Re: [ccp4bb] Protein expression

Hi Reza, In addition to the many useful suggestions already made, I would suggest lowering the final concentrations of IPTG. In many cases, 1mM IPTG interferes with expression levels and/or solubility. This suggestion does not address your concern for why things become ugly in going from 3mL to

Re: [ccp4bb] Searching for DNA structures in the PDB

, Sheffield University -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Difficult MR with MBP fusion protein

nonetheless. Also, I'm curious whether you ran Phaser jobs with the default settings or whether you tried tweaking some of the parameters? I'm happy to speak further about this offline. Good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham

Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein

On Sun, Feb 16, 2014 at 10:58 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Everyone, After several attempts to cleave the SUMO tag off my membrane protein under various conditions (different reducing agents, enzyme-to-substrate ratios, etc.) and after reading the manual

Re: [ccp4bb] Determining concentration of membrane protein

-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my membrane protein. My purified membrane protein is in 20mM Tris pH 7, 150mM

[ccp4bb] Trouble cleaving SUMO tag off of membrane protein

a SUMO tag and the expression is abysmal. Thanks very much for your time and suggestions! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

[ccp4bb] Determining concentration of membrane protein

to know what other folks working on membrane proteins are doing. Thanks very much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Sister CCPs

that a global and diverse group of researchers from various interrelated disciplines can have on one other. Many thanks to the amazing members of the ccp4bb! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research

Re: [ccp4bb] Determining concentration of membrane protein

University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote: Dear CC4BBers, I am trying to figure out what is the best way to determine the protein concentration of my

Re: [ccp4bb] A question on protein-DNA complex crystallization

...@yahoo.com wrote: Dear All, I am now working on the crystallization of a complex of protein-16 bp DNA by co-crystallization. In the screening very small needle-like crystal occurs. If not salt crystal, is there a method to know it is not the crystal of the DNA? Cheers, Acoot -- Raji

Re: [ccp4bb] Membrane fractionation

for one of my membrane proteins because the pellet from the second round is invisible and the protein is pure and functional after purification. Good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research

Re: [ccp4bb] PDB structure validation

Hills RoadE-mail: rj...@cam.ac.uk Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk ** ** -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital

Re: [ccp4bb] About molecular replacement

?. -- *Dhanasekaran Varudharasu* Post-Doctoral Fellow Department of Oral Biology Rutgers school of Dental Medicine Rutgers Biomedical and Health Sciences Newark, NJ 07103 USA -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's

[ccp4bb] What kind of reflection data to deposit to PDB

approach. Many thanks and cheers, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Xtal formed during purification

not diffract at all but after he optimized his buffer conditions to prevent those non-diffracting crystals and screened for optimal crystallization conditions, he got hits from the screens that diffracted to 1.8 Ang. Cheers and good luck! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard

Re: [ccp4bb] Off topic: Gel filtration of membrane protein

particular coloumns meant for such runs. Thanks -- Nazia Nasir PhD Scholar Protein Crystallography Lab National Institute of Immunology New Delhi -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research

[ccp4bb] Off-topic post: Inverted DNA repeats vs direct repeats

thus far haven't yielded much more than what I've shared above. Many thanks! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Concentrating purified membrane protein

. Just occurred to me that I could simply dialyze out the imidazole after the affinity step. Thanks again! Raji On Sat, Jul 13, 2013 at 8:47 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry for the non-ccp4 post. I have purified an 18kDa membrane protein and want

[ccp4bb] Concentrating purified membrane protein

to concentrate my low MW protein without concentrating the DDM? Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

[ccp4bb] Detergent solubilization step (membrane proteins)

be misfolded, just cut my losses and grow tons more bacterial cultures. Many thanks for sharing your successes and heartaches on this matter! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar

Re: [ccp4bb] Detergent solubilization step (membrane proteins)

of total detergent and will definitely try that. Jim, thanks for suggesting Elugent. Never heard about it before so great to know. Many thanks and cheers, Raji On Wed, Jul 10, 2013 at 5:23 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Dear BBers, Sorry for the non-ccp4 post. I'd

