these mutations but they're not the same as Derewenda's
surface entropy method (which also works) or some of the existing servers
dedicated to the same purpose.
If it helps, you can look at my recent short blurb for other ideas :)
http://www.xtals.org/pdfs/rescue_crystals.pdf
Artem
> Dear
Hi,
Don't forget system losses (friction in the tubing). An empty AKTA prime
typically eats up about 0.25 MPa so your maximum is closer to 0.7MPa loss
on column.
Artem
>
>
> Dear Frank,
> AKTA prime delivers a max. pressure of 1 MPa. The 24 ml SEC columns can
> be run at pr
27;d be silly (I've seen
people do this, and the result isn't pretty). Filter your solutions and
everything would be OK :)
Cheers!
Artem
nd place a hold on the structure - it's not really a choice at
all. Up until now, all my structures are always released *immediately*
(yes, before publication) - so please try to avoid negative comments!
Many thanks,
Artem
choice at the moment - not to publish at all, versus (maybe) to
publish but keep the structure on hold for a year. Since a whole lot of us
are obliged to look for jobs (yours truly included), publications are
important. Hopefully this explains things a bit better.
Best regards,
Artem
that you still have to buy primers, use PCR reagents and
cleanup/cloning kits, etc. etc. and at the end also would want to sequence
the products.
How many genes are you trying to get?
Artem
> Sorry a little offtopic...
>
> We'r trying to clones a number of putative huma
me sort (such as ammonium sulfate or PEG
precipitation, or perhaps bulk ion exchange etc.) prior to your IMAC step.
Best regards,
Artem
> Dear CCP4 users,
>
> I'm purifying a kind of protease having His-tag. The protein is expressed
> in
> insect cells and broken by sonication.
> I used
nusually high protease
load in your lysate
Caveat emptor :)
Artem
> Hi,
>
>
>
> We got some good experiences using the IBA streptag for baculo-expressed
> proteins. You'll have to redo the cloning, but it will be worth your while
> when you see the first purification
TVMV, TEV, and Thrombin. With the first two enzymes, you
have no worries whatsoever about non-specific digestion, whereas Thrombin
is pretty gentle but has been reported to eat things up sometimes.
Cheers,
Artem
> Hi,
>
> A non-ccp4 Q. Sorry.
>
> I would like to use a cleavable puri
even none if you have the right N-terminus).
Artem
> I'd like to third TeV & second 3C ("PreScission").
>
> Both have high specificity, good processivity and I have had a lot of
> success with 3C.
>
> I have _never_ got Thrombin to cut cleanly - but I guess I co
Since people asked:
http://www.usbweb.com/category.asp?cat=118&id=22293
is my preferred source. Please note that I don't have any relation to USB
whatsoever (but large amounts of money sent to my unnamed Swiss bank
account are always appreciated. The password for the account is BACON
provisions of this kind.
Are the journals trying to exclude industrial submissions?
Artem
elf!
Good luck :)
Artem
> Hi Jobi
>
> Sounds like you need to explore your protein vs PEG concentration, and my
> guess (directly contradicting Artem's) is that chances are you need to
> pump
> [protein] way up and drop [PEG] way down.
>
> Like in [protein]=>30-40
f the drop is pulled around by fluxes of evaporating
iPrOH.
There are numerous other things you can try, including the one mentioned
here: http://www.xtals.org/ under 'stuff'.
Regards,
Artem
> Hi,
> l have a crystal grow at condition screen l 40:
> 0.1M tri-sodium citrate di
rotein. I would be glad to help you
with that.
Artem
llary - it still
can be saved sometimes :) Or not.
Artem
> diffracts in general. It is true it may be a cryo problem, but if
> the non cryo protected crystals do not diffract then why would one
> expect the cryo protected one to.
ifact?
Regards,
Artem
> Dear all
> in my structure I think I can see an oxidised Cys in cys-SO. Refining
> cys-SO
> I observe a residual density between the oxigen of one oxidised cys and
> the
> one of the other molecule in AU.
> I'd like to try to refine it as cys-SO-O
Hi,
We use additives extensively. Pretty much all available kits, as well as
some of our own design. My favorite is Nextal (Qiagen) kit named the Salt
Optimizer.
