[ccp4bb] French Press valve replacement
Dear CCP4 users, I apologize for the non-crystallography question. Our French Press valve have just been broken and we are looking for a replacement (see link for valve's image): http://www.cgp.co.il/Documentary/Zarivach-Lab/Misc/i-rcxfFhp/0/M/French-Press-Valve-M.jpg Please email me or zarivach[at]bgu.ac.il in case someone has such a replacement valve. Thank you, Chen --- Chen Guttman The Zarivach laboratory for Macromolecular Crystallography Building 39, Room 009B Ben-Gurion University of the Negev POBox 653 Zip Code 84105 Beer-Sheva Israel http://lifeserv.bgu.ac.il/wb/zarivach Tel. +972-8-6479519 Fax. +972-8-6472970
Re: [ccp4bb] Crystal structure and NMR structure
It's not a protein, but here's another example: the PreQ1 Riboswitch. In this case the two methods revealed different structures, but the NMR group was able to determine that the differences were due to calcium in the crystallization condition. Crystal Structure: Nat Struct Mol Biol. 2009 Mar;16(3):343-4. Epub 2009 Feb 22. Cocrystal structure of a class I preQ1 riboswitch reveals a pseudoknot recognizing an essential hypermodified nucleobase. Klein DJ, Edwards TE, Ferré-D'Amaré AR. NMR Structure: Mol Cell. 2009 Mar 27;33(6):784-90. Epub 2009 Mar 12. Structural Insights into riboswitch control of the biosynthesis of queuosine, a modified nucleotide found in the anticodon of tRNA. Kang M, Peterson R, Feigon J. Erratum in Mol Cell. 2010 Aug 27;39(4):653-5. An explanation of the differences: J Am Chem Soc. 2011 Apr 13;133(14):5190-3. Epub 2011 Mar 16. Comparison of solution and crystal structures of preQ1 riboswitch reveals calcium-induced changes in conformation and dynamics. Zhang Q, Kang M, Peterson RD, Feigon J. Cheers, Mike - Original Message - From: "Vandu Murugan" To: CCP4BB@JISCMAIL.AC.UK Sent: Friday, May 20, 2011 11:34:31 PM GMT -08:00 US/Canada Pacific Subject: [ccp4bb] Crystal structure and NMR structure Dear all, I would like to get some information on proteins where there is conformation/structural change between the crystal structure and solution structure of the same protein. Do anybody came across such situations? Thanks in advance.. cheers, Vandu -- Michael C. Thompson Graduate Student Biochemistry & Molecular Biology Division Department of Chemistry & Biochemistry University of California, Los Angeles mi...@chem.ucla.edu
Re: [ccp4bb] crystal bent once open cover slip
Weikai, What you might be experiencing is a detergent effect, i.e., you are near a detergent-dependent crystallization boundary. We have been hit with this many times. Under vapor diffusion conditions, sitting or hanging drop, the protein-detergent complex crystallizes and the free/ bound detergent reaches an equilibrium, but when you open the well, the drop begins to dry out. Hence, the detergent concentration increases, which dissolves/cracks the crystals. We also found that this also happened when our stabilization/freezing buffers had too high a detergent concentration (lower didn't hurt nearly as badly). Anecdotally, we have experienced that too high detergent concentrations inhibited crystallization, perhaps by having too high a concentration of free detergent micelles, which may interfere with the crystallization of the protein-detergent complex (there is alway detergent exchange between the solvent and the crystal). The way we solved the problem is by setting up the crystals at lower initial detergent concentrations. Note that as the concentrations of salt and PEG increases (like during crystallization), the detergent CMC decreases, even for non-ionic detergents. Therefore, by dropping the detergent concentration, often to just below the apparent CMC, we could grow nice stable crystals. Hope this helps, Michael R. Michael Garavito, Ph.D. Professor of Biochemistry & Molecular Biology 513 Biochemistry Bldg. Michigan State University East Lansing, MI 48824-1319 Office: (517) 355-9724 Lab: (517) 353-9125 FAX: (517) 353-9334Email: rmgarav...@gmail.com On May 24, 2011, at 1:19 PM, weikai wrote: Hi Folks, We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are long rods and grown under room temperature in a hanging drop set up. But once we open the cover slip, we see the rods start to break and bend in a few seconds. Since it is in high PEG400, we just directly freeze the crystals. The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar problem before and any suggestions? Thanks a lot, Weikai
