Re: [ccp4bb] Against Method (R)

Hi Bart, I agree with the building strategy you propose, but at some point it stops helping and a bit more attention to detail is needed. Reciprocal space refinement doesn't seem to do the fine details. It always surprises me how much atoms still move when you real-space refine a refined model,

[ccp4bb] POST-DOCTORAL POSITION in STRUCTURAL BIOLOGY

Dear Friends: One post-doctoral position to work on structural biology project is available immediately at RIKEN SPring-8 Center, Harima Institute, Japan. For details, please click here : http://www.riken.go.jp/engn/r-world/info/recruit/k101029_s_rsc.html With regards, Kumarevel

[ccp4bb] Post-Doctoral Position in Structural Biology

Dear Friends: One post-doctoral position to work on structural biology project is available immediately at RIKEN SPring-8 Center, Harima Institute, Japan. With regards, Kumarevel **

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

Lake Wobegon!!! For those outside the US and/or otherwise not familiar with that small town, check out: http://en.wikipedia.org/wiki/Lake_Wobegon "Lake Wobegon, where all the women are strong, all the men are good looking, and all the children are above average" The best use of modern statisti

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

Zbyszek, Since you mention I/sigmaI in your PDF, do you mean or /? Do you mean I/sigmaI (in whatever rendition you choose) for the averaged unique reflections or the I/sigmaI for the observations? Also since one can adjust sigmaI in your scalepack program through the use of the Error Scale Fac

Re: [ccp4bb] Against Method (R)

On 10-10-28 04:09 PM, Ethan Merritt wrote: This I can answer based on experience. One can take the coordinates from a structure refined at near atomic resolution (~1.0A), including multiple conformations, partial occupancy waters, etc, and use it to calculate R factors against a lower resolutio

Re: [ccp4bb] Against Method (R)

You're second suggestion would be a good test because you are dealing with data from the same crystal and can thus assume the structures are identical (radiation damage excluded). So, take a highly diffracting crystal and collect a short-exposure low resolution data set and long exposure high r

Re: [ccp4bb] oligomer ligand building

Hi As I see you want to use link between ligands. You need to create this link description first. It can be done using JLigand that is available from: www.ysbl.york.ac.uk/mxstat/ There are tutorials how to create ligands and links. It should help you to create links We are updating at the mom

Re: [ccp4bb] Against Method (R)

Bart Hazes wrote > > There are many cases where people use a structure refined at high > resolution as a starting molecular replacement structure for a closely > related/same protein with a lower resolution data set and get substantially > better R statistics than you would expect for that re

Re: [ccp4bb] Against Method (R)

Yes, but even the high-resolution structures cannot explain THEIR data to within experimental error. You can see this if you download the CIF file for one of the highest-resolution structures there is: 2vb1 (triclinic lysozyme at 0.6 A), which contains both I and FC: http://www.rcsb.org/pdb/downlo

Re: [ccp4bb] Against Method (R)

So let's say I take a 0.6 Ang structure, artificially introduce noise into corresponding Fobs to make the resolution go down to 2 Ang, and refine using the 0.6 Ang model--do I actually get R's better than the artificially-inflated sigmas? Or let's say I experimentally decrease I/sigma by attenua

Re: [ccp4bb] oligomer ligand building

On 28/10/10 20:12, Changyi Xue wrote: Hi, all I am trying to build an oligomer ligand. I have obtained all the cif files for the monomers from HIC-UP. I tried to build several monomers in using coot. Then I modified the pdb file and stated the link in the pdb head (LINKR X ABC 1 Y AB

Re: [ccp4bb] Against Method (R)

There are many cases where people use a structure refined at high resolution as a starting molecular replacement structure for a closely related/same protein with a lower resolution data set and get substantially better R statistics than you would expect for that resolution. So one factor in th

Re: [ccp4bb] Babinet solvent correction [WAS: R-free flag problem]

On Thu, 28 Oct 2010 16:56:42 +0200 Dirk Kostrewa wrote: > > In the Babinet bulk solvent correction, no bulk solvent phases are > used, it is entirely based on amplitudes and strictly only valid if > the phases of the bulk solvent are opposite to the ones of the > protein. And as Sasha Urzhumtsev

