Re: [ccp4bb] Off topic - transformation problems

In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm Not quite. pGEX vectors carry a copy of lacI^q, resulting in very low leak expression from PTAC promoter. Definitely low

Re: [ccp4bb] Off topic - transformation problems

Thank you for your kind advices. In this case, pGEX4T3 vector also expressed inserts constitutively. http://www.biovisualtech.com/bvplasmid/pGEX-4T-3.htm I also thought that target protein may have tocicity on host strain. But, why pGEX4T3-target protein was not show the same phenomena. Now, we

Re: [ccp4bb] Off topic - transformation problems

I never used pET20b, but I found on the Internet that it expresses inserts constitutively. Since the cloned protease should be toxic to cells, its constitutive expression might prevent the formation of colonies. But there might be millions of other reasons. Why did you use pET20b? http://www

Re: [ccp4bb] Map Using Both Bijvoet and Dispersive Differences with Model Phases

I believe the OP was asking how to best make an "element density map" where the map value is proportional to the occupancy of not just any anomalous scatterer, but a specific element of interest. For example, suppose you have Zn and Ni in your protein, but you are not sure which atom is which. If

Re: [ccp4bb] How to evaluate Fourier transform ripples

I think it is important to remember here that although 2Fo-Fc maps can have "Fourier ripples" (AKA series termination errors) difference maps are "resistant" to them. This is because "leaving out" data in a map calculation is equivalent to setting the coefficient to zero, and for a difference map

[ccp4bb] Off topic - transformation problems

Hi, all Recently, some bactrial source enzyme(sialidase) was subcloned pET20b and pGEX4T3 vector. Then, these two construct were transformed to BL21(DE3) expression host. DNA sequencing results were accurate. In case of pGEX, many colony was formed and shown the high level of expression. B

Re: [ccp4bb] large R-Rfree difference in "final" structure

Hi Careina, in our lab we once had the problem, that the asymmetric unit contained 8 molecules, whereas 7 had only been modeled. Somehow the 8th monomer had evaded detection. So be careful not to miss density. Matthias On 7/13/2011 7:54 PM, Robbie Joosten wrote: Hi Careina, Assuming you

Re: [ccp4bb] large R-Rfree difference in "final" structure

In my experience, the most common cause of this is the "locally correct, globally wrong" issue. You could be in the wrong space group (i.e P21212 when you should be in P212121). Another possibility is missing some element of local symmetry. Is the packing in the unit cell appropriate or are y

Re: [ccp4bb] large R-Rfree difference in "final" structure

Hi Careina, Assuming you don't suffer from a very poor data parameter ratio that would lead to such a large R-free/R, you need to improve your refinement. If you have NCS you should use local NCS restraints. You could also try jelly-body restraints, although they may not work at your resolut

Re: [ccp4bb] large R-Rfree difference in "final" structure

Careina, I recommend to compare the quality of your data (Rmerge) to that of an average data set of same resolution. Do you have meaningful data to 2.3A resolution? Another possibility is the anisotropicity of your data. Try this server http://services.mbi.ucla.edu/anisoscale/, if the anisotropi

Re: [ccp4bb] large R-Rfree difference in "final" structure

Hi Careina, Your starting R/Rfree values seem just fine. The reason for the large disparity after refinement is because the weighting factor is way too large during the first rounds of refinement. For each round of refinement, have a few different runs which test weighting factors between 0.01

Re: [ccp4bb] large R-Rfree difference in "final" structure

What leads you to believe Rfree is too high? - without knowing your data/parameter ratio it's impossible to tell. If you look at our paper Acta Cryst. (1998). D54, 547-557 (see Fig 1), then calculate the ratio y = Rfree/Rwork: in this case it is 0.33/0.24 = 1.375. Now look at the curve labelled '

