[ccp4bb] HTP ligand screening
Dear Crystallographers, I have ~30 data sets from ligand soaks of my protein of known structure (all approximately the same cell. Can anyone suggest a high throughput method which would do molecular replacement, refine, then output new blobs, perhaps as a water pdb file? I am sure they do this or better in pharma every day... Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Windows 7 and Xtal Software
Dear Crystallographers, are there any additional problems or known issues running ccp4 or other xtal software on windows 7 (beyond those of Vista, etc.?) Your input would be really appreciate before I sink my own personal $$$ into a new laptop Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Windows 7 and Xtal Software
Dear Crystallographers, once again I am filled with graditude to this list--there were many helpful responses and even some geek humor. I have decided to go with a dual-boot windows7/linux, which seems easy enough. All the best, and thanks everyone for your quick and helpful advice. Jacob Keller
[ccp4bb] No Cl- or S Anomalous Signal
Dear Crystallographers, I recently have been working with a 2.5 Ang SeMet peak wavelength dataset which contains 2 cys's and also a couple of bona fide Cl ions (reasonable b-factor/site is semi-buried/water does not work). In the FFT anomalous difference map using PhiC from the refined model and Dano, I can see the MSE's at ~10 sigma, but no Cl ions, even though Cl should have f" = ~0.3 versus Se's f" = ~4, and no S's in the cys, despite f" = 0.23e. There is really no anomalous peak at all--is it just the smallness of the signal, or are the Se's somehow "swamping out" the other signal? Perhaps the phases are tainted by the presence of semet in the model? Looking for suggestions, Jacob Keller *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] No Cl- or S Anomalous Signal
Update: I tried more anomalous maps, this time with the originally-deposited data at 1.8 Ang (mine were similar, substrate-soaked crystals) and phases from the refined model, and the Se sites are now ~40-50 sigma, and there is still totally nothing at the Cl and S sites, even though in 2Fo-Fc the Cl is ~9 sigma, and the S is 8 sigma (the Se is ~15 sigma). If it has reasonably-high electron density, shouldn't it have at least some anomalous scattering? I am wondering whether somehow the model phases are biasing the map, but I can't really imagine how that would be... JPK On Thu, Sep 1, 2011 at 3:15 PM, Bosch, Juergen wrote: > Where in refinement of your model are you ? > At an early stage I wouldn't be surprised to only see SeMets but once you've > refined your structure and go back to calculate an anomalous map with the > improved phases you might double your signal for SeMet and start seeing > sulfurs. > An alternative explanation, you've blasted your crystals at the synchrotron > and the remaining anomalous signal is too weak to show the sulfurs. > Just two thoughts, > Jürgen > On Sep 1, 2011, at 4:03 PM, Jacob Keller wrote: > > Dear Crystallographers, > > I recently have been working with a 2.5 Ang SeMet peak wavelength > dataset which contains 2 cys's and also a couple of bona fide Cl ions > (reasonable b-factor/site is semi-buried/water does not work). In the > FFT anomalous difference map using PhiC from the refined model and > Dano, I can see the MSE's at ~10 sigma, but no Cl ions, even though Cl > should have f" = ~0.3 versus Se's f" = ~4, and no S's in the cys, > despite f" = 0.23e. There is really no anomalous peak at all--is it > just the smallness of the signal, or are the Se's somehow "swamping > out" the other signal? Perhaps the phases are tainted by the presence > of semet in the model? > > Looking for suggestions, > > Jacob Keller > > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** > > .. > Jürgen Bosch > Johns Hopkins University > Bloomberg School of Public Health > Department of Biochemistry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Office: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-2926 > http://web.mac.com/bosch_lab/ > > > > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to "digest" PISA results
Well, I guess I have always been curious what is the gold standard here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a polydisperse sample with weak oligomerization, or SPR a very weak binding constant? Do we then revert to a functional assay? Or what if the functional assay does not show anything, but the binding constant is really strong? Or vice versa, the binding is completely undetectable, but the functional assay shows something? JPK On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans wrote: > I get confused by these figures. As I understand it the "interface area" > given in Pisa is half the loss of accessible area on forming the complex: is > that right? > > We're working on a complex with interface area ~500A^2, where