Hi Flavio
While i am not aware of any commercial vector with cleavable c
terminal tag, you can easily make your own by introducing protease cleavage
site of your choice. However this strategy will results in quite a few extra
residues from protease site.
An alternative, which is
While I understand that you want to have protein concentration at its
solubility limit, i had several proteins which can go to 60-80 mg/ml, they
seems to get crystallized at 10-15 mg/ml. All you want is saturation in
crystallization conditions.
Dhiraj srivastava
, December 22, 2023 5:35 PM
To: Srivastava, Dhiraj
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: [External] Re: [ccp4bb] Relion issue with MPI
Hi,
First of all, please report details of your hardware and your job.
- Type of GPU
- Number of GPU
- GPU memory size
- Box size
- Number of threads
- Number
Hi
I am trying to use relion and I am getting error when trying to use mpi (for 3d
classification and 3D auto-refine).
ERROR: out of memory in
/home/lvantol/relion5/relion/src/acc/cuda/custom_allocator.cuh at line 436
(error-code 2)
in:
Hi All
sorry for off topic question. does anyone have script for rmsf
calculation of multistate pdb file in pymol? There used to be a script
rmsf_states written by Robert Campbell but with newer version of pymol, it's
not working.
Thank you
Dhiraj
(301) 846-1823
On Sun, 2 Apr 2023 at 10:44, jacinto.ls<http://jacinto.ls>
mailto:jlopez.sagas...@gmail.com>> wrote:
I am also not sure whether AlphaFold can address the impact of ions and other
cofactors on the fold of many proteins.
Best wishes,
Jacinto
On 2/4/23 16:20, Srivas
May be this article is of some help suggesting the need of experimental
structures despite excellent alphafold model.
https://www.nature.com/articles/s41401-022-00938-y
From: CCP4 bulletin board on behalf of Ian Tickle
Sent: Sunday, April 2, 2023 8:28 AM
To:
No offense to anyone but most of these systematic studies are often on small
not so flexible, single domain, easy to crystallize proteins with little
conformational variability and that’s where alpha fold excel. It fails with
(may not be all though) multidomain proteins with conformational
When I tested alpha fold on some of my proteins, it failed to predict the
intramolecular interactions needed for their functions. Alphafold predicts the
folds and overall structure of single domain and may be simple multi domain
proteins but when conformational changes are needed for protein
You can make molecule in chemdraw or chemdraw 3d with proper bond order and
save it in sdf or .mol format or any other format compatible with schrodinger.
Schrodinger should be able to read .sdf or .mol format.
Dhiraj
From: CCP4 bulletin board on behalf of
Thank you every one for all your input. Our concern was whether there is any
new advanced system than Bac-to-Bac or Bac-to-Bac is still pretty standard
system for baculovirus expression. Based on all your inputs, it appears that we
are not very outdated in our information about baculovirus
Hi All
sorry for the question not related to crystallography. What is the
baculovirus expression system that people use these days to get good yield in
less time?
Thank you
Dhiraj
To unsubscribe from the CCP4BB
ase to cleave a tag from
recombinantly expressed protein.
Kind regards,
Nikolay
On 02/03/2022 6:25 PM Srivastava, Dhiraj wrote:
Hi
sorry for off topic question. does anyone know which protease inhibitors we
can include safely while cleaving with 3C protease?
Thank you
Dhi
Hi
sorry for off topic question. does anyone know which protease inhibitors we
can include safely while cleaving with 3C protease?
Thank you
Dhiraj
To unsubscribe from the CCP4BB list, click the following link:
is bend, I can not simply extend it using ideal
DNA double helix conformation. How can I extend it computationally? x3DNA will
work but needs pdb id.
Thanks
Dhiraj
From: Phoebe A. Rice
Sent: Saturday, January 1, 2022 11:01 AM
To: Srivastava, Dhiraj ; CCP4BB
Hi
I solved the structure of protein DNA complex where the DNA molecule is
bent. I want to extend the DNA computationally so I can model two DNA binding
proteins together on same DNA molecule. is there a way by which I can extend
the DNA in both direction? its easy to do it with ideal DNA
Recently there were few articles where synthetic symmetrization was used to
enhance the crystallizability. proteins used were MBP, lysozyme and GFP to name
a few. you can search for it. one of them is -
https://www.sciencedirect.com/science/article/pii/S0969212615002890
I am posting this advertisement on behalf of Prof. Nikolai Artemyev.
A postdoctoral position is available immediately at the Department of Molecular
Physiology and Biophysics, University of Iowa Carver College of Medicine to
study the molecular mechanisms of vertebrate visual transduction. We
Even pymol and chimera can add hydrogen. Sorry I understand it’s ccp4bb forum
and i am talking about chimera and pymol.
Depending on you need, you can do energy minimization in chimera as well.
Dhiraj
From: CCP4 bulletin board on behalf of Sam Tang
Sent:
Hi Rohit
Since you are saying the C terminus in crystal structure is not
end of your protein in crystal, and you are worried about its charge status, is
in't a good idea to model few residues beyond the C terminus you see in crystal
structure using weak electron density and crystal
Sorry I got confused with the wrong labeling. But the stoichiometry issue and
poor expression is still the main problem. As Tom suggested, you should try to
improve the expression. Poor expression often results in impurities
copurifying. Also, if the interaction is not tight, why are you
Hi Dilip
It seems like your problem is poor stoichiometry. 37 kda protein
is poorly expressed. I wouldn’t be worried too much about 60 kda protein.