Re: [ccp4bb] off topic

a quick question, does anybody know if ethidium bromide binds to poly(dI-dC)? Careina -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Missing DNA density in Protein DNA complex structure

? How can we explain the missing DNA in the structure? I will appreciate any kind of explanation and suggestions. Thanks Ashok -- Ashok kumar patel Department of Biophysics Johns Hopkins University Baltimore, MD 21218 -- Raji Edayathumangalam Instructor in Neurology, Harvard

[ccp4bb] Off-topic: PDB statistics

I am searching. Maybe the way I am doing the searches is no good. Does someone have a better way to do this? Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Off-topic: PDB statistics

phasing really means, but I've been too lazy to mine the actual entries for details. Phil Jeffrey Princeton On 4/15/13 9:48 AM, Raji Edayathumangalam wrote: Hi Folks, Does anyone know of an accurate way to mine the PDB for what percent of total X-ray structures deposited as on date

Re: [ccp4bb] plate crystal optimization

to 135 degrees, the spots become streaky. should i try different cryo-MPD/ PEG400 etc? to circuvent the problem i did some additive screen but was not of much help. any valuable suggestion willbe handfull. thanx in advance rakesh -- Raji Edayathumangalam Instructor in Neurology, Harvard

Re: [ccp4bb] delete subject

to provide data to those willing to take a closer look. Cheers, Ed. -- Hurry up before we all come back to our senses! Julian, King of Lemurs -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital

Re: [ccp4bb] How to calculate data collection strategy manually?

? regards Saleem Harry -- ** note change of address ** Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH Chairman of European Crystallographic Association SIG9 (Crystallographic Computing) -- Raji Edayathumangalam

[ccp4bb] Fwd: [Err] Re: [ccp4bb] How to calculate data collection strategy manually?

Local User Inbox Full (yangr...@korea.ac.kr) 8,61440,240522(163.152.6.98) Action: failed Status: 4.0.0 -- Forwarded message -- From: Raji Edayathumangalam r...@brandeis.edu To: CCP4BB@JISCMAIL.AC.UK Cc: Date: Tue, 26 Mar 2013 10:17:35 -0400 Subject: Re: [ccp4bb] How to calculate data

Re: [ccp4bb] molecular replacement problem.

effort. Thank you Appu On 24 March 2013 02:38, Raji Edayathumangalam r...@brandeis.edu wrote: Dear Appu, I am not sure that I have a complete sense of the issue at hand since some of the information needed to think your issue through is missing in your email. For example, to what high

Re: [ccp4bb] molecular replacement problem.

Content Analysis: $SCATTER :N*Composition vs Probability:0|3x0|1:1,2: $$ N*Composition Probability $$ loggraph $$ 1 0.306066 2 0.00141804 $$ Most probable VM for resolution = 2.27817 Most probable MW of protein in asu for resolution = 92664.2 Thank a lot in advance -- Raji

Re: [ccp4bb] Oligomerization state

to be ambiguity about detergent and lipid effects. Is Thermofluor a right method? Does oligomerization require special assembly proteins, which will mean that tag cleavage is not useful to obtain native state? Thank you. Theresa -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School

[ccp4bb] Need specific molecular replacement test cases

(25% or less). (2) A case in which the search and target models share 80-100% sequence identity but where conformational changes in the target relative to the search model prevented a successful MR solution. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical

Re: [ccp4bb] How to slow down crystallization? Need hep!

some suggestion on how to slow down the process? I used lower conc. of potein, lower conc. of PEG ( 10%), it helped a little bit, giving me small rod crystal. but no improvement after that. Thank you very much for your suggestions -- Raji Edayathumangalam Instructor in Neurology, Harvard

[ccp4bb] Thrombin cleavage of membrane protein with fusion tag

large-scale thrombin cleavage experiments with their favorite membrane proteins. Many thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Thrombin cleavage of membrane protein with fusion tag

) will cleave may mostly depend on your protein/fusion type/protein-micelle complex structure/access to the site... You just have to try. Best wishes. toufic On Wed, Feb 20, 2013 at 5:15 PM, Raji Edayathumangalam r...@brandeis.eduwrote: Hi Folks, Sorry this isn't a non-ccp4 post. I am