Re: [ccp4bb] large R-Rfree difference in "final" structure

Hi Careina, I just have a slight concern regarding my Rwork Rfree difference. I have a > structure that I have solved. I am reasonably content that it is complete > because it has refined well, it no longer has bad geometries and contacts > and all the rotamers, ramachandra, bond lengths etc are g

[ccp4bb] large R-Rfree difference in "final" structure

Dear ccp4 bulletin board I just have a slight concern regarding my Rwork Rfree difference. I have a structure that I have solved. I am reasonably content that it is complete because it has refined well, it no longer has bad geometries and contacts and all the rotamers, ramachandra, bond lengths

[ccp4bb] occupancy-refinement

Dear CCP4ers, While trying to refine a protein-ligand structure (reso=2.9A) I notice that the density (2Fo-Fc)for the ligand is discontinuous. I also notice that the density for the residues in the ligand binding pocket (LBP) is also very feeble. And when I refine the ligand occupancy the density

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

Dear All, Thank you so much. Because the data quality is not so good (P1 space group, Rmerge 0.19, redundancy 3.9). I would like to try all the methods one by one to see which is better for my case. Thanks again. On Wed, Jul 13, 2011 at 8:01 AM, Soisson, Stephen M < stephen_sois...@merck.com> wro

[ccp4bb] PhD and Postdoctoral Position / Helmholtz Centre for Infection Research

Applications are invited for a PhD and a postdoctoral position in Protein Crystallography and Biochemistry at the Helmholtz-Centre for Infection Research, Braunschweig, Germany. Topic: Structural characterization of protein complexes involved in the regulation of autophagy Methods: protein expr

[ccp4bb] Advanced Light Microscopy Facility at EMBL Heidelberg

Light microscopy and fluorescence techniques permit the analysis not only of individual proteins but also functional macromolecular complexes, which have increasingly come into the focus of modern structural biology. The Advanced Light Microscopy Facility (ALMF) at European Molecular Biology L

[ccp4bb] how to define coordinate number of Heavy atom and identity of attached atoms

  Dear all i have protein soaked in Dimethyltindibromide in the active site 2 cysteine and 1 aspartic acid by help of anomalous map i can see 2 Sn atoms in each subunit in AU there is difference in both subunits, Sub-A shows acetate near Sn and Sub-B show phosphate ion, even the position of one

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

Seconding David's suggestion, I have had this issue on several occasions and I would highly recommend using SHARP. In my experience, SHARP does a superior job handling this type of data. Best of luck- Steve From: CCP4 bulletin board [mailto:CCP4BB@JISCMAI

Re: [ccp4bb] Off topic - Streptactin Column

We have been using Strep-Tactin SuperFlow (high capacity) columns more than 100 times, even when run under preparative conditions with protein extracts from E. coli fermentations. Biotin binding to the engineered streptavidin is not irreversible but can be cured by extended washing. To check th

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

structure, and refined simultaneously, the phase information is combined naturally, without having to export part of the information through Hendrickson-Lattman coefficients.   I am very happy to see that phenix and phaser have adopted and are promoting using the SAD function. Although, I

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

Hi, Most of the answers are interpreting your question as follows, that you have a single Se-SAD data set that goes to 3.3A but only has anomalous signal to 3.8A. Is that correct? If so, then there are various ways to combine the MR and SAD phase information. As you might imagine, we like th

[ccp4bb] Postdoc position and R&D position at the MAX IV Laboratory

TWO POSITIONS AVAILABLE AT THE MAX IV LABORATORY POSTDOC POSITION (2-year position, application deadline September 1st 2011) A postdoc position focusing on studies of how to best take radiation damage into account when collecting data, in particular at microfocus beamlines. This project is in c

Re: [ccp4bb] how to combine the experimental phase and molecular replacement phase

Dear Jiamu, Try out MRSAD protocol of Auto-Rickshaw (http://www.embl-hamburg.de/Auto-Rickshaw). You can start this just by providing your selenomet data, starting model, sequence information and space group on the Auto-Rickshaw server. It would do substructure determination based on your