Re: [ccp4bb] cryoprotection ideas for salt based condition

[Mahesh Lingaraju]

Hi all

Just wanted to thank you all for all the suggestions and tell you of all
the things i tried what finally worked, if anyones interested.

Among the several things mentioned here, the crystals diffracted the best
(~ 2.9 Å at the synchrotron) when grown in a solution containing either 5%
ethylene glycol and cryo protected in 20% ethylene glycol or 15% glycerol
and protected in 25% glycerol.

Thanks for all the help

Mahesh


On Thu, Jan 23, 2014 at 7:04 AM, Elizabeth Morris <
emorr...@staffmail.ed.ac.uk> wrote:

> Hi Mahesh,
>
> I have experienced similar situations with AmSO4 as a precipitant. One
> thing I found worked is to optimise crystallization in a lower
> concentration of AmSO4 so that you reduce the amount of salt crystals you
> get when you try and loop your crystals. I got hits in 2 M AmSO4, but I
> have managed to reduce this to ~1 M and still get crystals by:
>
> - increasing protein concentration
> - varying pH/buffer
> - using microseeding to obtain crystals
>
> I have less experience with different types of cryoprotectants, but in my
> opinion, if you problem is that you see salt crystals when you open your
> droplet to the air, try reducing the salt concentration in your drop.
>
> Alternatively, perhaps your microscope is heating up your sample and
> increasing the rate of evaporation from your drop. If that is the case, try
> to work at lower intensities of the lamp, or turn it off more frequently.
>
> Finally, I have heard of people putting a damp tissue or a humidifier near
> to where you are looping your crystals to reduce evaporation of water from
> your drop.
>
> Good luck!
>
> Liz
>
>
>
>
>
>
> Quoting Mahesh Lingaraju  on Wed, 22 Jan 2014 12:28:15
> -0500:
>
>  Hello folks,
>>
>> I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10%
>> dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have
>> finding the right cryoprotection for salt based conditions; I have tried
>> glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing
>> ammonium sulphate concentration in presence of 5-10% glycerol. The
>> crystals
>> disintegrate in any kind of PEG based cryo. I made all these solutions in
>> the mother liquor and tested if they freeze clearly before using them.
>> However when I loop the crystals and try to soak them in these cryo-
>> mother
>> liquor, a lot of salt crystals suddenly form around the protein crystal
>> and
>> I see diffraction only from these salt crystals. The best I have been able
>> to get so far is ~ 9Å diffraction (Home-source) with DMSO as the
>> cryoprotectant.
>>
>> I have also tried using 1 M sodium malonate as the cryoprotectant but my
>> crystals are not too stable in this mother liquor probably because I had
>> to
>> lower the ammonium sulphate by ~ 7-10% to make the drop not form salt
>> crystals instantly when exposed to air.
>>
>> Other than trying to make the crystals more cryo-ready by finding other
>> hits or may be growing the same crystals with some cryoprotectant, I was
>> wondering if any of you have ideas based on your experience or suggestions
>> in case I am doing everything wrong in the first place.
>>
>> Thanks for all the help,
>>
>> Mahesh
>>
>>
>
>
> --
> The University of Edinburgh is a charitable body, registered in
> Scotland, with registration number SC005336.
>
>
>

Re: [ccp4bb] cryoprotection ideas for salt based condition

[Mahesh Lingaraju]

Thanks for all the suggestions. Ill try these and post an update here
regarding what happens.

Thank you so much !!

Mahesh


On Wed, Jan 22, 2014 at 1:23 PM, Mark van der Woerd
wrote:

> Mahesh,
>
> Have you tried to see what happens when you do not freeze the crystals at
> all? Do you get good resolution data? Do the crystals suffer radiation
> damage?
>
> Those are two important questions. First, if your crystals do not diffract
> well before you freeze them, usually (but not always) they will also not
> diffract well after you freeze them and you can try cryo-conditions until
> you see blue in the face, but they will never improve. In that case, you
> need new/better crystals. Second, many well-diffracting crystals do not
> necessarily need to be frozen at the home source. Of course they do need to
> be frozen at the synchrotron, but if you can get good (enough) data at
> home...
>
> On to recipes: try increasing dioxane. Try adding alcohols (somewhat
> similar to dioxane, maybe iso-propanol). Expect this to be a pain: very
> volatile agents make it difficult to harvest crystals. Of course try the
> mixtures without protein or crystals for freezing conditions and how well
> you can mix the components. Salt and organics are often not compatible.
>
> Try to regrow your crystals in sodium malonate (replace Ammonium sulfate
> with malonate). Try to regrow them in the presence of small amounts of the
> cryo-protectants you have already tried. For the ones that work, repeat
> with higher amounts. Very often crystals are much happier when you don't
> soak/tinker with them and freeze them straight out of the drop.
>
> Good luck.
>
> Mark
>
>
>
>  -Original Message-
> From: Mahesh Lingaraju 
> To: CCP4BB 
> Sent: Wed, Jan 22, 2014 10:38 am
> Subject: [ccp4bb] cryoprotection ideas for salt based condition
>
>  Hello folks,
>
>  I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10%
> dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have
> finding the right cryoprotection for salt based conditions; I have tried
> glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing
> ammonium sulphate concentration in presence of 5-10% glycerol. The crystals
> disintegrate in any kind of PEG based cryo. I made all these solutions in
> the mother liquor and tested if they freeze clearly before using them.
> However when I loop the crystals and try to soak them in these cryo- mother
> liquor, a lot of salt crystals suddenly form around the protein crystal and
> I see diffraction only from these salt crystals. The best I have been able
> to get so far is ~ 9Å diffraction (Home-source) with DMSO as the
> cryoprotectant.
>
>  I have also tried using 1 M sodium malonate as the cryoprotectant but my
> crystals are not too stable in this mother liquor probably because I had to
> lower the ammonium sulphate by ~ 7-10% to make the drop not form salt
> crystals instantly when exposed to air.
>
>  Other than trying to make the crystals more cryo-ready by finding other
> hits or may be growing the same crystals with some cryoprotectant, I was
> wondering if any of you have ideas based on your experience or suggestions
> in case I am doing everything wrong in the first place.
>
>  Thanks for all the help,
>
>  Mahesh
>