Re: [ccp4bb] Help with Optimizing Crystals

Hi. Thanks for all of the helpful advice. Below is a summary of the suggestions, along with some things that I have tried and the results thus far. 1) Make sure that the crystals are protein and not salt. My crystals absorb Izit dye well and shooting some initial crystals did not produce any di

Re: [ccp4bb] oligomer ligand building

Hi Changyi, I'm not sure if this will answer your question, but it is likely that the cif file you are reading into Refmac is not in the correct format from Hic-Cup. I would suggest reading your coordinate file for the ligand into the Dundee server which will output a cif formats for refineme

[ccp4bb] oligomer ligand building

Hi, all I am trying to build an oligomer ligand. I have obtained all the cif files for the monomers from HIC-UP. I tried to build several monomers in using coot. Then I modified the pdb file and stated the link in the pdb head (LINKR X ABC 1 Y ABC 2X-Y). However, when using re

[ccp4bb] Free R with doubled cell edge

Dear BB Sages, I have a problem where I think I could very easily do the wrong thing. And I don't really want to do that... We have solved a new structure using zinc SAD phases (1 zinc in 27kD, 2 Zn/AU - Shelx, RESOLVE, ARPwARP. Cool.). In p21 30 109 65 90 105 90 at 2.5A However, we have now co

Re: [ccp4bb] Bug in c_truncate?

The GUI task has the option to run (c)truncate after f2mtz (if you have intensities in the input hkl file), and then uniqueify after that. I can reproduce this problem. ctruncate is losing the freeR column. At the moment, I don't know if this is a bug or a feature. As a work around, you can run c

Re: [ccp4bb] Against Method (R)

It is important to remember that if you have Gaussian-distributed errors and you plot error bars between +1 sigma and -1 sigma (where "sigma" is the rms error), then you expect the "right" curve to miss the error bars about 30% of the time. This is just a property of the Gaussian distribution: you

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

>2) when I submit this to referees will they think my structure is appropriate to draw these conclusions?". Particularly question 2) can rarely be answered w/o looking at electron density. Binding site details, ligand geometry, all have practically no effect on the Rules of Thumb. > It is whil

Re: [ccp4bb] Bug in c_truncate?

Why are you running [c]truncate? this is used to convert I -> F and I would be surprised if it recognised or preserved a FreeR column Phil On 28 Oct 2010, at 17:48, Peter Chan wrote: > Hello Tim, > > Thank you for the suggestion. I have now tagged the working set as "1" and > test set as "0".

Re: [ccp4bb] Bug in c_truncate?

Hello Tim, Thank you for the suggestion. I have now tagged the working set as "1" and test set as "0". Unfortunately, it still gives the same error about all Rfree being the same, and only in c-truncate but not old-truncate. Perhaps I should install 6.1.3 and see if the problem still persist.

Re: [ccp4bb] Against Method (R)

So I guess there is never a case in crystallography in which our models predict the data to within the errors of data collection? I guess the situation might be similar to fitting a Michaelis-Menten curve, in which the fitted line often misses the error bars of the individual points, but gets the o

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

THERMATOGA MARITIMA IscU IS A STRUCTURED IRON-SULFUR CLUSTER ASSEMBLY PROTEIN June 14, 2002 The Journal of Biological Chemistry, 277, 21397-21404. His-tagged Iron cluster that runs as a doublet. Mass-spec show they are the same species. They concluded "that the protein binds SDS in two stoichio

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

Dear Sebastian, Having personally purified upwards of 500 (I lost count really) of His-tagged proteins, I can't say that I have the same awareness as you with respect to the additional band being 'very common'. Depending on the kind of expression system, size of your protein, conditions of purific

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

Please let me clarify that it is by no means my intention to be rude to any referees, nor to round up alternative explanations for the extra band. The only thing I am after is a "proper" reference for the phenomenon of his-tagged proteins producing an extra band at slightly higher apparent molecula

Re: [ccp4bb] Babinet solvent correction [WAS: R-free flag problem]

Hi Tim, sorry for my late reply - I just came back to the lab. In the Babinet bulk solvent correction, no bulk solvent phases are used, it is entirely based on amplitudes and strictly only valid if the phases of the bulk solvent are opposite to the ones of the protein. And as Sasha Urzhumtsev

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

Hi Seb, I'm not aware of the notion (and neither are your reviewers apparently) that a His tag often results in two bands on a lane in SDS page. Why would that be? Extra SDS binding to the positive patch? Just wondering if there's any truth to your statement. Also, since in this case there seem

