Re: [ccp4bb] [off topic] Recovering pET expression plasmid from BL21 strain

Hi Javier, For a few years (during an industry job) we regularly cloned directly into BL21 (we made our own high competency cells) and confirmed via expression before plasmid sequencing. Only after sequencing did we transform into a cloning strain for miniprep and “long term storage”. We didn’t

Re: [ccp4bb] [off topic] Recovering pET expression plasmid from BL21 strain

This should all go fine. You can maxi- or mini-prep the plasmid DNA from the expression strain and transform it back into a cloning strain for sequencing, etc. Best wishes, Jon Cooper. jon.b.coo...@protonmail.com Sent from Proton Mail mobile Original Message On 19 Feb 2024,

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

tage (post AmS?), in any case use IDA instead of EDTA! that's pretty obvious from the affinities of EDTA/NTA/IDA. cheers jon Von: CCP4 bulletin board Im Auftrag von Jonathan Bailey Gesendet: Mittwoch, 1. November 2023 19:53 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] [OFF TOPIC] On Ni-NTA

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

I have always performed TEV cleavage after eluting my protein from the Ni-NTA column (TEV cleavage performed during overnight dialysis in imidazole free buffer as imidazole inhibits TEV activity at high concentrations). I use TCEP as reducing agent and have never included EDTA, by this method I've

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

Hi Rafael, In this case I recommend the use of Zn-charged Chelating Sepharose™ Fast Flow (Cytiva), as we have established it in the early days when first applying IMAC to the purification of native antibody fragments (see PMID: 1367302 and PMID: 8163179). While the Zn-IDA matrix has

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

Dear Rafael In addition to the excellent suggestions already offered by previous responders, I can attest that Ni-penta resin (sold among others by a company with an odd name Marvelgent) is resistant to EDTA and DTT, and it leaks very little Ni (almost none). Plus, it elutes with low inidazole,

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

Hi Rafael, Dom et al, Indeed nickel eluted with imidazole stays on all your proteins with a His tag, and addition of DTT will make almost all proteins go brown and crap out at this point. But, as Dom says, addition of EDTA before the DTT will solve the problem. Most of the time… If your

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

Hi Rafael, For completeness - there are alternatives to imidazole for eluting proteins from Ni-NTA resins: EDTA - (strips Ni2+, and therefore everything bound to the Ni2+) - 50mM should be sufficient, in your favourite buffer/salt system. Obviously not appropriate for metalloproteins. You'll

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

Hi Rafael, Once I inherited a protocol with a problem similar to yours and they told me that the precipitation was caused by nickel leaking out during the elution with 250 mM imidazole. I am not sure whether this was true, however, their fix was to place something like 20 ul of 200 mM EDTA at

Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

Hi Rafael, the simple answer is that the EDTA and DTT( or any other reducing agent) is not required for the TEV activity. You can have your TEV protease in 20 mM Tris pH 7.5 (RT) and 150 mM NaCl, plus either 10% Glycerol or 50% glycerol depending on storage conditions preference - 80 or -20C

Re: [ccp4bb] Off topic - 3D models demonstrating drug binding to students

These magnetically-linking atomically accurate single-atom models look interesting - but I understand they might be too detailed or might shift around inadvertently :Snatoms Online Store - Magnetic Molecular Models for Educationsnatoms.comI also cannot say I used them (but I am always looking for

Re: [ccp4bb] Off topic - 3D models demonstrating drug binding to students

Hi Ed, The future is already here... and virtual reality is your friend. This is a field that will likely be revolutionized in the near future, and your students would get an advantage by being exposed to these new technologies. You could check the following:

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hi Oleksiy, If the Akta Start is just for loading affinity columns (without air sensor or software), why not to get a Cyvita peristaltic pump for £3k rather than £9k? They

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

.jpg@01D73AFE.96DB0800] From: CCP4 bulletin board On Behalf Of Kovtun, Oleksiy Sent: Friday, September 16, 2022 8:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure Hi Eike, Instead of AKTA Pure I purchased a combo of AKTA Go (€24k with air sensor

