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 50

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

forward to hearing your suggestions. > > Sophie, > 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 Associat

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

On Sun, Feb 16, 2014 at 10:58 AM, Raji Edayathumangalam wrote: > 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 and troubl

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

gain! Raji On Sun, Feb 16, 2014 at 10:58 AM, Raji Edayathumangalam wrote: > 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

[ccp4bb] Trouble cleaving SUMO tag off of membrane protein

membrane protein without 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

Re: [ccp4bb] Determining concentration of membrane protein

t;> Cheers, >> >> ___ >> Roger S. Rowlett >> Gordon & Dorothy Kline Professor >> Department of Chemistry >> Colgate University >> 13 Oak Drive >> Hamilton, NY 13346 >> >> tel: (315)-228-7245 >> of

Re: [ccp4bb] Determining concentration of membrane protein

nably pure. > > Cheers, > > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > > tel: (315)-228-7245 > ofc: (315)-228-7395 &g

Re: [ccp4bb] Sister CCPs

ntific impact 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 H

[ccp4bb] Determining concentration of membrane protein

would like 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] A question on protein-DNA complex crystallization

rett 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 DN

Re: [ccp4bb] Membrane fractionation

ultracentrifugation 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

Re: [ccp4bb] PDB structure validation

*** > > ** ** > > -- > > Randy J. Read > > Department of Haematology, University of Cambridge > > Cambridge Institute for Medical Research Tel: + 44 1223 336500 > > Wellcome Trust/MRC Building Fax: + 44 1223 336827 &

Re: [ccp4bb] About molecular replacement

y molecular replacement failed thought > over-all fold is same?. > > > -- > *Dhanasekaran Varudharasu* > Post-Doctoral Fellow > Department of Oral Biology > Rutgers school of Dental Medicine > Rutgers Biomedical and Health Sciences > Newark, NJ 07103 > USA > >

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

ght be the best 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

d 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, Ha

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

cult. the > > Has anyone faced a similar proble? Or is there a way that buffers with > detergents are supposed to be made? Or are there any particular coloumns > meant for such runs. > > Thanks > > -- > Nazia Nasir > PhD Scholar > Protein Crystallography Lab > Natio

[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

otein. 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 wrote: > Hi Folks, > > Sorry for the non-ccp4 post. > > I have purified an 18kDa membrane protein and wan

[ccp4bb] Concentrating purified membrane protein

ow 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

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

mount 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 wrote: > Dear BBers, > > Sorry for the non-ccp4 post. > &

[ccp4bb] Detergent solubilization step (membrane proteins)

lubilize may 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 Visit

Re: [ccp4bb] off topic

oes 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

in crystallization. > > 3. Is anything has to do with ODD and EVEN duplex DNA. When odd 17 base > duplex was used, it has 17 bases in the structure, while in all EVEN case > of 18, 20 or 20, only 12 bases in the structure. > > 4. The complex having odd DNA length 17 has 2 molecules in ASU while all > other has 1. > > > > Why only 12 mer DNA density in the complex? Why I am missing 6 or 8 bases > in the density? 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 Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Off-topic: PDB statistics

in some versions of ADIT the guidance > that RCSB gives about this field is very weak, which accounts for the > variation. > > I'm interested in what "ab initio phasing" really means, but I've been too > lazy to mine the actual entries for details. > > Phil Jeffrey

[ccp4bb] Off-topic: PDB statistics

nted for in the way 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 Resear

Re: [ccp4bb] plate crystal optimization

> upto 2.6 Angstroms in home source Cu K-alpha. however when the crystal > rotates between 90 degrees 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 suggestio

Re: [ccp4bb] delete subject

actual issue was not > realizing there could be more than one molecule in the asymmetric unit. > > More traditional route is to describe your situation in general terms > and offer to provide data to those willing to take a closer look. > > Cheers, > > Ed. > > > --

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

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

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

answer this, if we have 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 > Ch

Re: [ccp4bb] molecular replacement problem.

I have initially indexed the data in C2221 >> but Rfree was not decreasing so i reindexed the data in data in P121 space >> group keeping the Rfree flag of C2221. While analysing the symmetry mates , >> i found large space but no density. structure of Ligand binding domain is >>

Re: [ccp4bb] molecular replacement problem.

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

Re: [ccp4bb] Oligomerization state

but there seems 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 > > --

[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!

ive me 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 I

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

thrombin (or most other proteases) 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 > wrote: &

[ccp4bb] Thrombin cleavage of membrane protein with fusion tag

ions about 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] protein crystals or salt crystals

rotein has zero tryptophan so i could distinguish by UV camera. >> the condition was conditions: >> 0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM. >> >> >> best regards >> Amr >> >> >> >> >> >> >>

Re: [ccp4bb] need some suggestions for crystallization

mind knowing unit cell parameters as well (just a citation works, > I can have them figure it out). I have about 7 weeks 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 v

Re: [ccp4bb] Problems in scaling up expression

don't get strong > 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 Bi

Re: [ccp4bb] Today ...

gt; > > Happy Christmas 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

> -- > Sebastiano Pasqualato, PhD > Crystallography Unit > Department of Experimental Oncology > European Institute of Oncology > IFOM-IEO Campus > via Adamello, 16 > 20139 - Milano > Italy > > tel +39 02 9437 5167 > fax +39 02 9437 5990 > > please note t

Re: [ccp4bb] usefulness of cacodylate?

