Re: [ccp4bb] crystallizing fusion proteins

should be bright yellow so that speeds up the screening process a bit. I hope this helps. Good luck Best regards, Pascal Egea, PhD UCLA School of Medicine > > > -- > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscma

Re: [ccp4bb] Data 3 A

offense intended). Best, Pascal Egea On Tue, Jan 12, 2021 at 3:22 PM rohit kumar wrote: > Dear all, > > I am trying to solve a data with 3 A resolution, however data quality is > very bad and mathews coffi. suggest two molecules per ASU but It always > gives one molecule in AU after

Re: [ccp4bb] Cell disruption

Dear Bernhard , I would recommend the emulsified from Avestin. It is great . Depending on your budget you can ask for stainless steel (probe to slow tear ) or ceramic (longer lasting but a bit more expensive). We have the ceramic but I have worked with the stainless steel one and it is really goo

Re: [ccp4bb] Structural Importance of Methionine

Hi Samer, In addition to what has been already mentioned to you, there are also the two well illustrated cases of methionine rich domains. - in the case of the M (for Met rich) domain of the Signal Recognition Particle protein SRP54/Ffh involved in promiscuous binding of the N-terminal hydrophobic

[ccp4bb] Post Doctoral Position at UCLA

three potential references to *pe...@mednet.ucla.edu .* Thanks. All the best, Pascal Egea -- Pascal F. Egea, PhD UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310

Re: [ccp4bb] Should I optimize these crystals?

Hi Marta, 1-- There is always a possibility of forming detergent/lipid (cholesterol in your case) -containing crystals combined with other small molecule(s) (organic or not). Although you did not specify the width of the oscillation used to collect the frame displayed in your mail (was it 45 degre

Re: [ccp4bb] suggestions for cryoprotectant

ually easier to cryo-protect. Hope this helps Pascal Egea On Fri, Oct 19, 2018 at 2:57 PM Firdous Tarique wrote: > Dear members > > I have got beautiful crystal hits in SaltRx screens which are not > diffracting to a good resoultion. All of them are salt based condition and >

Re: [ccp4bb] Protein toxicity or low expression

Hi Firdous,, As you mentioned they are many things to change and try. before changing vector it is more efficient in my opinion to change expression conditions (temperature, amount of IPTG, length of culture), culture medium (regular ones against M9 or auto induction media) and expression strains

Re: [ccp4bb] the purification process of protein, used for crystallization

advantage to remove some large molecular weight contaminants ( usually DNA) and some aggregates that are annoying. These choices depend on your target of course and the level of abundance too. Best, Pascal Egea, PHD UCLA Geffen School of Medicine On Fri, Nov 17, 2017 at 5:46 AM Liuqing Chen <519

[ccp4bb] MR phasing using Negative Stain EM reconstruction

Dear All, I would like to know if it is possible to use a low resolution EM reconstruction of a complex obtained in negative stain EM (not cryo EM) to help molecular replacement in a 4.5A resolution X-ray diffraction data set of the same complex I am aware of the possibility of using low resolutio

[ccp4bb] fluorescence question

’ fluorescence that interferes severely with our measurements (we are using tetramethyl rhodamine as fluorescent reporter). I was curious to know if anyone else had encountered this problem and figured out a solution. Any suggestion will be greatly appreciated. Many thanks in advance. Pascal Egea Assistant

Re: [ccp4bb] Zinc binding protein expressed from insect cells

ve managed to purify some protein I would try to do some emission spectroscopy to see what ions are bound (zinc and or iron ) you may be surprised by what you will see. sorry for the lengthy response but I hope this helps. all the best, Pascal Egea On Fri, Aug 15, 2014 at 8:03 AM, Harvey

Re: [ccp4bb] Difficult MR with MBP fusion protein

going on in your crystals. Good luck Pascal Egea On Fri, May 16, 2014 at 8:03 AM, Niu Tou wrote: > Dear All, > > Recently we collected some data of a MBP fusion protein, at around 4A > resolution. The protein itself is about half of the MBP size. However when > we tried to solve

Re: [ccp4bb] off-topic: bug busting

with this since my post-doc Hope this helps. Best regards, Pascal Egea On Tue, Feb 4, 2014 at 8:49 AM, Phoebe A. Rice wrote: > Some time ago, there was a nice discussion of cost-effective, wimpy > protein-friendly ways to break open E. coli. We're thinking about > replacing an a

[ccp4bb] membrane protein and phase separation

Dear All, I have a question tailored for the membrane protein and detergent folks. We are purifying a membrane protein that associates into an homoligomeric pore and we have been successfully preparing it in two detergents: FC-12 or a mild lipid. The two Protein Detergent Complexes look very homog