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hi Eike, Instead of AKTA Pure I purchased a combo of AKTA Go (€24k with air sensor) and AKta start (€9k with frac collector). It provides two parallel workstations with AKTA start handling the dirty job of loading lysates onto affinity cartridges and the GO doing clean SEC stages. For multiple

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hi Stephan, You’re mostly correct, however, gradients made on instrument with one pump are less reliable. Thank you. Vaheh From: CCP4 bulletin board On Behalf Of Stephan Rempel Sent: Friday, September 16, 2022 7:38 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off-topic: experience

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hi Eike, we bought a Biorad back in 2005 or so, an Akta Purifier in 2011 and recently an Akta Go (2020). All work(ed) well, the latter two are in use. Don't know about the Biorad, it's in Santiago de Compostela. Both the Biorad and Purifier have been used a lot, the Akta Go not that much so

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

mber 2022 13:17 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure Hello Eike, A couple of comments on the AKTA Pures (while I have not experience with the BioRad systems). We have a couple of AKTA Pures and we (and another lab close to us) had a

Re: [ccp4bb] off-topic: experience BioRad NGC vs Aekta Pure

Hello Eike, A couple of comments on the AKTA Pures (while I have not experience with the BioRad systems). We have a couple of AKTA Pures and we (and another lab close to us) had a catastrophic failure of the 3-wavelength detector "block". Price tag for the replacement part (apparently a black

Re: [ccp4bb] Off-topic question related to ITC binding studies

Hi Gergő, Thank you for discussing it in detail. It is a great help. Based on the curve fit, we also believe that the two proteins bind independently to the DNA duplex with the same affinity. The DNA indeed contains 2 sites for protein binding. As far as the dimerization of protein is concerned,

Re: [ccp4bb] Off topic: Are all storage dewars equal?

Hi Carmien, It appears to me that the dewars in your first link are those made by Taylor Wharton. You can do a search with that name and hopefully find less expensive options. We've used them for many years and they are good. Another one, you could try, is VME https://mvebio.com/aluminum-dewars/

Re: [ccp4bb] Off-topic: Mol* Viewer re-centring hack

Hi Jon, Glad to see you are making use of the Mol* viewer. I am also replying on-list as others will hopefully find my response useful. For your specific case, I would recommend using the 'selection mode' - activated by clicking the cursor icon at the top right of the viewport. This mode

Re: [ccp4bb] off topic -- pymol error message

This means that you have an O-umlaut in your PDB file. That should never happen! PDB files should only have basic ASCII characters, not UTF-8. Cheers, Robbie > -Original Message- > From: CCP4 bulletin board On Behalf Of 陈成 > Sent: Tuesday, April 5, 2022 13:12 > To:

Re: [ccp4bb] Off topic - gene toxic for expression strains

Hi there, Somehow I've missed the original email :) Sorry! There are options for expressing really toxic genes, some of which have already been mentioned and others perhaps not: 1. tight regulation of expression (promoter, repressor, other regulatory elements, or a combination thereof). Beyond

Re: [ccp4bb] Off topic - gene toxic for expression strains

Hi Andy, just to follow up on Christian suggestion, which is exactly the way to go. In case you are using an already pET based vector, simply try BL21-AI (https://www.thermofisher.com/order/catalog/product/C607003), which has the T7 RNA polymerase under arabinose promoter should do the trick.

Re: [ccp4bb] Off topic - gene toxic for expression strains

Hi Andy, have you tried another promotor? Arabinose is much tighter, just to be sure that it is really not leaking. Cheers Christian On Mon, Apr 4, 2022 at 10:20 AM Andrew Lovering wrote: > Dear Board, > > > > Perhaps off-topic, but in the wider scope it’s relevant to many on here. > > > > We

Re: [ccp4bb] Off-topic: happy 2022

For those who want to riddle it out (and as structural biologists hopefully don’t come across mirror and glide planes) a ppt where you can take the International Tables and superimpose the tetragonal PG diagrams on the Kaleidoscope. Find the unit cell first, and then ferret out the rest.