Re: [ccp4bb] protein crystals or salt crystals

Stewart http://www.douglas.co.uk Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034 Regd. England 2177994, VAT Reg. GB 480 7371 36 -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar

Re: [ccp4bb] need some suggestions for crystallization

to get everything grown and frozen and ready to go. Any help would be greatly appreciated. It always amazes me how helpful this group is. Thank you very much. Dave -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital

Re: [ccp4bb] Problems in scaling up expression

over expression. Has anyone else experienced problems when scaling up expression? (and more importantly, solved them?) best wishes James -- Dr. James W. Murray David Phillips Research Fellow Division of Molecular Biosciences Imperial College, LONDON Tel: +44 (0)20 759 48895 -- Raji

Re: [ccp4bb] Today ...

everyone! And Merry Christmas to those on the left side of the pond! ...dac -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] vitrification vs freezing

20139 - Milano Italy tel +39 02 9437 5167 fax +39 02 9437 5990 please note the change in email address! sebastiano.pasqual...@ieo.eu -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar

Re: [ccp4bb] usefulness of cacodylate?

-- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

[ccp4bb] Resuspension of bacterial cell pellets

, including a high number of impurities in her elution from affinity columns. I'm curious to hear what other folks do and recommend. Cheers, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar

Re: [ccp4bb] Resuspension of bacterial cell pellets

to be an issue. Thanks. Raji On Thu, Oct 25, 2012 at 8:15 AM, Raji Edayathumangalam r...@brandeis.eduwrote: Hello Everyone, Sorry for this rather naive and non-CCP4 question but I am very curious. My rule of thumb is to resuspend bacterial cell pellets in about 1-2% of the original culture

Re: [ccp4bb] Resuspension of bacterial cell pellets

wrote: It makes sense to use a fixed ratio of resuspension buffer to cell weight; we weigh the pellets after centrifugation, then suspend in at least 4-5 volumes (ml/gr) of buffer. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's

Re: [ccp4bb] Plate crystals

notify us immediately by reply e-mail and then delete it from your system. -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Professor Dame Louise Johnson

and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

[ccp4bb] Detergent and protein oligomerization

? How should one interpret the 100kDa mass estimate from the gel filtration? Thanks. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

[ccp4bb] Einstein would be proud

or imagination or philosophy or some vague combination of the five... Enjoy! Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] weird--No protein expression using pET30a

give some instruction? Thanks a lot and have a nice weekend, Jerry -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

[ccp4bb] Human multi-pass integral membrane protein crystal structures

following criteria: human + multi-pass + alpha-helical + integral membrane protein. If anyone can provide the answer, that would be very helpful. I need this information for a fellowship application. Thanks much. Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research

[ccp4bb] Aggregated protein for crystallization

and set up some crystal trays (after perhaps testing by CD). So I'd like to hear from folks who have been successful in solving structures from aggregates when many many known and tested optimization methods still leave one with aggregated protein. Thanks. Raji -- Raji Edayathumangalam Instructor

[ccp4bb] Fwd: HR3699, Research Works Act

#!/petition/oppose-hr3699-research-works-act/vKMhCX9k -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] [OFF-TOPIC] Site-Directed Mutagenesis [OFF-TOPIC]

increased the extension time up to 9 min. Is there anything else I can try? Any help is appreciated! Regards. Fred P.S.: Agilent's e-mail support is not working. P.P.S.: this might not be of other's interest, address the answers, please, to my e-mail only. -- Raji Edayathumangalam Instructor

[ccp4bb] THANK YOU: Crack-resistant tubes for centrifugation

and a bunch of folks suggested that breakage may have AS MUCH to do with centrifuge and shape-complementarity (understandably) as much as with the centrifuge tubes. Many thanks for your time and help. Go CCP4BB! Raji -- Forwarded message -- From: Raji Edayathumangalam r

[ccp4bb] Crack-resistant tubes for centrifugation

without problems and I want to be able to spin down bacterial lysates without a mess. Any suggestions for tubes that have worked well in your experience? Thanks, Raji -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting

Re: [ccp4bb] Crack-resistant tubes for centrifugation

: To how many g does your 9000 rpm translate ? Perhaps that's the problem ? 10 minutes @ 5000xg for pelleting cells is more than enough in my opinion. Jürgen On Jan 31, 2012, at 11:59 AM, Raji Edayathumangalam wrote: Hi Folks, Are you any favorite brands out there for crack-resistant 50mL

[ccp4bb] TCA or acetone precipitation of proteins

in the gel. Unfortunately, I already added protein dye with SDS and all. Cheers and thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

[ccp4bb] Coot File Save Coordinates

upgraded Coot or anything. Am using Coot 0.6.2-pre-1 (revision 3468) [with guile 1.8.7 embedded] [with python 2.7.1 embedded]. Help? Thanks. Raji -- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital

Re: [ccp4bb] Coot File Save Coordinates

:) Raji On Mon, Aug 15, 2011 at 3:56 PM, Bosch, Juergen jubo...@jhsph.edu wrote: Have you moved your primary window away ? I mean just in case the pop up window opened behind the actual scene window. Jürgen On Aug 15, 2011, at 3:54 PM, Raji Edayathumangalam wrote: Hi Folks, Apologies

Re: [ccp4bb] Coot File Save Coordinates

, at 21:25, Raji Edayathumangalam r...@brandeis.edu wrote: Thanks Mischa and Juergen. That was probably my most ridiculous post to the CCP4BB!! I found the pop-up dialog box hiding behind all my zillion windows. Now why the pop-up window would not pop up actively on top of all other windows

Re: [ccp4bb] Pymol question

-- -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Potential Space Group Issue

Oops sorry for the slippery fingers. I meant h00, 0k0 and 00l in my original email and NOT 00h, 00k, 00l. Note the correction especially if you are a first-year graduate student trying to learn stuff from these emails :) Raji On Thu, Jul 7, 2011 at 11:30 PM, Raji Edayathumangalam r

[ccp4bb] Potential Space Group Issue

a p212121 cell to a p21 cell with almost identical unit cell parameters as that of the p212121 cell and leave all systematic absences intact? Thanks much. Raji --- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital

Re: [ccp4bb] immobilized DNA resin

in this case... Thanks a lot, Alex -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

Program cel: 773.608.9185 email: j-kell...@northwestern.edu *** -- --- Raji Edayathumangalam Research Fellow in Neurology, Harvard Medical School Postdoctoral Fellow, Center for Neurologic Diseases, Brigham and Women's Hospital Visiting Research

[ccp4bb] Protein melting temperatures

. Please could you share some examples. Many thanks. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University

[ccp4bb] Petsko-Ringe Symposium on June 18-19, 2010

/ Please post your congratulatory messages for Greg Petsko and Dagmar Ringe on our online Message Board at: http://prsymposium2010.blogspot.com/2010/05/celebration-of-dynamic-duo.html With Warm Regards, Members of the Petsko-Ringe Lab --- Raji Edayathumangalam Joint Research Fellow Harvard

[ccp4bb] Symposium Celebration of Greg Petsko and Dagmar Ringe

--- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University

Re: [ccp4bb] Expression of large proteins in E. coli

autoinduction protocol 5. Try expression with chaperone kit, trigger factor (Takara) 6. You don't mention whether the protein is human etc., but you may have to move to yeast or insect cells, in the worst case. DISCLAIMER: I am not paid by Takara to mention their kit. Good luck! Raji --- Raji

Re: [ccp4bb] align DNA structures

Hi Mike, By 'align', if you mean superimposition, lsqman will do the job. Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Oct 21, 2009, at 11:06 AM, Mike England wrote: Hi all, I will highly

Re: [ccp4bb] Weird expression behavior

Hi Ezra and others, Just thought I'd let you know that I have noticed that one or two of those E. coli proteins that love to bind Ni-affinity resin do not bind to Cobalt resin. Of course, there is always a price to pay (for the Co resin, in this case) :)! Cheers, Raji --- Raji

