent in the drops I
would run a gel with two samples (with and without a reducing agent such as
DTT or beta-mercaptoethanol for example).
you could also, if you had crystals to spare (although from what you say it
does not seem the case), run a gel on a crystal (it takes a little bit of
practice) to characterize what is in your crystal or if you have a mass spec
at hand look at the content of a crystal.
How long did those crystals take to grow? Is there a skin covering your
drops?

Hope this helps

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


[ccp4bb] Protease inhibitor cocktails and protein crystallization.

2011-03-10 Thread Pascal Egea
Dear All,

I apologize if the questions has already been asked on this forum.
We are purifying a membrane that seems prone to proteolysis. Although we use
Protease Inhibitor cocktails during lysis and the first step of purification
we get rid of them after and only keep PMSF and EDTA as general
anti-protease control agents.
I am considering reincluding the cocktail of inhibitors at the last
purification step (a size exclusion in our case) and was wondering if having
this infamous mixture of peptidic inhibitors (for the most part) around
during crystallization would be a problem: specifically getting crystals of
these inhibitors.
Does anyone have extended experience in this matter.

Many thanks in advance.

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] off topic: GPCR membane insertion/orientation

2011-03-04 Thread Pascal Egea
Hi Justin,

Since GPCRs are polytopic a-helical transmembrane proteins, it is very
likely that (1) insertion into the membrane is primarily performed by the
Sec61 complex AKA translocon and (2) targeting to the membrane would be
controlled by the signal recongition particle and its receptor. the latter
implies that a N-terminal signal sequence (that may very well be the first
TM of a GPCR) would control the insertion process. Does your favorite GPCR
have a predicetd  signal sequence?
Sec61 in theory contributes to signal sequence orientation according to the
positive-inside end rulebut as for any rule they are exceptions.

there is a set of excellent papers dissecting this mechanism by

Skach WR NSMB (2009) 16:6 606-12 (review)
Pitonzo  Skach Mol Biol Cell (2009) 20(2) 685-698 (article)
Sadlish H  Skach NSMB (2005) 12(10) 870-878 (article)
Sadlish and Skach J Membrane Biol 202 115-126 (2004) (review)

You may also want to look in the work of the group of Art Johnson (paper by
Woolhead et al)

describing the insertion process of aquaporin by the sec61 complex. they are
polytopic a-helical membrane proteins and you may want to look into these
articles since they dissect the process of TM insertion, orientation and
protein maturation quite well.

Hope this helps,

Best regards


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Lysis of Pichia pastoris

2010-11-02 Thread Pascal Egea
Hi Cory,
I am afraid to say that Pichia and Saccharomyces are tough cells to break
compared to bacteria. We also purifiy membrane proteins in yeast in my lab
so we have gone through this process.
With regular yeast we can break in an emulsiflex but it takes some hard work
and this is not really viable option on a large scale once you process
several liters of cell culture.
So we use a bead-beater ( from BIOSPEC) and glass beads (0.5 mm for yeast,
the diameter depends on the kind of organism you want to disrupt). it works
well and is quite fast , however you have to deal with the bead cleaning
part and the losses due to the mass of liquid trapped in the beads (rinsing
of beads with buffer is fine but it results in an increase burden at the
membrane centrifugation step)
It takes some optimization (mass of beads/mass of cells processed), but once
this is is set this is probably the way to go,
Bead beaters come in various sizes depending on the volumes you want to
process.
It is just a blender after all.

Hope this helps.

Best regards

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Confirming expression of a GPCR in HEK293

2010-09-02 Thread Pascal Egea
Hi Qing,

I would recommend either the use of GFP as mentioned by Jacob or a his-tag
or a flag-tag. C-terminal tagging is preferred to prevent interference with
signal sequences at the N-terminus of the protein.
Flag tag is really good for detection , the commercial antibodies for
detection are really great, however it is not that great, in my hands, when
it comes to purification of a membrane protein (in presence of detergent) I
prefer his-tags to flag-tags in this case. You can use a his-flag to combine
both advantages.