Re: [ccp4bb] Off-topic: happy 2022

Looks like no. 12 p4g to me: http://www2.clarku.edu/faculty/djoyce/wallpaper/wall12.html Happy New Year. On Sat, 1 Jan 2022 at 20:52, Bernhard Rupp wrote: > ..and which one of the 17 plane groups do we see here? > > Cherrs br > > On Sat, Jan 1, 2022, 12:47 Phoebe A. Rice wrote: > >> Apologies

Re: [ccp4bb] Off Topic: CCP4MG Help

Hi Or make your first object residues 1-50 & 60-100 and the second object 50-60 (i.e. don't leave a gap between them). ISTR there's a way to join two atoms, but I can't remember off-hand. Harry -- Dr Harry Powell > On 5 Nov 2021, at 19:10, Denis Rousseau > wrote: > > Hi Matthew > > Try

Re: [ccp4bb] Off Topic: CCP4MG Help

Hi Matthew Try making one set from 1-100 and a separate set from 50-60. Then change the color of the 50-60. It worked on my computer. Best Denis From: CCP4 bulletin board on behalf of Whitley, Matthew J Sent: Friday, November 5, 2021 2:21 PM To:

Re: [ccp4bb] off-topic: pH meters

The meter doesn't matter so much as the electrode. For the meter, anything with at least 3 pH multipoint calibration is sufficient for most purposes. I usually bought something from our preferred university vendor a with very large LCD displays for my aging eyes. For protein work, and especially

Re: [ccp4bb] Off topic, protein characterization and binding job opportunity in Boston area

Here is the right link - https://www.linkedin.com/jobs/view/2662640332/?refId=cCDvahCrRPGWslMQWMgEOQ%3D%3D Thank you everyone for pointing this out. On Mon, Aug 23, 2021 at 4:18 PM Yanfeng Zhou wrote: > Hi, > > Sorry for the off topic post. If anyone is passionate about working in an >

Re: [ccp4bb] off-topic: structural motif / domain comparison

A lesser known service with very powerful search across domains and chains is TopSearch by Manfred Sippl & Cie.: https://topsearch.services.came.sbg.ac.at/ Its training set includes PDB entries up to 2018. Best, BR From: CCP4 bulletin board On Behalf Of Sam Tang Sent: Thursday,

Re: [ccp4bb] off-topic: structural motif / domain comparison

You might also find use in databases such as pfam, ecod, cath, scop. Orly On Fri, Aug 6, 2021, 09:43 Sam Tang wrote: > Dear all > > Sorry for an off-topic question here. I wonder if anyone may be aware of > any search program which allows one to 'blast' a protein domain just like > we 'blast'

Re: [ccp4bb] off-topic: structural motif / domain comparison

Perhaps DALI can be of use http://ekhidna2.biocenter.helsinki.fi/dali/ Dal. pá 6. 8. 2021 v 8:42 odesílatel Sam Tang napsal: > Dear all > > Sorry for an off-topic question here. I wonder if anyone may be aware of > any search program which allows one to 'blast' a protein domain just like > we

Re: [ccp4bb] off-topic: structural motif / domain comparison

Hi Sam, Perhaps the Modeller service is what you’re looking for? https://salilab.org/modeller/ Best wishes, Chris On Fri, 6 Aug 2021 at 07:42 Sam Tang wrote: > Dear all > > Sorry for an off-topic question here. I wonder if anyone may be aware of > any search program which allows one to

Re: [ccp4bb] off-topic: structural motif / domain comparison

Like PDBeFOLD search? https://www.ebi.ac.uk/msd-srv/ssm/ Jan On Fri, Aug 6, 2021 at 8:43 AM Sam Tang wrote: > Dear all > > Sorry for an off-topic question here. I wonder if anyone may be aware of > any search program which allows one to 'blast' a protein domain just like > we 'blast' a