Artem
> Hello everyone,
>
> Does anyone have suggestions or comments on the various additive
> screens that are availabl
is a small error in unit cell parameters for instance, etc.
Artem
>
> Dear all
>
> I am doing one structure at 2.6A resolution where I found unusual density
> near methionines. Actually three methionines come close together nearby
> and I get difference even at 5sigma cutoff. I don&
like new
year's tree lights.
Artem
> The obvious solution is go to a synchrotron and scan around the Mn edge
> and
> see what happens, however, whilst waiting for beam time, is there any way
- have the selenium
safe and the disulphides oxidized.
Not to mention that there always are the good old heavy atom derivatives
to fall back on if the selenium data does not pan out :)
Artem
27;stick a fork in it' method: stick a needle in
your rod-like crystals and push. If you hear a crack, and see sharp clean
edges on the break - it's salt. If you feel the crystal 'give' and see
bending or pitting - it's probably protein.
Good luck!
Artem
> Hi All!
>
rowth conditions (which is how PKA was done)
etc.
Artem
> Dear all,
>
> Below is a question my friend asked me, but I have never worked on
> phosphorylated proteins.
> Has anyone worked on crystallizing phosphorylated proteins and can you
> comment on it?
>
ds on your protein and on conditions.
b. I am surprised that mercury derivatives did not work. Have you tried
increasing amounts of ethyl mercury phosphate?
c. Have you tried my favorite platinum derivative - KPt(NO2)3? You can add
a LOT of this stuff - it's very mild on the protein.
Artem
&
procedures for rational protein crystal
engineering and now need more Guinea pigs to test them.
Artem
> Greetings,
>
> I've taken a protein (structure known) with some disordered termini and
> created a new truncated construct that lacks these disordered bits
> (approx.
>
ld be relatively small so it will all get
eaten up while the cells are still in early log phase.
Regards,
Artem
> Hello everybody,
>
> Sorry for an offtopic question. I am trying to express a protein in M9
> minimal media for Selenomet incorporation. When grown in LB this protein
> express
ike a halide
soak...
Let me know if you need more detailed help :)
Artem
> Dear all,
>
> Could any one recommend some heavy atoms used for crystals grown in 0.1M
> tri-sodium citrate pH 5.6, 1M Lithium sulfate and 0.5M ammonium sulfate? I
> read from Hampton user guide of heavy atom
everything works, you should be able to easily detect 5 ug
of protein in a *reasonable* peak volume. This would require a true
injection of perhaps 15 ul assuming a dilution down to about 50 ul in the
peak. Oviously if you have multiple smeary peaks than you won't get any
signal at all :)
with lower concntrations of
chaotropes.
Good luck,
Artem
> Hi,
>
> I have a membrane protein with a C-terminal S-tag, and there is no
> cleavage
> site for the S-tag (novagen, e.g, thrombin, EK). I wonder whether I still
> can use this for purification without cleavage of the S-tag. Here ar
econd approach, since soaking can and does generate scary artifacts.
Artem
> Hi, all,
> I met some crystal structures with disordered active sites. Soaking
> common ligands can not make it become ordered. I am wondering what
> people generally do in such situation.
>
> Thanks,
>
> Nian Huang
>
To steal the words from one of my favorite cartoon shows, to me stereo is
just a headache with pictures.
Artem
(the show is Futurama)
> I was just curious to see how popular stereo graphics is for
> crystallographers. I personally think its a wonderful teaching tool which
> is
> cur
'll gladly remove it so as not to cause confusion.
Artem
> Yes, this is how a wiki page for CCP4 might look like. However, to make a
> wiki successful we have to make sure it is secure and that there is a
> dedicated core team to maintain it. Kevin was asked by the CCP4 to lead
>
more
"open" approach to wiki - such as articles could be written by anyone who
has a 'real name registration'. Any kind of rigorous procedure designed to
select 'worthy' contributors goes against the spirit of the whole
enterprise, in my opinion :)
Artem
> Kevin Co
Does this happen in darkness as well as under light? Can
you repeat this in an oxygen-free environment? Does this work with linear
DNA or synthetic oligos? Can you cleave single-stranded DNA (i.e. just one
oligo without any self-complementarity).
Artem
existing style that is identical to the one I look, I
would very much appreciate this!