Re: [ccp4bb] crystal bent once open cover slip
you could try sitting drops. Perhaps you can fish a crystal quicker, before it degrades. In sitting drops the crystals may also be further away from the drop surface and take longer to degrade. Or quickly add mineral oil to cover the sitting drop and fish the crystals through the oil. If this is still too slow, you could try microbatch under oil and you should be able to fish the crystals without previous exposure to air. You may need to adapt the mother liquor somewhat. The mineral oil may not be compatible with your membrane protein and detergent, but without trying you will never know. Mark J van Raaij Laboratorio M-4 Dpto de Estructura de Macromoleculas Centro Nacional de Biotecnologia - CSIC c/Darwin 3, Campus Cantoblanco E-28049 Madrid, Spain tel. (+34) 91 585 4616 http://www.cnb.csic.es/content/research/macromolecular/mvraaij On 24 May 2011, at 19:19, weikai wrote: > Hi Folks, > > We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na > Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are > long rods and grown under room temperature in a hanging drop set up. But > once we open the cover slip, we see the rods start to break and bend in a few > seconds. Since it is in high PEG400, we just directly freeze the crystals. > The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar > problem before and any suggestions? > > Thanks a lot, > > Weikai > >
Re: [ccp4bb] crystal bent once open cover slip
You probably use hanging drops. It's the surface tension effect. Check if sitting drops are better. Maia Sitting drops weikai wrote: Hi Folks, We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are long rods and grown under room temperature in a hanging drop set up. But once we open the cover slip, we see the rods start to break and bend in a few seconds. Since it is in high PEG400, we just directly freeze the crystals. The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar problem before and any suggestions? Thanks a lot, Weikai
[ccp4bb] crystal bent once open cover slip
Hi Folks, We have some membrane protein crystals that are grown in 30%PEG400, 0.1M Na Citrate pH 4.5, 0.1M LiCl. The protein is purified in DDM. The crystals are long rods and grown under room temperature in a hanging drop set up. But once we open the cover slip, we see the rods start to break and bend in a few seconds. Since it is in high PEG400, we just directly freeze the crystals. The diffraction only goes to 10 Ang at synchrotron. Have anybody had similar problem before and any suggestions? Thanks a lot, Weikai
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
On 5/24/2011 2:35 AM, herman.schreu...@sanofi-aventis.com wrote: Dear Clement, In case of a noisy experimental map, you have to do explicit solvent flattening. However, in case of molecular replacement, if the model occupies only say 30% of the asymmetric unit, the solvent where there is no model, will be flattened automatically. You can also view it like this: if 70% of the asymmetric unit is featureless solvent, the model at hand (=flat bulk solvent model), will be very accurate. I never really tested this, but in the cases where I had a very high solvent content, I was always surprised by the quality of the electron density maps. Off If you choose your contour level based on the map rms (often inappropriately called "sigma") the 2Fo-Fc density of a high-solvent-content map will appear stronger even when the absolute quality is the same. All that flat space will cause the overall rms to be low even if the rms calculated over the protein is the same. Dale Tronrud course, crystals with a high solvent content tend to diffract poorly and if the solvent is not featureless, this will not work either. If you get high Rfree values for a structure with high solvent content, I would get suspicious and look for extra molecule(s), which may have been overlooked. If these extra molecule(s) are disordered, this will off course lead to high Rfree values. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 11:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman
Re: [ccp4bb] Mosflm problem with CBF file
Hi Allan Luke left us nearly two years ago, so if you got that message you must be running a very old copy of iMosflm! The cure to your problem is to install the latest versions of iMosflm (1.0.5) and Mosflm (7.0.7). On 24 May 2011, at 16:51, Allan Pang wrote: > Dear everyone, > > I sent this email to lu...@mrc-lmb.cam.ac.uk but it appears that the email is > no longer active, so I'm hoping to get an input from you guys to troubleshoot > this problem. > > I tried adding (.CBF) images onto Mosflm, which I recently acquired from > Diamond. And this message immediately appears: > > > Error message: > > Mosflm has issued an badly formatted xml message. > An error log has been compiled in file: > /home/allan/.mosflm/comms_2011.05.24.1740 > > The developers would be extremely grateful if you could send this report to: > lu...@mrc-lmb.cam.ac.uk > > Thank you. > > > Please help! > > Thanks, > > Allan > > > > -- > Allan Pang > > PhD Student > > G35 Joseph Priestley Building > Queen Mary University of London > London > E1 4NS > > Phone number: 02078828480 Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, Cambridge, CB2 0QH http://www.iucr.org/resources/commissions/crystallographic-computing/schools/mieres2011
[ccp4bb] Mosflm problem with CBF file
Dear everyone, I sent this email to lu...@mrc-lmb.cam.ac.uk but it appears that the email is no longer active, so I'm hoping to get an input from you guys to troubleshoot this problem. I tried adding (.CBF) images onto Mosflm, which I recently acquired from Diamond. And this message immediately appears: Error message: Mosflm has issued an badly formatted xml message. An error log has been compiled in file: /home/allan/.mosflm/comms_2011.05.24.1740 The developers would be extremely grateful if you could send this report to: lu...@mrc-lmb.cam.ac.uk Thank you. Please help! Thanks, Allan -- Allan Pang PhD Student G35 Joseph Priestley Building Queen Mary University of London London E1 4NS Phone number: 02078828480
Re: [ccp4bb] Ubiquitin E2-E3 co-crystallization
Hi Manjeet, You are describing a rather complicated situation but you can try to sort it out in the following ways: 1. If you can make all the reagents i.e. your E3 ligase, all the putative E2s and E1, then you can do biochemical assays to figure out which E2's work. Boston Biochem sells E1 and a variety of E2's. The easiest assay would be a thio-ester release assay from E2. Basically you incubate E1 with E2, Ub and ATP to generate E2~S~Ub thioester intermediate which you can see on a non-reducing SDS-PAGE gel. Then you will have to block this reaction with NEM so that further charging of E2 is blocked. You can then test whether addition of your E3 and substrate results in the loss of Ub from the E2~S~Ub substrate. Please look into papers from Ray Deshaies lab to see how this is done. 2. Alternately you can test if any of the E2's make a stable complex with your E3. You can do this by a pull-down assay or you can incubate your E3 and the E2's and run them over a sizing column to see what makes a complex. 2. By Ub-E2-E3 complex do you mean a complex of E3 with a E2 which has a Ub linked to the E2 active site? If so, this has been done before by mutating the active site Cys of E2 to a Ser which substantially increases the stability of the E2~Ub ester bond. Regardless, you should always test this for your specific system. Try purifying the E2~Ub complex and then add the E3 and test if the complex is stable. It is perfectly possible that this will be a stable intermediate in the absence of substrate. I hope this helps. You can email me directly if you have further questions. Good luck! -Anirban On Tue, May 24, 2011 at 6:48 AM, manjeet mukherjee < manjeet.mukher...