Hope this helps



-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

2010-09-01 Thread Pascal Egea
Hi Qiangmin,

All the comments and references that were already mentioned to you are
excellent,

I would stress 3 points.
1- The detergent.
A clear distinction should be made between the detergent used for
extraction/solubilization and the detergent (or cocktail of
detergents/lipids) used for crystallization. These are two very different
things.
If you are lucky you may not need to change, but once you have extracted
your rmembrane protein in one detergent, you should try to characterize its
homogeneity by size exclusion chromatography in different detergents ( with
shorter or longer chains and/or belonging to a different class (change from
a choline or a phospho-glycero-lipid to an alkyloside or from a charged to
an uncharged detergent etc). This scouting is tedious but is extremely
informative and it can be done on analytical scales (so it does not require
too much protein).

If you like statistics about detergent use you can look there.
*http://www.mpdb.tcd.ie/*

depending on the class of membrane protein beta-barrel versus all-alpha
helical etc etc you can initially concentrate your efforts on a subset of
detergents.

2- The diffraction.
As mentioned, starting with very poorly diffracting crystals is not uncommon
(as it is for RNA crystals). My own personal experience is that you can get
from 40 A resolution to the dreadful 6-4.0 A resolution barrier by tweaking
the purification/extraction conditions (1/ changing detergent (shorter
chain) and 2/ carefully controlling the amount of detergent present in the
sample used for crystallization (to avoid or at least minimize the phase
separation problem)).

3- The cryo conditions.
Crystallization drops in presence of detergent are actually not as
homogenous at it seems. Within the same drop you may have crystals of
identical size and morphology and freeze them in the same condition and
still get very different diffraction limits. When you freeze your
crystals matching the detergent concentration in your cryo-condition with
the 'expected' concentration in the drop  can be extremely important
especially with alkylosides (personal experience).

Good luck,

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] problem in annealing

2010-07-26 Thread Pascal Egea
Hi Hussain,

I think you need to edit your input file and increase the max number of
chain and the max number of tree . I had this problem several times
especially if the structure is big or the chain(s) is(are) fragmented. The
default values in CNS usually work but you can increase them and it should
go through.

HTH





-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


[ccp4bb] metal-chelating affinity chromatography and FosCholine detergents

2010-07-12 Thread Pascal Egea
Dear All,

I apologize for the not strictly crystallography-related query.
I am currently purifying several membrane proteins solubilized in
fos-cholines detergents and I consistently observe a significant loss of
protein at the binding step (done in absence of imidazole). Has anyone else
experience the same quite systematic (so far in my hands) problem with this
class of detergents.
I would appreciate any comments or advices from biochemists that face(d) the
same situation.

Thanks in advance


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] question - GFP fusion - cleavage sites

2010-05-24 Thread Pascal Egea
Hi Celina,

I cannot answer to your question concerning the GFP-related problem.
Fot the thrombin vs TEV protease related question I can tell you that in my
hands thrombin works really very well in most detergents (TEV is somehow
more sensitive). I am working on membrane proteins purified in very
different detergents such as OG, DM, DDM, FosCholines and mixed
lipids/detergents mixture and I see good (very good) cleavage with thrombin.
We buy it from Sigma ( bovine thrombin) and it can be stored for years at
-20C in a suitable buffer without loss of efficiency.

Hope this helps,

Cheers

Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu

n Mon, May 24, 2010 at 5:05 AM, Celina R. r.g.cel...@gmail.com wrote:

 Dear CCP4er's,
 Sorry for the non-crystallography related question and was hoping someone
 on the bulletin board might have some suggestions to overcome my peculiar
 protein purification problem.

