Re: [ccp4bb] Off topic - fluorescence plate reader in cold room or cold box

Dear Markus, I've had to solve this exact issue in the past. Putting a 'simple' (nothing is simple these days, everything has some kind of AI or other evil gnomes in it) spectrofluorimeter into the cold room *should* be OK. Caveat #1: I don't know if this will work with your specific device

Re: [ccp4bb] minor off topic protein symmetry

It is possible that the following tetramer displays the features you seek: https://www.rcsb.org/structure/6v1v You will notice that this is P1. When I solved it, I had at first some trouble recognizing why this molecule does not have a P4 (I am slow, I guess) but then it dawned on me :) It

Re: [ccp4bb] Ligand identification in X-ray density

At this resolution you should be able to infer differences between C and O based on the map levels at each location. Another dead giveaway is the bond lengths and the asymmetry of the top region - that is not a carboxylic acid to me. This looks lime MPD to me, very classic shape. Artem On Wed,

Re: [ccp4bb] Skimming crystal

Hello This is a common issue with volatile organics in the drop. Solutions that have worked in the past include: 1. covering the drop in oil (some people prefer light oil, I personally prefer heavy/viscous oil, as it makes harvesting easier) 2. diluting the drop with a much larger volume of

Re: [ccp4bb] Difficult Molecular replacement

where you absolutely have to treat domains as separately mobile rigid bodies. I would love to help more but it would require more sharing that you might be justifiably unwilling or unable to do :) Best of luck, Artem On Mon, Feb 19, 2024 at 6:51 PM Artem Evdokimov wrote: > Hello Ma

Re: [ccp4bb] Difficult Molecular replacement

Hello Marco, First: compare your unit cell parameters with RCSB (there is an option for that in the search, very helpful). Give it say 5% error margin. Worst case scenario - cry a little, having found that your protein is something else... Next, check in depth for space group oddities,

Re: [ccp4bb] Äktaxpress uninet connector : Off topic

Ebay to the rescue: https://www.ebay.com/itm/185248167872 is this the correct connector? Once you have a single connector, you can pin-trace it to your hearts' delight and make new ones for pennies, since the cable and the plugs are standard... Artem - Cosmic Cats approve of this message On

Re: [ccp4bb] What could these crystals be?

Could be dna crystals. They often do not diffract. Artem On Wed, Nov 8, 2023, 11:18 AM careinaedgo...@yahoo.com < 02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote: > > The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG > > Protein buffer is 300 mM NaCl, 20 mM HEPES

Re: [ccp4bb] What could these crystals be?

Dear Careina These look like crystals indeed. Without more information, I can make some educated guesses but the ultimate proof is in your hands :) 1. they don't have to be spherulites. Note that low-symmetry crystals can have edge-less habits, especially P1 are notorious for that - I've had

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] Autoinduction Medium

- Cosmic Cats approve of this message On Mon, Oct 2, 2023 at 10:15 AM Artem Evdokimov wrote: > Dear Jinhui Dong > > You can simply make swimming-pool size amounts of it, as long as you have > basic lab equipment and supplies including an autoclave, and access to ~8 > chemic

Re: [ccp4bb] Autoinduction Medium

Dear Jinhui Dong You can simply make swimming-pool size amounts of it, as long as you have basic lab equipment and supplies including an autoclave, and access to ~8 chemicals. The recipe is in the Studier papers, but I am happy to share it with you here if you prefer. Artem - Cosmic Cats

Re: [ccp4bb] Adding metal ions

Take a protein structure with waters, replace surface waters with Cu++ and run some very basic MD to optimize distances. Many of the atoms will move since Cu++ will find itself in an 'unfriendly' environment, as always caveat emptor :) Artem - Cosmic Cats approve of this message On Fri, Jun

Re: [ccp4bb] Regarding the diffraction image

With 50 mM Zn++ in solution, whatever it is will probably have a Zn salt in it. So if you wanted to solve it by direct methods or via SAD - that should do well. Sadly (hur hur) it's probably quite small, whatever it is. Artem - Cosmic Cats approve of this message On Fri, Feb 3, 2023 at 6:20 AM

Re: [ccp4bb] small scale -15C chiller

erm solution to make overnight > measurements. > > Thanks to everyone. I feel overwhelmed with the large number of quick > responses. > > Best, > Tim > > On Thu, 2 Feb 2023 20:19:32 -0500 > Artem Evdokimov wrote: > > > A basic Peltier element will likely wor