[ccp4bb] cryoprotection ideas for salt based condition

[Mahesh Lingaraju]

Hello folks,

I have crystals for a protein which form in 0.1 M MES pH 6-6.75, 10%
dioxane and 1.6-2 M Ammonium sulphate. Based on little experience I have
finding the right cryoprotection for salt based conditions; I have tried
glycerol (15-25%), Ethylene glycol (20-25%), DMSO (15-20%) and increasing
ammonium sulphate concentration in presence of 5-10% glycerol. The crystals
disintegrate in any kind of PEG based cryo. I made all these solutions in
the mother liquor and tested if they freeze clearly before using them.
However when I loop the crystals and try to soak them in these cryo- mother
liquor, a lot of salt crystals suddenly form around the protein crystal and
I see diffraction only from these salt crystals. The best I have been able
to get so far is ~ 9Å diffraction (Home-source) with DMSO as the
cryoprotectant.

I have also tried using 1 M sodium malonate as the cryoprotectant but my
crystals are not too stable in this mother liquor probably because I had to
lower the ammonium sulphate by ~ 7-10% to make the drop not form salt
crystals instantly when exposed to air.

Other than trying to make the crystals more cryo-ready by finding other
hits or may be growing the same crystals with some cryoprotectant, I was
wondering if any of you have ideas based on your experience or suggestions
in case I am doing everything wrong in the first place.

Thanks for all the help,

Mahesh

Re: [ccp4bb] seeding, reproducibility

[Mahesh Lingaraju]

Thanks you for the suggestions, Patrick and Matthias. I was actually
wondering if any of the components from the seeding solution actually were
important but your explanations sound more logical.

I apologize for the large attachment. I did not realize that it was so big.

Have a nice weekend !

Mahesh


On Fri, Jan 17, 2014 at 6:18 PM, Patrick Shaw Stewart  wrote:

>
> Mahesh, this is a very interesting and slightly controversial question.
>
> One approach is to mix together all of the crystals that you have in the
> initial screen.  The idea at the beginning of the project is to get as many
> diverse hits as possible - you can worry about crystal size, space group
> and quality later on.
>
> If you can collect data from crystals and determine the unit cell then you
> can be more rational.  You can construct a "library" of polymorphs having
> different unit cells.  This *may *allow you to push the space group in a
> given condition to the unit cell or space group that you want - maybe you
> find e.g. that your ligand can only diffuse into the active site with a
> certain unit cell.
>
> There is a very interesting paper with several examples of how to use
> polymorphs by the Stura group, see below.
>
> However, seeding can give rise to crystals with different but related
> space groups.  A very nice example is shown in the wikipedia article about
> epitaxy - titanium oxide growing on iron oxide.  Also Stura's paper on
> epitaxial jumps is interesting and relevant.
>
> Make sure you dilute your seed stock in a systematic way as part of your
> final optimization - one great advantage of microseeding is that oyu can
> control the number of crystals per drop by diluting the seed stock.
>
> Also, make sure that you use fresh crystals to make the seed stock.  For
> some strange reason old crystals sometimes fail to act as seeds, even
> though they can be crushed and still diffract.  Maybe the unit cell
> shrinks, or maybe the crystals become cross-linked.  Make the seed stock as
> soon as your crystals stop growing, then freeze them.  Seed stocks can
> almost always be frozen.
>
> Best wishes, Patrick
>
>
>
> *Library of polymorphs:*
> Vera, Laura, Claudia Antoni, Laurent Devel, Bertrand Czarny, Evelyn
> Cassar-Lajeunesse, Armando Rossello, Vincent Dive, and Enrico A. Stura.
> "Screening Using Polymorphs for the Crystallization of Protein–Ligand
> Complexes." Crystal Growth & Design 13, no. 5 (2013): 1878-1888.
>
> Stura, Enrico A., Jean-Baptiste Charbonnier, and Michael J. Taussig.
> "Epitaxial jumps." Journal of crystal growth 196, no. 2 (1999): 250-260.
>
>
> *Cross-seeding:*
> Obmolova, Galina, Thomas J. Malia, Alexey Teplyakov, Raymond Sweet, and
> Gary L. Gilliland. "Promoting crystallization of antibody-antigen complexes
> via microseed matrix screening." Acta Crystallographica Section D:
> Biological Crystallography 66, no. 8 (2010): 927-933.
>
> Abuhammad, Areej, Edward D. Lowe, Michael A. McDonough, P. D. Shaw
> Stewart, Stefan A. Kolek, Edith Sim, and Elspeth F. Garman. "Structure of
> arylamine N-acetyltransferase from Mycobacterium tuberculosis determined by
> cross-seeding with the homologous protein from M. marinum: triumph over
> adversity." Acta Crystallographica Section D: Biological Crystallography
> 69, no. 8 (2013): 1433-1446.
>
>
> *Seed stability etc*
> Shaw Stewart, Patrick D., Stefan A. Kolek, Richard A. Briggs, Naomi E.
> Chayen, and Peter FM Baldock. "Random microseeding: a theoretical and
> practical exploration of seed stability and seeding techniques for
> successful protein crystallization." Crystal Growth & Design 11, no. 8
> (2011): 3432-3441.
>
>
>
> *Original description of the "random" microseeding method*D'Arcy, Allan,
> Frederic Villard, and May Marsh. "An automated microseed matrix-screening
> method for protein crystallization." Acta Crystallographica Section D:
> Biological Crystallography 63, no. 4 (2007): 550-554.
>
>
>
>
>
>
> On 17 January 2014 22:24, Mahesh Lingaraju  wrote:
> >
> > Hi Folks
> >
> > The protein that I am working on gives several initial hits which are
> needles. And at random, I picked the needles from a condition (0.2 M cacl2,
> 0.1 M HEPES pH 7.5 & 28% PEG 400) and seeded into another screen where I
> added 1µl protein + 1µl reservoir solution + 0.3 µl seed stock. I prepared
> the seed stock fresh by adding 36 µl of the reservoir solution to
> preexisting 2µl drop.
> > One of the conditions from the seeded screen gave me a hit that looks
> really promising ( see attached). I am sort of positive that these crystals
> are protein as they are UV active.
>