@gmail.com> wrote: > Hi all, > > I am working on a newly identified E3 ubiquitin ligase (RING type). I am > interested to co-crystallize it with E2 enzyme. Among the papers reported > so far, which are just a few, people have used a mixture of E2s including > UbcH1, UbcH5 and UbcH6 for the ubiquitination assays. However, there is no > clear evidence on E2 enzyme functions with this E3 ubiquitin ligase in vivo. > > > Can someone suggest how can I choose one particular E2 (among > UbcH1/UbcH5/UbcH6/UncH7...) for co-crystallization studies. It should be > noted that all these E2 enzymes have a similar fold, so will it make sense > to just randomly choose any one of these E2 enzymes and get started. > > Also, if someone with experience in this area suggest weather a Ub-E2-E3 > complex is stable enough and suitable for ternary complexes. > > Thanks in advance. > -Geet > -- Anirban Adhikari Postdoctoral Researcher Dept. of Biochemistry & Biophysics University of California San Francisco 600 16th Street Genentech Hall Room S414 San Francisco, CA 94158-2517 http://www.linkedin.com/in/anirbanadhikari
[ccp4bb] Ubiquitin E2-E3 co-crystallization
Hi all, I am working on a newly identified E3 ubiquitin ligase (RING type). I am interested to co-crystallize it with E2 enzyme. Among the papers reported so far, which are just a few, people have used a mixture of E2s including UbcH1, UbcH5 and UbcH6 for the ubiquitination assays. However, there is no clear evidence on E2 enzyme functions with this E3 ubiquitin ligase in vivo. Can someone suggest how can I choose one particular E2 (among UbcH1/UbcH5/UbcH6/UncH7...) for co-crystallization studies. It should be noted that all these E2 enzymes have a similar fold, so will it make sense to just randomly choose any one of these E2 enzymes and get started. Also, if someone with experience in this area suggest weather a Ub-E2-E3 complex is stable enough and suitable for ternary complexes. Thanks in advance. -Geet
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Dear Herman, You are right. Thank you for the explanation. Clement > Dear Clement, > > In case of a noisy experimental map, you have to do explicit solvent > flattening. However, in case of molecular replacement, if the model > occupies only say 30% of the asymmetric unit, the solvent where there is > no model, will be flattened automatically. You can also view it like > this: if 70% of the asymmetric unit is featureless solvent, the model at > hand (=flat bulk solvent model), will be very accurate. I never really > tested this, but in the cases where I had a very high solvent content, I > was always surprised by the quality of the electron density maps. Off > course, crystals with a high solvent content tend to diffract poorly and > if the solvent is not featureless, this will not work either. > > If you get high Rfree values for a structure with high solvent content, > I would get suspicious and look for extra molecule(s), which may have > been overlooked. If these extra molecule(s) are disordered, this will > off course lead to high Rfree values. > > Best, > Herman > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Clement Angkawidjaja > Sent: Tuesday, May 24, 2011 11:19 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] how to remove part of data with bad signal to > noise ratio > > But you have to do solvent flattening (density modification), which > people often (unintentionally?) skip for structures solved with > molecular replacement. Please correct me if I am wrong. > > Clement > > On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: > >> This is not my experience. Provided the solvent is featureless, I find > >> that a high solvent contents leads to a lower Rfree due to a kind of >> solvent flattening effect. Of course, if a significant part of the >> molecule(s) is/are disordered, this will lead to a degradation of the >> Rfree. >> >> My 2 cents, >> Herman >
Re: [ccp4bb] do we have to exclude Rfree columns when generating the real space density maps?
If you run coot with the FWT/PHWT columns from refmac, you are fine because refmac omits the observed F and substitutes a calculated F, giving a map which is rather less biased by omission of the free set while not contaminating the free set. The buccaneer/refmac pipeline does the same. On Tue, 24 May 2011 09:02:20 +0900, Keitaro Yamashita wrote: > Dear all, > > I'm very interested in this topic. > I have a question about the default behaviors in output reflection > files of each refinement softwares. > Are test set reflections excluded from the columns for calculating > electron density maps? > > I found in phenix.refine documentation the option > electron_density_maps.exclude_free_r_reflections was equal to False by > default. > (Does this option affect real space refinement in phenix.refine?) > > And I don't think Coot excludes test set reflections when opening MTZ > file... because there's no option to specify the flag number, right? > > Thanks in advance, > > Keitaro > > > 2011/5/24 Pavel Afonine : >> Hailiang, >> r-free reflections should not participate in >> refinement, regardless whether >> it is real or reciprocal space one, done with machine driven >> minimizers or >> your hands moving atoms in Coot. Period. The issue of correcting the >> map appearance for missing data (resolution or completeness) is >> relevant but >> different. Removing the data is noticeable, but most of the time putting >> aside test set is not critical for map appearance (given that >> reflections >> are selected randomly and do not exceed a reasonable fraction of >> available >> data); and when it is critical, the cross validation should be done >> differently anyway. >> So the answer to your question is: every time you compute a map not just >> to >> enjoy its appearance but to use it to improve your model, do not >> include >> test flagged reflections into it. >> Pavel. >> >> On Mon, May 23, 2011 at 1:02 PM, Hailiang Zhang wrote: >>> >>> Hi, >>> >>> I have a preliminary question. I understand Rfree reflection sets are >>> never used during automatic refinement, but, when generating the real >>> space density maps, do we have to exclude Rfree columns? Any references >>> will also be greatly appreciated! >>> >>> Best Regards, Hailiang >> >>
Re: [ccp4bb] problem with ccp4 6.1.13
Dear Seema Nath, Following that thread, Refmac5 is the name you gave to CCP4i to refer to the program. The important thing is which binary is linked to that name. Run a Refmac job and check the log. The version number is at the top. Cheers -- David On 24 May 2011 10:14, Seema Nath wrote: > Recently I've installed CCP4 6.1.13 (RHL4) to use refmac 5.5 twinning > refinement (as stated in the current version) but the program list shows > refmac5 instead of the current one. So I followed the thread- > > >Re: [ccp4bb] refmac 5.6 and the ccp4i task interface > > >Garib N Murshudov > >Wed, 29 Sep 2010 11:50:14 -0700 > > >Yes. There are two ways: > > >1) replace $CBIN/refmac5 with refmac5.6 > >2) on ccp4i click "System administration", select "configure interface" > and jus > >below > >"Give full path name for CCP4 programs to overcome name conflicts" click > "Add a > >program" > >there will appear two fields. On the right field type refmac5 and on the > left > >field actual address of the program. > > > >I hope it helps > >regards > >Garib > > On 29 Sep 2010, at 16:39, Ben Eisenbraun wrote: > > > Is it possible to use refmac 5.6 with the ccp4i Refmac5 task? > > > > Can I just copy the 5.6 binary over the 5.5 binary distributed with CCP4? > > It looks like the new refmac has a number of new keywords which wouldn't > be > > accessible doing this, but would it otherwise be compatible? If so, > where > > does the dictionary go? > > > > Is there some elegant way of letting users choose between the two? > > > > Thanks. > > > > -ben > > > > -- > > | Ben Eisenbraun | Software Sysadmin | > > | Structural Biology Grid | http://sbgrid.org | > > | Harvard Medical School | http://hms.harvard.edu | > > I followed both the option still there is refmac5 in place of the current > version. Please suggest possible solution of the problem. > Thanking you > > > > Regards, > Seema Nath >
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
I think you are just confused. The solvent flattening is just a step to make your map clearer. You do not carry the modified phases from solvent flattening to refinement (and I sincerely hope you don't refine against the solvent flattened amplitudes but against the original data!) Herman's observation is right, i think. One of the reasons that high solvent content will give better Rfree is also that this wY you have more reflections than usual per atom: considering two asymmetric units with the same volume, they have the same number of reflections at a given resolution, but if one has higher solvent it has less atoms so you refine better. A. Sent from my iPhone On 24 May 2011, at 11:18, Clement Angkawidjaja wrote: > But you have to do solvent flattening (density modification), which people > often (unintentionally?) skip for structures solved with molecular > replacement. Please correct me if I am wrong. > > Clement > > On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: > >> This is not my experience. Provided the solvent is featureless, I find >> that a high solvent contents leads to a lower Rfree due to a kind of >> solvent flattening effect. Of course, if a significant part of the >> molecule(s) is/are disordered, this will lead to a degradation of the >> Rfree. >> >> My 2 cents, >> Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
It's also possible that a lower Rfree is the result of reduced overfitting, because increased solvent content pushes up the obs/param ratio, i.e. the unit-cell volume is greater so you get more reflections for a given resolution cutoff, while the no of parameters stays about the same (i.e. there's a greater volume of solvent but you use the same no of parameters to describe it). Of course this assumes you get data to the same resolution with the increased solvent content, which may not be the case if there's increased thermal motion/disorder. Cheers -- Ian On Tue, May 24, 2011 at 10:01 AM, wrote: > This is not my experience. Provided the solvent is featureless, I find > that a high solvent contents leads to a lower Rfree due to a kind of > solvent flattening effect. Of course, if a significant part of the > molecule(s) is/are disordered, this will lead to a degradation of the > Rfree. > > My 2 cents, > Herman > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of > Clement Angkawidjaja > Sent: Tuesday, May 24, 2011 10:47 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] how to remove part of data with bad signal to > noise ratio > > Hi Seema, > > Small addition to the already abundant suggestions, if you have high > solvent content or significant portion of non-observable density, you > normally get higher R-free. > > Clement > > > Clement Angkawidjaja, PhD. > G30 Assistant Professor > > --- > Chemistry-Biology Combined Major Program International College, Osaka > University 1-30 Machikaneyama-cho Toyonaka, Osaka 560-0043, Japan > http://cmp.sci.osaka-u.ac.jp/CMP/ Tel. +81-6-6850-5952 Fax > +81-6-6850-5961 > > --- > Laboratory of Molecular Biotechnology > Graduate School of Engineering Osaka University > 2-1 Yamadaoka U1E-804 > Suita, Osaka 565-0871, japan > http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html > Tel/Fax +81-6-6879-4157 > >
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Dear Clement, In case of a noisy experimental map, you have to do explicit solvent flattening. However, in case of molecular replacement, if the model occupies only say 30% of the asymmetric unit, the solvent where there is no model, will be flattened automatically. You can also view it like this: if 70% of the asymmetric unit is featureless solvent, the model at hand (=flat bulk solvent model), will be very accurate. I never really tested this, but in the cases where I had a very high solvent content, I was always surprised by the quality of the electron density maps. Off course, crystals with a high solvent content tend to diffract poorly and if the solvent is not featureless, this will not work either. If you get high Rfree values for a structure with high solvent content, I would get suspicious and look for extra molecule(s), which may have been overlooked. If these extra molecule(s) are disordered, this will off course lead to high Rfree values. Best, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 11:19 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: > This is not my experience. Provided the solvent is featureless, I find > that a high solvent contents leads to a lower Rfree due to a kind of > solvent flattening effect. Of course, if a significant part of the > molecule(s) is/are disordered, this will lead to a degradation of the > Rfree. > > My 2 cents, > Herman
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
But you have to do solvent flattening (density modification), which people often (unintentionally?) skip for structures solved with molecular replacement. Please correct me if I am wrong. Clement On May 24, 2011, at 6:01 PM, herman.schreu...@sanofi-aventis.com wrote: > This is not my experience. Provided the solvent is featureless, I find > that a high solvent contents leads to a lower Rfree due to a kind of > solvent flattening effect. Of course, if a significant part of the > molecule(s) is/are disordered, this will lead to a degradation of the > Rfree. > > My 2 cents, > Herman
[ccp4bb] problem with ccp4 6.1.13
Recently I've installed CCP4 6.1.13 (RHL4) to use refmac 5.5 twinning refinement (as stated in the current version) but the program list shows refmac5 instead of the current one. So I followed the thread- >Re: [ccp4bb] refmac 5.6 and the ccp4i task interface >Garib N Murshudov >Wed, 29 Sep 2010 11:50:14 -0700 >Yes. There are two ways: >1) replace $CBIN/refmac5 with refmac5.6 >2) on ccp4i click "System administration", select "configure interface" and >jus >below >"Give full path name for CCP4 programs to overcome name conflicts" click "Add >a >program" >there will appear two fields. On the right field type refmac5 and on the left >field actual address of the program. >I hope it helps >regards >Garib On 29 Sep 2010, at 16:39, Ben Eisenbraun wrote: > Is it possible to use refmac 5.6 with the ccp4i Refmac5 task? > > Can I just copy the 5.6 binary over the 5.5 binary distributed with CCP4? > It looks like the new refmac has a number of new keywords which wouldn't be > accessible doing this, but would it otherwise be compatible? If so, where > does the dictionary go? > > Is there some elegant way of letting users choose between the two? > > Thanks. > > -ben > > -- > | Ben Eisenbraun | Software Sysadmin | > | Structural Biology Grid | http://sbgrid.org | > | Harvard Medical School | http://hms.harvard.edu | I followed both the option still there is refmac5 in place of the current version. Please suggest possible solution of the problem. Thanking you Regards, Seema Nath
Re: [ccp4bb] do we have to exclude Rfree columns when generating the real space density maps?
