Dear All,
The CCP4 document says refmac can process electron microscopy map for
refinement. But I cannot localize that function in the refmac5 of the CCP4.
Will you please advise how to have the refmac process the electron microscopy
map?
Smith
Thanks everyone for your insights,
I really appreciate it.
Cheers,
ivan
On Mon, Apr 20, 2015 at 5:38 PM, Edward A. Berry wrote:
> On 04/20/2015 06:44 PM, xaravich ivan wrote:
>
>> Hi CCP4eans,
>>
>> solvent based HPLC system?
>>
>
> Do you mean like acetonitrile:water:TFA on reverse-phase column
On 04/20/2015 06:44 PM, xaravich ivan wrote:
Hi CCP4eans,
solvent based HPLC system?
Do you mean like acetonitrile:water:TFA on reverse-phase columns? I think this
is
routinely used for analytical runs or to obtain material for protein chemistry,
but the concentration of acetonitrile required
On Apr 20, 2015 8:28 PM, "Roger Rowlett" wrote:
> Depends on what you want to accomplish... If you have a liter of crude
> lysate, "capacity" should be one of the choices. A step gradient is fast
> but low resolution; a gradient elution has more resolution but will eat
> buffer and take much long
Many protein purification media have large particle sizes or will not
mechanically stand excessive pressure without crushing. (Superdex is an
example of the latter.) In general, smaller stationary phase particle sizes
give rise to higher selectivity and separation efficiency at the expense of
flow
>I have processed test data sets (measured at the SLS) which have only a few
>non-zero pixels on each frame. Can be nicely integrated - but it was insulin.
What were the values of the non-zero pixels, I wonder? How many total counts
per image? And this was, I assume, Pilatus? Sounds pretty amazi
Hi CCP4eans,
Do you guys have any preference in purifying a protein by SEC in FPLC
system or using a solvent based HPLC system after the initial affinity
column purification. Where would you prefer HPLC purification over standard
FPLC?
I have routinely seen HPLC based purification of organic molec
On Fri, 17 Apr 2015 15:00:12 +, Keller, Jacob
wrote:
>>Finally, there is simply no downside in collecting more degrees with
>>proportionally lower dose on the Pilatus. Merging the data recovers the
>>_same_ signal. It has only advantages - so many that I won't write them up
>>here with 1
Thank you Rajesh,
That could be a possibility though. I am planning to do some MS analysis
from the extra gel bands, if I get any by running SDS-PAGE on the purified
protein.
Best,
Dipankar
On Mon, Apr 20, 2015 at 10:42 PM, rajesh ponnusamy <
ponnusam...@googlemail.com> wrote:
> just to add u
Dipankar,
It is possible that the peptide was cleaved by other proteases present in the
soaking solution as impurities. The crystallized protein simply selected that
fragment that binds the best. You can try soaking with a lower amount of the
peptide (use a smaller drop and concentration).
Alex
Dear Barbel,
Thank you!
Yes you are right that I did the SDS-PAGE with bigger substrate. Regarding
peptide, we did check the MS and HPLC profile for the peptides which
clearly shows that there should not be any cleaved peptides!
Best,
Dipankar
On Mon, Apr 20, 2015 at 9:46 PM, Bärbel Blaum <
ba
On Fri, 17 Apr 2015 13:02:01 +, Antonio Ariza
wrote:
>Hi,
>
>I agree with Kay, try to fry your native crystals to get the highest overall
>resolution possible,
since you mention my name in your sentence: no, this is not what I think (nor
said). Burning/frying the crystal is never the rig
I suppose you do the SDS PAGE test not with the peptide but some
bigger substrate. Are you sure your peptide is intact *before*
soaking? I.e. have you checked the batch yourself with MS or NMR? We
regularly get small compounds (sugars) that turn out not to be what
the label says.
Bärbel
Dear Bonsor,
Thanks for your suggestions!
It need 2-3 weeks to get the fully grown crystals. And harvest, freeze and
data collection just take usually next 3-5 days. I usually incubated
substrate overnight. Initially I was purifying with the same column as the
WT but in the next batch I used new
First of all, you don't say how long it took to first set up crystals, for them
to grow, harvest, freeze and collect data on. Secondly how long did leave the
peptide/substrate for your SDS PAGE experiment? If they are of a different time
scale e.g. 6 hours v.s. 30 days, it may be that your enzym
Dear Crystallographers,
I am working with a cysteine protease. I co-crystallized the protease with
some small chemically synthesised peptides of 7 amino acid residues. I
mutated the active Cysteine residue with Alanine to avoid the peptide
cleavage so that I can get the whole peptide bound with my
Hi all,
We have an opening for a scientific programmer to work (1) on the
incorporation of small-angle scattering (SAS) data into the wwPDB Deposition
and Annotation system and (2) on presentation and dissemination of SAS data
through the PDBe website. The project (which will last for 18 month
My understanding is that NCS restraint can significantly enhance the speed
> of calculation, but considering the subunits even with the eactly same
> sequence may not be identical, to have NCS restraint may be not necessary
> or may be not good for the refinement, am I right?
>
Non-crystallograph
Hi Herman,
Strictncs is still used for viral capsids and other high NCS structures. It
works very well in Refmac as long as your MTRIX records (in the PDB file) are
correct and you have the identity MTRIX as well. The keyword is simply
'strictncs'. You can even combine strict and local NCS if y
Hi,
The purpose of NCS is to reduce the degrees of freedom in order to avoid over
refinement -not only to expedite refinement. Strict or restrained NCS should be
applied at lower resolutions (<2.7Å) or data completeness. Forgo NCS If you
have a complete and better than 2.5Å dataset. Also, you c
To
Ed Pozharski,
Thanks for suggestion. I have checked the electron density of all the residues
of the refined pdb ( till now, with R(work)/ R(free) = 0.24/0.30) and also the
ones showing as "Ramachandran outliers" in different loop region. it seems the
residues fit well to the electron densi
Dear Smith,
There used to be something called "strict NCS" which meant that instead of many
identical subunits, only one "average" subunit was refined, which would speed
up the refinement significantly, at the expense of requiring that all subunits
are exactly identical.
I do not think that th
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