[ccp4bb] best strategy to establish stable expression in mammalian cells

recommend a best strategy for mammalian cell expression? Thank you so much and have a nice weekend, Jerry McCully

[ccp4bb] co-express two proteins in E.coli

Dear ALL, We are planning to co-express two proteins in E.coli. Could anyone suggest a good dual set plasmid or a proper insertion sequence between two genes, including the Shine-Dalgarno sequence? Thank you very much and have a nice summer. Jerry McCully

[ccp4bb] how to interpret DALI search results

since our structure only contains 130 residues. Sequence identity is between 5 to 15%. The RMSD of structural alignment is between 2.5 to 6 angstrom. Any suggests to interpret the DALI results? Many thanks, Jerry McCully

[ccp4bb] difference between PEG 4000 and polyacrylate 5100

Dear ALL; Both PEG and polyacrylate are polymers. Although their formulations are different, Could they be exchangeable in crystallization conditions? Thanks a lot, Jerry McCully

[ccp4bb] minimum protein concentration for NI-NTA column

. Thanks a lot and best regards, Jerry McCully

[ccp4bb] weird--No protein expression using pET30a

Dear ALL; Recently, one of my colleagues cloned a gene (200aa) into pET30a vectors with either a N-ter or C-ter His6 tag. The correct reading frame was confirmed by sequencing. However, it is weird that there was no protein expression either in the soluble fraction or as inclusio

[ccp4bb] mammalian expression vector

Dear ALL; As an alternative strategy to avoid endotoxin, I plan to express the protein in mammalian cells. As suggested by others, the typical vector is pcDNA3.1(+). Does anyone have comments on this vector or recommend some other powerful vectors? I am new to mammalian exp

[ccp4bb] endotoxin removal: Triton X-114 dissociates protein oligomers?

Dear ALL; Thanks a lot for all the instructive suggestions. As my first trial, I tried 0.1%Triton X-114 plus Ni-column binding buffer. However, the oligomer of my protein got dissociated in to monomers as indicated by gel-filtration. Does anyone know how to resc

[ccp4bb] FW: endotoxin removal

protein contains quite a few aromatic residues and has a pI around 6. Any ideas to remove the endotoxins will be highly appreciated. best regards, Jerry McCully

[ccp4bb] scalepack--scale data in .sca format with original index

Dear ALL; I am sorry for this "HKL2000 scalepack" question. To confirm the scape group using Pointless, I processed the images using HKL2000 but kept the original index using "No merge original index". Now Pointless gave the right space group, and I want to mer

[ccp4bb] prescission protease cutting site

over concerned. Thanks a lot and have a nice summer. Jerry McCully

Re: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation

CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jerry McCully Sent: Friday, May 27, 2011 10:17 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] larger molecular weight shown by analytic ultracentrifugation Dear ALL; I am sorry for this off-topic question about analytic

[ccp4bb] larger molecular weight shown by analytic ultracentrifugation

some comments on this discrepancy? Thanks a lot and have a nice weekend! Jerry McCully

[ccp4bb] insert a cleavage site between scondary structures

artificial cleavage site, say TEV or thrombin site, to at least prove the idea at the biochemistry level. There are some short loops between strands and helices. Can anyone suggest a good way of adding in the cleavage site without changing the native structures? Thanks a lot. Jerry McCully

[ccp4bb] E. coli mutant strains

Dear ALL: Recently I am expressing one protein in BL21(DE3) and the protein undergoes N-terminal degradation. I am trying to keep this crucial N-terminal tail on the protein, which has MRS at the first 3 positions. Digging in to the literatures, I found the N-end rule, wh

[ccp4bb] NMR structure of 30KD protein, doable?

refinement software? Welcome any comments and Have a nice weekend. Jerry McCully

[ccp4bb] increase the solubility of proteins

heavy aggregates. I guess the problem is due to the missing of glycosylation. Does anyone have suggestions to increase the current solubility? Thanks a lot, Jerry McCully

[ccp4bb] update-MBP fusion protein

portion of MBP fusion protein exsited as monomers even in the expression condition with low temperature and 0.1mM IPTG. Does anyone experience the oligomerization of MBP fusion protein? Thanks again and have a nice weekend! Jerry McCully

[ccp4bb] autobuster problem

formation in mtzchk.2.log: >>>>>> CCP4 library signal ccp4_general:Cannot open environ.def (Error) raised in ccp4fyp <<<<<< mtzdump: Cannot open environ.def mtzdump: Cannot open environ.def How can we fix the problem? Thanks a lot, Jerry McCully

[ccp4bb] update--degradation of MBP fusion protein

omers. Does anyone have the experience to increase the yield of the monomeric proteins? Thanks a lot, Jerry McCully

[ccp4bb] **Possible spam**cryoprotetant for 35% Dioxane

Dear All; We just got some crystals from 35% (v/v) Dioxane. We are going to collect some data soon. Does anyone have the experience with the cryoprotectant in this condition? Thanks a lot, Jerry McCully

[ccp4bb] degradation of MBP fusion protein

a nice day! Jerry McCully By the way, thank folks for the answers of my another question about setting up view point in Pymol along axis in the unit cell.