Re: [ccp4bb] Against Method (R)

Not quite. I was trying to say that for good small molecule data, R1 is usally significantly less than Rmerge, but never less than the precision of the experimental data measured by 0.5*/ = 0.5*Rsigma (or the very similar 0.5*Rpim). George Prof. George M. Sheldrick FRS Dept. Structural Chemistr

Re: [ccp4bb] Against Method (R)

In addition to bulk solvent, the other well recognized problem with macromolecular structures is the inadequate description of disorder. With small molecules, the Debye-Waller works much better because the harmonic oscillator is indeed a good model there. Note that the problem is not anisotropy (w

Re: [ccp4bb] Bug in c_truncate?

Hello Peter, I faintly rememeber a similar kind of problem, and think that if you replace "-1" with "0", the problem should go away. It seemed that "-1" is not an allowed flag for (some) ccp4 programs. Please let us know if this resolves the issue. Tim On Thu, Oct 28, 2010 at 10:21:20AM -0400,

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

It can sometimes be struggle to find the boundary between cynicism and pragmatism! I was, however, rather bemused by Dr Joosten's 7 rules of thumb - probably all of which I use and have seen used by referees. Of course I wouldn't want to blindly advocate any of them, however their use doe

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

Hi Sebastian, Under the assumption that the SDS in your assay does not completely unfold the protein during electrophoresis (chemical impurity can be excluded because of MS experiments, right?), how about adding some urea additionally to the SDS-PAGE (or changing SDS concentration)? GL Jan

Re: [ccp4bb] Bug in c_truncate?

Dear Crystallographers, Thank you all for the emails. Below are some details of the procedures I performed leading up to the problem. The reflection file is my own data, processed in XDS and then flagging FreeR's in XPREP in thin resolution shells. I am using CCP4i version 6.1.2. I tried l

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

Additional band on a gel might not be caused by his-tag. It is often a result of different conformation/molecular shape and so the molecule travels with different speed in the gel. We may wish for a homogenous sample (chemically and structurally) but this is seldom true See example: Int J

Re: [ccp4bb] Against Method (R)

So I guess a consequence of what you say is that since in cases where there is no solvent the R values are often better than the precision of the actual measurements (never true with macromolecular crystals involving solvent), perhaps our real problem might be modelling solvent? Alternatively/a

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

Dear Sebastiaan, isn't it the editor rather than the referees whom you have to convince? And if the editor does not even understand how SDS-PAGE works and still considers this a reason not to publish your article against your own expertise, maybe it is worth changing the journal. Finally, since r

Re: [ccp4bb] Bug in c_truncate?

Dear Peter, it seems to me that you are having trouble with f2mtz and not with ctruncate, so I am confused by the subject. Can you please post - the error message, - the first couple of lines of the hkl-file you are trying to import (including one or two reflections which are flagged for Rfre

[ccp4bb] Additional band on gel due to his-tag: any references?

Dear all, we have a his-tagged protein that shows a minor accompanying band in SDS-PAGE, just above the main band. According to all other methods available to us the material is homogeneous, the protein has the correct mass in MALDI-TOF, epitopes are recognized, etc. etc. I know that the addit

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

I do not know if that's really cynical: I've had the case of a referee recommending manuscript rejection because the title of the manuscript was not appropriate. The editor followed the advice of the referee. A proper refereeing job would have been to suggest that the authors change the title o

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

Oh cynic! Eleanor On 10/27/2010 09:01 PM, Simon Kolstoe wrote: Surely the "best" model is the one that the referees for your paper are happy with? I have found referees to impose seemingly random and arbitrary standards that sometime require a lot of effort to comply with but result in little

Re: [ccp4bb] Against Method (R)

It is instructive to look at what happens for small molecules where there is often no solvent to worry about. They are often refined using SHELXL, which does indeed print out the weighted R-value based on intensities (wR2), the conventional unweighted R-value R1 (based on F) and /, which it call

Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree) [SEC=UNCLASSIFIED]

I reckon you could share hypothetical review comments for educational purposes. -Original Message- From: CCP4 bulletin board on behalf of Bernhard Rupp (Hofkristallrat a.D.) Sent: Thu 10/28/2010 12:22 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst