Re: [ccp4bb] Coming July 29: Improved Carbohydrate Data at the PDB -- N-glycans are now separate chains if more than one residue

And, to me at least, this change makes no sense. Protein chains with covalently attached glycans are one biochemical, structural and functional unit. Best wishes, Radu > This suggestion violates a basic principle of data base theory. A > single data item cannot encode two pieces of info

Re: [ccp4bb] Highest resolution of X-ray / neutron / electron crystallography, cryo EM

Hi Tobias,For what is worth, we report 1.22 A for single particle cryo-EM ;-)) But very likely there is more in that dataset, we should know soon.Best wishes,RaduOn 9 Jun 2020 3:11 pm, Tobias Beck wrote:Dear all,Thanks a lot! (I should have used the PDB query myself for neutrons, sry, my bad)As

Re: [ccp4bb] E. coli BirA biotin ligase expression/purification

plasmids are also available on Addgene. Some extra biotin has to be added to culture medium in my experience. Examples from our lab (HEK cells) are in PMID: 27418511 and 28817804. Best wishes, Radu > Dear friends in crystallography, > I know this seems unrelated, but it really isn't ...

Re: [ccp4bb] challenges in structural biology

Hi Paul, Fair point, apologies if anyone was offended by my comments! I simply thought that such matters are meaningful for this forum. I am just as guilty as everyone, and it is important to put our work into the broader perspective from time to time. Best wishes, Radu > Hi Radu and

Re: [ccp4bb] challenges in structural biology

of the structures solved on synchrotrons worldwide and of the zillions in the PDB are of any use or biological relevance (original question)? There is an enormous amount of waste, including the nasty chemicals use to grow crystals and to phase pointless structures, let's be honest. Best wishes, Radu

Re: [ccp4bb] challenges in structural biology

). But it doesn't really ask or answer biological questions... for these, whether we like it or not, macromolecular crystallography (or NMR, even in cell) cannot be the future. In my opinion :-) Best wishes, Radu > Stating the crystallography is dead might be a bit premature, it is still king > for depos

Re: [ccp4bb] Non-specific disulfides in a secreted protein

, or higher, dilutions in empty (pLEXm or any irrelevant plasmid) to keep total DNA amounts constant. Is there no hint of monomer whatsoever in your prep? Best wishes, Radu -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K

Re: [ccp4bb] Large scale insect cell expression

, especially for membrane and secreted proteins. All vectors are with Addgene, early passage cells from ATCC. Best wishes, Radu -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. tel: +44-(0)1223-267049 fax: +44-(0)1223-268305

Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

Oops, I made a mistake in my previous email, apologies! Griffithsin would also require high-Man glycans, so kifunensine treatment or GnTI-/- cells So yes, it's either PNGase or cryo-EM... Best wishes, Radu > Hi Savvas, > > Many Thanks for your inputs and references. This cell

Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

ize proteins these days anyway :-) Best wishes, Radu -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. tel: +44-(0)1223-267049 fax: +44-(0)1223-268305 www: http://www2.mrc-lmb.cam.ac.uk/group-leaders/a-to-g/radu-aricescu &

Re: [ccp4bb] new ContaMiner features

Hi Stefan, I also owe you an apology for my bad choice of words. One can debate about usefulness, but Pavel is right, the software is not "bizarre". And, as several people pointed out, it certainly addresses a need (which I didn't know about). Mea culpa. Best wishes, Radu > Dear

Re: [ccp4bb] new ContaMiner features

s can only help up to a point... Best wishes, Radu PS: At least, one day, maximum-likelihood refinement programs will deal with weak data satisfactorily :-) Nobody likes to throw data away. > Dear all, > > to join Pierre's comments on what 'strange' things happen at the beamlines... &g

Re: [ccp4bb] new ContaMiner features

r, all negative (i.e. thankfully haven't encountered of such accidents). I'm wondering whether "crystallisation contaminants" might be related to a particular type of targets, or expression system, or purification strategies... I'll check the paper Tristan suggested when I get the chance.

Re: [ccp4bb] new ContaMiner features

rification tag or whatever. I suppose this might have happened to somebody you know, hence the motivation to spend time on the bizarre ContaMiner. Which is a pity, a silly outcome would only teach people to do their job (or train their robots) properly. Best wishes, Radu -- Radu Aricescu MRC

Re: [ccp4bb] Large scale mammalian expression system recommendation?