the complex is > stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa > estimate of DelG -2.3. Does that sound sensible? > > Phil > > On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: > >> 720 is not an impressive size for a stable interface, but it may do >> depending on molecule size and exact chemistry of the interface (h-bonds, >> salt bridges, disulphides, charges etc etc). Everything is subject to >> chemical environment and concentration, as usual. For these entries, PISA >> gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol >> estimated (guessed) accuracy of PISA, this may or may not be a stable thing. >> And yes, it has about 70-80% chances to be simply an artefact of crystal >> packing, according to some sort of derivations that I did in 2nd PISA paper >> in J.Comp.Chem. in January last year. >> >> Having said all this, PISA is not an oracle and does not pretend to be >> correct in 100% of instances. >> >> Eugene. >> >> >> On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: >> >>> Like Jan, I find it very useful to sort out the clear cut cases. Otherwise >>> it is easy to get things wrong.. >>> >>> But isnt a buried surface area of 720 rather small for a stable interface? >>> If there is other confirming evidence like 2 diff space groups then you >>> feel more secure!! >>> >>> On 09/01/2011 02:27 PM, Yuri Pompeu wrote: This is regarding Ethan´s point, particularly: > 2) the protein has crystallized as a monomer even though it > [sometimes] exists in solution as a dimer. The interface > seen in the crystal is not the "real" dimer interface and > thus the PISA score is correct. I see the same exact interface in a crystal of a close homologue that belongs to a different space group (hexagonal vs tetragonal system) > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to "digest" PISA results
mea culpa! How about FRET? JPK On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen wrote: > Hi Jacob, > you forgot cross-linking to stabilize a weak complex and verify that it > exists. > Jürgen > On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: > > Well, I guess I have always been curious what is the gold standard > here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a > polydisperse sample with weak oligomerization, or SPR a very weak > binding constant? Do we then revert to a functional assay? Or what if > the functional assay does not show anything, but the binding constant > is really strong? Or vice versa, the binding is completely > undetectable, but the functional assay shows something? > > JPK > > On Mon, Sep 5, 2011 at 6:45 AM, Phil Evans wrote: > > I get confused by these figures. As I understand it the "interface area" > given in Pisa is half the loss of accessible area on forming the complex: is > that right? > > We're working on a complex with interface area ~500A^2, where the complex is > stable enough for gel filtration, and with a measured Kd of ~2microM, Pisa > estimate of DelG -2.3. Does that sound sensible? > > Phil > > On 5 Sep 2011, at 10:33, Eugene Krissinel wrote: > > 720 is not an impressive size for a stable interface, but it may do > depending on molecule size and exact chemistry of the interface (h-bonds, > salt bridges, disulphides, charges etc etc). Everything is subject to > chemical environment and concentration, as usual. For these entries, PISA > gives dissociation free energy of -1 kcal/mol. Given some +/- 5 kcal/mol > estimated (guessed) accuracy of PISA, this may or may not be a stable thing. > And yes, it has about 70-80% chances to be simply an artefact of crystal > packing, according to some sort of derivations that I did in 2nd PISA paper > in J.Comp.Chem. in January last year. > > Having said all this, PISA is not an oracle and does not pretend to be > correct in 100% of instances. > > Eugene. > > > On 5 Sep 2011, at 10:14, Eleanor Dodson wrote: > > Like Jan, I find it very useful to sort out the clear cut cases. Otherwise > it is easy to get things wrong.. > > But isnt a buried surface area of 720 rather small for a stable interface? > If there is other confirming evidence like 2 diff space groups then you > feel more secure!! > > On 09/01/2011 02:27 PM, Yuri Pompeu wrote: > > This is regarding Ethan´s point, particularly: > > 2) the protein has crystallized as a monomer even though it > > [sometimes] exists in solution as a dimer. The interface > > seen in the crystal is not the "real" dimer interface and > > thus the PISA score is correct. > > I see the same exact interface in a crystal of a close homologue that > belongs to a different space group (hexagonal vs tetragonal system) > > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** > > .. > Jürgen Bosch > Johns Hopkins University > Bloomberg School of Public Health > Department of Biochemistry & Molecular Biology > Johns Hopkins Malaria Research Institute > 615 North Wolfe Street, W8708 > Baltimore, MD 21205 > Office: +1-410-614-4742 > Lab: +1-410-614-4894 > Fax: +1-410-955-2926 > http://web.mac.com/bosch_lab/ > > > > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to "digest" PISA results