Re: [ccp4bb] off-topic: glycans

Thanks David! This is exactly what I am looking for. Sam On Tue, 29 Jun 2021 at 19:35, David Briggs wrote: > Hi Sam, > > GlycoMod from Expasy sounds like it might do what you want to do. > > https://web.expasy.org/glycomod/ > > D > > -- > > *Dr David C. Briggs* > > Senior Laboratory Research

Re: [ccp4bb] off-topic: glycans

Hi Sam, GlycoMod from Expasy sounds like it might do what you want to do. https://web.expasy.org/glycomod/ D -- Dr David C. Briggs Senior Laboratory Research Scientist Signalling and Structural Biology Lab The Francis Crick Institute London, UK == Diamond User Committee (MX) CCP4 WG2

Re: [ccp4bb] OFF TOPIC question

Hello Anamika, >From the information you gave, you have two different promoters- araC and the >lac promoter. LacI is the lac inhibitor. The lac promoter is a two part >system- when lactose is not present, the inhibitor sits near the promoter and >blocks transcription of genes downstream.

Re: [ccp4bb] OFF TOPIC question

, November 26, 2020 2:48:24 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] AW: [ccp4bb] OFF TOPIC question Bl21Pro j Von: CCP4 bulletin board Im Auftrag von Anamika Singh Gesendet: Donnerstag, 26. November 2020 11:07 An: CCP4BB@JISCMAIL.AC.UK Betreff: Re: [ccp4bb] OFF TOPIC question One

Re: [ccp4bb] OFF TOPIC question

*One correction to the previous question:* I have two constructs having different ori, p15ori and M13 ori, different promoters *araBAD promoter* and LacI, and different antibiotic resistance chloramphenicol and Ampicillin respectively. I would like to know which E. coli host cells will be good for

Re: [ccp4bb] (off-topic) beamline for XAFS

Dear Banu, I would recommend looking also at SSRL beam line 7-3 and 9-3 as they are well set up for protein EXAFS. Greetings, Jan On Tue, Nov 10, 2020 at 11:03 AM PULSARSTRIAN wrote: > Dear all, > Sorry for the off topic. > Looking for suggestions on beamlines for XAFS on proteins

Re: [ccp4bb] Off-topic: Mild cross-linking protocol

If you have hopes for cysteine residues in reasonable proximity, then bis-iodoacetamide (with a suitable spacer, commercially available is the ethylenediamine spacer). Any reasonable chemist can make you other spacer lengths to order. Homobifunctional PEG with maleimide, N-hydroxy succinimide

Re: [ccp4bb] Off-topic: (micro)array image analysis software

Hello, You could run Image Quant TL in a VM (parallels, vmware or virtual box). https://bmi.cchmc.org/resources/software/imagequant-tl For this appliation (arrays), we use an old program called VisualGrid that is no longer available and run it an isolated XP VM via virtual box (in linux).

Re: [ccp4bb] Off-topic (somewhat): Call for papers on structure-guided kinase drug discovery

Dear Isabel, I strongly support and **heartily thankyou** for your email below in support of IUCr Journals. A wide range of readerships are covered, now also including IUCrJ with its articles describing results aimed at highly diverse readerships ie well beyond crystallography. I would also add

Re: [ccp4bb] Off-topic (somewhat): Call for papers on structure-guided kinase drug discovery

Yes, this issue will definitely compete with IUCr's many journals dedicated to kinase drug discovery. /sarcasm On a more serious note: nobody should be "loyal" to a journal or scientific society; both are servants of the scientific community, not the other way around. If they stop supporting

Re: [ccp4bb] Off-topic (somewhat): Call for papers on structure-guided kinase drug discovery

Dear Julie and Mathew, I feel advertisement on behalf of professional publishers is not appropriate for the bulletin board. MDPI should pay for its advertisements, rather than get them for free. (Being a for-profit firm, they should also pay for, rather than invite editing, but this is of course

Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

Thanks to everyone who gave helpful suggestions; I now have stereo working on Ubuntu 18.04. To help anyone who comes across this in the CCP4 archives in the future, it was necessary to: 1) Install lightdm and set it as the default display manager. Other display managers that don't do

Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

I used to use Ubuntu Mate OS to get 3D work for coot and pymol. I am not sure if the latest version still works. Regards Xiao On Fri, Nov 8, 2019, 7:19 AM Chris Richardson wrote: > Apologies for the only slightly relevant question. > > Does anyone know the correct incantations to get nVidia

Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

Hi Chris We have it working with the Mate desktop. Composite disabled. Let me know if you would like more details Regards Christine. Sent from my iPad > On Nov 8, 2019, at 4:18 AM, Chris Richardson > wrote: > > Apologies for the only slightly relevant question. > > Does anyone know the

Re: [ccp4bb] [Off-Topic] 3D Vision stereo with Ubuntu 18.04

Hello, The desktop changed in the passage from Ubuntu16 to Ubuntu18. I think Nvidia stereo now works only with a xfce desktop. The passage from debian 8 to debian 9 was not a problem as long as xfce is kept. Best regards Wim - Mail original - De: "Chris Richardson" À: "CCP4BB" Envoyé:

Re: [ccp4bb] off topic: microscope in glove box

Short version: It should be OK, especially since the vacuum is transient and not particularly 'strong*' :) Long version: if this is an older microscope there may be further delamination of optically bonded components if air is already admitted between glass planes (i.e. the optical cement is worn

Re: [ccp4bb] Off-topic question

Hi, there is a guide here, but you should get the proper script for Pymol from the Consurf server. http://www.protein.osaka-u.ac.jp/rcsfp/supracryst/suzuki/jpxtal/Katsutani/en/consurf.php Jan On 6/22/19 10:24 PM, khaja faisal tarique wrote: > Hi everyone, > > I was wondering can anyone suggest

Re: [ccp4bb] Off topic: room temp FPLC column cooling

Bear in mind that if you have cold buffers going into a room temperature (or warmer) pumps, you can get outgasing that will cause inaccurate flow or loss of priming for the pumps. You may be able to work around by using freshly degassed eluents, but the outgasing will still be a problem for long

Re: [ccp4bb] Off topic: room temp FPLC column cooling

Hi, a cheap solution would be to put buffers, fraction collector, and the column (if not too big) inside a cold cabinet, next to the FPLC. you probably need to use longer pipes but it works Best regards, GIA Le 07/06/2019 16:51, - - a écrit : Dear all, our cold cabinet FPLC is rotting

Re: [ccp4bb] off topic: Membrane protein "into" soluble protein

In theory yes, you can fuse the termini to restrain the protein. You could use T4 lysozyme and insert in the loop which they use for crystallizing GPCRs (many references, including https://www.ncbi.nlm.nih.gov/pubmed/25450769). The potential problems are that you have no idea where the termini

Re: [ccp4bb] Off topic question

Dear Reza, CD hit will do exactly that. Cheers, Georg Sent from my iPhone On Jan 3, 2019, at 2:41 PM, Reza Khayat mailto:rkha...@ccny.cuny.edu>> wrote: ​Hi, Happy new year to all! A bit of an off topic question. Does anyone know of a method/program to extract the most distinct "n"

Re: [ccp4bb] Off topic question

Hi Reza, happy new year! The choice would depend on your alignment (aminoacid or nucleotides? are the sequences closely or distantly related? is it a large alignment? are there many gaps?)... Anyway, I think the safest, unbiased way to determine a group of outliers might be to compute a

Re: [ccp4bb] Off topic question

On Thursday, January 3, 2019 12:40:05 PM PST Reza Khayat wrote: > ?Hi, > > > Happy new year to all! A bit of an off topic question. Does anyone know of > a method/program to extract the most distinct "n" (n>2) sequences from a > sequence alignment? Thanks. If these putative "most distinct"