Re: [ccp4bb] structural homologs as cross seeds

[Mahesh Lingaraju]

Thanks for the input folks. Ill surely try it. Hopefully some christmas/new
year miracles will happen.

Thanks again

Mahesh



> Dear all,
>
> I was wondering if it sounds logical to use the crystals from a possible
> structural homolog as seeds to induce nucleation ? (in terms of overall
> sequence, the proteins are considerably different but based on sequence
> alignment and structures from other related proteins, it is highly likely
> the protein would have the same structure.)
>
> Please comment if any of you had experience with this.
>
> Thank you
>
> Happy holidays :)
>
> Mahesh
>
>
>
>
>
>
>
>

[ccp4bb] structural homologs as cross seeds

[Mahesh Lingaraju]

Dear all,

I was wondering if it sounds logical to use the crystals from a possible
structural homolog as seeds to induce nucleation ? (in terms of overall
sequence, the proteins are considerably different but based on sequence
alignment and structures from other related proteins, it is highly likely
the protein would have the same structure.)

Please comment if any of you had experience with this.

Thank you

Happy holidays :)

Mahesh

Re: [ccp4bb] small crystals

[Mahesh Lingaraju]

Hi All


On similar lines, I have been trying to optimize crystallization conditions
for my protein. Initially, I had showers of needles in a PEG screen which
did not really improve after screening around the condition. So, I seeded
these needles into all the screens that I have available and I have plate
like crystals which do not diffract at home in MPD (~15%), 0.1 M sodium
acetate, 0.2 Mgcl2/cacl2 at 294 K. I tried incubating at 287 K, but that
did not yield any useful results.The protein itself is in 50mM MOPS, 10%
glycerol pH 7.5. I could try to take of glycerol but i cannot concentrate
the protein more than ~ 5mg/ml which clearly was not sufficient to achieve
crystallization.

Any advice is deeply appreciated.

Thank you

cheers
Mahesh


On Thu, Dec 5, 2013 at 10:03 AM, mesters wrote:

>  Hi,
>
> can you give a bit more information...
>
> Can you concentrate the protein easily to a higher concentration, let's
> say 2-3 times from what you have now, without precipitation?
>
> What is the buffer of your protein stock solution at the moment?
>
> At what temperature and what precipitant are you using?
>
> - Jeroen -
>
>
> showers of crystals
>
>
>  Original message 
>
>   Subject:[SURESPAM] [ccp4bb] small crystals
> From:Careina Edgooms 
> To:CCP4BB@JISCMAIL.AC.UK
> Cc:
>
>
>  Hi all
>
>  Any advice on how to get bigger crystals from conditions that give
> showers of tiny crystals? I am getting small pretty looking individual
> crystals but they are too small and they don't seem to grow. In fact, in
> some instances if left for a couple of days they actually dissolve. I have
> fiddled around with mother liquor volume, protein concentration as well as
> drop volume (I am using hanging drop method) but none seem to make any
> difference and I always get the same tiny crystals. I think I might try
> microseeding but I haven't tried that yet.
>
>  Any suggestions or tricks would be welcome
>  Careina.
>
>
> *TotalCare* Message Security: Check 
> Authenticity
>
>
>
> --
> Dr. Jeroen R. Mesters
> Deputy and Group Leader
>
> Institute of Biochemistry, University of Lübeck
> Ratzeburger Allee 160, 23538 Lübeck, Germany
>
> phone: +49-451-5004065 (secretariate 5004061)
> fax: +49-451-5004068
>
> http://www.biochem.uni-luebeck.de 
> http://www.iobcr.org 
>
>
>  --
> If you can look into the seeds of time and tell which grain will grow and
> which will not, speak then to me who neither beg nor fear (Shakespeare's
> Macbeth, Act I, Scene 3)
> --
> Disclaimer
> * This message contains confidential information and is intended only for
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>

Re: [ccp4bb] changes in small sections of secondary structure

[Mahesh Lingaraju]

Hello experts

Thanks for your insights.
For one of the structures, it turned out to be a rendering issue by pymol
like matt pointed out. For the other, the residues are clearly in a less
than ideal position. Even if I see deviation from the RMSD plots, i cannot
be sure that the structure were refined ideally at those positions ( those
are not my structures, i just have the pdb files from my collaborator).