NMR...take that! JPK 2011/9/5 Andreas Förster : > AUC ! > > > Andreas > > > > On 05/09/2011 6:00, Jacob Keller wrote: >> >> mea culpa! How about FRET? >> >> JPK >> >> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen wrote: >>> >>> Hi Jacob, >>> you forgot cross-linking to stabilize a weak complex and verify that it >>> exists. >>> Jürgen >>> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: >>> >>> Well, I guess I have always been curious what is the gold standard >>> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a >>> polydisperse sample with weak oligomerization, or SPR a very weak >>> binding constant? Do we then revert to a functional assay? Or what if >>> the functional assay does not show anything, but the binding constant >>> is really strong? Or vice versa, the binding is completely >>> undetectable, but the functional assay shows something? >>> >>> JPK >>> > > > -- > Andreas Förster, Research Associate > Paul Freemont & Xiaodong Zhang Labs > Department of Biochemistry, Imperial College London > http://www.msf.bio.ic.ac.uk > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to "digest" PISA results
I did a similar assay years ago, but since the results were negative, never published anything--it was seeing whether nucleotides bound to my protein of interest by time courses of proteolysis +/- nucleotide. One tricky part of the assay, however, is to be sure that the compound of interest doesn't inhibit the protease--did you address that? I guess you would have to have some control proteins for that... Jacob g > > -- > Jing Huang, Ph.D. > Associate Professor > UCLA > Department of Molecular & Medical Pharmacology > 310-825-4329 > jinghuang at mednet dot ucla dot edu > http://labs.pharmacology.ucla.edu/huanglab/ > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller > [j-kell...@fsm.northwestern.edu] > Sent: Monday, September 05, 2011 10:10 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Trying to "digest" PISA results > > NMR...take that! > > JPK > > 2011/9/5 Andreas Förster : >> AUC ! >> >> >> Andreas >> >> >> >> On 05/09/2011 6:00, Jacob Keller wrote: >>> >>> mea culpa! How about FRET? >>> >>> JPK >>> >>> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen wrote: >>>> >>>> Hi Jacob, >>>> you forgot cross-linking to stabilize a weak complex and verify that it >>>> exists. >>>> Jürgen >>>> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: >>>> >>>> Well, I guess I have always been curious what is the gold standard >>>> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a >>>> polydisperse sample with weak oligomerization, or SPR a very weak >>>> binding constant? Do we then revert to a functional assay? Or what if >>>> the functional assay does not show anything, but the binding constant >>>> is really strong? Or vice versa, the binding is completely >>>> undetectable, but the functional assay shows something? >>>> >>>> JPK >>>> >> >> >> -- >> Andreas Förster, Research Associate >> Paul Freemont & Xiaodong Zhang Labs >> Department of Biochemistry, Imperial College London >> http://www.msf.bio.ic.ac.uk >> > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** > > IMPORTANT WARNING: This email (and any attachments) is only intended for the > use of the person or entity to which it is addressed, and may contain > information that is privileged and confidential. You, the recipient, are > obligated to maintain it in a safe, secure and confidential manner. > Unauthorized redisclosure or failure to maintain confidentiality may subject > you to federal and state penalties. If you are not the intended recipient, > please immediately notify us by return email, and delete this message from > your computer. > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Trying to "digest" PISA results
Good ol' trypsin--any reason why not? JPK On Mon, Sep 5, 2011 at 5:40 PM, Huang, Jing wrote: > Excellent point indeed. We always include (at least one, preferably multiple) > control proteins that are proteolysed equally across to make sure that the > observed "target stabilization" is not due to fortuitous protease inhibition. > > What protease did you use for your nucleotide binding case? The protease > sometimes matters. For example, thermolysin mainly only digests proteins that > are unfolded, whereas pronase, which is a mixture of various proteases, can > digest both folded and unfolded proteins. > > Best, > Jing > > -- > Jing Huang, Ph.D. > Associate Professor > UCLA > Department of Molecular & Medical Pharmacology > 310-825-4329 > jinghuang at mednet dot ucla dot edu > http://labs.pharmacology.ucla.edu/huanglab/ > > > From: Jacob Keller [j-kell...