[ccp4bb] DM and MR
Hello All, What is the current state of the art with respect to density modification after molecular replacement? In other words, what programs are people having the best luck with these days? Thanks in advance for any hints. James
Re: [ccp4bb] DM and MR
Hi James, I usually use DM in teh CCP4 suite to do DM. It gives you options to do solvent flattening, Histogram matching and NCS averaging. If you compare the FOM to the DMFOM in the mtz files, you can usually see and improvement in the score. That is usually all I bother my arse to look at. Any comments as to what else I should be looking at generally, would be welcomed. I am currently working on a difficult structure at 2.9 Angstrom that I cant get to lower the RFree any more and am stuck at 32% with a R fact of about 21%. I went back and performed density modification right at the start, then performed MR and the same refinement as before and saw absolutely no improvement in refinement statistics at all. They were identical using the non-DM modified mtz file. I was surprised, but this may be due more to the fact that I don't fully appreciate what is going on here. For the record I performed flattening and matching and tried this and then both plus NCS (there is NCS in my model) averaging and still absolutely identical refinement statistics. This is using refmac by the way TLS + restrained refinement. Sorry for the thread hijack James, but at least it is related! ;) cheers andy 2009/4/15 James Stroud xtald...@gmail.com: Hello All, What is the current state of the art with respect to density modification after molecular replacement? In other words, what programs are people having the best luck with these days? Thanks in advance for any hints. James
Re: [ccp4bb] DM and MR
Your split between Rfree and R suggests you are using refmac. (I also got a hint when you explicitly said you were using refmac.) Although there are many efficient ways to get stuck in a refinement, I have found the most effective by far is underweighting the structure factors and falling into the viscous cycle of model bias, which is why I have religiously avoided refmac in the past. Try upping the weighting term from the default of 0.3 and building into the maps that result. I got this latter advice recently from our x-ray admin who was also kind enough not to give me a forehead slap for my cluelessness. James On Apr 15, 2009, at 2:39 AM, ANDY DODDS wrote: Hi James, I usually use DM in teh CCP4 suite to do DM. It gives you options to do solvent flattening, Histogram matching and NCS averaging. If you compare the FOM to the DMFOM in the mtz files, you can usually see and improvement in the score. That is usually all I bother my arse to look at. Any comments as to what else I should be looking at generally, would be welcomed. I am currently working on a difficult structure at 2.9 Angstrom that I cant get to lower the RFree any more and am stuck at 32% with a R fact of about 21%. I went back and performed density modification right at the start, then performed MR and the same refinement as before and saw absolutely no improvement in refinement statistics at all. They were identical using the non-DM modified mtz file. I was surprised, but this may be due more to the fact that I don't fully appreciate what is going on here. For the record I performed flattening and matching and tried this and then both plus NCS (there is NCS in my model) averaging and still absolutely identical refinement statistics. This is using refmac by the way TLS + restrained refinement. Sorry for the thread hijack James, but at least it is related! ;) cheers andy 2009/4/15 James Stroud xtald...@gmail.com: Hello All, What is the current state of the art with respect to density modification after molecular replacement? In other words, what programs are people having the best luck with these days? Thanks in advance for any hints. James
Re: [ccp4bb] DM and MR