 I am working on several membrane proteins (for crystallization trials) that
 have a C-Terminal eGFP fusion partner followed by a His-tag. The membrane
 protein and the GFP-His tag are separated by a TEV protease site. After
 purifying the fusion protein by IMAC, I add TEV protease to cleave the
 linkage between the membrane protein and the GFP-His tag.The cleavage
 reaction is also dialyzed to get rid of the imidazole. This cleavage seems
 to go to completion as judged by SDS-PAGE. However, when I try to separate
 the membrane protein from the GFP-His tag by passing through a IMAC column
 twice (excess nickel resin), a significant amount (about 1 mg) of  the
 GFP-His tag doesn't bind the IMAC column and flows through along with my
 protein. In addition, other methods such as centricons (30, 50 or 100 kDa
 M.W.C.O.), Gel Filtration and Ion-Exchange are also not able to separate
 them. All my buffers have 5 mM reducing agent and 500 mM NaCl to try and
 prevent any non-specific interaction between my protein and the GFP-His tag.
 It appears that the GFP-His tag is somehow stuck to my protein and co-elutes
 on any chromatographic column that i use.

 Has anyone encountered such a problem and managed to overcome it? Any
 suggestions/tricks would be helpful.
 I also have a question: Which is better to use for the cleavage of
 His-tags, in case I want to clone the membrane protein without GFP: TEV
 protease or thrombin?

 Thanks in advance.
 C.




--


[ccp4bb] continuous flow centrifuge

2010-02-16 Thread Pascal Egea
Dear All,

Sorry for the not strictly crystallography-related question.
We are currently setting up a fermentation core and are considering
purchasing a T1 Sharples continuous flow centrifuge. It is a mid-range
capacity instrument. We will be processing bacteria and yeast.

I was wondering if anyone had experience with this type of instrument and
would be willing to share his/her thoughts about it.

Thank you very much in advance.

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] UV microscope for screening

2010-01-20 Thread Pascal Egea
Dear Scott,

We had a UV inspection microscope from KORIMA to look at crystals by UV
fluorescence. It works relatively well but it is expensive ( way too much in
my opinion), you pay for the microscope, the UV source and the software
(named Wasabi) that comes with it
An alternative to that is described in the paper from Alan D'Arcy and coll.
Acta Cryst D63 (2007) p550-554. They use a DUVI 204 LIght source (from PLS
design GmbH , in germany) adapted to their Crystal Score system. If I
understood well, this is a just a UV source light adapted to your microscope
of choice. It is probably much cheaper and as efficient.

Hope this helps.


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] Expression of large proteins in E. coli

2010-01-13 Thread Pascal Egea
Hi Nick,

Some success has been reported for large soluble proteins using the C41(DE3)
and C43(DE3) *E. coli* strains (see the paper by Miroux  Walker). Also you
can try another promoter/expression system , the pBAD expression system
based on arabinose induction.

Hope this helps.

Best

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu


[ccp4bb] Post Doctoral Position at UCLA. Membrane Protein Complexes.

2009-12-13 Thread Pascal Egea
*Post-Doctoral Researcher. Structure and Function of Membrane Protein
Complexes*.

We seek a motivated individual to join our laboratory in the Department of
Biological Chemistry at the University of California in Los Angeles. We are
interested in studying the mechanisms of protein folding/translocation and
organelle biogenesis in diverse systems. Research projects involve the study
of various multi-subunits membrane protein complexes using X-ray
crystallography and cryoEM. State-of-the-art equipment is available in a
modern research setting including robotic crystallization, cryoEM and
fermentation facilities. Synchrotron radiation time is available at the
Advance Light Source in Berkeley and also at the Advanced Photon Source in
Chicago.

*Qualifications. *Applicants should have received a Ph.D degree in a field
relevant to structural biology (X-ray crystallography or cryoEM) and/or
molecular biophysics and have a good background in protein expression and
purification. Crystallographers applying should have some experience in
experimental phasing.