Re: [ccp4bb] Off topic question

PCA? On Jan 3, 2019, at 3:41 PM, Reza Khayat mailto:rkha...@ccny.cuny.edu>> wrote: ​Hi, Happy new year to all! A bit of an off topic question. Does anyone know of a method/program to extract the most distinct "n" (n>2) sequences from a sequence alignment? Thanks. Best wishes, Reza

Re: [ccp4bb] OFF TOPIC

Hi Anamika, As far as I understood, the biotin in the elution buffer is helping your protein to get stripped off from the Avidin column. So, maybe you dialyze your purified protein (or run FPLC) and get rid of biotin completely, before you load the protein on to strptavidin

Re: [ccp4bb] OFF TOPIC

Hi Anamika, - Have you double-checked that the sequence of your cDNA is correct and includes the biotin acceptor peptide tag (BAP tag aka AviTag; GLNDIFEAQKIEWHE in single-letter amino acid code)? - Are you using a dedicated bacterial strain that over-expresses BirA enzyme? This may not be

Re: [ccp4bb] OFF TOPIC

You don’t say so, but one assumes that you have a BAP tag on the protein, and co-express a biotin ligase such as BirA? Ed T.A.Edwards Ph.D. Associate Professor of Biochemistry Deputy Head of School _ Astbury Centre for Structural Molecular Biology School of

Re: [ccp4bb] OFF TOPIC

Hello, you probably purified a contaminant. Do a blot with an anti-biotin antibody or get electro-spray mass spectrometry done in order to confirm the identity of your protein. Wim On 13/11/2018 11:13, Anamika Singh wrote: Hi All,

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

r 27, 2018 5:41 AM To: Whitley, Matthew J Cc: ccp4bb Subject: Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice Dear Matthew, I am also late in responding to this, but as part of a Nature Protocols paper on iMosflm (Supplementary Information for Na

Re: [ccp4bb] Off-topic, protein in dye-front (ion front?) on native-PAGE

Hi All, Sorry to bring this old topic up again. I planned to run tricine gels but I found a possible error in table 2 (4% stacking gel formula) in Hermann Schägger protocol (Nature Protocols volume 1, pages 16–22 (2006), the author wrote 3ml 3X gel buffer in a total of 12 ml solution, it should

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi Matthew, I am also a bit late in responding, I have a few incommensurately modulated protein crystal datasets that you would be welcome to use in your course. It would be neat for students to at least know that this type of diffraction exists. As far as I know, they can only be processed

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear Matthew, I am also late in responding to this, but as part of a Nature Protocols paper on iMosflm (Supplementary Information for Nature Protocols 12, 1310-1325, 2017) I provided a number of examples of “problem datasets”. Some of these are just two images, to show

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

For some reason, the September 19th ccp4bb digest got caught in my spam filter and didn't come through until a few minutes ago, so I didn't see several responses concerning interesting datasets for processing until just now. Therefore, thanks also to Kay Diederichs, Eugene Osipov, and David

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi Matthew, I'm a little late to the thread, but I thought I would still like to add DPF3b, kindly provided by Wolfram Tempel. This dataset is available on zenodo and forms the basis of a tutorial for using DIALS:

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear colleagues, I want to thank the following people for providing suggestions and comments about ‘difficult’ datasets suitable for teaching data processing: Tim Craig Jacob Keller Graeme Winter Aleksandar Bijelic Clemens Vonrhein Loes Kroon-Batenburg James Holton If anyone else has

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

It was brought to my attention that the link to the preprint I provided below doesn't work, but this one does: https://www.biorxiv.org/content/early/2018/08/18/394965 Thanks to Folmer Fredslund for pointing this out to me! -James Holton MAD Scientist On 9/21/2018 3:50 PM, James Holton wrote:

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi James, you’re probably aware of this but you can edit CBF headers in place with sed. That’s what I do when I make the detector on my diffractometer go closer than the hardware limit. All best - Andreas On Sat, 22 Sep 2018 at 00:51, James Holton wrote: > For teaching purposes I have found

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

For teaching purposes I have found that controlled pairs of data sets are most instructive.  You are right that an easy one-button-push processing run tells you nothing, but so does a bang-it-crashed-now-what data set.  Most useful are two data sets that are identical in every respect but one,

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Dear Matthew, In my search for the validity of meta data, I went through several data sets in SBGrid and proteindiffraction.org (IRRMC), especially those where automatic processing did not succeed are gave different results with different processing software. We reprocessed those data sets

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Hi Matthew, I have some notes which indicate that SBgrid data sets 5, 62, 78, 117, 218 posed problems for "automatic" processing using generate_XDS.INP / XDS when I saw them for the first time. Some of these problems (mainly the conversion of header values to ORGX ORGY) are taken care of in

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

Matthew, One SBGrid example I used for a workshop was https://data.sbgrid.org/dataset/218/ This has the “wrong” beam centre as understood by (me/dials/xia2) which causes a certain amount of fun, nice example for use of the reciprocal lattice viewer and image viewer in DIALS to make sensible

Re: [ccp4bb] Off topic: 'Difficult' Datasets for Processing Practice

I deposited a dataset on SBGrid from a calmodulin-peptide complex which has some "nice" features: merohedral twinning (with variable twin fraction in the same dataset) and unavoidable detector cutoffs. It's relatively easy to integrate, but solving is somewhat harder. There's a paper on it too

Re: [ccp4bb] Off-topic: protein 2d diagrams

Batch mode generation of residue-based diagrams of proteins Fabien CampagneEmmanuel BettlerGert VriendHarel Weinstein /Bioinformatics/, Volume 19, Issue 14, 22 September 2003, Pages 1854–1855,https://doi.org/10.1093/bioinformatics/btg236 Published: 22 September 2003 On 4-8-2018 10:38, Joana

Re: [ccp4bb] Off-topic: protein 2d diagrams

Try http://gpcrdb.org/ They have a tool for snake plots of GPCRs Best Gebhard Prof. Gebhard F.X. Schertler Structural Biology ETH Zürich D-BIOL Head of Biology and Chemistry Division Paul Scherrer Institut Laboratory of Biomolecular Research, LBR OFLC 109 CH-5232 Villigen PSI

Re: [ccp4bb] Off-topic question

Hi, I was going to suggest the same but since it has already been said, here’s a cheeky suggestion: you could try determining the crystal structure of the complex and get a direct sequence readout for the nuclei acid. Best, Debanu > On Mar 5, 2018, at 9:34 PM, Philippe BENAS >

Re: [ccp4bb] Off-topic question

Hi Jobi, Phenol/CHCl3 extraction, iPrOH precipitation and then nucleic acid sequencing. Best regards,Philippe Philippe BENAS, Ph.D. Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS Faculté de Pharmacie, Université Paris Descartes Case 48 Av, de l'Observatoire F-75270 PARIS cedex

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

Perhaps this can be automated: https://www.phenix-online.org/papers/wd5073_reprint.pdf Software doesn't get tired or bored, and thus potentially can try more and produce more plausible interretations. Then one can hire a number of people of various expertise to choose "best" result according to

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

It sounds a bit like this paper from a year ago: https://www.nature.com/articles/ncomms12549 apart from the conclusion: here the point is that amateurs build higher quality models than crystallographers. On 20 November 2017 at 20:25, Shintaro Aibara wrote: > Dear All,

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

Thanks Mark for the paper, it kind of catches the essence of what I was looking for. I do however remember it was more people and I think I'm looking for a figure where all the builds (which if I remember correctly was in the 10's rather than 3) were superimposed over each other to make what

Re: [ccp4bb] [Off-topic] Comparison of the same structure built by many people

This one?http://scripts.iucr.org/cgi-bin/paper?SE0260 Mark J van RaaijCNB-CSICwwwuser.csic.es/~mjvanraaij Original message From: Shintaro Aibara Date: 20/11/2017 21:25 (GMT+01:00) To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] [Off-topic] Comparison

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

Hi Anamika, As it was said before, a cocktail of protease inhibitors should be fine. But let`s say you can not overcome the problem. It is not specified if you are doing this dialysis to cleave or not your His-tagged protein but even this you could try getting samples in 1 hour

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

suppose protease is the issue, then avoid overnight dialysis 发自网易邮箱大师 在2017年10月26日 22:18，Anamika Singh 写道： Dear All, I am purifying the His-tagged proteins ( 21Kda) using buffer 20mM Tris, 150mM Nacl, and 5mM MgCl2 with 500mM imidazole. Earlier I was facing the precipitation problem during

Re: [ccp4bb] OFF topic (Protein degrades during Dailysis)

Hello, PMSF inhibits only 1 class of proteinases You can try to add other inhibitors : e.g. for purification from yeast I used the following in addition to PMSF: 1000 x stock individual inhibitors pepstatin5 mg/ml methanol chymostatin1 mg/mlDMSO antipain3 mg/mlmethanol (sonicate to

Re: [ccp4bb] Off topic: denaturing urea gels

; Molecular Biology > The University of Chicago > pr...@uchicago.edu > > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Opher > Gileadi [opher.gile...@sgc.ox.ac.uk] > Sent: Saturday, September 30, 2017 3:44 PM > To

Re: [ccp4bb] Off topic: denaturing urea gels

C.UK Subject: Re: [ccp4bb] Off topic: denaturing urea gels In addition to the previous suggestions: With very small gels, the sample composition and depth (in the well) have a strong effect on the resolution. Rinse the wells with TBE buffer just before loading, as urea from the gel diff

Re: [ccp4bb] Off topic: denaturing urea gels

In addition to the previous suggestions: With very small gels, the sample composition and depth (in the well) have a strong effect on the resolution. Rinse the wells with TBE buffer just before loading, as urea from the gel diffuses into the well and may prevent the sample from settling at the

Re: [ccp4bb] Off topic: denaturing urea gels

Smith: One should expect to see ladders each separated by 1 nt in such cases. Mohammed: My suggestions: 1) running at 70V seems low. I do not see a reason for not using higher voltages. 200Vx45min should be OK. IT is better to not let the BPB front run out of the gel - so that you know whether

Re: [ccp4bb] Off topic: denaturing urea gels

your enzyme cannot give definite fragment. thus smear 发自网易邮箱大师 在2017年09月30日 19:28，Mohammad Khan 写道： Dear all, I am working with an exonuclease and I run the digested DNA on a 8Murea-20%acrylamide gel in TBE buffer. I use the Mini-Protean BioRad system and cast gels of about 8.6x6.5 cm

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear Thomas, I do all my experiments in a 384 well, non-binding plates. I have a salt concentration of 50 mM NaCl, with BSA. i will try the other suggestions as well. >From a couple of experiments, I could determine the Kd to be approx. 10 nM. Thanks for all the suggestions! On Fri, Jul 21,

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear Didier and Opher, The difference in polarities can reach upto 60 units. I have tried concentrations between 1-5 nM DNA. Maybe I will give higher concentrations a shot. Thanks! On Fri, Jul 21, 2017 at 4:02 PM, Didier Spittler wrote: > Hello, > > What is your

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Dear Julius, That is a good suggestion. I will definitely try this. Thanks! On Fri, Jul 21, 2017 at 3:41 PM, Rabl, Julius wrote: > Dear Mohammad, > your buffer is key to success in biophysical measurements. While your > protein may be totally fine in standard buffer, the

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

, 2017 9:44 AM To:CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement Completely agree, you need a higher DNA concentration. We have had luck with 10 nM DNA. Also, bubbles have a HUGE impact on how the fluorescent signal is measured. Make sure you spin yo

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

r...@uchicago.edu> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Morgan Milton [eilise.mil...@gmail.com] Sent: Friday, July 21, 2017 9:44 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Off topic: Flourescence anisotropy measurement Comple

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