Re: [ccp4bb] small scale -15C chiller

A basic Peltier element will likely work (may need a stack of two to reach -20) however the simpler option indeed would be to 'fake out' thermocouple input using a voltage, as described by Ivan. https://us.flukecal.com/Thermocouple-Temperature-Calculator And for $38 one can apparently purchase a

Re: [ccp4bb] Alternative server for: Surface Entropy Reduction prediction (SERp)

Hi Yong Tang, I am not sure if there is another SER server out there, but if you can share the sequence in a private email I can run it through my code that does a similar type of analysis (it's not user friendly so I never did the right thing and put it up as a server). All the best, Artem -

Re: [ccp4bb] TEV vs HRV3C

One final comment: if you need orthogonal proteases, TEV and TVMV do not seem to cut each others' sites (at least for me they don't). No idea if 3C will cut both, or only TEV site. Artem - Cosmic Cats approve of this message On Thu, Dec 8, 2022 at 5:26 AM Dom Bellini - MRC LMB <

[ccp4bb] hiring (off-topic but could be of interest)

Dear CCP4-ers, We're hiring :) In case you're interested, please visit the link below. http://careers.animol-discovery.com/apply/fUyc5HcbL0/Head-Of-Medicinal-Chemistry?referrer=2022083118532124ZTDOKKYYDQHFBP Artem VP@ Animol Cheesecake aficionado

[ccp4bb] help request (synchrotron schedules)

Dear CCP4-ers :) I used to have a handy chart where major synchrotron schedules were all drawn out as lines (across an entire year) with shutdowns and other blackout dates marked such that it was pretty easy to find what synchrotron is open for business at what week/month. This schedule took me a

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] Strategies for increasing Trp content of proteins

elinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fprofile%2FCasper_Wilkens=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJX

Re: [ccp4bb] Strategies for increasing Trp content of proteins

We have had success placing single trp on the outside of proteins, as weird as it sounds it works pretty well. If your protein is easy to express you can try a few choices. If your protein is difficult to express, a fusion with a trp-positive domain or with gfp may be a better option. There are

Re: [ccp4bb] Baculovirus expression system

There are several but if you discount the transfection based ones (i.e. direct DNA, no virus at all) then the speed is roughly the same - about four weeks from clone to process. FlashBac works a bit faster because it avoids the use of ecoli to recombine the bacmid but the time savings is about a

Re: [ccp4bb] OFF_TOPIC: Should you be worried about BPA from plastics? Yes, if you store alkaline reagents in polycarbonate bottles!

Interesting note - and a reminder that alkaline solutions are tricky :) By personal preference, HDPE bottles are probably the best for storing strongly alkaline solutions (XLPE also is very good, but harder to get lab-grade bottles). Here's a handy chart for plastic vs stuff resistance. Note that

[ccp4bb] multiple openings in my team @ Roivant

Friends, Sorry for the off-topic message :) We're advertising three positions, and anticipating a posting on the fourth shortly. *The three jobs with links should be applied to directly* whereas for non-posted one please write to me directly. I will do my best to reply to everyone. Thank you

[ccp4bb] (off topic) Job posting - Associate Director, Protein Science

https://boards.greenhouse.io/roivantsciences/jobs/3549863 Finally posted! Thank you for looking. Artem - Cosmic Cats approve of this message To unsubscribe from the CCP4BB list, click the following link:

[ccp4bb] job opening: Associate Director, Protein Sciences

Dear CCP4-ers I would like to share a preview of a job opening we're about to post for a senior Protein Sciences research professional. This is an Associate Director level position, with some wiggle room if exceptional candidates materialize. Rather than posting explicit job description, here

Re: [ccp4bb] RNA-PROTEIN purification on FPLC

Hi 1. Lysate on fplc is messy :) which is why I always do the first step in batch. 2. A good scrubbing with NaOH, trypsin, and a strong chaotropic agent (in series) should take care of the contamination, however it may be easier to find e.g. an old HPLC never used for protein work in order to

Re: [ccp4bb] Enzyme Vmax and Km

Is your enzyme monomeric or multimeric? What time exactly does it mean when you say unprocessed substrate versus processed? What are your error margins (no error bars evident on your plots)? Depending on your errors, your second plot is not a 'steep decline' but instead could be a constant line.