*Contact.* To apply please email the following to pe...@mednet.ucla.edu: a
CV, the contact information for 2 or 3   references (e-mail, address,
phone), a list of experimental expertise and a brief research statement
describing your past research and future goals.
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] off topic, design of a self-cleaving tag

2009-10-28 Thread Pascal Egea
Hi Daniel,

look at this
the Profinity eXact Fusion-tag system from BioRad* *

the protease i fused to your protein and self activated by halides (F or I I
think). Cleavage in on column. The principles is clever, now the cleavage
conditions may not suit to your protein, but it seems to work.
This is for expression and purification purposes only.

Hope this helps

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] small molecule library

2009-10-27 Thread Pascal Egea
I can suggest the following reading
Discovering novel ligands for macromolecules uisng X-ray crystallographic
screening
Nienaber VL et al, Nature Biotechnology vol 18 october 2000 pp1105



-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab (310)-983-3516
email pe...@mednet.ucla.edu


Re: [ccp4bb] lysozyme

2009-09-18 Thread Pascal Egea
Hi Camille,
I don't know if you have any protein labs around you but if someone is using
rosetta or codon-plus type expression Ecoli strains those cells usually
contain a plysS plasmid derivative that is chloramphenicol resistant and
carries the gene encoding lysozyme among other things (plus the rare tRNA
genes). If they don't have the plasmid at hand you can still grow the cells
and make a miniprep of the plasmid (low copy) to have a lysozyme gene in
your hands.

Hope this helps.
Let me know if you can't find it.

-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-825-1013
lab (310)-825-8722
email pe...@mednet.ucla.edu


Re: [ccp4bb] Adding a transmembrane segment

2009-09-06 Thread Pascal Egea
Hi,if you add the TM segment, I would expect that the protein will then get
targeted to the membrane and hopefully inserted correctly. My guess is that
you will have to treat your protein as a membrane protein or a
membrane-anchored protein...which means prepare a membrane fraction, use
detergent and look for your protein in there.
Is your ectodomain N or C-terminal (I assume it is N from your message)?
Have you looked at the targeting sequences present in the protein (signal
sequence, reverse anchoring motifs)?

Hope this helps

Pascal F. Egea, PhD


Re: [ccp4bb] Cryoprotection in 3M ammonium sulfate

2009-08-19 Thread Pascal Egea
Hi Brenda,
You can try sugars like glucose, trehalose and sucrose for high AS contents.
It has been succesfully used in really hard cases such at protein RNA
crystals grown in AS.  see Acta Cryst (2002) D58 1664-1669 Garber et al.

HTH

Pascal Egea


Re: [ccp4bb] DNA binding protein

2009-08-10 Thread Pascal Egea
Hi Neeraj,
An absorption spectra between 220 and 400 nm (for example) should show you
if there is DNA coming along with your protein. In theory A280 is about 1.7
times A260 for a pure protein sample. This is a rough estimate. If your peak
is shifted towards 260 instead of 280 then you can suspect the presence of
contaminating DNA or RNA.

As to get rid of the DNA, I can suggest several ways to do it.
1-An heparin affinity column could out-compete your contaminating DNA while
binding your protein. They work really well.

2-Ion exchange is worth trying. It has worked for me with an extremely basic
archeal RNA binding protein purified in E.coli and bringing along some
endogenous RNA/DNA. An ion exchange ( Mono-S type) got rid of the
contaminating nucleic acid, however with some loss of protein.

3-DNAse treatment may be worth trying but the presence of traces of DNase
may be a problem with subsequent crystallization trials in presence of the
bona-fide target DNA sequence.

4-Precipitation of the DNA with streptomycine sulfate. You could also loss
some protein.

 1 and 2 are in my opinion the less invasive soutions.

Hope this helps.