Thanks again,

Mahesh

P.S from what all of you are saying it sounds like those changes are not
real, if I find that they could be Ill let everyone know.




>

Re: [ccp4bb] R and R free from CIF file deposited in PDB

[Mahesh Lingaraju]

Thanks Robbie
I found what I was looking for in PDB_REDO.

Thanks

Mahesh


On Fri, Oct 11, 2013 at 4:49 PM, Robbie Joosten
wrote:

> Hi Mahesh,
>
> They will be close but not the same. RMSDs should be very close normally,
> but different treatment of hetero compounds (ligands and such), outliers
> and
> LINKs added during annotation may cause small deviations. The deviations in
> R-factors can be much larger for all sorts of reasons. The data that is
> used
> now can be (slightly) different from what was used before due to different
> scaling, merging of I+ and I-, conversion from F to I, outlier rejection,
> R-free set annotation, deposition errors (i.e. missing data), or deposition
> of all measured data, not just the data used in the final refinement (this
> is actually a good thing). Different programs have different treatment of
> bulk solvent. There can be rounding errors is the conversion from TLS to
> anisotropic B-factors. The use of riding hydrogens can also make a big
> difference.
> There are several papers that discuss the sources of these R-factor
> deviations, e.g. papers about EDS, PDB_REDO and phenix.model_vs_data.
>
> Cheers,
> Robbie
>
> > -Original Message-
> > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> > Mahesh Lingaraju
> > Sent: Friday, October 11, 2013 21:59
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [ccp4bb] R and R free from CIF file deposited in PDB
> >
> > Hello Experts,
> >
> > I was wondering if the R, Rfree, RMSD for bonds and angles calculated by
> > polygon plug in in PHENIX GUI using the cif file & pdb file deposited in
> protein
> > structure database would be the same as reported in database/publication
> ?
> >
> > I apologize in advance if this is a PHENIX specific question.
> >
> > Many thanks
> >
> > Mahesh
>

[ccp4bb] R and R free from CIF file deposited in PDB

[Mahesh Lingaraju]

Hello Experts,

I was wondering if the R, Rfree, RMSD for bonds and angles calculated by
polygon plug in in PHENIX GUI using the cif file & pdb file deposited in
protein structure database would be the same as reported in
database/publication ?

I apologize in advance if this is a PHENIX specific question.

Many thanks

Mahesh

Re: [ccp4bb] Stuck rfree - possible non merohedral twinning ?

[Mahesh Lingaraju]

Hi Juergen & other experts

Thanks for the suggestions.  I was under the impression that the twin
laws/operators are to be used if the twinning is merohedral. In my case, it
appears as if the twinning is non-merohedral and more over the data i have
is processed as P422 which does not have twin operators ( probably because
all the axial pairs are same anyway). Probably because of the same reason,
xtriage did not give any operators for me to use. In such cases, do people
usually try to process the data in another space group of lower symmetry
which has a twin law and is there a definite way to distinguish twinning by
merohedry vs non-merohedry other than just looking at the diffraction
pattern ( is it crucial to make that differentiation ?) ?

Thanks

Mahesh


On Sun, Aug 25, 2013 at 10:24 PM, Bosch, Juergen  wrote:

> Hi Mahesh,
>
> if you use Refmac, then you can tell it to refine the twin fraction, no
> need to tell it the twin law as Refmac will figure it out. If you use
> phenix, you explicitly tell it the twin law and refine then with it. You
> can get the possible twin laws by running phenix.xtriage and looking at the
> log file.
>
> For a 1.7 Å dataset you should see excellent holes in the Phe and Tyr,
> even though your Rfactors are high. If that is the case then you are likely
> correct with the twin (if nothing else is wrong, Cbeta, Ramas etc). And you
> did add some waters to your structure already right ? if not the go water
> picking via Coot.
>
> Have you been converted to XDS now ? Welcome to the club.
>
> Jürgen
>
> On Aug 25, 2013, at 8:35 PM, Mahesh Lingaraju wrote:
>
> Hello everyone,
>
> I collected a dataset which looked like it is twinned ( or a really long
> axis in the cell)  and did not process in HKL2000 and MOSFLM but with some
> of help and suggestions from CCP4BB, XDS was able to process it. The data
> looks good upto 1.7 Å. However, the rfree is stuck at 0.34 even though my
> model is almost complete. I am beginning to wonder if the data is really
> twinned as it has the characteristics of non- merohedral twinning:
> In the images some of the reflections are sharp while some are split and one
> of the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c=
> 400.34; 90 90 90)
>
> is there anyway to work around this ? or collecting better data is the
> only solution ?
>
> Any help is deeply appreciated
>
> Thanks
>
> Mahesh
>
>
>  ..
> Jürgen Bosch
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
> Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
> http://lupo.jhsph.edu
>
>
>
>
>

[ccp4bb] Stuck rfree - possible non merohedral twinning ?