Regards,
Artem
ly, did you try seeding? If seeding does not work then you should
take a really close look at the above :)
Good luck!
Artem
> hi all,
> I am working on a protein for which we get nicely diffracting
> crystals but the problem is that the crystals grow from anywhere between
> 4-6
Thank you all who sent in the link to the correct endnote library -
somehow I failed to find it on the endnote website, which proves that not
only I am a lazy slob, but a blind one as well :)
Thank you,
Artem
ion
tends to produce complex mixtures of products, unless you do it with a
huge excess of the reagent and allow the reaction to proceed to
exhaustion.
Artem
> Our lab is trying to crystallize a highly soluble (100+ mg/ml) protein
> with a molecular weight of 35 kd.
>
> The protein was scr
a few ratios in a simple screen (say,
one 96-well screen over 6 or 9 ratios) to see how things develop from
there.
Good luck,
Artem
> Dear CCP4 Users,
> I'd like to solve the structure of the protein-protein complex. I
> intend to purify and incubate the complex then run gel-filtrati
ts".
Good luck,
Artem
> Hi all,
>
> I have a 1.8A structure for which I am in the final stages of refinement.
> In a critical hinge region there are two unknown molecules bound to my
> protein, which appear to induce the formation of a domain swap. I am
> totally out of ideas as
exceptions, such
as iodine [not iodide!] soak to iodinate tyrosines which is typically
better done during a day or so with very low amount of I2, via vapor
phase.
Artem
> Hi,
>
> I'd like to find out how successful the "quick soak" method for heavy
> atom derivatisation p
es) have been proposed for
kinases with a Cys in the ATP site:
http://www.freshpatents.com/Specific-kinase-inhibitors-dt20060413ptan20060079494.php
Good luck,
Artem
> Hi all
>
> I have searched the PDB without much luck and wondered whether anyone out
> there could confirm:
>
> I
C-terminal 'clasp' domain of any bacterial gyrA or parC - very small,
pretty tight dimers.
Artem
> This is not strictly a crystallography question, but I imagine (and
> hope) that some of you would be able to advise me. In short, I would
> like to add to t7he N-terminal par
ou've hit on something
very essential since you're going from good expression down to no
expression.
Lastly, you could try insect cells :)
Artem
>
> Dear all,
>
> Our lab is new to working with Pichia pastoris, also new to working with
> glycosylated proteins. We have a co
ction
curves of quadric surfaces'. There are seventeen types of quadric surfaces
(including real and imaginary ones) and they're a right pain in the neck
to calculate intersections of.
Artem
> Dear All -
>
> if memory serves me right, the intersection curve of
> a cone with a c
What you need is a tubing and connector kit. Every decent lab has one, and
you also usually can order these in part or whole from FPLC/HPLC
manufacturers. The two types of female fittings are then attached to a
short length of tubing and presto.
Artem
> Dear All,
>
> I have econo co
Any basic chromatography (for proteins) text will have those. It's
generally not hard - if you have dry sephadex just resuspend it in 10-20%
alcohol, stir well (but gently!) and let sit overnight, then pack the
column, wash, and go.
Artem
> does anyone have protocols / method of sephad
tp://www.jbc.org/cgi/content/full/278/3/2008/F1
4. in theory this could be a particularly stable calcium complex (what's
the distance between the center of the fat blob in the middle and the N of
His?)
Cheers,
Artem
> Hi -- I am close to finishing up a protein structure of an ald
small amount - enough to drive the displacement at the end appreciably.
Depending on the amount of ice you may experience angular, lateral, or
vertical movement.
Drying the pins before use eliminated most of the issues.
Artem
> Dear all,
>
> in a recent synchrotron trip we had a problem
Yes, I did that recently and it worked although I found SOLOMON maps to be
(subjectively) better. You should probably use the 'centroid' phases from
the eden.mtz (SHARP gurus might want to correct me :))
Artem
> Hi
>
> Sorry for a mixed up question about two great software.
&g
You could try Coomassie Blue Native gel. It's a very neat technique and it
worked for me on a couple of occasions. In one unfortunate case, it
resulted in dissociation of a heterotetrameric complex, though.