Re: [ccp4bb] Looking for proteins for undergraduate biochemistry lab

All the previously suggested proteins are awesome candidates! Here are a few more in case you want to add some extra 'zing' to your curriculum: - luciferase - magneto reactive protein(s) that can be isolated with strong magnets - spy-tag and spycatcher (covalently reactive proteins!) - split GFP

Re: [ccp4bb] [OUT-OF-TOPIC] Compound service management CRO

Frontier Pretty good service, but definitely not the cheapest. Artem On Tue, Jun 1, 2021, 4:54 AM Wim Zhong wrote: > Hi Group, > > Does anyone have experience with CROs that can provide good compound > service management in terms of quality and cost? We are looking for a > partner to manage

Re: [ccp4bb] crystallizing fusion proteins

If you have a biophysical way to confirm functionality and/or folding it would go a long way towards helping you make this choice. Does your domain bind a ligand of any kind? Is it significantly stable in a simple thermal melt experiment (of course you have to account for the melting of the

[ccp4bb] Off-topic: research associate job posting

Dear CCP4 friends We are looking for fresh talent :) please see the posting below or directly at MassBio.

Re: [ccp4bb] OFFTOPIC question "Two plasmids in one host cell"

Hi An easy fix is to PCR amplify your low copy number plasmid (modern polymerases have no issues amplifying up to 20 kB if you have a largeish vector) and then sequence the product. Alternatively you can amplify just a portion of interest, of course (you said sequence the plasmid so I went with

Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

Probably a good idea to share an image :) worth many words... Artem On Wed, Dec 9, 2020, 9:17 AM wrote: > Dear All > There is a 43kd protein purified via Ni-chelating affinity > chromatography, anion exchange chromatography and gel filtration > chromatography in sequence. However the

Re: [ccp4bb] AW: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

Well that is sad, and true, and also very common. I have personally experienced dozens of cases where methods from literature do not reproduce because (and this is important) the authors "just slap some generic boilerplate" instead of the actual methods. My favorite is always to read stuff like

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

[ccp4bb] offtopic: three job postings @ Silicon Therapeutics

Dear CCP4 friends, I would like to let you know that we have just posted three jobs open within my team at Silicon Therapeutics. Qualified inquiries are welcome! Artem 1. Senior position in Biochemistry

[ccp4bb] slightly offtopic: antibody and nanobody development providers

Dear CPP4ers Once again I would like to refill my cup of borrowed wisdom from the collective fountain that is this excellent list :) I am looking to make antibodies/nanobodies for crystal-chaperone (nano/Fab/ScFv etc) kind of a deal. I have a few providers from my past endeavors, but really

Re: [ccp4bb] opinions about crystal soaking service providers

will post it here. Cheers, Artem - Cosmic Cats approve of this message On Fri, Sep 11, 2020 at 10:26 AM Artem Evdokimov wrote: > Dear CCP4ers! > > I would like to once again benefit from the communal wisdom and experience > of our community. Thank you in advance for your advice! >

[ccp4bb] opinions about crystal soaking service providers

Dear CCP4ers! I would like to once again benefit from the communal wisdom and experience of our community. Thank you in advance for your advice! Question: we are considering engaging an external partner to perform crystal soaking campaign(s) on our targets. In the past I've successfully done

Re: [ccp4bb] Advice on DNA negative staining

Hiya *Half an hour in the library saves a month of research.* That's what my late advisor Dr. Frolow used to say a lot and he was not wrong. Erenpreisa J. 1981. Staining of DNA with uranlyacetate in hydrolyzed ultrathin sections. Acta histochem 68(1): 22-26 there are several very clever

Re: [ccp4bb] Protocol for bacmid transformation in E.coli

Yes It requires electroporation and very careful handling of the bacmid. Other than that it is a fairly simple process. Here is an example reference: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067234/ Artem On Sat, Jul 18, 2020, 9:12 PM Digant Nayak wrote: > Dear all, > > Sorry for the

Re: [ccp4bb] Looking for suggestions with protein expression

Hi It is hard to give specific advice without knowing details about your protein. There are multiple reasons that may be responsible for it's behavior (fundamentally the simplest reason is that proteins by and large are not designed by Nature to be purified and handled) so the more you share with