..and I may add that for DMMULTI two rather nonisomorphous data sets of the same crystal form can work like a dream, in particular at low resolution where solvent flattening and histogram matching can be tricky, see for example Morth et al. 2007, Nature 450, 1043 Pedersen et al. 2007, Nature 450, Poul On 15/04/2009, at 12.02, Pietro Roversi wrote: Dear James and Andy, my twopenny worth. I am using dm and dmmulti to solvent flatten after molecular replacement phases when no shred of experimental phases is available. If the model is severely incomplete (say at least 25% missing or more) I use phases from Buster-TNT refinement with missing atoms modelling switched on (this reduces the model bias imp[roves the scaling and helps bringing up weaker features from the yet unmodelled bits). I also blur the phase distribution either by multiplying the FOMs by a number less than one (say 0.6-0.8) or by doing the same to the Hendrickson-Lattmann coefficients (the idea is driectly inspired from this paper: Perrakis, A., Harkiolaki, M., Wilson, K. S. Lamzin, V. S. (2001). Acta Crystallogr D Biol Crystallogr 57, 1445-1450. I tend to try a few values of the multiplicative factor and choose the one that gives the maps that I like the best. Ah: it may be trivial again, but for the project I am working on at the moment even a 6.7 A dataset in a different crystal form has made a big difference! the dmmulti fourfold averaging across 2 crystal forms being much better than the twofold averaging in dm in the high-resolution form. Admittedly this extra crystal form has 90% solvent! So if possible at all use dmmulti and multi-crystal averaging. I hope this helps! Pietro -- Pietro Roversi EP Abraham Fellow in Biochemistry - Lincoln College - Oxford Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3RE, England UK Tel. 0044-1865-275385
Re: [ccp4bb] DM and MR
Jusht my two penneth... Any list of DM programs worth a punt after MR *musht* *shurely* include *resholve*? Resolve ± 'primeswitch' ± NCS pretty much always comes up with the goods for me. Dave 2009/4/15 James Stroud xtald...@gmail.com: Hello All, What is the current state of the art with respect to density modification after molecular replacement? In other words, what programs are people having the best luck with these days? Thanks in advance for any hints. James -- David C. Briggs PhD Father Crystallographer http://drdavidcbriggs.googlepages.com/home Skype: DocDCB
Re: [ccp4bb] DM and MR
Dear James and Andy, my twopenny worth. I am using dm and dmmulti to solvent flatten after molecular replacement phases when no shred of experimental phases is available. If the model is severely incomplete (say at least 25% missing or more) I use phases from Buster-TNT refinement with missing atoms modelling switched on (this reduces the model bias imp[roves the scaling and helps bringing up weaker features from the yet unmodelled bits). I also blur the phase distribution either by multiplying the FOMs by a number less than one (say 0.6-0.8) or by doing the same to the Hendrickson-Lattmann coefficients (the idea is driectly inspired from this paper: Perrakis, A., Harkiolaki, M., Wilson, K. S. Lamzin, V. S. (2001). Acta Crystallogr D Biol Crystallogr 57, 1445-1450. I tend to try a few values of the multiplicative factor and choose the one that gives the maps that I like the best. Ah: it may be trivial again, but for the project I am working on at the moment even a 6.7 A dataset in a different crystal form has made a big difference! the dmmulti fourfold averaging across 2 crystal forms being much better than the twofold averaging in dm in the high-resolution form. Admittedly this extra crystal form has 90% solvent! So if possible at all use dmmulti and multi-crystal averaging. I hope this helps! Pietro -- Pietro Roversi EP Abraham Fellow in Biochemistry - Lincoln College - Oxford Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3RE, England UK Tel. 0044-1865-275385
Re: [ccp4bb] DM and MR
I have to use refmac for licencing reasons, sadly, unless someone can recommend an entirely free refinement program to anyone including industry/people in limbo etc. Actually, I was just thinking. In the new MTZ files (which helpfully I dont have on me) there are new values with 'DM' added to them in the columns e.g. FOMDM instead of DM. If this applied to other labels in the MTZ, would I have to explicitly tell refmac to use them instead of the non-DM label columns? Probably. Blast me leaving the flash drive on my dresser. cheers Andy PS - re:weighting, I have messed about with them quite a bit reducing them to 0.1 seems to get the best results. I struggle onward.thanks for the replies so far. 2009/4/15 Poul Nissen p...