). Also, I'd use different systems depending whether the recombinant protein is intended for functional, fluorescence microscopy, structural analyses (say X-ray vs cryo-EM). Best wishes, radu -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus

Re: [ccp4bb] Error in assignment of symmetry operators

Dear Gerard, Thank you for the answer, absolutely not a problem! I am very grateful for all the great software from Global Phasing and I know that support is great. Enjoying the STARANISO server for example atm. Best wishes, Radu > Dear Radu, > Nice to know that your problem is

Re: [ccp4bb] Error in assignment of symmetry operators

wishes, Radu > Hi Radu, > This may be caused by the coordinates being too many unit cells away from the > origin. In COOT you can easily ‘symmetry move coordinates here’ to a place closer to the origin. > Hope this helps, > Robbie > Sent from my Windows 10 phone > Van: r

[ccp4bb] Error in assignment of symmetry operators

in. I tried everything I could think of to fix this, no luck so far I'm probably making a very silly mistake... But would be very grateful for any advice! Best wishes, Radu PS: space group is C2 -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Camp

[ccp4bb] Fwd: Re: [ccp4bb] Glycoprotein expression question

solving meaningless structures? Best wishes, Radu -- Radu Aricescu MRC Laboratory of Molecular Biology Francis Crick Avenue Cambridge Biomedical Campus Cambridge CB2 0QH, U.K. tel: +44-(0)1223-267049 fax: +44-(0)1223-268305 Original message >Date: Wed, 12 Apr 2017 10:24:10 +0200 &g

Re: [ccp4bb] FW: [ccp4bb] Fwd: [ccp4bb] just out of totally idle curiosity ...

Hi JPK, Not sure about them, but I was a bit surprised to get this ID a couple of years ago: 4COF! Nice structure nevertheless :-) Best wishes, Radu On 9 Nov 2016 10:40 p.m., "Keller, Jacob" <kell...@janelia.hhmi.org> wrote: > > I recently was surprised to be

Re: [ccp4bb] Trimming of carbohydrate chains

Dear Herman, Assuming that the protein is eukaryotic, and glycans N-linked, various ways of dealing with the issue are described in PMID: 17355862. Best wishes, radu -- A. Radu Aricescu, PhD MRC Senior Research Fellow Associate Professor University

Re: [ccp4bb] Sighting of Protein Crystals in Vivo?!

or more cells in the field? This does not look like transient expression using a normal plasmid... Very intriguing nevertheless! radu -- A. Radu Aricescu, PhD University Research Lecturer University of Oxford Wellcome Trust Centre for Human Genetics Division

[ccp4bb] Postdoctoral position, C2D2, University of York

-Discipline-hopping-internships or http://www.york.ac.uk/c2d2/internships/ For informal enquiries contact: radu.caline...@york.ac.uk -- A. Radu Aricescu, PhD University Research Lecturer University of Oxford Wellcome Trust Centre for Human Genetics

Re: [ccp4bb] install crystallography software on mac

Best place to start: http://scottlab.ucsc.edu/~wgscott/xtal/wiki/index.php/Crystallography_on_OS_X -- A. Radu Aricescu, PhD University Research Lecturer University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology

Re: [ccp4bb] The effect of His-tag location on crystallization

Or indeed support surreal structures :-)) (PMID: 11853672 vs PMID: 14749821 and so on) -- A. Radu Aricescu, PhD University Research Lecturer MRC Career Development Award Fellow University of Oxford Wellcome Trust Centre for Human Genetics Division

Re: [ccp4bb] mammalian expression vector

Hi Jerry, You may find that pcDNA3.1 won't give you the protein yields needed for crystallization. Have a look at PMID: 17001101 for an alternative. Your Kozak sequence looks good, radu -- A. Radu Aricescu, PhD University Research Lecturer MRC Career

Re: [ccp4bb] FW: [ccp4bb] highly glycosylated protein

, play with deglycosylation conditions etc) and produce a more homogeneous sample than CHOs (which are leaky, see Chang et al 2007, PMID: 17355862, for details). Best wishes, radu -- A. Radu Aricescu, PhD University Research Lecturer MRC Career

Re: [ccp4bb] Deglycosylation enzymes

not a bad deal in long term. Best wishes, radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom Phone: +44-1865-287564 Fax: +44-1865-287547

Re: [ccp4bb] update on SeMet production

others followed. More recently, people have moved to transient expression and probably the easiest labelling method for HEK293 cells can be found in PMID: 17001101. Best wishes, radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre

[ccp4bb] OXION Postdoc fellowships and DPhil/PhD studentships

expression, purification and crystallization are warmly invited to apply :-) Best wishes, radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive, Oxford OX3 7BN United Kingdom

Re: [ccp4bb] Deglycosylation

design (eliminate Ser/Thr/Pro-rich domains), for other types have a look at http://www.prozyme.com/pdf/gk80110.pdf for example. Best wishes, radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division

[ccp4bb] Fwd: [ccp4bb] HEK293S

Dear Vaheh, I'm not aware of a commercial or cell bank source, but you can get these cells from HG Khorana's lab at MIT. Best wishes, radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural

Re: [ccp4bb] insect expression system

Se-Met labelling and so on. Here are some references (PMID): 17355862, 17001101, 16823037, 11788735, 16082028. Radu -- A. Radu Aricescu, PhD University of Oxford Wellcome Trust Centre for Human Genetics Division of Structural Biology Roosevelt Drive