Artem
> Dear CCP4 community,
>
> Sorry for the off-topic subject, but
are starting at fairly high salt (1-2 M NaCl or KCl
typically). But at least you won't have to pass the pI.
Cheers,
Artem
> Dear All,
>
> This is with reference to the purification of our recombinant protein
> sample
> expressed in E.coli as inclusion bodies. After Solubil
Yes, we use them all the time and they're great. Stuff does not slip on
the bottom like it does in the glass Dewars and the formation of ice is
greatly reduced. Plus, they're much easier to dry.
Artem
> Does anyone in the biocrystallogaphy community use foam dewars for
> handly
mage
of the e.d. that I saw does not support this.
Artem
> Dear Colleagues,
>
> The amidine originating from the condensation reaction between the
> Met N-formyl group and the His side chain should be the other way
> around! This would also be roughly consistent with the repor
To quote Terry Pratchett:
+++ Divide By Cucumber Error. Please Reinstall Universe And Reboot +++
Couldn't resist :)
Artem
> You have an error in your mtz file..
>
> OK- so I am sorry! But the Warning will not disappear..
> Eleanor
>
May be something that tagged along from purification or one of the
impurities in the chemicals... nitrate, carbonate, etc.
Incidentally, a 3.9 M BMP file was a nasty surprise for my mail box. A
JPEG or PNG file of similar quality would have taken less than 100K...
Artem
>
> Hi all,
>
heers,
Artem
>
> Dear Members,
>
> I am getting crystals of my protein. The secondary structure prediction
> implies that it has N-terminal with high degree of loop regions. I also
> get some mountable crystals yielding weak diffraction pattern(10 A). The
> quality of the crystal
- Ni-NTA is just one of many.
I would be glad to help you further off the main list if you want to share
more details.
Cheers,
Artem
Hi,
It could be all sorts of things, but the one that for some reason is stuck
in my mind is isopentenyl phosphate (phosphate, not pyrophosphate!). Of
course w/o seeing the density in 3D this is just a guess.
Artem
> Dear All,
>
> When I was refining my structure, I found some unmode
Sure, it's not always 'disastrously bad' to have EDTA (hence my use of the
word 'bad' rather than a more categorical statement. Donuts are bad for me
yet I can't stop eating them :)
Yes, you can take a risk. However since periplasmic isolation is already a
PITA, wh
ted the two processes.
Synthetic DNA is cheap these days. If you can afford it - it's useful to
try before taking the next step. In this case the obvious next step is
attempt at expression in insect cells - kinases usually work out really
well in IC.
Artem
argU (AGA, AGG), ileY (AUA), leuW (CUA)
BL21-CodonPlus-RP strain argU (AGA, AGG), proL (CCC)
BL21-CodonPlus(DE3)-RP strain argU (AGA, AGG), proL (CCC)
BL21-CodonPlus(DE3)-RP-X strain argU (AGA, AGG), proL (CCC)
Artem
> Thanks for the reply.
>
> I've checked my sequence for rare codons;
e trouble? You decide :)
Artem
> Some thoughts about SUMO tags and fusion tags in general.
>
> Fusion tags also follow the "Garbage In, Garbage Out" philosophy.
> Yes, if for many of the reasons already hashed out extensively on
> CCP4BB, one is dealing with lack of expr
:)
Is this 'normal' and 'expected'? I would say that yes, if you are working
with a large human protein that has potentially disordered regions and is
normally a part of a stable complex with other stuff - yes you can expect
problems exactly like these (or worse).
Artem
>
Hello,
1. As long as all proteins have seventy amino-acids or less and express in
E. coli in mM concentrations - we're in business.
2. As for the question below - my favorite answer is 'It will take a week
and ten million dollars in unmarked bills. We begin as soon as the money
arrives
ect
matter is 'hot' and interesting, and doubly so if the results are
unexpected or remarkable in some other way. I doubt that people would go
to the trouble of faking routine structures, since those generally do not
make their authors any money/fame/recognition.
Artem
> After consul
synthesis/folding or in secretion
itself.
More details would be awesome!
Artem
> Hi,
>
> We are trying to express a protein in insect cells as secreted. However,
> the yield is VERY low using either pFastBAC or pAcGP67A. We have tried
> many different constructs and this is th
secretion process itself?
More details would be great to have!