[Mahesh Lingaraju]

Hello everyone,

I collected a dataset which looked like it is twinned ( or a really long
axis in the cell)  and did not process in HKL2000 and MOSFLM but with some
of help and suggestions from CCP4BB, XDS was able to process it. The data
looks good upto 1.7 Å. However, the rfree is stuck at 0.34 even though my
model is almost complete. I am beginning to wonder if the data is really
twinned as it has the characteristics of non- merohedral twinning:
In the images some of the reflections are sharp while some are split and one
of the axis in the cell is usually long ( the cell is a= 46.78 b= 46.78 c=
400.34; 90 90 90)

is there anyway to work around this ? or collecting better data is the only
solution ?

Any help is deeply appreciated

Thanks

Mahesh

Re: [ccp4bb] Purification of a protein

[Mahesh Lingaraju]

i am not sure this would work as his protein seems to be degraded by the n
end degradation pathway. i feel like it almost needs to be expressed as a
fusion protein with some stabilizing sequence


On Fri, Aug 23, 2013 at 10:08 PM, Roger Rowlett wrote:

> Why not adopt a classical purification strategy? IEX-HIC-GEC. His-tag not
> required. With your protein strongly basic, anion exchange seems like a
> likely first step. After IEX, hydrophobic interaction or salt precipitation
> followed by gel exclusion is normally enough for well-expressed proteins.
>
> Roger Rowlett
> On Aug 23, 2013 9:27 PM, "Jahan Alikhajeh"  wrote:
>
>> Dear Friends,
>>
>> I have been trying to purify a protein (27 kDa) which has a sumo plus 6
>> His-tag at N-ter giving totaly a 45 kDa protein.
>> This protein does not express without sumo tag and has a poly basic tail
>> (Arg and Lys) at its N-ter. It does polymerize at acidic pHs.
>> When I tried to purify it with chelating Ni-NTA, it did not bind to the
>> column. I thought perhaps His-tag hided somewhere in the protein and is not
>> accessible thus, I repeated the experiments at 8 M urea. It did not make
>> any difference; very low binding to the column with high amount of unbound
>> protein in FT. Your advice is highly appreciated.
>>
>> Regards,
>> Jahan
>>
>>
>>
>

[ccp4bb] Twinabs-cell_now

[Mahesh Lingaraju]

hello everyone

Can anyone point to me where i could obtain the programs twinabs, cell_now
? i cannot seem to locate them by simple googling.

Thanks

mahesh

Re: [ccp4bb] Data processing - twinned xtals

[Mahesh Lingaraju]

Hi Everyone,

Just wanted to let everyone know that i was able to process this dataset
with XDS ( and lots help from experts !)

Thanks again

Mahesh


On Mon, Aug 19, 2013 at 5:07 PM, Petri Kursula wrote:

>  I have often processed images like this with XDS. Of course, you will get
> a better quality of data with a more optimal strategy, but I would never
> say never. If I had a penny every time people told me 'you cannot process
> that'…
>
>  Petri
>
>  On Aug 19, 2013, at 11:40 PM, Mahesh Lingaraju 
>  wrote:
>
>
> Thank you experts for your valuable suggestions. I think Ill try to solve
> it by proper data collection strategy the next time as i am unable to
> process my current data even with the tricks that were mentioned here.
>
>  Thanks again
>
>  Mahesh
>
> On Fri, Aug 16, 2013 at 2:04 PM, Bosch, Juergen  wrote:
>
>> tilted is what I meant at an angle of e.g. 30 or 60 degrees. Works fine
>> with most SSRL beamlines except of the 12-2 microfocus - but that might
>> have been fixed in the meantime.
>>
>>  Jürgen
>>
>>  On Aug 16, 2013, at 1:57 PM, Bosch, Juergen wrote:
>>
>>  for #2)
>>
>>  I'd suggest get some of those Mitigen loops that are titled. I assume
>> you have hexagonal plates as crystals and you really want to shoot along
>> the thin area of the crystal down the sixfold. With normal loops it's an
>> art to get that crystal to sit upright in the loop but not impossible if
>> you take smaller loops.
>>
>>  My longest axis collected was 420 Å to ~2 Å resolution by this method.
>>
>>  Jürgen
>>
>>   On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote:
>>
>> This is clearly a case of a crystal with a very long unit cell; a case
>> which should be approached mindfully.
>>
>> HKL2000 has a default search for indexing solutions such that diffraction
>> along the longest unit cell will be resolved, with the assumed spot size.
>>
>> The problem with such diffraction has 2 aspects:
>> 1) how to process the already collected data where the spots are close to
>> each other;
>> 2) how to collect future data.
>>
>> Ad 1) The best solution is to reduce the spot size, so the spots are
>> resolved. This may require an adjustment of spot size by a single pixel;
>> one should not only change spot radius, but also change the box size
>> between even and odd number of pixels in the box dimensions.
>>
>> Just changing the spot radius changes the spot diameter by an even number
>> of pixels, so if one wants to change the spot diameter by one pixel, one
>> has to change the box size. This is the consequence of the spot being in
>> the center of the box.
>>
>> Just during indexing, there is also a workaround by specifying the command
>> before indexing: longest vector followed by a number that defines the
>> upper limit of the cell size. This may help finding indexing, but will
>> create overlaps between spots during refinement and integration.
>>
>> This dataset presents a problem of collecting data by rotating on the axis
>> perpendicular to the long unit cell. In consequence, the Image 1 has
>> essentially (barely differing in centroid position) overlapping spots, so
>> it would be hard to process them meaningfully by any program.
>>
>> Ad. 2) What would be a better way to collect data in the future?
>>
>>
>> Hi CCP4 folks
>>
>>
>>  I have a data set which is looks twinned ( see the image-1  - I zoomed
>> on
>>
>> to the image so that one can spot the twinning. Furthermore, the spots are
>>
>> very smeary from ~ 30 - 120 degrees of data collection, see image 2) I
>>
>> tried using HKL2000 and mosflm to process this data but i cannot process
>>
>> it. I was wondering if anyone has any ideas as to how to process this data
>>
>> or comments on whether this data is even useful. Also, I would really
>>
>> appreciate if someone could share their experiences on solving twinning
>>
>> issues during crystal growth
>>
>>
>>  Thanks in advance !
>>
>>
>>  Mahesh[image: Inline image 2][image: Inline image 3]
>>
>>
>>
>>
>> Zbyszek Otwinowski
>> UT Southwestern Medical Center at Dallas
>> 5323 Harry Hines Blvd.
>> Dallas, TX 75390-8816
>> Tel. 214-645-6385
>> Fax. 214-645-6353
>>
>>
>>  ..
>> Jürgen Bosch
>> Johns Hopkins University
>> Bloomberg School of Public Health
>> Department of Biochemistry & Molecular Biology
>> Johns Hopkins Malaria Research Institute
>> 615 North Wolfe Street, W8708
>> Baltimore, MD 21205
>> Office: +1-410-614-4742
>> Lab:  +1-410-614-4894
>> Fax:  +1-410-955-2926
>> http://lupo.jhsph.edu
>>
>>
>>
>>
>>
>>  ..
>> Jürgen Bosch
>> Johns Hopkins University
>> Bloomberg School of Public Health
>> Department of Biochemistry & Molecular Biology
>> Johns Hopkins Malaria Research Institute
>> 615 North Wolfe Street, W8708
>> Baltimore, MD 21205
>> Office: +1-410-614-4742
>> Lab:  +1-410-614-4894
>> Fax:  +1-410-955-2926
>> http://lupo.jhsph.edu
>>
>>
>>
>>
>>
>
>

