Re: [ccp4bb] Total occupancy of two conformations of one nucleotide is over 1.0? Need help!

Dear Wei, remember that you might have "something else" in the location occupied by each alternate conformation when it is not occupied by that particular conformation. If you only model two alternate conformations you are saying something like that space A is occupied 50% of the time by this A

Re: [ccp4bb] ample

Dear Patrick, Apologies for a partly non-CCP4 answer! You can use the HHPRED alignments in some of the software available in Phenix. One option is to provide the .hhr file to MRage, which will fetch the PDB entries, run Sculptor to modify the templates with one or more protocols, based on

Re: [ccp4bb] AW: [ccp4bb] high Rfree

Hi there AMPLE works very well for coiled-coil structures, irrespective of parallel/anti-parallel etc http://journals.iucr.org/m/issues/2015/02/00/lz5005/ Resolution permitting, SHELXE tracing and phase modification, built into the AMPLE pipeline, gives statistics that differentiate very

Re: [ccp4bb] high Rfree

Have you looked for other possible models in the PDB? Eleanor On 21 July 2017 at 02:16, 张士军 <21620150150...@stu.xmu.edu.cn> wrote: > Hi everyone > >I have got a anti-parallel coiled-coil structure in a short fragment > recently, then I want to solve a longer fragment structure with phenix-MR

Re: [ccp4bb] ample

Dear Pat Yes, AMPLE does it's own, purely structure-based, alignment of the homologs provided using GESAMT. You provide a directory of structures to work with and, crucially, give AMPLE the -homologs True flag so that it knows the inputs have different sequences (unlike in modelling mode),

[ccp4bb] AW: [ccp4bb] high Rfree

Hi ???, Coiled coil structures can be very tricky with MR, so your solution may not be correct. You could try to split your search model and run MR with the separate coils. If you have high enough resolution (> ~2.3 Å?) and your MR solution is basically correct, you may be able to solve your

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

You might also be getting aggregation. If you do an old-fashioned EMSA ("gel shift") assay, does it hang up in the well? ++ Phoebe A. Rice Dept. of Biochemistry & Molecular Biology The University of Chicago pr...@uchicago.edu

[ccp4bb] AW: AW: [ccp4bb] high Rfree

Hi ???, I would first try AMPLE, as was suggested by Daniel. If separate helices still give you an anti-parallel coiled coil, your structure may in fact be anti-parallel. Your problem is not that the structure has another parallelity than you would like, but that you cannot solve the MR

Re: [ccp4bb] Total occupancy of two conformations of one nucleotide is over 1.0? Need help!

Hi Wei, thanks for sharing the data (off-list). I did some detective work and yes, Clemens is correct: what you see is the effect of bulk-solvent. After adjusting the solvent contribution (mask) in regions occupied by altlocs A and B the positive density mostly disappears. Pavel On Fri, Jul 21,

[ccp4bb] PhD student position available in CBB Poznan, Poland

Dear All, I'd like to draw your attention to the opening for PhD student position at Center for Biocrystallographic Research (Poznan, Poland), to work on a combination of protein crystallography and supramolecular chemistry (project PI prof. Mariusz Jaskolski) More information at:

[ccp4bb] Off topic: Flourescence anisotropy measurement

Dear all, I am trying to measure the difference in polarization upon the binding of the DNA to my protein. I take 1-5 nM of Cy3-labelled DNA and add varying dilutions of my protein to it (100 microM to 1 nM). I do get a decrease in difference of polarization with decrease in protein

[ccp4bb] postdoc position in protein MS

On behalf of Clemens Steegborn please note this job offer: Dear colleagues, Sorry for the slightly off-topic posting, but since almost everyone here does use MS occasionally I consider it not too far off: We have a postdoc / senior postdoc position in protein MS requiring a rather experienced

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

Hello, What is your difference between the maximum and minimum value ? Have you try to change the probe concentration? Best, Didier Le 21 juil. 2017 15:34, "Mohammad Khan" a écrit : Dear all, I am trying to measure the difference in polarization upon the binding of

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

A few tips, some/all of which may be relevant. Or not… If you are in 96 or 384 well plates, they vary. We tried quite a few batches and brands before settling on one. Keep your salt concentration as low as possible. You should be able to measure Kd in a range of about 1nM to low uM. Outside

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

In addition to everyone else's suggestions (especially trying greater [probe]), I would look at optimizing the following: - Sufficient rxn volume. In the 384 well plates, I found that consistency between replicates improved with greater volume - Read height - Scaling Corbin Black, BSc MSc

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 your plates down (assuming you are using them) to remove any bubbles. We just had an undergrad read his anisotropy

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

FP is the ratio between two fluorescence measurements; if the fluorescence signal is too low, you will still get a ratio but it will be essentially noise. Try to perform the measurements at 10-50 nM DNA. If your binding affinity is in the low nM range, you may have to use other methods to

[ccp4bb] eBIC centre coordinator vacancy

Dear all, reminder that we have a vacancy at eBIC for a centre coordinator Full details here: http://www.diamond.ac.uk/Careers/Vacancies/All/061-17-CH.html Please do contact Peijun Zhang for informal discussions Best, Martin -- Martin A. Walsh, Diamond Light Source Ltd Diamond House, Harwell

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

With this type of behavior, one suspicion is that you didn’t let the reaction come to equilibrium prior to taking the measurement and the variability between time of mixing and measuring between individual replicates is introducing extra variability. Take a concentration at which you know there

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

>Ideally you should convert polarization to anisotropy. Simple enough – but >some referees can get picky… What is the argument for anisotropy being better? JPK

Re: [ccp4bb] Off topic: Flourescence anisotropy measurement

if one has multiple species , e.g. bound and free, with each a different relative brightness and anisotropy/polarisation, then the use ( and interpretation) of anisotropy is straight forward; just a sum weighted by molecular fraction and the relative brightness. For polarisation is far more