Best regards

Pascal Egea


Re: [ccp4bb] detergent crystals

2009-08-04 Thread Pascal Egea
Hi Jose,
It is quite difficult to crystallize DDM. The main problem is to estimate
detergent concentration in the final sample.
One method requires the use of chromatographic workstation coupled to a
light scattering detector and refraction index measurement unit. In this
case you can know if there is an excess detergent (particularly likely to be
the case if you are using a low CMC detergent like DDM and if you have been
concentrating your sample on a membrane with a cutoff smaller than the empty
micelles). This is more involved and requires a specialized equipment. But
you can detect both your protein and your excess detergent (if there is).
Also to test you crystals, you may want to see if you can access a
fluorescence microscope. If your protein contains tryptophan (and you have
the correct wavelength) then you protein crystals should glow. Your
detergent crystals should not. Those microscopes are getting more and more
popular so maybe you have one next by. It is very convenient.

Hope this helps

Pascal Egea, PhD
University of California Los Angeles
Department of Biological Chemistry


Re: [ccp4bb] citrate blocks the active site

2009-06-11 Thread Pascal Egea
Dear Samy,
You mentioned that your enzyme crystallizes in 0.1M citric acid pH3.5 plus
25% PEG 3,350.
I wonder if you have tried to systematically scan for other carboxylic acids
as buffer/co-precipitating salts. I would suggest you to try the serie of
carboxylic acids, citrate, acetate, formate and also the magic carboxylate
malonate; a little bit like the Hofmeister series.
You maybe able to find a surrogate to citrate that will able you to either
soak your crystals or co-crystallize successfully with  your substrate(s).

Hope this helps

Cheers,

Pascal Egea
University of California San Francisco
Department of Biochemistry and Biophysics


Re: [ccp4bb] Tips on fitting poorly defined loop regions

2009-05-20 Thread Pascal Egea
Hi Drew,
Have you tried arp/wARP (the LOOPY option) or the AutoBuild option in Phenix
? If you haven't tried this you can try a complete rebuilding using you
model as it stands and providing the complete sequence of your protein.
At this resolution (2.1A) , they may be able to rescue your loops at least
partially.
There is another program called XPLEO (and a derivation of it called LoopTK)
and is available from the Stanford site at the Synchrotron Linear
Accelerator that has been helpful in our lab in the case of a loop region in
a membrane protein.

Hope this helps. Good luck

Pascal F. Egea, PhD
University of California San Francisco
Department of Biophysics and Biochemistry





Re: [ccp4bb] Acrylamide in RNA crystallization

2009-04-01 Thread Pascal Egea
Hi Vanessa
It is better to get rid of traces of residual acrylamide that may
contaminate your final purified and refolded RNA.
It is usual to have contaminations with monomeric acrylamide. NMR
spectroscopists studying RNA can usually detect its presence on their
spectra.
If you can dialyze your purified product to try to get rid of it it would be
the best. Traces (sometimes it is not a negligible amount) are  not good
because you may not be able to reproduce your results and optimize eventual
crystals. And for RNA this can be an excruciating pain.
When we transcribe RNA, we usually run the preparative acrylamide-urea gels
and elute the RNA out of the gel (most of the time by electroelution). The
RNA usually contains urea and acrylamide so I either precipitate using the
salt/ethanol technique and then resuspend the pellet and dialyze/refold or I
further purifiy on an ion exchange (Q type column) to try to clean it up.

If you have an NMR spectroscopist friend around, try to look at the presence
of acrylamide before and after these steps and see what works the best for
you.

Hope this helps

Pascal Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics


Re: [ccp4bb] OT: Crystallisation compatible detergents

2009-03-24 Thread Pascal Egea
Hi Darren
I believe that the most frequently used detergent for protein
crystallization (not including membrane proteins) is octyl-glucoside.
The most important parameter is the CMC of the detergent and the size of the
micelles of free detergent if you have micelles around. These considerations
do not apply if you are well under the CMC (only free molecules of detergent
are present in solution). I believe tween-20 CMC is roughly 0.006% so you
are at the border.
The problem is you have micelles of detergent is that depending on their
size and the MW cutoff of the device you use for protein concentration you
may end up concentrating both components protein and detergent and then this
is when trouble starts. You may have an unknown amount of concentrated
detergent above the CMC, this can be very problematic for crystallization
because you may have a lot of phase separation in your drops; phase
separation are usually (but not always) not desirable.