Re: [ccp4bb] non-diffracting xtals

[Mahesh Lingaraju]

Hi Evgeny

I do have a few free cysteine residues but i am not sure if this is the
problem in my case, I say that because i have crystallized this protein
with another ligand without any issues and if oxidation of cysteine
residues is a problem, i would imagine that it would have happened in the
case where it crystallized properly. furthermore, i have other hits with
the protein which gives very mozaic and twinned crystals which diffract
till 2.3 Å. I am having lots of trouble processing that data and for the
work i am trying to do i need to be able to produce and reproduce well
diffracting crystals in a robust manner.
My protein elutes as a single peak on an s-200 once i got rid of
aggregates.
Thanks

Mahesh


On Tue, Aug 20, 2013 at 11:09 AM, Evgeny Osipov wrote:

>  It is very similar with my situation: we are trying to crystallize Fab
> fragments. All we got is spherulites. After rMMS-seeding we got better
> shaped crystals with  only 5 A resolution. So now I think that this caused
> by free cysteine near Fab-Fc knee. Oxidation of them leads to uncontrolled
> aggregation and I am trying to figure out how to protect free cysteine
> without reduction of disulphide bridges between heavy and light chains.
> So, here is the question: do you have free cys residues and are your
> protein is monodisperse (SE-chromatography or DLS)?
>
> P.S.: I am sorry for any mistakes in my letter because Thunderbird does
> not provide any grammar checking tool.
>
> 20.08.2013 04:13, Mahesh Lingaraju пишет:
>
> Hi Jürgen
>
>  you are right, I did not try any major optimization yet. I only tried to
> vary PEG and protein concentration. That did not really improve things too
> much. The protein mostly forms spherulites beyond 25% PEG. I am also
> thinking that these crystals are poorly diffracting/not diffracting as they
> might be growing from sub-microscopic spherulites ? Thanks for the insights
>
>  Mahesh
>
>
> On Mon, Aug 19, 2013 at 8:00 PM, Bosch, Juergen  wrote:
>
>> Well said Petri,
>>
>> also how much PEG3350 do you have in your conditions ? More than 25% ?
>> I'm going after cryo-conditions at this point, you might want to replace
>> your PEG3350 with smaller PEGs or a mixture of PEG400 and PEG3350.
>> Almost sounds as if no optimization of the original conditions was
>> performed yet.
>>
>>  Plenty to do for you, also since you have some crystal use them for
>> seeding into your new screens.
>>
>>  Jürgen
>>
>>  On Aug 19, 2013, at 5:46 PM, Mahesh Lingaraju wrote:
>>
>>  Hi Petri
>>
>>  They are non-diffracting at the home source and they are cryo cooled.
>> Like david suggested I guess ill try introducing a buffer as my condition
>> does not have a buffer. it is ammonium acetate and PEG 3350.
>>
>>  Thanks for the encouragement !
>>
>>  Mahesh
>>
>>
>> On Mon, Aug 19, 2013 at 5:37 PM, Petri Kursula 
>> wrote:
>>
>>> Hi,
>>>
>>> non-diffracting on the home source or state-of-the-art synchrotron?
>>> Cryocooled or room-temperature? What happens if you change the buffer but
>>> keep your pH? etc etc...
>>>
>>>  For an important project, one should never ever give up.
>>>
>>>  Petri
>>>
>>>
>>>  ---
>>> Petri Kursula, PhD
>>> project leader, adjunct professor
>>> Department of Biochemistry & Biocenter Oulu, University of Oulu, Finland
>>> Department of Chemistry, University of Hamburg/DESY, Germany
>>> www.biochem.oulu.fi/kursula
>>> www.desy.de/~petri/research <http://www.desy.de/%7Epetri/research>
>>> petri.kurs...@oulu.fi
>>> ---
>>>
>>>
>>>
>>>
>>>
>>>  On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju  wrote:
>>>
>>>  Hello people
>>>
>>>  I recently obtained hexagonal rod like crystals (150x50x20 um) which
>>> turned out to be non diffracting. What is the usual convention for cases
>>> like this ? do people usually give up on the condition or still try to
>>> optimize it ?
>>>
>>>  The crystals are also not very reproducible. I believe it is because
>>> of ammonium acetate in the condition causing fluctuations in the pH because
>>> of its volatility. Is there any way to work around such a problem ?
>>>
>>>  Thanks
>>>
>>>  Mahesh
>>>
>>>
>>>
>>>
>>
>>..
>> Jürgen Bosch
>> Johns Hopkins University
>> Bloomberg School of Public Health
>> Department of Biochemistry & Molecular Biology
>> Johns Hopkins Malaria Research Institute
>> 615 North Wolfe Street, W8708
>> Baltimore, MD 21205
>> Office: +1-410-614-4742
>> Lab:  +1-410-614-4894
>> Fax:  +1-410-955-2926
>> http://lupo.jhsph.edu
>>
>>
>>
>>
>>
>
>
> --
> Eugene Osipov
> Junior Research Scientist
> Laboratory of Enzyme Engineering
> A.N. Bach Institute of Biochemistry
> Russian Academy of Sciences
> Leninsky pr. 33, 119071 Moscow, Russia
> e-mail: e.m.osi...@gmail.com
>
>