Artem
> Hi,
>
> We are trying to express a protein in insect cells as secreted. However,
> the yield is VERY low using either pFastBAC or pAcGP67A. We have tried
> many different constructs and this is the only on
ren't always the same which
(in my experience) is responsible for 40-50% of cases when screening
conditions do not reproduce.
Old PEG solutions can go very acidic, for instance - see previous CCP4bb
exchanges on this subject.
Good luck!
Artem
> Hello,
>
> I am trying to reproduce two
to go
out of the window and one has to painstakingly scan the termini in small
increments.
If you're interested in further discussion, we should probably take this
out of the list. Feel free to drop me a note.
Artem
> Hello
>
> I am designing a protein construct for structural biolo
e several times :)
Artem
> You always get the entry of Bromide into crystal by quick soaking,
> because it does not require the incorporation of Bromide into the
> protein. But whether the signal is good enough for phasing is another
> story. You have to collect the full data set to know th
odified method that I will be posting shortly does not
involve adding iodine directly to the crystals, which is its sole (but I
think significant) benefit :)
Artem
> Hi everyone,
> the in situ iodination reaction described in the following classic paper
> by
> the late Paul Sigler works
Hey,
This is *the* place to report my experiments with Uranium (IV) Astatide
(UAt4) and Radon for phasing!
Artem
> Dear Crystallography Community:
>
... good stuff ...
> Sehl Oediter
> Chief Guy in Charge
> Journal of Failed Crystallization Trials
> Sell Press
> Boston, MA
>
there's considerably fewer human proteins than theer are stars.
Imagine weary crystallographer coming home:
Darling, we finally solved the structure of Edgar Allan Poe!
What, full-length?
No, unfortunately we had to chop the head off - it was wobbling all over
the place.
Artem
> H...
The impact factor of UAt4 has recently been upgraded from 'huge' to 'all
over the place' after the unfortunate accident at the Miscatonic
university labs which left half the building as glowing multi-colored
glass puddle.
Artem
> What is the impact factor for that? 100
We often have good luck using protease cleavage sites as linkers. They're
evolved to have certain flexibility to them and they usually have a
healthy mixture of hydrophobic and hydrophilic amino acids. As a
delightful bonus they also offer the option to separate the two partners
at will.
have come from.
Pure speculation, of course!
Artem
> Dear all,
> i am working on a structure of M.tb, solved at 2.18 A. In my structure
> there
> is a density near to the active site and we modelled it as a malonate (C3
> O4
> H2) molecule acorrding to the density, which was
om purifying protein
from transfected CHO, HEK, etc. cells where you can expect reasonable
abundance.
Details are very important :)
Artem
>
> Hi, All:
>
>Although it is off-topic, definitely I think I can get some help
> here because we crystallographers are dealing with
n get
polyclonals made very rapidly. Polyclonals are potentially even better for
immunocapture as some of them will recognize natively folded epitopes,
unlike monoclonals that may be raised against a peptide from deep inside
the protein.
Again, more details are needed :)
Artem
> The holy tr
.
Your solutions are
1) to use cells with higher levels of lac repressor (XL1-blue for example)
2) to re-clone this ORF under some tightly controlled promoter
Artem
>
> i have cloned my gene successfully into qiagen vector into pqe30 but i do
> transformation of this into BL21,pLys,Rosseta,C4
at
contain this promoter.
I've expressed many proteins in XL1-Blue and I see no reason why you can't
express yours, either.
Artem
> xl1-blue is not an expression host,since i have cloned it successfully,i
> need to transform into expression host, i am able to transform it into dh5
>
d
old days pQE series of vectors was quite commonly used and I had things in
that were inherited from others - these 'things' were fairly simple to
express in XL1-blue whereas they gave me loads of trouble in other strains
and I was too busy/lazy to re-clone them.
Artem
> Hello Artem,
&
, exchange with a 'milder' detergent, and so forth.
Good luck!
Artem
>
> dear sanjiv
> i dont think that the removal of detergent is more difficult than
> urea,since u are purifying ur protein over Ni-NTA column,so after a few
> wash over there u can remove the detergent c
glucose in the growth medium; there are other options
available to you. Hopefully once you induce transcription, the gene
products do not shut down protein synthesis - which would be a disaster
since it would likely shut down its own synthesis as well. Since I don't
know what you're growin
ions = lovely salt crystals. It all usually ends up in
tears.