@fsm.northwestern.edu] > Sent: Monday, September 05, 2011 12:18 PM > To: Huang, Jing > Cc: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Trying to "digest" PISA results > > I did a similar assay years ago, but since the results were negative, > never published anything--it was seeing whether nucleotides bound to > my protein of interest by time courses of proteolysis +/- nucleotide. > One tricky part of the assay, however, is to be sure that the compound > of interest doesn't inhibit the protease--did you address that? I > guess you would have to have some control proteins for that... > > Jacob > > > g >> >> -- >> Jing Huang, Ph.D. >> Associate Professor >> UCLA >> Department of Molecular & Medical Pharmacology >> 310-825-4329 >> jinghuang at mednet dot ucla dot edu >> http://labs.pharmacology.ucla.edu/huanglab/ >> >> >> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob Keller >> [j-kell...@fsm.northwestern.edu] >> Sent: Monday, September 05, 2011 10:10 AM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] Trying to "digest" PISA results >> >> NMR...take that! >> >> JPK >> >> 2011/9/5 Andreas Förster : >>> AUC ! >>> >>> >>> Andreas >>> >>> >>> >>> On 05/09/2011 6:00, Jacob Keller wrote: >>>> >>>> mea culpa! How about FRET? >>>> >>>> JPK >>>> >>>> On Mon, Sep 5, 2011 at 11:39 AM, Bosch, Juergen wrote: >>>>> >>>>> Hi Jacob, >>>>> you forgot cross-linking to stabilize a weak complex and verify that it >>>>> exists. >>>>> Jürgen >>>>> On Sep 5, 2011, at 11:45 AM, Jacob Keller wrote: >>>>> >>>>> Well, I guess I have always been curious what is the gold standard >>>>> here--perhaps SEC, ITC, SPR, pulldowns? What if SEC shows a >>>>> polydisperse sample with weak oligomerization, or SPR a very weak >>>>> binding constant? Do we then revert to a functional assay? Or what if >>>>> the functional assay does not show anything, but the binding constant >>>>> is really strong? Or vice versa, the binding is completely >>>>> undetectable, but the functional assay shows something? >>>>> >>>>> JPK >>>>> >>> >>> >>> -- >>> Andreas Förster, Research Associate >>> Paul Freemont & Xiaodong Zhang Labs >>> Department of Biochemistry, Imperial College London >>> http://www.msf.bio.ic.ac.uk >>> >> >> >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> cel: 773.608.9185 >> email: j-kell...@northwestern.edu >> *** >> >> IMPORTANT WARNING: This email (and any attachments) is only intended for >> the use of the person or entity to which it is addressed, and may contain >> information that is privileged and confidential. You, the recipient, are >> obligated to maintain it in a safe, secure and confidential manner. >> Unauthorized redisclosure or failure to maintain confidentiality may subject >> you to federal and state penalties. If you are not the intended recipient, >> please immediately notify us by return email, and delete this message from >> your computer. >> > > > > -- > ***
[ccp4bb] converting mmcif to mtz failure
Dear Crystallographers, in trying to convert a mmcif to mtz, I get the logfile below. I looked in the directory, and there is a file cf_mm.dic, but this is presumably not the same as the similar .lib file. Any thoughts about this? Did the file somehow get lost? Also, I recently did this same conversion to another file without problems... Jacob Keller #CCP4I VERSION CCP4Interface 2.1.0 #CCP4I SCRIPT LOG import #CCP4I DATE 06 Sep 2011 16:29:11 #CCP4I USER 'UNKNOWN' #CCP4I PROJECT 3mgl #CCP4I JOB_ID 3 #CCP4I SCRATCH C:/Ccp4Temp #CCP4I HOSTNAME chloe #CCP4I PID 2728 ### ### ### ### CCP4 6.2: cif2mtz version 6.2 : 16/11/09## ### User: Jacob Run date: 6/ 9/2011 Run time: 16:29:19 Please reference: Collaborative Computational Project, Number 4. 1994. "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Data line--- title [No title given] Data line--- symmetry P4212 Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib Data line--- cell 90.91 90.91 65.21 90.0 90.0 90.0 Data line--- end >>>> CCIF signal CCIF_FOPEN (severity: SEVERE ERROR/FATAL) <<<< (Raised in zzs_undump) Cannot open file C:\CCP4-Packages\ccp4-6.2.0\lib\data\cif_mmdic.lib for reading! *** * Information from CCP4Interface script *** The program run with command: cif2mtz HKLIN "C:/Users/Jacob/Desktop/structures/PDB_3mgl/3mgl-sf.cif" HKLOUT "C:/Ccp4Temp/3mgl_3_1_mtz.tmp" has failed with error message child process exited abnormally *** #CCP4I TERMINATION STATUS 0 "child process exited abnormally" #CCP4I TERMINATION TIME 06 Sep 2011 16:29:19 #CCP4I MESSAGE Task failed -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] converting mmcif to mtz failure