At-least at this stage. I might be mistaken but based on previous post, it
seems like your protein was aggregated. If it is so, you
Hi all
I am trying to express my protein in HEK293 cells for crystallization
purpose but getting poor expression. I am using cmv promoter. Which
promoter/vector people use to get good expression in eukaryotic expression
system?
Thank you
Dhiraj
Thank you everyone for the help. I am sorry about phenix related question. But
since the question was refinement related I thought it will be ok to ask on
ccp4bb.
Thank you
Dhiraj
From: Oleg Sobolev
Sent: Monday, March 29, 2021 6:18 PM
To: Srivastava, Dhiraj
Hi
I have structure with bent DNA. I am trying to refine the structure using
phenix. do I need to turn off the DNA secondary structure restraints during
refinement?
Thank you
Dhiraj
To unsubscribe from the CCP4BB
Hi Jon
there is no surface exposed cysteine nearby which can potentially
make disulfide bond with the modified cysteine intramolecularily.
Dhiraj
From: Jon Cooper
Sent: Sunday, November 1, 2020 7:41 AM
To: Srivastava, Dhiraj ; CCP4BB@JISCMAIL.AC.UK
-migrating.
Dhiraj
From: Barone, Matthias
Sent: Saturday, October 31, 2020 1:29 PM
To: CCP4BB@JISCMAIL.AC.UK ; Srivastava, Dhiraj
Subject: Re: Cysteine oxidation product
Hi Dhiraj
how did you measure the affinities? What are the two affinities, incl standard
Hi All
I added an extra cysteine residue to one of my protein interactions
partners and somehow it increased the affinity in the absence of reducing agent
however in the presence of reducing agent, the affinity is same. there is no
disulfide bond formation between the two interaction
P53 tetramerization domain might be a way to go.
From: CCP4 bulletin board on behalf of Comolettis
<85822939...@gmail.com>
Sent: Friday, September 25, 2020 9:59 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] tetrameric tag for extracellular proteins
Hi all,
.tomch...@utsouthwestern.edu<mailto:diana.tomch...@utsouthwestern.edu>
(214) 645-6383 (phone)
(214) 645-6353 (fax)
On Sep 22, 2020, at 12:08 PM, Srivastava, Dhiraj
mailto:dhiraj-srivast...@uiowa.edu>> wrote:
EXTERNAL MAIL
Hi
I want to make my protein dimeric to increase its affinity fo
Hi
I want to make my protein dimeric to increase its affinity for its
interaction partner which is a dimer. does anyone know a suitable tag/fusion
protein which can be used as C terminal fusion for this purpose? I can not use
any of the leucine zipper as I am looking for the distance
Well, its not no. of frames but minimum degree of crystal rotation. A goggle
search gave me this article.
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557013/
Dhiraj
From: CCP4 bulletin board on behalf of Murpholino
Peligro
Sent: Monday, June 22, 2020 5:03
As other said, having co-crystal doesn’t mean you will have measurable
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which
buffer you used during previous ITC experiment? If heat in the previous
experiment (wild type) was due to heat of ionization and you removed the
As other said, having co-crystal doesn’t mean you will have measurable
affinity. Further no heat in ITC experiments doesn’t mean no binding. Which
buffer you used during previous ITC experiment? If heat in the previous
experiment (wild type) was due to heat of ionization and you removed the
Pyruvate kinase specially M2 isoform is one example which exists in tetramer
and dimer/monomer state. Several ligands affect its shape and oligomeric state,
thus affecting its activity.
Dhiraj
From: CCP4 bulletin board on behalf of Gabriela GARCIA
RODRIGUEZ
I am not much aware of pichia system but in general, its quite possible your
protein is getting protelysed and all that’s leftover is gfp. Although I expect
mbp to be there as well as its quite well behaved tag. You might want to
analyze whole cell lysate to see if your protein is there. A time
Hi All
sorry for asking non-crystallography related question. Is it possible
to integrate crysol or FoxS in python and pymol? I have generated thousands of
models of my protein that I want to test against SAXS and other biochemical
data. The ability to integrate the comparison of
Probably you can not use thiocyanate as cryoprotectant because its a chaotropic
agent. Although its a very mild denaturant (destabilizer) but at high
concentration, it can destroy your crystals. I have recently used 30 % sucrose
for cryo protection of crystals growing under same condition and
Hi
I have a protein with two substrate. when I am doing the binding studies
with the two substrate separately, I am finding one of the substrate to have
similar kd and Km. however the km and kd values are almost 30 fold different
for the other substrate. it binds 30 fold more tightly then
Thank you Prof Lewis.
Its serine residue which is getting phosphorylated. the pI of my protein is
around 8.2. So native gel ( around pH 8.3) is also not working for me. I tried
ion exchange at pH 7.0, 7.5 and 8.5 but my protein is either coming in flow
through or very poorly binding to Q and SP
See the following reference-
Shen A, Lupardus PJ, Morell M, Ponder EL, Sadaghiani AM, et al. 2009
Simplified, Enhanced Protein Purification Using an Inducible, Autoprocessing
Enzyme Tag. PLoS ONE 4(12): e8119. doi:10.1371/journal.pone.0008119
From: CCP4
Hi All
does anyone know any software that can calculate and print out RMSD of
every residue (c alpha will be good) for homologous structures which has only
30-40 % sequence similarity? I looked on the web but all the software that I
found require the sequence to be the same for both
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