Re: [ccp4bb] problems of co-crystallization of protein-DNA complex

that helps. Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Aug 13, 2009, at 12:56 AM, ruheng wrote: Dear CCP4bbers, I am now working on a DNA binding protein and the purity of the protein is quite good

Re: [ccp4bb] How to express a 95KD FAD protein

Hi Wei Yong, Sounds like a tricky situation. A couple of things come to mind: 1) Have you tried expression from a synthetic gene? Sometimes the mRNA is unstable and improving mRNA stability through optimization (synthetic gene) helps. 2) Are you able to look at either human isoforms or

Re: [ccp4bb] desktop models

I've seen some very pretty 3D models in optical crystal glass made and sold by http://www.luminorum.com/ Cheers, Raji Disclaimer: I have nothing to do with this company. On May 5, 2009, at 2:41 PM, Christopher Rife wrote: Hi, I am looking to have a model produced from a PDB, i.e.

Re: [ccp4bb] problem in transformation of pqe 30 clone

One thing to check is whether there is too much DNA in the transformation reaction. This is sometimes a reason for failed transformations, be it DNA from regular minipreps, PCR DNA or ligation reactions etc. Raji On May 4, 2009, at 3:57 PM, b...@freesurf.fr wrote: This story is rather

[ccp4bb] SUMMARY (Link two proteins into one polypeptide)

Hi Folks, First of all, thanks to all the people that responded. Many of you asked me for a summary of responses. So, below is a concise summary containing mainly the references that people pointed me to: Cheers, Raji --- ORIGINAL POST: Hi People, Could

[ccp4bb] Link two proteins into one polypeptide

Hi People, Could anyone point me to successful examples for two unrelated proteins that have been stitched together into one single polypeptide chain with flexible amino acids to create a functional chimera that was subsequently crystallized. I've looked up a few. I am particularly

Re: [ccp4bb] Lowest resolution you can do MR with

If you look at the molecular replacement search parameters, you will find that the rotational and translational searches can be done at 4 Angstrom or lower values assigned to the 'high resolution' values. So the real worry in your case, in all likelihood, is not whether MR will work for

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

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 expression or miniscule expression, often tagging the protein with

[ccp4bb] Pymol: Zoom without Mouse

How does one zoom into the molecule in Pymol without a mouse and with just the Mac trackpad and keyboard? Have tried to look it up in the manual and on the web. No success finding it yet; I did figure it out once before but can't redo it now for the life of me. Need to know how to do it

[ccp4bb] Summary: Phylogenetic analysis??

Hi Everyone, Since some folks have been asking me, below is my original question and here's a summary of the responses. I had already googled a bunch of these links before posting to ccp4bb but still lots of useful info in responses, as always! Raji DNAStar will do

[ccp4bb] Thanks: Phylogenetic analysis??

Thanks to all who responded. I was able to somewhat generate the info I needed. Raji

[ccp4bb] Phylogenetic analysis??

Hi Everyone, Could someone please tell me how to display the evolutionary/ phylogenetic tree of the homologs of my protein of interest. When I perform a PSI-BLAST search for my protein, I receive about 130 top hits for homologs. The NCBI or EBI tools that I've laid my hands on seem to

Re: [ccp4bb] Troubleshooting protein purification cation IEX

Hi, Many things can lead to your observation. Please outline all steps of your purification procedure as it is not clear what is done before and after the Ion Exchange steps. I am not sure if IEF in your emails refers to Isoelectric focusing, as the acronym is usually used?? Couple of

Re: [ccp4bb] regarding cloning

Hi Vijay, I have heard of TOPO-TA cloning. Not sure what T/A cloning is. I have a couple to check based on your description: 1) Do you CIP-treat your vector? If not, that might be a step to add. Also, you could include a 'vector only' transformation control to determine how many colonies are

Re: [ccp4bb] Anion binding sites in proteins

Hi Albert, Please refer to the following paper (and references therein) for a description of the four chloride located in the nucleosome structure.. 1: Davey CA, Sargent DF, Luger K, Maeder AW, Richmond TJ. Solvent mediated interactions in the structure of the nucleosome core particle at 1.9 a

Re: [ccp4bb] Two off-topic questions

hloy cow! taht msut be vrey crortcet idened ! rjai -Included Message-- Date: 12-may-2008 14:15:03 -0400 From: Scapin, Giovanna [EMAIL PROTECTED] To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Two off-topic questions Hello there, I don't usually do this, but the missing letter

Re: [ccp4bb] sa_omit_map

To me, it seems that your syntax for chain and residue definitions is incorrect. See the 'Tutorial' section on the CNS website, which gives some very nice examples. For example, if you want to select chain K and residues 2 and 15, chain L and residues 1, 2, and 14, and so on, here's pne

[ccp4bb] Bacterial induction at 18C

Hi Folks, I am working with E. coli cells co-transformed with two plasmids and I find that my cells lyse following overnight inductions at 18C. I suspect (among many things) that Ampicillin+ Chloramphenicol+ Kanamycin in the medium may be the source of my woes. My colleagues have suggested

Re: [ccp4bb] Bacterial induction at 18C

Thanks to everyone for all your suggestions. I am growing the cultures as we speak and have increased the temp to 22C and plan to harvest in about 6-8 hrs. Thanks for the Q7 rule. I read it before but I couldn't remember exactly and a quick-and-dirty Google and Pubmed search did not bring it

Re: [ccp4bb] Co-expression plasmids

I do use the Duet vectors extensively and they work just fine. I have trouble with my protein complex, but that is solely related to my proteins. My lab mate has used these vectors very successfully. I have been using pRSF-Duet and pETDuet1 more than pCDFDuet1 or pACYCDuet1 for no specific

Re: [ccp4bb] Co-expression plasmids

I thought only I was having trouble with cloning into pETDuet1. But just to add to what someone just said Yes, I had hell with trying to get one of my ~2kb fragments into pETDuet1 (5.4kb). Could be a combination of vector size and insert size.. Who knows! Sometimes, I do what Tasos says:

Re: [ccp4bb] Tough Low-Res MR

Absolutely try Phaser! See Phaser documents for all the nifty combinations. Multiple molecules in MR model; break down molecule by domains etc etc. You can trim down side chains, make hybrid models and what not. All easy to get up and going through the GUI. Once the GUI drove me insane because

Re: [ccp4bb] secondary structure restraints

Hi Sean, Not an answer to your question but have you looked into model building with TEXTAL? I am not sure about 3.6Ang resolution data but it might be worth looking into. Raji -Included Message-- Date: 13-feb-2008 16:54:33 -0500 From: Sean Johnson [EMAIL PROTECTED] To:

[ccp4bb] Solved: PDB file column-cut-paste issues

Thanks everyone for all your suggestions. What's the issue? Each residue has one CSV value (per residue value) and the PDB line contains many lines of B-values per residue. This would leave us with no easy one-to-one line correlation between the B-value column and the CSV column. From what I

[ccp4bb] PDB file column-cut-paste issues

Hi People, I post this on behalf of my colleague. My colleague has a file containing chemical-shift values for the 150 aa in his structure. He also has the PDB file for the crystal structure. Now, he would like to replace the B-factor column with the CS values to make some figures. It would

Re: [ccp4bb] Solved: PDB file column-cut-paste issues

Ooh..la la! Where were you 12 hrs ago when we were suffering brain damage! Cheers! Raji PS: Thanks! Oh dear - too late :-(. You can do it in Coot too! (The solution is on the Coot Wiki now)

Re: [ccp4bb] Web site GOOD, Attachment BAD (was: salt sensitive complex)

Can the CCP4BB provide something like a website to upload pictures and then have the BB-ers just post the link in their email. Please! These attachments are clogging my inbox... Thanks much. Raji -Included Message-- Date: 31-jan-2008 03:58:44 -0500 From: Frank von Delft

Re: [ccp4bb] protein expression problem

I wholly agree with the below. I am not sure how well E.coli can correctly fold snaky misfolded/unfolded protein that are chaperoned by folded tags! Not to rule out that tags do it sometimes... Folded by association for insoluble proteins has often not worked well for me. Sometimes, when it

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