The default output for REFMAC Missing Data: For those reflections where the FP are missing, mFo is set equal to dFc. Hence the terms become FWT=dFC and DELFWT=0.0. the Rfree reflections are counted as "missing" hence there shouldnt be any bias intoroduced towards those Fobs assigned as free I dont think.. Of course all maps use the PhiCalc so there is inevitably bias towards to current model.. eleanor On 05/23/2011 11:03 PM, Pavel Afonine wrote: Hailiang, r-free reflections should not participate in refinement, regardless whether it is real or reciprocal space one, done with machine driven minimizers or your hands moving atoms in Coot. Period. The issue of correcting the map appearance for missing data (resolution or completeness) is relevant but different. Removing the data is noticeable, but most of the time putting aside test set is not critical for map appearance (given that reflections are selected randomly and do not exceed a reasonable fraction of available data); and when it is critical, the cross validation should be done differently anyway. So the answer to your question is: every time you compute a map not just to enjoy its appearance but to use it to improve your model, do not include test flagged reflections into it. Pavel. On Mon, May 23, 2011 at 1:02 PM, Hailiang Zhang wrote: Hi, I have a preliminary question. I understand Rfree reflection sets are never used during automatic refinement, but, when generating the real space density maps, do we have to exclude Rfree columns? Any references will also be greatly appreciated! Best Regards, Hailiang
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
This is not my experience. Provided the solvent is featureless, I find that a high solvent contents leads to a lower Rfree due to a kind of solvent flattening effect. Of course, if a significant part of the molecule(s) is/are disordered, this will lead to a degradation of the Rfree. My 2 cents, Herman -Original Message- From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Clement Angkawidjaja Sent: Tuesday, May 24, 2011 10:47 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] how to remove part of data with bad signal to noise ratio Hi Seema, Small addition to the already abundant suggestions, if you have high solvent content or significant portion of non-observable density, you normally get higher R-free. Clement Clement Angkawidjaja, PhD. G30 Assistant Professor --- Chemistry-Biology Combined Major Program International College, Osaka University 1-30 Machikaneyama-cho Toyonaka, Osaka 560-0043, Japan http://cmp.sci.osaka-u.ac.jp/CMP/ Tel. +81-6-6850-5952 Fax +81-6-6850-5961 --- Laboratory of Molecular Biotechnology Graduate School of Engineering Osaka University 2-1 Yamadaoka U1E-804 Suita, Osaka 565-0871, japan http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html Tel/Fax +81-6-6879-4157
Re: [ccp4bb] how to remove part of data with bad signal to noise ratio
Hi Seema, Small addition to the already abundant suggestions, if you have high solvent content or significant portion of non-observable density, you normally get higher R-free. Clement Clement Angkawidjaja, PhD. G30 Assistant Professor --- Chemistry-Biology Combined Major Program International College, Osaka University 1-30 Machikaneyama-cho Toyonaka, Osaka 560-0043, Japan http://cmp.sci.osaka-u.ac.jp/CMP/ Tel. +81-6-6850-5952 Fax +81-6-6850-5961 --- Laboratory of Molecular Biotechnology Graduate School of Engineering Osaka University 2-1 Yamadaoka U1E-804 Suita, Osaka 565-0871, japan http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html Tel/Fax +81-6-6879-4157