> screening a "hazardous activity". (We're being subjected to a safety > review.) > > > Thoughts welcome. > phx > -- Raji Edayathumangalam Instructor in Neurology, Harvard Medical School Research Associate, Brigham and Women's Hospital Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Resuspension of bacterial cell pellets

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

Re: [ccp4bb] Resuspension of bacterial cell pellets

pension volumes to be an issue. Thanks. Raji On Thu, Oct 25, 2012 at 8:15 AM, Raji Edayathumangalam wrote: > 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 >

[ccp4bb] Resuspension of bacterial cell pellets

ssues, 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 R

Re: [ccp4bb] Plate crystals

this message. If you are > not the intended recipient, and have received this message in error, > please 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

rce Ltd. cannot guarantee that this e-mail or any >> attachments are free from viruses and we cannot accept liability for any >> damage which you may sustain as a result of software viruses which may be >> transmitted in or with the message. >> Diamond Light Source Limit

[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 art or music 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

; the soluble fraction or as inclusion bodies. > > Could anyone 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

atisfy all 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, Ha

[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 Instr

[ccp4bb] Fwd: HR3699, Research Works Act

ose-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]

7;s temperature > annealing (best is 62 oC) and I've 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

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

stion 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 Date

Re: [ccp4bb] Crack-resistant tubes for centrifugation

oes 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

[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 Vis

[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

Re: [ccp4bb] Coot "File Save Coordinates"

from my iPhone > > On 15 Aug 2011, at 21:25, "Raji Edayathumangalam" > 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 n

Re: [ccp4bb] Coot "File Save Coordinates"

:) Raji On Mon, Aug 15, 2011 at 3:56 PM, Bosch, Juergen 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 Fo

[ccp4bb] Coot "File Save Coordinates"

at just happened now! Haven't 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, Bri

Re: [ccp4bb] Pymol question

ellow, (HISA and elem S) > set stick_radius=0.20, HISA > #hide everything, HISA > > > -- > Dr. Christopher Browning > Post-Doctor to Prof. Petr Leiman > EPFL > BSP-416 > 1015 Lausanne > Switzerland > Tel: 0041 (0) 02 16 93 04 40 > -- -- 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 Edayathumang

[ccp4bb] Potential Space Group Issue

tors can relate 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 W

Re: [ccp4bb] immobilized DNA resin

ve a tag 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 ?

index .we think it is because that > there > > is a long unit cell axes. so is there any method to solve this problem? > > > > best wishes. > > > > 2011-04-05 > > ____ > > dengzq1987 > > > > -- > *

[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

Re: [ccp4bb] Elspeth Garman's husband

My deepest and heartfelt condolences to Elspeth and her family at this very difficult time. May his soul rest in peace. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University On Jul 3, 2010, at 6:28 AM,

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

posium2010/ 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 Resea

[ccp4bb] Symposium & Celebration of Greg Petsko and Dagmar Ringe

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

Re: [ccp4bb] Reg Protein purification

(21):4624-8 It is a great method when many others fail to efficiently remove DNA. Hope that helps. Raji --- Raji Edayathumangalam Joint Research Fellow Harvard Medical School/ Brigham and Women's Hospital Brandeis University On Mar 6, 2010, at 7:23 AM, Sivaraman Padavattan

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

. Try Studier's 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 luc

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,

Re: [ccp4bb] Nobel prize for chemistry 2009

This makes for a great week for crystallography along with the 2009 Nobel Prize in Physics awarded "for the invention of an imaging semiconductor circuit – the CCD sensor"!! Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard

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 ---

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

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 ortho

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. somethin

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 puz

[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 any

[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 intere

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 3.

[ccp4bb] Eukaryotic Membrane Proteins

Hello Folks, My enquiry pertains to membrane proteins. If you have been able to successfully express a eukaryotic integral MEMBRANE protein in E. coli or know of such a case, would you please provide some details. Thanks. Raji

Re: [ccp4bb] how to show double conformation in PyMol

Although there are more elegant ways to do the same, an easy way is to simply write out either just the loop region or the entire molecule containing the second conformation into a second PDB file and to simply read in the two PDB files as two separate molecules in PyMol. Raji On Feb 26

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 wit

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

Did you not know that the CCP4BB-- even when all else appears to be melting down-- is Nature's sustained gift to humankind? Did you also not know that astronauts, astronomers, astrologers, artists, musicians, sportsmen, scientists, politicians, writers and everyone else subscribes to the bo

[ccp4bb] Quikchange cloning: Insert length

Hi Folks, Sorry for the non-xtallo posting. I am curious to hear what is the longest insert anyone has cloned using a modification of the Quikchange cloning strategy. Basically, ligation-independent cloning by strapping on homologous regions of the vector onto the primers which also genera

[ccp4bb] Thanks: Pymol: Zoom without Mouse

lickpad. 3. With the clickpad depressed, remove ring finger from pad and move your middle finger. You should be zooming. 4. Practice steps 1-3 over and over. 5. Buy a mouse. From: Raji Edayathumangalam <[EMAIL PROTECTED]> Reply-To: Raji Edayathumangalam <[EMAIL PROTECTED]>

[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 wit

[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 t

[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 only

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 s

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 ob

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] DLS and SEC-LS facility

The facility at Yale University is very good. http://keck.med.yale.edu/biophysics/ Don't know about current wait times. I got excellent service. Good luck! Raji -Included Message-- >Date: 1-jun-2008 22:48:00 -0400 >From: "Michael Colaneri" <[EMAIL PROTECTED]> >To: >Subject:

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: >Subject: Re: [ccp4bb] Two off-topic questions > >Hello there, I don't usually do this, but the missing letter reminded me

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 possib

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 up

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