If you absolutely must make phosphate buffers - just open up any basic
practical biochemistry book, there are standard ratios (both by weight and
by volumes of molar solutions) that produce specified pH in a wide range.
Artem
> Dear all,
&
Use the FRET channel - it's ideal for SYPRO orange.
Artem
> Sorry Charlie for the late reply.
> I'm using the CFX96 5 channel with Sypro and other dyes. I tested the
> machine against the Eppendorf product nd decided to go for the Biorad
> version as running identical sampl
Hi,
If you're worried - instead of transfering the crystal, just flood the
entire drop with excess of your cryo buffer. No exposure to air this way.
I bet that the bubbles won't go away though :)
Artem
> Dear members,
> I have protein crystals grown in a condition with Isopr
Hello,
Van der Waals Forces: A Handbook for Biologists, Chemists, Engineers, and
Physicists by V. Adrian Parsegian
Give it a try!
Artem
> Hi CCP4ers
>
> Perhaps I am hashing over old news.but
>
> We are having a discussion about Van Der Waals contacts and effective
>
7;s safety precautions are different.
Artem
>
> Does mercury tends to get into the protein core to denature protein or
> not?
>
> For gel-shift assay, do people normally use a special gel tank for heavy
> metal work?
>
> thanks in advance
>
> Xiaoli Xiong (Alex)
>
&
's
kind of high for disposables) and also they only come in 1000-ml capacity.
I wonder if anyone on this list have seen/used something like that but a)
cheaper and b) in a wider variety of volumes.
Thank you,
Artem
I second Phil's opinion - it is better to scan and be sure - as long as
the scan results are not hideously abnormal. If you cannot scan for
whatever reason but are sure that the X-ray optical system is properly
calibrated - then use Phil's numbers below :)
Artem
> Always take the
Hi,
Barring orientation mishaps - it almost sounds like you have an ethyl
rather than a methyl ester...
Could it be that your compound is an ester of ethanol rather than methanol?
Artem
> Dear ccp4 experts,
> I am trying to solve the structure of one esterase with its substrat
wap a cloned drive in and your computer
won't know the difference!
Artem
>
>
>
> Dear List,
>
>
>
> I'm planning to migrate soon from Red Hat Linux 7.0 on an HP xw6000
> workstation with dual Xeon processor.
>
>
>
> Please, any suggestion for the best
My suggestion would be to attempt finding Se atoms in the MAD dataset and
then use these coordinates to do sulphur phasing - assuming of course that
you can get the native crystals to diffract at an appropriately low-energy
X-ray source.
Artem
>
> Dear all,
>
>
>
> I hav
Beyond improvement via 'chemical' means, I would heartily recommend
mutagenesis for crystal packing improvement. I would be glad to help you
with the latter if you're interested.
Artem
> Dear All:
> We have been struggling to improve the crystals shown in the attachment.
pLysS contains a phage lysozyme.
You can get it from any pLysS cells. Ditto pLysE
Artem
> Dear crystallographers,
>
> does anyone happen to have a plasmid containing a lysozyme gene (any
> naturally occurring sequence) that would be suitable for use as a PCR
> template? We'
As far as systematic absences lost due to special orientation - yes,
although if you have a bent loop (similar to the cheater pins from the
bygone days) that's not much of an issue. Additionally, precise selection
of space group can be always made as the structure's solved :)
Artem
> Useful
Good times :)
When you patent this design, be sure to give royalties to all the
contributors. Daddy needs a new Ferrari.
I assume you've seen Inteins already, right? Cleavage induced by DTT or BME.
Artem
> Hi,
>
> We would like to design a self-cleaving tag. It will be simi
Hi,
This is not typical. Try different cell lines for propagation and also add
more sugar and glycerol to your medium - this helps.
Artem
> Dear all,
>
> We are attempting to clone into pETDuet, pACYCDuet, pCola1Duet and
> pCDFDuet
> and we are encountering numerous difficult
d together in a single
tube with air trapped between them - if the 'vent' is blocked (by e.g. old
grease or something) then these may pop.
https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/oculars/
But the short version is right 99% of the time.
Artem
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