[ccp4bb] post-doctoral in membrane protein structural biology

We are seeking a post-doctoral researcher to join my research group at UCLA. The lab focuses on the structural studies of challenging targets: membrane protein complexes and channels from eukaryotes (yeast and several parasites) and seeks to characterize their architecture and molecular mechani

Re: [ccp4bb] Membrane Protein Optimisation

Hi Rhys, I suspect that what you call a gel might be phase separation (correct me if this is wrong) like an oily phase enriched in protein and detergent. you may have too much detergent in your drop. may I ask what detergent you are using (low or high CMC) and at what concentration of detergent do

Re: [ccp4bb] gelification of a pure protein

Thanks to All for the diligent answers to my query, The protein is not thermophilic and has only one cysteine. We are working in presence of freshly added reducing agent and glycerol to promote solubility (well kinda it seems). This is not an RNA or DNA binding protein and it has no low-complexity

[ccp4bb] gelification of a pure protein

Dear All, I am presently faced with a peculiar case in the lab. We are expressing a protein in E. coli and we are able to express it as a fusion protein without problems . Fusion cleavage goes well and the final product looks homogenous by size-exclusion chromatography with the expected molecular

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

Hi Raji, Thrombin is a rather good protease and behaves well in a large set of different detergents ( there is a paper by Michael Wiener that describes the relative efficiencies of several usual proteases, amongst those thrombin is inlcuded, used routinely for cleavage of membrane protein fusions

Re: [ccp4bb] Off-topic: heterologous co-expression in yeast

Hi Andre, There is set of plasmids allowing coexpression in yeast they are described in the following article Constructionand characterization of bidirectional expression vectors in Saccharomyces cerevisiae Aimin Li1,2, Zengshan Liu1, Qianxue Li2, Lu Yu1, Dacheng Wang2 & Xuming Deng 1,2 in FE

Re: [ccp4bb] 24 well screw-cap crystallization plates

Hi Brad, I am afraid that there is no alternate source for these plates. The screw cap system , I believe, was patented by the canadian company NEXTAL that was then assimilated by Q...N and the patent is probably still holding. Pascal -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geff

[ccp4bb] recombinant sources of N-glycanase

Hi All, We are trying to deal with a eukaryotic membrane protein that crystallizes but, as usual, diffracts poorly so far. As it is heavily glycosylated, we are considering enzymatic deglycosylation. Does anyone know about a recombinant source of N-glycanase ; we would like to prepare it ourselves

[ccp4bb] Subject: [ccp4bb] Soaking Kinase Crystals with ATP analogues

Hi , To add to the previous comments, crystallization of GTP or ATP (or their analogues) with their kinase/ A- or G-tpases can depend on a lot of factors that were mentioned (such as packing). A simple common problem is that ATP solutions should be carefully buffered prior to their use for soakin

Re: [ccp4bb] Nanodrop versus Nanophotomter Pearl versus good old Bradford.

I would like to add something about the NanoDrop versus NanoPearl, I don't think that the path length is fixed on this instrument (the NanoDrop) since if I recall well, the instruments sets the path length as it scans through the droplet, hence the characteristic clicky noise that you hear as the

Re: [ccp4bb] GTP Agarose Resin

Hi Matthew, Most GTPases required Magnesium to hydrolyze so you maybe able to reduce this by working in presence of EDTA and absence of magnesium. This may promote removal of traces of GTP or GDP ( from previous experience with SRP GTPases I would be more worried about residual GDP). Having EDTA a

[ccp4bb] expression of membrane proteins as GST fusions

Dear All, This is not strictly a crystallography related question. We are trying to express several membrane proteins and were considering the use of GST as a fusion partner instead of the HIS or FLAG purification tags. I would like to know if anyone had any experience (positive or negative) to sh

Re: [ccp4bb] Hydrophobic protein surface and SDS page

Hi Debajyoti, Migration of proteins in SDS containing gels is dependent on hydrophobicity, the amount of SDS that your protein binds (despite the fact that in theory all proteins should behave the same way under these conditions) and charge. Highly basic or acidic proteins will migrate anomalously

Re: [ccp4bb] crystallization of a weird protein

Hi Wei, this milk is precipitated/microcrystals of SDS probably with some phosphate since you are in PBS buffer. I would have two suggestions. 1-Can't you find a better detergent than SDS for your membrane protein? Have you run a detergent screen for this protein to find a milder and more crystalli

Re: [ccp4bb] highly glycosylated protein

Hi Wei, Glycosylation usually stabilize proteins although it is a source of structural heterogeneity for us crystallographers.Since you are expressing in HEK293 cells, there is a strain of cells that is deficient for glycosylation (it was designed by Gobind Khorana at the MIT I believe). You may w

Re: [ccp4bb] reproducibility of protein crystals

Anita, Proteolysis and oxydation are the most common alteration affecting proteins during the course of crystallization. If you have drops of the trays that yielded crystals I would run a gel on those drops and look at the aspect of protein still around in the drop. That would give you some clues.

[ccp4bb] Protease inhibitor cocktails and protein crystallization.

Dear All, I apologize if the questions has already been asked on this forum. We are purifying a membrane that seems prone to proteolysis. Although we use Protease Inhibitor cocktails during lysis and the first step of purification we get rid of them after and only keep PMSF and EDTA as general ant

Re: [ccp4bb] off topic: GPCR membane insertion/orientation

Hi Justin, Since GPCRs are polytopic a-helical transmembrane proteins, it is very likely that (1) insertion into the membrane is primarily performed by the Sec61 complex AKA translocon and (2) targeting to the membrane would be controlled by the signal recongition particle and its receptor. the la

Re: [ccp4bb] Lysis of Pichia pastoris

Hi Cory, I am afraid to say that Pichia and Saccharomyces are tough cells to break compared to bacteria. We also purifiy membrane proteins in yeast in my lab so we have gone through this process. With regular yeast we can break in an emulsiflex but it takes some hard work and this is not really via

Re: [ccp4bb] Confirming expression of a GPCR in HEK293

Hi Qing, I would recommend either the use of GFP as mentioned by Jacob or a his-tag or a flag-tag. C-terminal tagging is preferred to prevent interference with signal sequences at the N-terminus of the protein. Flag tag is really good for detection , the commercial antibodies for detection are rea

Re: [ccp4bb] how to optimize crystallization of a membrane proteinf

Hi Qiangmin, All the comments and references that were already mentioned to you are excellent, I would stress 3 points. 1- The detergent. A clear distinction should be made between the detergent used for extraction/solubilization and the detergent (or cocktail of detergents/lipids) used for cryst

Re: [ccp4bb] problem in annealing

Hi Hussain, I think you need to edit your input file and increase the max number of chain and the max number of tree . I had this problem several times especially if the structure is big or the chain(s) is(are) fragmented. The default values in CNS usually work but you can increase them and it sho

Re: [ccp4bb] SAXS on a coiled coil protein

Hi Rongjin, During the SAXS experiment, have you noticed X-ray induced damage on your samples that could explain this? If you are characterizing your protein in solution you may also be able to pinpoint conformational flexibility using other techniques. - analytical sedimentation. since you have

[ccp4bb] metal-chelating affinity chromatography and FosCholine detergents

Dear All, I apologize for the not strictly crystallography-related query. I am currently purifying several membrane proteins solubilized in fos-cholines detergents and I consistently observe a significant loss of protein at the binding step (done in absence of imidazole). Has anyone else experienc

Re: [ccp4bb] question - GFP fusion - cleavage sites

Hi Celina, I cannot answer to your question concerning the GFP-related problem. Fot the thrombin vs TEV protease related question I can tell you that in my hands thrombin works really very well in most detergents (TEV is somehow more sensitive). I am working on membrane proteins purified in very d

[ccp4bb] continuous flow centrifuge

Dear All, Sorry for the not strictly crystallography-related question. We are currently setting up a fermentation core and are considering purchasing a T1 Sharples continuous flow centrifuge. It is a mid-range capacity instrument. We will be processing bacteria and yeast. I was wondering if anyon

Re: [ccp4bb] UV microscope for screening

Dear Scott, We had a UV inspection microscope from KORIMA to look at crystals by UV fluorescence. It works relatively well but it is expensive ( way too much in my opinion), you pay for the microscope, the UV source and the software (named Wasabi) that comes with it An alternative to that is descr

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

Hi Nick, Some success has been reported for large soluble proteins using the C41(DE3) and C43(DE3) *E. coli* strains (see the paper by Miroux & Walker). Also you can try another promoter/expression system , the pBAD expression system based on arabinose induction. Hope this helps. Best -- Pasca

[ccp4bb] Post Doctoral Position at UCLA. Membrane Protein Complexes.

*Post-Doctoral Researcher. Structure and Function of Membrane Protein Complexes*. We seek a motivated individual to join our laboratory in the Department of Biological Chemistry at the University of California in Los Angeles. We are interested in studying the mechanisms of protein folding/transloc

Re: [ccp4bb] off topic, design of a self-cleaving tag

Hi Daniel, look at this the Profinity eXact Fusion-tag system from BioRad* * the protease i fused to your protein and self activated by halides (F or I I think). Cleavage in on column. The principles is clever, now the cleavage conditions may not suit to your protein, but it seems to work. This i

Re: [ccp4bb] small molecule library

I can suggest the following reading Discovering novel ligands for macromolecules uisng X-ray crystallographic screening Nienaber VL et al, Nature Biotechnology vol 18 october 2000 pp1105 -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Ch