[ccp4bb] pyMol--set up view through one axis of the unit cell

Dear All, It is a Pymol question. How can I set up the view through one axis of the unit cell? Thanks a lot and have a nice weekend, Jerry

[ccp4bb] autoBuster--Rfree_falg

Dear All: I am currently using autoBuster to refine my structure. I notice that autobuster generates a new column in the MTZ data file with the label of "FreeR_flag". Because my MTZ file has already had the FreeRflag, I am wondering whether autoBuster generated a new set of Rfreeflag

Re: [ccp4bb] to what extent bacterial expression reveals the native oligomerization state of mammalian proteins.

.coli expression of a human protein without disulfide bonds, to what extent do you believe that the oligomerization state of this bacterial expression will reflect the real physiological state of this protein in humans? Can someone give comments or refer some literature? Thanks a lot,

[ccp4bb] to what extent bacterial expression reveals the native oligomerization state of mammalian proteins.

state of this bacterial expression will reflect the real physiological state of this protein in humans? Can someone give comments or refer some literature? Thanks a lot, Jerry McCully

[ccp4bb] how to make cholesterol solution

100% ethanol at a concentration of 10mM. However, it is very difficult to make the dilution into 5% ethanol either just in water or some buffers. Does anyone have such experience to make cholesterol solution in normal buffers plus some ethanol? Thanks a lot, Jerry McCully

[ccp4bb] crystallization of highly-glycosylated protein complex-avoid phase separation

some guidance here? Thanks a lot, Jerry McCully _ Hotmail: Powerful Free email with security by Microsoft. http://clk.atdmt.com/GBL/go/201469230/direct/01/

[ccp4bb] phenix refinement --set cif for sugar links

Dear folks: I am currently using Phenix to refine a structure that has many carbohydrate chains on it. I created the cif file according to an old message from Dr.Ralf W like the following: refinement.pdb_interpretation { apply_cif_link { data_link = NAG-ASN residue_se

[ccp4bb] Difficult molecular replacement in R32 with pseudo translation

I got good solutions in Phaser. I think they are correct solutions because my protein was a hexamer and the crystal packing in P1 was formed by hexamers. How can I get correct solutions in R32 and finish the refinement? Many thanks in advance. Jerry McCully

[ccp4bb] FW: [ccp4bb] Rfactor got stuck with a data having alternate strong and weak reflections.

> From: c...@ysbl.york.ac.uk > Subject: Re: [ccp4bb] Rfactor got stuck with a data having alternate strong > and weak reflections. > To: CCP4BB@JISCMAIL.AC.UK > > Ben Ammar Youssef wrote: > > Hi all, > > I just want to ask another question related to this topic: >

Re: [ccp4bb] Rfactor got stuck with a data having alternate strong and weak reflections.

gt; >> Hi all, > >> I just want to ask another question related to this topic: > >> Based on the example given by Jerry McCully and if the data has > >> alternate strong and weak reflections, how can we split it into two > >> different files; one containi

Re: [ccp4bb] Rfactor got stuck with a data having alternate strong and weak reflections

Dear all: Thanks a lot for the valuable suggestions I've got so far. I will try TLS refinement, anisotropy and super lattice check and then get back to you all with more information. Thanks again and best regards, Jerry _

[ccp4bb] Rfactor got stuck with a data having alternate strong and weak reflections.

I remember there is a discussion in CCP4bb about the same topic with the focus of pseudo-symmetry or translational pseudo-symmetry. Can anybody give some troubleshooting about my issue? Thanks a lot and have a nice weekend, Jerry McCully

[ccp4bb] model completion after MR with partial model

2.6A data. Any suggestions will be highly appreciated. Jerry McCully _ Windows Live™ Hotmail®: Celebrate the moment with your favorite sports pics. Check it out. http://www.windowslive.com/Online/Hotmail/Campaign/QuickAdd?ocid

Re: [ccp4bb] EPMR statistics

landscape look? Discriminate or flat? The R is scaling dependent in contrast to CC, so for incomplete models it can be nonsensical. BR From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jerry McCully Sent: Friday, July 17, 2009 10:46 AM To: CCP4BB@JISCMAIL.AC.UK

[ccp4bb] FW: EPMR statistics

definitive correct solutions? Thanks a lot for the help. Have a nice weekend! Jerry McCully Lauren found her dream laptop. Find the PC that’s right for you. _ Lauren found her dream laptop. Find the PC that’s right

[ccp4bb] EPMR statistics

weekend! Jerry McCully _ Lauren found her dream laptop. Find the PC that’s right for you. http://www.microsoft.com/windows/choosepc/?ocid=ftp_val_wl_290

[ccp4bb] MAD wavelength

due to the discrepancy between the fluorescence scanning and the theoretical vaules of f' and f''. When we collect the data, which wavelength should we use? Should we trust the scanning results? Thanks a lot for your comments. All the best,

[ccp4bb] space group R32: H settings

e some guidance here? Thanks a million. Jerry McCully _ Hotmail® has ever-growing storage! Don’t worry about storage limits. http://windowslive.com/Tutorial/Hotmail/Storage?ocid=TXT_TAGLM_WL_HM_Tutorial_Storage_062009

[ccp4bb] deglycosylation of proteins expressed in mammalian cells at room temperature or lower

aggregate at 37 degree. I am not sure whether EndoH and PNGase can work at 4 degree. Can anyone give some suggestion? Thanks a lot, Jerry McCully _ Hotmail® has ever-growing storage! Don’t worry about

[ccp4bb] how to optimize crystal without sharp boundary

Dear All: Recently we got some crystals from ammonium sulfate and PEG400. They look like hexagonal single crystals but without sharp boundary. They did not diffract well. This protein can also be crystallized from PEG6000 in star shape. We tried hampton additive screen but can no

[ccp4bb] how to purify protein in its native form

them can be crystallized. Now I am wonderring whether its native form in human is an octamer or tetramer. I am planning to purify a little protein from human cells to verify its native form. Can anybody recommend some protocols? Thanks a million, Jerry McCully

[ccp4bb] selenomethionine labeling

Dear All: I got a problem with the expression of one N-terminal His6 tagged protein in B834 cells when I tried to do selenomethionine labeling. However, the protein can be expressed in BL21{DE3) cells. Welcome any troubleshooting tips. All the best, Jerry __

[ccp4bb] Pymol movie ration along any axis

Hi , all: Sorry for this off-topic question. When you make movies, Pymol usually allows you rotate molecules along x, y, or z axis. The view point is always from Z axis. Does anyone have this experience of rotating molecules along any other specified axis and keeping the s

Re: [ccp4bb] protein-protein docking programs

kind of ideal for your case. A. On May 2, 2008, at 17:18, Jerry McCully wrote:Hi, All: Recently we solved a complex structure between two proteins, which indicated the interaction was kind of rigid. Therefore we want to test the binding mode of the receptor with another homologous

[ccp4bb] protein-protein docking programs

a sophisticated docking program to do this? Thanks a lot. Jerry McCully _ With Windows Live for mobile, your contacts travel with you. http://www.windowslive.com/mobile/overview.html?ocid=TXT_TAGLM_WL_Refresh_mobile_052008

Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex

Dear All: I am sorry that I did not know the policy. And thanks a lot for the kind reminder. Jerry CC: CCP4BB@JISCMAIL.AC.UK From: [EMAIL PROTECTED] Subject: Re: [ccp4bb] FW: [ccp4bb] salt sensitive complex Date: Thu, 31 Jan 2008 09:37:01 +0100 To: [EMAIL PROTECTED] Dear all -

Re: [ccp4bb] salt sensitive complex

and suggestions on how to improve the ITC experiments.have a nice weekend. Jerry > > Jerry McCully wrote: > > Dear All: > > > > Recently I am pursuing the crystallziation of a complex formd > > by two individual proteins and I met several in

[ccp4bb] salt sensitive complex

ere some better ways that I can validate the binding affinity? Thanks again for your great ideas. Jerry McCully _ Need to know the score, the latest news, or you need your Hotmail®-get your "fi

[ccp4bb] protein precipitated when they formed a complex

h for crystallization? By the way, there is some additional information about the individual components. One has a pI of 6.5, and the other has a pI of 10. Any suggestions will be highly appreciated. Jerry McCully _ Get the

Re: [ccp4bb] FW: protein induced precipitation of proteins?

sing > the two proteins as a single polypeptide. That way they aren't going > to let go easily! > > good luck > Jeremy Tame > > > > > On Dec 4, 2007, at 2:28 AM, Jerry McCully wrote: > > > sorry for the repeats. > > Jerry > > From: [E

[ccp4bb] FW: protein induced precipitation of proteins?

some tricks to avoid the precipitation so that the protein complex could be concentrated enough for the crystallization trials? Many thanks in advance. Jerry McCully Your smile counts. The more smiles you share, the more we donate. Jo

[ccp4bb] protein induced precipitation of proteins?

was a protein with the pI above 10. Did the smaller protein induce the precipitation of the bigger one? Does anybody know some tricks to avoid the precipitation so that the protein complex could be concentrated enough for the crystallization trials? Many thanks in advance. Jerry Mc