Re: [ccp4bb] non-diffracting xtals

[Mahesh Lingaraju]

Hi Jürgen

you are right, I did not try any major optimization yet. I only tried to
vary PEG and protein concentration. That did not really improve things too
much. The protein mostly forms spherulites beyond 25% PEG. I am also
thinking that these crystals are poorly diffracting/not diffracting as they
might be growing from sub-microscopic spherulites ? Thanks for the insights

Mahesh


On Mon, Aug 19, 2013 at 8:00 PM, Bosch, Juergen  wrote:

> Well said Petri,
>
> also how much PEG3350 do you have in your conditions ? More than 25% ? I'm
> going after cryo-conditions at this point, you might want to replace your
> PEG3350 with smaller PEGs or a mixture of PEG400 and PEG3350.
> Almost sounds as if no optimization of the original conditions was
> performed yet.
>
> Plenty to do for you, also since you have some crystal use them for
> seeding into your new screens.
>
> Jürgen
>
> On Aug 19, 2013, at 5:46 PM, Mahesh Lingaraju wrote:
>
> Hi Petri
>
> They are non-diffracting at the home source and they are cryo cooled. Like
> david suggested I guess ill try introducing a buffer as my condition does
> not have a buffer. it is ammonium acetate and PEG 3350.
>
> Thanks for the encouragement !
>
> Mahesh
>
>
> On Mon, Aug 19, 2013 at 5:37 PM, Petri Kursula wrote:
>
>> Hi,
>>
>> non-diffracting on the home source or state-of-the-art synchrotron?
>> Cryocooled or room-temperature? What happens if you change the buffer but
>> keep your pH? etc etc...
>>
>> For an important project, one should never ever give up.
>>
>> Petri
>>
>>
>>  ---
>> Petri Kursula, PhD
>> project leader, adjunct professor
>> Department of Biochemistry & Biocenter Oulu, University of Oulu, Finland
>> Department of Chemistry, University of Hamburg/DESY, Germany
>> www.biochem.oulu.fi/kursula
>> www.desy.de/~petri/research
>> petri.kurs...@oulu.fi
>> ---
>>
>>
>>
>>
>>
>> On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju  wrote:
>>
>> Hello people
>>
>> I recently obtained hexagonal rod like crystals (150x50x20 um) which
>> turned out to be non diffracting. What is the usual convention for cases
>> like this ? do people usually give up on the condition or still try to
>> optimize it ?
>>
>> The crystals are also not very reproducible. I believe it is because of
>> ammonium acetate in the condition causing fluctuations in the pH because of
>> its volatility. Is there any way to work around such a problem ?
>>
>> Thanks
>>
>> Mahesh
>>
>>
>>
>>
>
> ..
> Jürgen Bosch
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
> Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
> http://lupo.jhsph.edu
>
>
>
>
>

Re: [ccp4bb] non-diffracting xtals

[Mahesh Lingaraju]

Hi Petri

They are non-diffracting at the home source and they are cryo cooled. Like
david suggested I guess ill try introducing a buffer as my condition does
not have a buffer. it is ammonium acetate and PEG 3350.

Thanks for the encouragement !