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

Recent cryoEM resolution in apoferritin (yes, the Lysozyme of cryoEM, but still) seems to be 1.2 A by two groups in the UK and Germany. Notably (yes, I am kidding, please don't think that I really believe this) the resolution of MODEL structures is ZERO angstroms :) It's perfectly good... as long

Re: [ccp4bb] Question about small molecule crystallography

Hi A small organic molecule is typically crystallized from organic solvents (or water, if soluble) by means of at least three main techniques: 1. slow evaporation of solvent leading to supersaturation and eventual crystallization 2. supersaturation at higher temperature followed by gradual drop

Re: [ccp4bb] Fusion proteins to solubilize your protein

Probably the easiest would be blasting sequences all by all and extracting peaks above certain height with a filter of some sorts. Not entirely trivial. I can add to your list, although not all of these proteins are in the PDB - some are from my own collection :) Lysozyme (s) several are used

Re: [ccp4bb] server to predict structural stability

Rosetta will do this. But it's not a server, and there is a fairly steep learning curve. Artem - Cosmic Cats approve of this message On Mon, May 4, 2020 at 6:04 AM Firdous Tarique wrote: > Hi > > I am trying to incorporate novel disulfide bond by introducing Cys > mutants at the interface

Re: [ccp4bb] somewhat off-topic :)

Thank you Artem On Tue, Apr 28, 2020, 7:22 PM Thomas Cleveland wrote: > Hi, > > There are tons of these listed on eBay, many in very good condition or > even unused. I used to get them there all the time. > > Best > > On Tue, Apr 28, 2020 at 8:51 AM Artem Evdokimov &g

Re: [ccp4bb] somewhat off-topic :)

Thank you very much! Artem On Tue, Apr 28, 2020, 1:03 PM Guenter Fritz < guenter.fritz.phenix.c...@gmail.com> wrote: > Dear Artem, > > I am a fan of the former "Superformance" column from Merck. The production > / selling of these columns is sourced out to a small company called > Goetec. The

[ccp4bb] somewhat off-topic :)

CCP4 friends, Sorry for the somewhat off-topic post! For many years I've been a fan of the Pharmacia XK columns and heavily relied on them for the bulk of my work. Lately, however, GE has increased the prices so much that I am reluctant to buy new columns from them. And I don't mean filled

Re: [ccp4bb] insect secretion recommendations?

Gentle spin followed by TFF - a cheap peristaltic pump plus $200 filter cartridge will do the trick. Artem On Mon, Apr 20, 2020, 11:56 AM Gloria Borgstahl wrote: > Hi Friends, We are secreting Spike Ecto domain into the media from insect > cells for purification. As we scale up I am

Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!

ee it as their job to ignore emails like yours > because that's far easier than dealing with them. > > Alternatively, go the Rupp Route: Name and Shame! ;) > > On 07/04/2020 16:08, Artem Evdokimov wrote: > > Dear CCP4ers, > > > > I would like to solicit your thoughts

[ccp4bb] on-topic: your opinions requested!

Dear CCP4ers, I would like to solicit your thoughts on the following (this is a real situation, but salient details are changed): Imagine that you're an industrial scientist in a small company, working on the Bavarian Sausage (Weisswurst) Esterase project. The overall structure is previously

[ccp4bb] off-topic: coronavirus test summaries

Just FYI, here are two useful links for those who would like to see what's being used to test for coronavirus: https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf

Re: [ccp4bb] SARS-CoV-2 test on a smartphone

y reagent, take your phone and detect. It still is challenging in most > African countries but more doable, there are more smartphones e.g. older > Android & iPhones available than one might anticipate. > > Jürgen > > On Apr 2, 2020, at 2:52 PM, Artem Evdokimov > wrote: &g

Re: [ccp4bb] SARS-CoV-2 test on a smartphone

For DIY fanatics out there: Step 1. Express reverse polymerase (single subunit one for simplicity, like the one from swine retrovirus). For hardcore fans also express Taq. That is easy. Purify both with minimal RNAse contamination. Less easy but we know how to do that. Step 2. Design and order a

[ccp4bb] off-topic: work opportunities at Enko

Hello! Sorry (not sorry) for the off-topic post. We have a few jobs posted and I'd like to re-post them here. Please see the link for full descriptions >>> https://www.enkochem.com/careers-enko -Scientist, Structural Biology & Protein Chemistry -Senior Scientist, Protein Target Discovery