For membrane protein crystallization we are always facing this problem
because we usually work in this regime of critical detergent concentrations.
However for soluble proteins that need a little bit of detergent to remain
stable this is not as usual.

OG (octyl glucoside) is a rather mild detergent with a CMC of about 20 mM
(check ANATRACE catalog).
To simplify, maybe a little bit too much, the most important is at first to
find the minimum amount of detergent you need to keep you sample stable and
possibly stay as low under the CMC. And then may be go up in detergent
concentration if you don't get the results expected. You can check that your
complex stays functional using Biacore (in your specific case)
Other detergents very popular for non membrane proteins are DDM (dodecyl
maltoside) or CHAPS (zwitterionic cholesterol derivative).
So in order of decreasing preference I would suggest OG, CHAPS (which is a
totally different type of detergent) and DDM (this one has a very low CMC
~0.15mM I believe, so it is difficult to get rid of it, but it is fairly
genlte).
An alternative to detergent are non-detergent sulfo-betaines they can
sometime have the same protective effect without the trouble of detergents.

I hope this helps,
Cheers

Pascal F. Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics





Re: [ccp4bb] precipitation of deglycosylated protein

2009-03-16 Thread Pascal Egea
Hi Simon,
Although they are a source of heterogeneity for
crystallization, glycosylations usually stabilize proteins.
There are a couple of things that may be important to consider before you
deglycosylate your protein.

Do you know how many natural sequons/glycosylation sites your protein has?
And in what system/organism are you expressing your protein?
I am saying that because I have the case of a bovine enzyme which has three
sites. Inactivation of all sequons from Asn to Asp ( they are standards
N-glycosylation sites) and expression in Pichia pastoris results in a
fully-deglycosylated protein which is unstable and precipitates. However if
one specific sequon is kept intact, the obtained protein is glycosylated by
Pichia at this functional site, behaves very well and yields good quality
crystals. The electron density maps show the two first N-acetyl glucosamine
units N-linked to the Asn residue. Interestingly the protein is pretty
homogenously glycosylated by Pichia.

I know this may sound a little bizarre but you may get around this problems
by keeping some sequons or the sequon active (if there is only one) and try
to deal with a protein, glycosylated-light as you express it. This depends
on what your expression system is.

You can try to add stabilizing agents like glycerol, ethylene glycol or some
di-sugars like trehalose.

I hope this helps,
Cheers,

Pascal F. Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics



On Mon, Mar 16, 2009 at 9:29 AM, Yue Li simon.yu...@yahoo.com wrote:

 Hi everyone,

 Recently, I obtained a soluble glyco-protein. Unfortunately, after I added
 PNGase or Endo Hf to remove the glycans, the deglycosylated protein is
 precipitated. Is there any method to avoid this kind of precipitation?

 Thanks,

 Simon




Re: [ccp4bb] precipitation of deglycosylated protein

2009-03-16 Thread Pascal Egea
My apologies Simon, I should have been more thorough answering your
question.
Yes the protein was shown to be quite homogenously glycosylated using
mass-spectrometry.
The ED maps showed the first two ordered fully occupied NAG units and
residual density for a third sugar unit although it is very poor defined.