Much obliged! Thanks, Jacob On Wed, Sep 7, 2011 at 3:37 AM, Martyn Winn wrote: > > Yes, the program does need the (binary) data file cif_mmdic.lib which is > created from the cif_mm.dic file. > > This should be in the directory C:\CCP4-Packages\ccp4-6.2.0\lib and NOT > C:\CCP4-Packages\ccp4-6.2.0\lib\data as you have. > > Which suggests that the environment variable MMCIFDIC is set wrongly. > And if I look at my windows installation mmm yes it is set wrong > and cif2mtz indeed fails. Edit it in system -> advanced -> environment > variables (or whatever ), restart ccp4i, and voila it works! > > So sorry, this looks like a cock-up in the windows distribution :( > > HTH > Martyn > > On Tue, 2011-09-06 at 16:34 -0500, Jacob Keller wrote: >> Dear Crystallographers, >> >> in trying to convert a mmcif to mtz, I get the logfile below. I looked >> in the directory, and there is a file cf_mm.dic, but this is >> presumably not the same as the similar .lib file. Any thoughts about >> this? Did the file somehow get lost? Also, I recently did this same >> conversion to another file without problems... >> >> Jacob Keller >> >> >> >> #CCP4I VERSION CCP4Interface 2.1.0 >> #CCP4I SCRIPT LOG import >> #CCP4I DATE 06 Sep 2011 16:29:11 >> #CCP4I USER 'UNKNOWN' >> #CCP4I PROJECT 3mgl >> #CCP4I JOB_ID 3 >> #CCP4I SCRATCH C:/Ccp4Temp >> #CCP4I HOSTNAME chloe >> #CCP4I PID 2728 >> >> >> >> >> >> ### >> ### >> ### >> ### CCP4 6.2: cif2mtz version 6.2 : 16/11/09## >> ### >> User: Jacob Run date: 6/ 9/2011 Run time: 16:29:19 >> >> >> Please reference: Collaborative Computational Project, Number 4. 1994. >> "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. >> D50, 760-763. >> as well as any specific reference in the program write-up. >> >> Data line--- title [No title given] >> Data line--- symmetry P4212 >> >> Spacegroup information obtained from library file: >> Logical Name: SYMINFO Filename: >> C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib >> >> Data line--- cell 90.91 90.91 65.21 90.0 90.0 90.0 >> Data line--- end >> >>>> CCIF signal CCIF_FOPEN (severity: SEVERE ERROR/FATAL) <<<< >> (Raised in zzs_undump) >> Cannot open file C:\CCP4-Packages\ccp4-6.2.0\lib\data\cif_mmdic.lib for >> reading! >> >> *** >> * Information from CCP4Interface script >> *** >> The program run with command: cif2mtz HKLIN >> "C:/Users/Jacob/Desktop/structures/PDB_3mgl/3mgl-sf.cif" HKLOUT >> "C:/Ccp4Temp/3mgl_3_1_mtz.tmp" >> has failed with error message >> child process exited abnormally >> *** >> >> >> #CCP4I TERMINATION STATUS 0 "child process exited abnormally" >> #CCP4I TERMINATION TIME 06 Sep 2011 16:29:19 >> #CCP4I MESSAGE Task failed >> > > -- > *** > * * > * Dr. Martyn Winn * > * * > * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * > * Tel: +44 1925 603455 E-mail: martyn.w...@stfc.ac.uk * > * Fax: +44 1925 603634 Skype name: martyn.winn * > * URL: http://www.ccp4.ac.uk/martyn/ * > *** > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] help offline for cns 1.3?
Is there a CNS BB? JPK On Thu, Sep 8, 2011 at 2:20 PM, Laurie Betts wrote: > Sorry - I already belong to 2 bbs and to add another would make me really a > mess. > > Can someone out there who is a CNS developer please send me an email off > this bb I need to ask a glibc question. > > Thanks, Laurie Betts > > laurie.betts0...@gmail.com > > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Mac OSX 10.7 Lion
I have noticed, in new versions of OSes, that there generally is rampant violation of the concept of "if it ain't broken, don't fix." Shouldn't there be more moments of delight, when you see they have solved a previous poorly-engineered feature with an elegant solution? But a lot of the time, you have to try to figure out how to do what you did in the last version in this version, albeit for no net gain. Back to punch cards! The Friday Curmudgeon On Fri, Sep 9, 2011 at 1:09 PM, William Scott wrote: > Hi Phil: > > I've found few, if any advantages. I fear for the future. > > I've had problems getting coot to run stereo due to the X11 implementation in > 10.7. Apart from that, no major problems with crystallographic software. > > Lion greedily uses memory, and any computer I have with less than 4 gig of > memory has become extremely sluggish as a consequence of the "upgrade." > Ideally, you need 8 gig. > > Even with that, on my 2010 mini that I use for music playback, I regressed to > 10.6.8, because of the audio interface. (It seems less robust, more prone to > dropouts and now lacks integer mode output). > > Sara has been screaming at me for the last two weeks (nothing us usual in of > itself) because Apple decided to get rid of "Save As". > > Xcode and the compiler set is free again on 10.7. > > I've put some suggestions here for how to get rid of the most annoying new > features: > http://sage.ucsc.edu/~wgscott/xtal/wiki/index.php/Lion_upgrade_notes > > All the best, > > Bill > > > > > > > On Sep 9, 2011, at 1:28 AM, Phil Evans wrote: > >> Is there any opinion or experience about whether Lion is ready for >> crystallographic use? Should I "upgrade"? >> >> Phil > > William G. Scott > > Contact info: > http://chemistry.ucsc.edu/~wgscott/ > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Why Does Cross-linking Mean Anything?
Dear Crystallographers and Biochemists, cross-linking, say with gluteraldehyde, is an oft-used method of demonstrating a protein's oligomeric state in solution. I have a difficulty with this, however: theoretically (and in practice!), one can tune the amount of cross-linker to get what ever result is desired, such that any protein with some exposed lysines can be cross-linked in any oligomeric state. How, then, does one evaluate the power of this evidence? Maybe one should do a gradient of gluteraldehyde concentrations, then plot the deviation of the observed cross-linked oligomerization from a theoretical null hypothesis? Seems like this could be done, but I have never seen this in the literature... Best, Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Why Does Cross-linking Mean Anything?
>> Maybe one should do a gradient of >> gluteraldehyde concentrations, then plot the deviation of the observed >> cross-linked oligomerization from a theoretical null hypothesis? > > Right - just do it side-by-side with a protein known to be monomeric of > roughly the same size/lysine content... And what is the "critical > concentration" of gutaraldehyde at which the false positives appear in > your experience? The critical concentration depends on protein concentration, time of reaction, brand of gluteraldehyde, day of week, color of my shirt No, I don't know--I have seen cross-linking gradients in Nature and such in which several oligomeric states can be seen up to the one the author asserts is the physiological one. This is a nice experiment for proving one's point on paper, but maybe not for establishing the truth? Maybe a control with some SDS would be appropriate (although this would probably perturb the lysines). Or maybe the experiment should be done in a lysate, and then western-blotted? Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Protein preps become a jelly
I think I know another protein that does this: gelatin! (Well, not the crystallization part...) Jacob On Fri, Sep 16, 2011 at 2:41 PM, Phoebe Rice wrote: > Gamma delta resolvase catalytic domain stock solutions used to make a nice > clear jelly at 4 degrees, but it was perfectly reversible by warming the > sample to room T. In fact, one mutant crystallized in the stock tube after a > few trips in and out of the fridge. The crystals didn't diffract very well > (so we never published that), but it was a fun way to grow them. > Phoebe > > = > Phoebe A. Rice > Dept. of Biochemistry & Molecular Biology > The University of Chicago > phone 773 834 1723 > http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 > http://www.rsc.org/shop/books/2008/9780854042722.asp > > > Original message >>Date: Tue, 30 Aug 2011 23:31:20 +0800 >>From: CCP4 bulletin board (on behalf of aidong >>) >>Subject: [ccp4bb] Protein preps become a jelly >>To: CCP4BB@JISCMAIL.AC.UK >> >>Dear Buddies, >> >>Sorry for bothering you with an off-ccp4 question. We recently are >>experiencing a very strange phenomena. A couple of protein preps with >>reasonably high concentration (10-20mg/ml) become a jelly after >>storages for overnight or a couple of days at 4C. All of them have >>been purified by gel filtration. Some of these proteins behave like >>this from very first preps but some of them had been very kind to us >>previously. We have googled extensively in CCP4BB and www but it >>appears this only happens to us. It would be highly appreciated that >>you could exchange their experiences or provide your suggestions. >> >>Aidong Han, Ph.D >> >>Department of Biomedical Sciences >>School of Life Sciences >>Xiamen University >>Xiamen, Fujian 361005 >>China >>Phone: 0592-218-8172 (O) >> 0592-218-8173 (L) >>Web: http://life.xmu.edu.cn/adhanlab/ > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] pdf search
Why not ask the authors for a reprint? JPK On Wed, Sep 21, 2011 at 10:49 AM, anna delprato wrote: > Hello All, > I apologize in advance for the off topic subject but does anyone out there > have access to a journal called Anticancer Agents Med Chem ? I am having > difficulty obtaining this article: > http://www.ncbi.nlm.nih.gov/pubmed/21707505 and I would really like to have > a look at it. Thanks for your attention. > > Cheers, > Anna Delprato > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Neutron data collection