@mb.au.dk: ..and I may add that for DMMULTI two rather nonisomorphous data sets of the same crystal form can work like a dream, in particular at low resolution where solvent flattening and histogram matching can be tricky, see for example Morth et al. 2007, Nature 450, 1043 Pedersen et al. 2007, Nature 450, Poul On 15/04/2009, at 12.02, Pietro Roversi wrote: Dear James and Andy, my twopenny worth. I am using dm and dmmulti to solvent flatten after molecular replacement phases when no shred of experimental phases is available. If the model is severely incomplete (say at least 25% missing or more) I use phases from Buster-TNT refinement with missing atoms modelling switched on (this reduces the model bias imp[roves the scaling and helps bringing up weaker features from the yet unmodelled bits). I also blur the phase distribution either by multiplying the FOMs by a number less than one (say 0.6-0.8) or by doing the same to the Hendrickson-Lattmann coefficients (the idea is driectly inspired from this paper: Perrakis, A., Harkiolaki, M., Wilson, K. S. Lamzin, V. S. (2001). Acta Crystallogr D Biol Crystallogr 57, 1445-1450. I tend to try a few values of the multiplicative factor and choose the one that gives the maps that I like the best. Ah: it may be trivial again, but for the project I am working on at the moment even a 6.7 A dataset in a different crystal form has made a big difference! the dmmulti fourfold averaging across 2 crystal forms being much better than the twofold averaging in dm in the high-resolution form. Admittedly this extra crystal form has 90% solvent! So if possible at all use dmmulti and multi-crystal averaging. I hope this helps! Pietro -- Pietro Roversi EP Abraham Fellow in Biochemistry - Lincoln College - Oxford Sir William Dunn School of Pathology, Oxford University South Parks Road, Oxford OX1 3RE, England UK Tel. 0044-1865-275385
[ccp4bb] how to purify protein in its native form
Hi, All: Although it is off-topic, definitely I think I can get some help here because we crystallographers are dealing with protein purification almost every day. My protein was expressed in E.coli as a soluble octamer with or without his-tag revealed by gel-filtration. For the his-tagged protein, the final product is an octamer after Ni-column and gel-fil. However, purification of the non-tagged protein results in a tetramer because of a partially denatured condition. When I tested the enzymatic activity , it turns out the tetramer was active although both of them can be crystallized. Now I am wonderring whether its native form in human is an octamer or tetramer. I am planning to purify a little protein from human cells to verify its native form. Can anybody recommend some protocols? Thanks a million, Jerry McCully _ Rediscover Hotmail®: Now available on your iPhone or BlackBerry http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Mobile1_042009
[ccp4bb] Calculation of angle between two helix of different subunits
Hello all I am interested to know about any programme which can calculate the angle between the helices of different subunit not the consecutive helices. I know about helixang which is a ccp4 supported programme but, i am not sure that does it calculate the relative angle between the two helix of different subunit in different orientation. Suggestiona would be appreciated. Thnaks in advance Peter
Re: [ccp4bb] how to purify protein in its native form
Hello Jerry, I would be glad to provide protocol suggestions, but it'd help to know a bit more regarding what you are working on. If you're planning to purify material from its native source (i.e. someone's chopped up sittin' muscle?) - this would be tremendously different from purifying protein from transfected CHO, HEK, etc. cells where you can expect reasonable abundance. Details are very important :) Artem Hi, All: Although it is off-topic, definitely I think I can get some help here because we crystallographers are dealing with protein purification almost every day. My protein was expressed in E.coli as a soluble octamer with or without his-tag revealed by gel-filtration. For the his-tagged protein, the final product is an octamer after Ni-column and gel-fil. However, purification of the non-tagged protein results in a tetramer because of a partially denatured condition. When I tested the enzymatic activity , it turns out the tetramer was active although both of them can be crystallized. Now I am wonderring whether its native form in human is an octamer or tetramer. I am planning to purify a little protein from human cells to verify its native form. Can anybody recommend some protocols? Thanks a million, Jerry McCully _ Rediscover Hotmail®: Now available on your iPhone or BlackBerry http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Mobile1_042009