Pascal Egea


Re: [ccp4bb] Purification with ligand

2009-02-26 Thread Pascal Egea
Hi Mariah,
  You may need to specify what type of ligand (is it a nucleotide, a
small synthetic molecule, a peptide etc ?) and also what is the affinity
 between your ligand and your protein.
I have purified several protein-ligand complexes, you can go several routes.
If you have a high affinity binder and fairly 'cheap' or abundant ligand it
is possible for example to add it in your crude extract (or even in your
bacterial culture in some extreme cases). It may end-up improving your
'expression yield' in terms of soluble protein readily available in your
initial extract.
I have used this approach successfully when purifying the
ligand-binding domain of a nuclear receptor with its very hydrophobic
natural ligand (retinoic acid) or a synthetic drug. In this case it was
giving a more homogenous population of protein at the start of the
purification. I included the hormone in the crude extract after sonication
and centrifugation (so at an early stage of the purification). After that I
kept ligand around in the buffer (Cobalt affinity chromatography and gel
filtration (at low concentration) and kept adding ligand to the concentrated
protein. If your ligand has some kind of specific UV absorption, it can be
very easy to monitor its presence and the 'saturation level' of your
protein. If you have a high-affinity binder, it can be a very efficient way
to start with homogenous population of protein-ligand complex.
This approach is really useful when your ligand happens to be only soluble
in protein-unfriendly solvents like ethanol or acetone (this was the case
for retinoic acid); in the crude extract despite the addition of alcohol,
your protein won't suffer too much from the presence of added alcohol and if
affinity is high and you add enough of it, you will efficiently saturate it.
In another case, a complex between two GTPases, I had to use a
non-hydrolyzable GTP analog. The compound was far too expensive to be used
in the crude extract. In this case , we purified the two apo-proteins
separately, formed the complex in presence of ligand and included ligand in
the ion-exchange chromatography buffers and in the gel filtration buffer (at
a low concentration though but it helped us to stabilize the complex). Again
it all depends on the affinity.If you decide to include ligand in your
gel-filtration buffer, keep also in mind that you will contaminate your
columns and it can be hard to get rid of some ligands sometimes.

Sorry, if all this was a little bit too long.
Hope this helps,

Pascal Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics
Laboratory of Robert Stroud




On Thu, Feb 26, 2009 at 2:58 PM, protein.chemist protein.chemist 
pp73...@gmail.com wrote:

 Hello,
 I wanted to know if there is a standard procedure for purification of
 protein with ligand.  I have never done this before so it will be nice to
 get some help.

 Thanks,
 Mariah

 --
 Mariah Jones
 Department of Biochemistry
 University of Florida



Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

2009-02-24 Thread Pascal Egea
Hi Mo,
   Gene synthesis is definitely something you should try if you can afford
it.
However, I would suggest also trying to change expression plasmid and in
particular induction system and promoter system.
For toxic proteins we had some success using the pBAD (invitrogen)
expression system using arabinose induction and the arabinose operon
promoter different from the classical T7 promoter. It is tightly regulated
and has a very fast kinetic of induction. It is worth trying even combined
with gene synthesis.
   I also remember that some kinases are efficiently expressed in Ecoli in
presence of another expression plasmid encoding two  chaperones , the
trigger factor and GroES/EL. This is worth trying too.

Hope this helps, cheers.

Pascal Egea, PhD
University of California San Francisco
Department of Biochemistry and Biophysics
Stroud Lab


Re: [ccp4bb] Se oxidation

2009-02-09 Thread Pascal Egea
Hi,I believe it is not important as long as you run a proper scan of the
crystal. Both forms will allow proper phasing.
This is very well described in a paper from Thomazeau et al.
here is the reference

MAD on threonine synthase: the phasing power of oxidized selenomethionine.
Acta Crystallogr D Biol
Crystallogr.javascript:AL_get(this,%20'jour',%20'Acta%20Crystallogr%20D%20Biol%20Crystallogr.');
2001
Sep;57(Pt 9):1337-40. Epub 2001 Aug 23.

Cheers,

Pascal Egea, PhD
Post Doctoral Researcher
UCSF Department of Biochemistry and Biophysics
Stroud laboratory






On Mon, Feb 9, 2009 at 10:27 AM, aka akaka druida...@hotmail.com wrote:

  Dear All
 I would like to know whether oxidation of Se entails any problem for SAD or
 MAD experiments and/ or how to resolve it. Cannot use DTT or reducing agents
 in my protein (extracellular and disulphide bonds are important).
 Thanks

 Dr. R.Depetris
 Weill Cornell Medical College



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