Re: [ccp4bb] Expi 293 (Met-) expression medium for selenomethionine labeling

with ExpiSf9 and baculovirus to see if they also perform > similarly. > > Best, > > Kelvin > > On Mar 11, 2020, at 5:49 PM, Artem Evdokimov > wrote: > > Hello > > Bad news: Expi cells work poorly in non-Expi medium :( > Good news: you can add SelenoMet and make partia

Re: [ccp4bb] Expi 293 (Met-) expression medium for selenomethionine labeling

Hello Bad news: Expi cells work poorly in non-Expi medium :( Good news: you can add SelenoMet and make partially seleniated protein, which is usually enough. Note that too much SeMet is pretty toxic, and that the toxicity is associated with BOTH enantiomers of the amino acid, so if you have the

Re: [ccp4bb] Mixed oligomeric states in crystallo

5FRT We got scooped on this one a long time ago - but back in 2015 it was very gratifying to see that the academic group who published this structure have also obtained an ASU with two dimers and one monomer - same exact story as what we saw. The difference can be attributed to the movement of a

Re: [ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

chaotrope than guanidinium/HCl, the > different chemical actions of the two might give a hint as to what causes > the aggregation. > > Cheers > > jon > > > > *Von:* CCP4 bulletin board *Im Auftrag von *Artem > Evdokimov > *Gesendet:* Sonntag, 2. Februar 2020 00

Re: [ccp4bb] Urea vs. Guanidinium/HCl

More details would be helpful. Do you know whether your protein is folded and active to begin with? Many partially folded proteins behave in a way that resembles your experience... Urea is a less potent denaturant mole for mole than GuHCl so it is not super surprising that it behaves differently.

Re: [ccp4bb] microscope camera

I bought a bunch of cheap cameras from Amscope. They were OK for the price. Artem On Wed, Nov 27, 2019, 12:46 Dean Derbyshire wrote: > Forgive the off topic subject: > > > > Has anyone got any experience with moticam microscope cameras? > > We are looking into cheap cameras for record keeping

Re: [ccp4bb] Fwd: [ccp4bb] suggestions on small proteins that for a complex

FliS and FliC Artem On Sun, Nov 10, 2019, 03:42 Gianluca Cioci wrote: > ps: the 3D structure > of the complex should > be solved at high resolution > > ;o) > > > > > Dear All, > > I am looking for examples of two small proteins A and B that can form a > tight complex AB. > Each protein

[ccp4bb] Off topic: 384 well stamper

Colleagues I have a quick question for you: we are in need of a small footprint 384 well plate stamper with dilution (optional but desired) capability. We are already evaluating Integra, Jena Analytic, and Apricot models but we are curious if there are other companies who sell this sort of

Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

I am willing to bet that the ketone is covalently reacting with the arginine and the result is what you see :) Artem - Cosmic Cats approve of this message On Thu, Nov 7, 2019 at 3:33 AM wrote: > Dear HK, > > In your case mass spec would be extremely valuable. Not only could it > prove or

Re: [ccp4bb] lysine methylation in E. coli?

It happens. Ef-Tu is methylated on a single lysine Also LafV is a putative methylase in E. Coli but its effects were not studied in detail. Salmonella methylates flagellin which is how lysine methylation was discovered - and E.coli is a kissing cousin so... Artem On Tue, Nov 5, 2019, 12:31

Re: [ccp4bb] Sodium Ion Binding?

Hi Jacob Not the easiest task... Based on past experience your major issue will be the incredible abundance of sodium ions in everything. So assuming you have high quality sodium free solutions and are willing to work exclusively in plastic, quartz or fused silica - here are a few thoughts: 1.