Re: [ccp4bb] Phase separation to crystals

Hi James, I would first ask you what is your crystallization condition? You mentioned MPD at 70%. Very high concentration (and this is very high) of organic non volatile alcohols like MPD or polymers like PEGs will promote phase separation if there is some salt around. The higher the polymer concen

Re: [ccp4bb] lysozyme

Hi Camille, I don't know if you have any protein labs around you but if someone is using rosetta or codon-plus type expression Ecoli strains those cells usually contain a plysS plasmid derivative that is chloramphenicol resistant and carries the gene encoding lysozyme among other things (plus the r

Re: [ccp4bb] Adding a transmembrane segment

Hi,if you add the TM segment, I would expect that the protein will then get targeted to the membrane and hopefully inserted correctly. My guess is that you will have to treat your protein as a membrane protein or a membrane-anchored protein...which means prepare a membrane fraction, use detergent a

Re: [ccp4bb] Cryoprotection in > 3M ammonium sulfate

Hi Brenda, You can try sugars like glucose, trehalose and sucrose for high AS contents. It has been succesfully used in really hard cases such at protein RNA crystals grown in AS. see Acta Cryst (2002) D58 1664-1669 Garber et al. HTH Pascal Egea

Re: [ccp4bb] DNA binding protein

with subsequent crystallization trials in presence of the bona-fide target DNA sequence. 4-Precipitation of the DNA with streptomycine sulfate. You could also loss some protein. 1 and 2 are in my opinion the less invasive soutions. Hope this helps. Best regards Pascal Egea

[ccp4bb] Electron Density Maps

between those programs? Can differences in the way to perform bulk solvent correction account for those differences? Thanks in advance for your suggestions. Pascal Egea

Re: [ccp4bb] detergent crystals

microscopes are getting more and more popular so maybe you have one next by. It is very convenient. Hope this helps Pascal Egea, PhD University of California Los Angeles Department of Biological Chemistry

Re: [ccp4bb] citrate blocks the active site

and also the magic carboxylate malonate; a little bit like the Hofmeister series. You maybe able to find a surrogate to citrate that will able you to either soak your crystals or co-crystallize successfully with your substrate(s). Hope this helps Cheers, Pascal Egea University of California San

Re: [ccp4bb] Tips on fitting poorly defined loop regions

Hi Drew, Have you tried arp/wARP (the LOOPY option) or the AutoBuild option in Phenix ? If you haven't tried this you can try a complete rebuilding using you model as it stands and providing the complete sequence of your protein. At this resolution (2.1A) , they may be able to rescue your loops at

Re: [ccp4bb] Acrylamide in RNA crystallization

type column) to try to clean it up. If you have an NMR spectroscopist friend around, try to look at the presence of acrylamide before and after these steps and see what works the best for you. Hope this helps Pascal Egea, PhD University of California San Francisco Department of Biochemistry and

Re: [ccp4bb] OT: Crystallisation compatible detergents

Hi Darren I believe that the most frequently used detergent for protein crystallization (not including membrane proteins) is octyl-glucoside. The most important parameter is the CMC of the detergent and the size of the micelles of free detergent if you have micelles around. These considerations do

Re: [ccp4bb] precipitation of deglycosylated protein

poor defined. Pascal Egea

Re: [ccp4bb] precipitation of deglycosylated protein

Hi Simon, Although they are a source of heterogeneity for crystallization, glycosylations usually stabilize proteins. There are a couple of things that may be important to consider before you deglycosylate your protein. Do you know how many natural sequons/glycosylation sites your protein has? And

Re: [ccp4bb] Purification with ligand

er, keep also in mind that you will contaminate your columns and it can be hard to get rid of some ligands sometimes. Sorry, if all this was a little bit too long. Hope this helps, Pascal Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics Laboratory of Rob

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

of another expression plasmid encoding two " chaperones ", the trigger factor and GroES/EL. This is worth trying too. Hope this helps, cheers. Pascal Egea, PhD University of California San Francisco Department of Biochemistry and Biophysics Stroud Lab

Re: [ccp4bb] Se oxidation

Biol Crystallogr. 2001 Sep;57(Pt 9):1337-40. Epub 2001 Aug 23. Cheers, Pascal Egea, PhD Post Doctoral Researcher UCSF Department of Biochemistry and Biophysics Stroud laboratory On Mon, Feb 9, 2009 at 10:27 AM, aka akaka wrote: > Dear All > I would like to know whether oxidation

Re: [ccp4bb] Visualization system for a Mosquito Robot

those, one at room temperature and one in the cold. They seem to work OK. I said OK not great so you can consider this type of system for your setup, depending on what level of high-throughput you are willing or able to reach. Hope this helps Best Pascal Egea Post Doctoral Fellow Robert