Mahesh


On Mon, Aug 19, 2013 at 5:37 PM, Petri Kursula wrote:

> Hi,
>
> non-diffracting on the home source or state-of-the-art synchrotron?
> Cryocooled or room-temperature? What happens if you change the buffer but
> keep your pH? etc etc...
>
> For an important project, one should never ever give up.
>
> Petri
>
>
> ---
> Petri Kursula, PhD
> project leader, adjunct professor
> Department of Biochemistry & Biocenter Oulu, University of Oulu, Finland
> Department of Chemistry, University of Hamburg/DESY, Germany
> www.biochem.oulu.fi/kursula
> www.desy.de/~petri/research
> petri.kurs...@oulu.fi
> ---
>
>
>
>
>
> On Aug 19, 2013, at 11:49 PM, Mahesh Lingaraju  wrote:
>
> Hello people
>
> I recently obtained hexagonal rod like crystals (150x50x20 um) which
> turned out to be non diffracting. What is the usual convention for cases
> like this ? do people usually give up on the condition or still try to
> optimize it ?
>
> The crystals are also not very reproducible. I believe it is because of
> ammonium acetate in the condition causing fluctuations in the pH because of
> its volatility. Is there any way to work around such a problem ?
>
> Thanks
>
> Mahesh
>
>
>
>

[ccp4bb] non-diffracting xtals

[Mahesh Lingaraju]

Hello people

I recently obtained hexagonal rod like crystals (150x50x20 um) which turned
out to be non diffracting. What is the usual convention for cases like this
? do people usually give up on the condition or still try to optimize it ?

The crystals are also not very reproducible. I believe it is because of
ammonium acetate in the condition causing fluctuations in the pH because of
its volatility. Is there any way to work around such a problem ?

Thanks

Mahesh

Re: [ccp4bb] Data processing - twinned xtals

[Mahesh Lingaraju]

Thank you experts for your valuable suggestions. I think Ill try to solve
it by proper data collection strategy the next time as i am unable to
process my current data even with the tricks that were mentioned here.

Thanks again

Mahesh

On Fri, Aug 16, 2013 at 2:04 PM, Bosch, Juergen  wrote:

> tilted is what I meant at an angle of e.g. 30 or 60 degrees. Works fine
> with most SSRL beamlines except of the 12-2 microfocus - but that might
> have been fixed in the meantime.
>
> Jürgen
>
> On Aug 16, 2013, at 1:57 PM, Bosch, Juergen wrote:
>
> for #2)
>
> I'd suggest get some of those Mitigen loops that are titled. I assume you
> have hexagonal plates as crystals and you really want to shoot along the
> thin area of the crystal down the sixfold. With normal loops it's an art to
> get that crystal to sit upright in the loop but not impossible if you take
> smaller loops.
>
> My longest axis collected was 420 Å to ~2 Å resolution by this method.
>
> Jürgen
>
> On Aug 16, 2013, at 1:46 PM, Zbyszek Otwinowski wrote:
>
> This is clearly a case of a crystal with a very long unit cell; a case
> which should be approached mindfully.
>
> HKL2000 has a default search for indexing solutions such that diffraction
> along the longest unit cell will be resolved, with the assumed spot size.
>
> The problem with such diffraction has 2 aspects:
> 1) how to process the already collected data where the spots are close to
> each other;
> 2) how to collect future data.
>
> Ad 1) The best solution is to reduce the spot size, so the spots are
> resolved. This may require an adjustment of spot size by a single pixel;
> one should not only change spot radius, but also change the box size
> between even and odd number of pixels in the box dimensions.
>
> Just changing the spot radius changes the spot diameter by an even number
> of pixels, so if one wants to change the spot diameter by one pixel, one
> has to change the box size. This is the consequence of the spot being in
> the center of the box.
>
> Just during indexing, there is also a workaround by specifying the command
> before indexing: longest vector followed by a number that defines the
> upper limit of the cell size. This may help finding indexing, but will
> create overlaps between spots during refinement and integration.
>
> This dataset presents a problem of collecting data by rotating on the axis
> perpendicular to the long unit cell. In consequence, the Image 1 has
> essentially (barely differing in centroid position) overlapping spots, so
> it would be hard to process them meaningfully by any program.
>
> Ad. 2) What would be a better way to collect data in the future?
>
>
> Hi CCP4 folks
>
>
> I have a data set which is looks twinned ( see the image-1  - I zoomed on
>
> to the image so that one can spot the twinning. Furthermore, the spots are
>
> very smeary from ~ 30 - 120 degrees of data collection, see image 2) I
>
> tried using HKL2000 and mosflm to process this data but i cannot process
>
> it. I was wondering if anyone has any ideas as to how to process this data
>
> or comments on whether this data is even useful. Also, I would really
>
> appreciate if someone could share their experiences on solving twinning
>
> issues during crystal growth
>
>
> Thanks in advance !
>
>
> Mahesh[image: Inline image 2][image: Inline image 3]
>
>
>
>
> Zbyszek Otwinowski
> UT Southwestern Medical Center at Dallas
> 5323 Harry Hines Blvd.
> Dallas, TX 75390-8816
> Tel. 214-645-6385
> Fax. 214-645-6353
>
>
> ..
> Jürgen Bosch
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
> Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
> http://lupo.jhsph.edu
>
>
>
>
>
> ..
> Jürgen Bosch
> Johns Hopkins University
> Bloomberg School of Public Health
> Department of Biochemistry & Molecular Biology
> Johns Hopkins Malaria Research Institute
> 615 North Wolfe Street, W8708
> Baltimore, MD 21205
> Office: +1-410-614-4742
> Lab:  +1-410-614-4894
> Fax:  +1-410-955-2926
> http://lupo.jhsph.edu
>
>
>
>
>