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

[ccp4bb] off topic: job posting

Good morning, We have two relevant job postingings that I would like to share. Thank you for your attention :) Artem https://careers.massbio.org/job/sr-scientistprincipal-scientist-computational-chemistry/49455467/ and

Re: [ccp4bb] challenges in structural biology

temptation to promise unrealistic outcomes when confronted with money > provided for political reasons, which ultimately undermines our > credibility. But this takes us away from James' points. > > best, > > Kay > > On Sun, 21 Jul 2019 16:06:48 -0400, Artem Evdokimov < >

Re: [ccp4bb] challenges in structural biology

expected to yield significant improvements. Personally, I think > that even huge funding would not result in methods that succeed in > crystallizing all molecules. > > best, > Kay > > On Sun, 21 Jul 2019 11:28:14 -0400, Artem Evdokimov < > artem.evdoki...@gmail.com> wro

[ccp4bb] multiple openings at Enko

Sorry for the off-topic post; only the first two out of the four positions below are related to structural work, I've included the other two for completeness' sake. We have several openings at Enko (a small crop protection company in Woburn, MA). - Principal Scientist, Discovery Chemistry

Re: [ccp4bb] Interesting pattern on a crystallization drop

Neat! Looks like multiple adjacent bubbles that were initially touching but eventually shrunk down to the central cores - the connectors are protein filaments (skin on the bubbles) left over from when bubbles had contact points. Artem On Wed, Mar 27, 2019, 19:39 Marshall, Bevan (Manufacturing,

Re: [ccp4bb] KCl in SDS-PAGE workarounds?

any third party, without a written consent of the sender. > If you received this message by mistake, please reply to this message and > follow with its deletion, so that we can ensure such a mistake does not > occur in the future. > > > > *From:* CCP4 bulletin board * On Behalf

Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins

Hi Alex, In my experience you just have to experiment with these parameters (in however of a limited space that is afforded by your experimental set-up). If you have the right tools you can slog through semifactorial or DoE-based scouting of various parameters with relative ease. These parameters

Re: [ccp4bb] Fwd: dry Shipper made in China

Dry shippers are often featured on auction sites for used equipment and sometimes can be found for huge discounts... Artem - Cosmic Cats approve of this message On Fri, Jan 25, 2019 at 8:36 AM Muhammad Imran wrote: > Dear Community members, > > We wanted to buy molecular dimension dry

Re: [ccp4bb] looking for 10-20 kDa proteins that express well and migrate appropriately on SDS-PAGE

Off the top of my head, for your mass range with gel behavior that you ask for: Human or mouse Synuclein (not folded, very heat stable, incredibly soluble) - interesting reaction with SDS but runs fine on the gel T4 Lysozyme with a double mutation (expresses mega-loads per gram of cell paste)

Re: [ccp4bb] Non-Cysteine modification with heavy atom

Off the top of my head, assuming nothing much about your protein(s) - some of this is only going to work if you can modify the protein(s) prior to crystallization. This excludes the 'normal' heavy atom soaking type stuff which can give rise to binding with covalent-like strength (while still being

Re: [ccp4bb] 2019 New Year’s resolutions of a cryo-EM newbie

My apologies for posting this on an open board :) I realize that this is completely off topic - but it is a pretty painful topic that affects us all regardless of academic or industrial affiliation, professional preferences etc. Artem --- do not read below unless you want to :) --- If an

Re: [ccp4bb] 2019 New Year’s resolutions of a cryo-EM newbie

https://www.unboundworlds.com/wp-content/uploads/2016/05/takei-oh-my.jpg oh, my... Happy new year! Artem - Cosmic Cats approve of this message On Tue, Jan 1, 2019 at 5:38 PM Marin van Heel < 057a89ab08a1-dmarc-requ...@jiscmail.ac.uk> wrote: > 1) I shall not make false FSC resolution

Re: [ccp4bb] opentrons pipetting robot

Good morning and Happy New Year to you In brief, it can be used to create screens although programming multiple ingredients with varying viscosities will initially be a fairly steep challenge since the software is not specific to crystallization. It can be done though and provided that you can

Re: [ccp4bb] Adding Zinc to Protein

Dear Nicola, Happy New Year It may or may not be possible to re-constitute Zn into purified protein. Some binding sites are very hard to fill back up once Zn is gone (for example sites with more than one -SH ligand can and often do undergo oxidative crosslinking in the absence of a stabilizing

[ccp4bb] off-topic: Laboratory RA job posting

Greetings Sorry for the off-topic post :) We are looking to fill an RA position in Enzymology/MolBio (junior or senior, depends on qualifications and experience). Full description follows. Interested candidates should forward their CV and letter of interest to: *i...@enkochem.com *