[ccp4bb] Postdoctoral Fellow: Protein expression and X-ray structural analysis at ZMBP, Tuebingen
Posted on behalf of ZMPB: # There is an opening for a Postdoctoral Student (Protein expression and X-ray structural analysis) in the Department of Plant Biochemistry at the Center for Plant Molecular Biology (ZMBP), University of Tübingen. The Center comprises four depart-ments with a total of approximately 200 staff members. The successful can-didate is expected to establish a research pipeline on the expression of mi-crobial and plant proteins and their structural analysis by X-ray crystallogra-phy. The latter will be conducted in close collaboration with the X-ray crystal-lography unit at the Interfaculty Institute of Biochemistry (IFIB). This institu-tion offers excellent conditions for atomic-scale resolution of protein struc-tures (headed by Thilo Stehle). The position is vacant immediately and is limited initially to 2 years. Salary and social benefits are according to the German E13 TV-L. The Center pro-vides lab space with full equipment, funding for staff (1 PhD student, 1 tech-nician), and shared equipment funds. For more information see http://www.zmbp.uni-tuebingen.de Applications including CV, publication list and research program should be sent immediately to: Prof. Thorsten Nürnberger, Universität Tübingen, Center for Plant Molecular Biology (ZMBP), Auf der Morgenstelle 5, 72076 Tübingen nuernber...@uni-tuebingen.de # Best Regards, Georg -- Universität Tübingen Interfakultäres Institut für Biochemie Dr. Georg Zocher Hoppe-Seyler-Str. 4 72076 Tuebingen Germany Fon: +49(0)-7071-2973374 Mail: georg.zoc...@uni-tuebingen.de http://www.ifib.uni-tuebingen.de
Re: [ccp4bb] how to purify protein in its native form
The holy trinity of protein chromatography is ion exchange (IEX), hydrophobic interaction chromatography (HIC), and gel exclusion (GEC), assuming no affinity purification is possible. Depending on the abundance of protein, these three steps, perhaps in concert with additional chemical steps, including but not limited to salt precipitation and/or thermal denaturation, are often sufficient to purify proteins to homogeneity from natural sources. For minisculely abundant proteins, purification can be nightmarish. (Been there done that, got the dirty T-shirt.) The basis for any protein purification is an efficient assay for determining the presence of the protein in a mixture, even at low concentration. If you don't have a good assay (activity, antibody binding, spectroscopic ligand, etc., you are in dire straits indeed. But if you have an assay, you may not need to purify your protein at all to determine its oligomerization state in vivo. You may only need to pass a suitably concentrated crude extract containing your protein over a calibrated gel exclusion column. The emergence of the active fractions will provide an elution volume that will point you to the native MW, +/- 20% or so. The difference between a tetramer and an octamer (2X) should be cake using GEC. (Caveats apply: some proteins like to stick to GEC columns, but this can be suppressed with 100 mM NaCl, normally, but YMMV!) Cheers, -- Roger S. Rowlett Professor Colgate University Presidential Scholar Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@mail.colgate.edu Jerry McCully wrote: Hi, All: Although it is off-topic, definitely I think I can get some help here because we crystallographers are dealing with protein purification almost every day. My protein was expressed in E.coli as a soluble octamer with or without his-tag revealed by gel-filtration. For the his-tagged protein, the final product is an octamer after Ni-column and gel-fil. However, purification of the non-tagged protein results in a tetramer because of a partially denatured condition. When I tested the enzymatic activity , it turns out the tetramer was active although both of them can be crystallized. Now I am wonderring whether its native form in human is an octamer or tetramer. I am planning to purify a little protein from human cells to verify its native form. Can anybody recommend some protocols? Thanks a million, Jerry McCully Rediscover Hotmail: Now available on your iPhone or BlackBerry Check it out.
Re: [ccp4bb] how to purify protein in its native form
Yes,
However the even holier single method suitable for purification of
proteins from native sources is immuno-capture. If you have a good
antibody (i.e. an antibody that recognizes a native epitope) you can get
very high purity very quickly. If you don't have an antibody - you can get
polyclonals made very rapidly. Polyclonals are potentially even better for
immunocapture as some of them will recognize natively folded epitopes,
unlike monoclonals that may be raised against a peptide from deep inside
the protein.
Again, more details are needed :)
Artem
The holy trinity of protein chromatography is ion exchange (IEX),
hydrophobic interaction chromatography (HIC), and gel exclusion
(GEC), assuming no affinity purification is possible. Depending on
the abundance of protein, these three steps, perhaps in concert
with additional chemical steps, including but not limited to salt
precipitation and/or thermal denaturation, are often sufficient to
purify proteins to homogeneity from natural sources. For
minisculely abundant proteins, purification can be nightmarish.
(Been there done that, got the dirty T-shirt.)
The basis for any protein purification is an efficient assay for
determining the presence of the protein in a mixture, even at low
concentration. If you don't have a good assay (activity, antibody
binding, spectroscopic ligand, etc., you are in dire straits indeed. But
if you have an assay, you may not need to purify your protein at all to
determine its oligomerization state in vivo. You may only need to pass a
suitably concentrated crude extract containing your protein over a
calibrated gel exclusion column. The emergence of the active fractions
will provide an elution volume that will point you to the native MW, +/-
20% or so. The difference between a tetramer and an octamer (2X) should
be cake using GEC. (Caveats apply: some proteins like to stick to GEC
columns, but this can be suppressed with 100 mM NaCl, normally, but
YMMV!)
Cheers,
--
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@mail.colgate.edu
Jerry McCully wrote: .hmmessage P { margin:0px; padding:0px }
body.hmmessage { font-size: 10pt; font-family:Verdana } Hi, All:
Although it is off-topic, definitely I think I can get some help
here because we crystallographers are dealing with protein
purification almost every day.
My protein was expressed in E.coli as a soluble octamer with or
without his-tag revealed by gel-filtration. For the his-tagged
protein, the final product is an octamer after Ni-column and
gel-fil. However, purification of the non-tagged protein results in
a tetramer because of a partially denatured condition.
When I tested the enzymatic activity , it turns out the tetramer
was active although both of them can be crystallized. Now I am
wonderring whether its native form in human is an octamer or
tetramer.
I am planning to purify a little protein from human cells to verify
its native form.
Can anybody recommend some protocols?
Thanks a million,
Jerry McCully
Rediscover Hotmailreg;: Now available on your iPhone or BlackBerry
Check it out.
[ccp4bb] problem with cns compile
Ok, I have tried sending this to the cns board and it has been reject with my old email and new email addresses so I will try here and sorry for the non ccp4 question. While compiling CNS 1.21 on an older systems running Redhat Enterp. 4 the following error comes up. machine_f.o(.text+0x11ca): In function `vopen_': : undefined reference to `rename_' collect2: ld returned 1 exit status problems with new executable - old version retained This is a fresh install on this machine. Any ideas would be appreciated. Thanks, Leonard Thomas Ph. D. Macromolecular Crystallography Laboratory Manager University of Oklahoma Department of Chemistry and Biochemistry 620 Parrington Oval Norman, OK 73019 lmtho...@ou.edu http://barlywine.chem.ou.edu Office: 405-325-1126 Lab: 405-325-7571
Re: [ccp4bb] how to purify protein in its native form
Hi: I have seen some protocols(IMPACT from New England Lab etc.) which can overexpress protein with a tag then cut or displace the tag off to get a native protein. Maybe those are the options. Best, Hongnan Cao UCR _ MSN 表情魔法书，改变你的对话时代！ http://im.live.cn/emoticons/