Re: [ccp4bb] Protein Purification- reg

Hi Amala, Depending on the resin you used there may be a conflict with BME and also 50 mM TRIS may be too much at the pH 7.5. Easy to test: use HEPES pH 7.5 and TCEP instead of BME, or use 30 mM TRIS at pH 8.0 Alternatively your protein is aggregated and does not bind well... Artem - Cosmic

Re: [ccp4bb] Zn+2 proteases

From personal experience, many of them really do need Zn++ and some of them can get by on Cd++ :) However, Zn++ can often be used in the micromolar range (so essentially quasi-stoichiometric with the enzyme) so it's not as much of a PITA as when you have millimolar quantities. HTH! Artem -

Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

Dear Partha Treat your culture with Kifunensine prior to transfection (or throughout growth if you're using stables) and then treat purified protein with EndoH. Pretty cheap and effective. PNGase F does not *require* denaturing conditions. It just likes the protein to be 'loose' - in my

Re: [ccp4bb] Glycoprotein expression strategy

Dear Wenhe, >From my perspective you have quite a few options. Notably, the presence of glycosylation in the native host does not always (or even often) mean that you *must* have the protein in the native glyco-state in order to be 'right'. Very often glycosylation sites can be trimmed (by e.g.

Re: [ccp4bb] disulfate bond ?

It seems that your CoA is one carbon out of reference. You have spotty difference density over the ligand. Shift it left by one carbon bond and redune to see if density fits better. Artem On Wed, Jul 4, 2018, 02:26 张士军 <21620150150...@stu.xmu.edu.cn> wrote: > Hi all > > I got a structure which

Re: [ccp4bb] Please share your experience about "ugly" crystals showing good diffraction

Good evening, Over the years I've seen great diffraction from any number of hideous brittle plates with perforated edges, 'Krypton skyscrapers', curved whiskers, 'hypodermic needles', even from crystals that looked like cat vomit (hairballs), and so forth. The structure *ab ovo* story is my

Re: [ccp4bb] suggestion of crystallization optimization

Janet, Patrick, and others beat me to it :) We have tremendous luck with MMS in cases like this. I would also suggest looking at 'atypical' additives - the word atypical is really not a good choice since the world of additives is pretty much infinite, but folks too often tend to take the lazy

Re: [ccp4bb] size exclusion columns

Xk10/300 packed with superdex of the appropriate pore size is by far the most economical and robust option :) pack your own and save. Artem On Thu, Apr 26, 2018, 12:06 Markus Heckmann wrote: > Dear all, > > We are looking for a size exclusion chromatography column >

Re: [ccp4bb] His-6 versus His-10 tag

Option 1: make two constructs and compare :) Option 2: put the his10 on the n-term of the protein with a cleavage site In my experience it matters. I had cases where his4 crystallized and his6 did not. I also had the opposite experience. Artem On Sep 19, 2017 6:11 AM,

Re: [ccp4bb] Difficult purification with imac columns

Hi This is a multidimensional problem, just like any other tricky protein purification. 1) IMAC columns are also ion exchangers (of both polarities!) so make sure you have adequate salt. 2) IMAC resins can be polysaccharide-based (agarose) and affinity to sugars can be an issue. Consider adding

Re: [ccp4bb] Primer design

Have to do primer walking in a cdna library first. Artem On Jul 24, 2017 7:26 AM, "syed ibrahim" < 048c02cac012-dmarc-requ...@jiscmail.ac.uk> wrote: Hello All I am interested in cloning a gene from a plant. I searched the database only partial sequence is available, ie: for 160 residues

Re: [ccp4bb] Unknown Ligand Density

Flying spaghetti monster. Ramen! Sorry. Could not resist. Artem www.harkerbio.com "...touched by His Noodly Appendage" On Jun 14, 2017 12:51 PM, "Nick Thomas" wrote: Dear CCP4bb, I am refining a structure and have come across strong electron density for an unknown

Re: [ccp4bb] Rhodamine in FPLC

30% acetic acid solution should wash it out (exercise some care, it's quite a strong vinegar). Hypochlorite and peroxide will bleach the stain, but technically speaking the leftover oxidized species will still be stuck in there, just colorless. Artem www.harkerbio.com "making lots of *really

Re: [ccp4bb] unknown ligand density

Hi It helps to see the environment of the blob, and to know what the resolution is - this *looks* like mid-range resolution (like maybe 2.4A) but because there is no reference in the image the size can be very misleading. Also, contour levels are good to know. Basically, more information. Artem

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