Wow, neutrons are pretty cool! No radiation damage--and time resolution? I guess this is since they have much higher energy, and are measurable individually? What are the numbers for fluxes (neutrons/sec)? Are the neutrons all at one energy, or is there a bandwidth? JPK > With X-rays, Laue diffraction leads to some systematic overlap as > reflections from different wavelengths fall on the same detector position, > and this cuts into completeness. > > With neutrons, it is possible to use a time-resolved detector such that all > events are time-stamped, and the reflections from lower energy neutrons do > not overlap with those of higher energy neutrons (neutrons having measurable > mass, and thus noticable velocity differences). I know that this is > possible, I do not know whether it is commonplace. > > See, for example: > Protein crystallography with spallation neutrons: the user facility at Los > Alamos Neutron Science Center (2004) P. Langan, G. Greene & B.P. Schoenborn, > J. Appl. Cryst. 37(1) 24-31. > > > -- > === > All Things Serve the Beam > === > David J. Schuller > modern man in a post-modern world > MacCHESS, Cornell University > schul...@cornell.edu > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Neutron data collection
Yes, I have read that paper (a seminal one and the source of the "Henderson limit," no?), and saw that the best "deal" is electrons, I think, but I was just delighted to learn that it doesn't happen in practice. As I recall, x-rays are the worst deal? JPK On Thu, Sep 22, 2011 at 11:58 AM, Murray, James W wrote: > >>Wow, neutrons are pretty cool! No radiation damage--and time >>resolution? > > Actually, as calculated by Richard Henderson in 1995, there is non-negligible > radiation damage from neutrons due to infrequent but energetic nuclear > reactions. The reason that radiation damage by neutrons is not observed in > practice is that neutron sources are so weak. > > The potential and limitations of neutrons, electrons and X-rays for atomic > resolution microscopy of unstained biological molecules. > Henderson R. > Q Rev Biophys. 1995 May;28(2):171-93. > > best wishes > > James > > -- > Dr. James W. Murray > David Phillips Research Fellow > Division of Molecular Biosciences > Imperial College, LONDON > Tel: +44 (0)20 759 48895 > ____ > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jacob Keller > [j-kell...@fsm.northwestern.edu] > Sent: Thursday, September 22, 2011 5:43 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] Neutron data collection > > Wow, neutrons are pretty cool! No radiation damage--and time > resolution? I guess this is since they have much higher energy, and > are measurable individually? What are the numbers for fluxes > (neutrons/sec)? Are the neutrons all at one energy, or is there a > bandwidth? > > JPK > >> With X-rays, Laue diffraction leads to some systematic overlap as >> reflections from different wavelengths fall on the same detector position, >> and this cuts into completeness. >> >> With neutrons, it is possible to use a time-resolved detector such that all >> events are time-stamped, and the reflections from lower energy neutrons do >> not overlap with those of higher energy neutrons (neutrons having measurable >> mass, and thus noticable velocity differences). I know that this is >> possible, I do not know whether it is commonplace. >> >> See, for example: >> Protein crystallography with spallation neutrons: the user facility at Los >> Alamos Neutron Science Center (2004) P. Langan, G. Greene & B.P. Schoenborn, >> J. Appl. Cryst. 37(1) 24-31. >> >> >> -- >> === >> All Things Serve the Beam >> === >> David J. Schuller >> modern man in a post-modern world >> MacCHESS, Cornell University >> schul...@cornell.edu >> > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Neutron data collection
That value, 2200m/s, is pretty slow--there are some bullets that go faster than that, I think... JPK On Fri, Sep 23, 2011 at 11:59 AM, Andreas Ostermann wrote: > Jacob Keller wrote: >> Wow, neutrons are pretty cool! No radiation damage--and time >> resolution? I guess this is since they have much higher energy, and >> are measurable individually? What are the numbers for fluxes >> (neutrons/sec)? Are the neutrons all at one energy, or is there a >> bandwidth? > > The energy of neutrons is even lower when compared to X-rays. > A neutron with a wavelength of 1.8A has an energy of about 25 meV. > The flux at neutron sources compared to synchrotrons is unfortunately low: > > Diffractometer "LADI III" reactor ILL/France: > 3 x 10^7 neutrons/sec/cm^2 (quasi-Laue, delta L / L = 20%) > > Diffractometer "BioDiff" reactor FRM II / Germany: > 1 x 10^7 neutrons/sec/cm^2 (monochromatic, delta L / L = 2.5%) > > Diffractometer "BIX4" reactor JRR3M / Japan: > 4 x 10^6 neutrons/sec/cm^2 (monochromatic, delta L / L = 2.0%) > > BUT you can detect hydrogen atoms even at a moderate resolution of > about 2A ! With neutrons the scattering power of hydrogen/deuterium > is "comparable" to the scattering power of carbon. You can even distinguish > between isotopes. Since the nucleus is a point scatterer the "form factor" > -for neutrons called scattering length- is not scattering angle depended. > A typical measurement time is about 2-3 weeks for a crystal of 1 mm^3. > I know...of course not every protein can be crystallized up to 1 mm^3 but > if you have such a system and you are interested in the protonation states > of amino acids in the active centre for example, than neutrons are worth a > try > for sure! If you fully deuterate your protein (which gets more and more > routine > work for example at the D-LAB at ILL/EMBL) you can even work with smaller > crystals. > > Because of the relative low flux most reactor based neutron diffractometers > for > proteins uses large cylindrical neutron image plate detector, which cover a > solid angle > of about 2 Pi. At spallation sources (which are pulsed neutron sources) > detectors > with time resolution are used. This instruments (PCS in Los Alamos; iBIX in > Japan > and MANDI in Oak Ridge) are time of flight instruments. They uses the fact > that > neutrons with different energy/wavelength show different velocities ( a 1.8A > neutron > has a velocity of about 2200 m/s). They measure different wavelength > neutrons at > different time at the detector. > > Hope to see some of you as new "neutron users" in the future, > cheers, > > Andreas > > -- > Dr. Andreas Ostermann > Technische Universität München > Research reactor FRM II > Instrument "BioDiff" > Lichtenbergstr. 1 > D-85747 Garching > Tel.: +49-89-289-14702 > Fax.: +49-89-289-14666 > Email: andreas.osterm...@frm2.tum.de > Web: http://www.frm2.tum.de/en/science/index.html > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Direct method solution at 1.15A
I don't really get your question: assuming the sigmas you mentioned are in an anomalous map and therefore have been located, why don't you just plug it into your usual phasing algorithm? Jacob On Fri, Sep 23, 2011 at 1:49 PM, Yuri Pompeu wrote: > Hello everyone, > I have a data set >99% completeness to 1.15A > This is a 400 amino acid long protein and it has 7 Met (Sulfur peaks around > 20sigma) > And a tightly bound phosphate (P peak around 22sigma) > Could I try and solve this directly or is it crazy idea? > If so what program should I try? > > thanks > Yuri > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Superpose, SSM
I vaguely recall notepad doing something wacky with files in certain cases...why don't you get the excellent text editor NoteTab Light [sic] (I use it all the time--free and works great), then take a look at your files and see whether MS notepad altered the files. JPK On Mon, Sep 26, 2011 at 2:42 PM, Matthias Zebisch wrote: > Dear CCP4 users, > > I am using the ccp4i version 6.2.0 under windows 7. I've come across a > problem with superpose. > The outputfile appears to have additional line feeds (see picture) which, > however are not seen in the windows notepad. > The structure can also be opened in coot and pymol. However, it is not > possible to use it within CCP4, eg. for a subsequent superposition. > > Is this problem known to anybody and is there a simple workaround available? > I need to compare hell of a lot of relative domain orientations... > > I did not have this problem on a second computer with ccp4 6.1.2. When I > updated to 6.2.0, the situation was as described above. > > Any help will be highly appreciated, > > Thanks, Matthias > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Superpose, SSM
I want to pitch again for NoteTab--it is really useful, can do almost everything a good text editor should. I particularly like to use regexes, and you can do that with NoteTab, along with a ton of other things. I am not affiliated in any way, and the program is totally free and does not install toolbars or anything. Try it! Maybe it should be part of ccp4! Jacob On Tue, Sep 27, 2011 at 5:08 AM, Pierre Rizkallah wrote: > Dear BB Readers, > > I have used CCP4 on Windows exclusively for nearly 10 years now. The text > editor of choice for PDB files coming out of CCP4 has always been WORDPAD, > which comes with Windows, no need to download. NOTEPAD causes me great > difficulty with PDB files, probably because of my age, but let's not get hung > up on that. I have never been interested enough in chasing up ways of using > NOTEPAD properly. > > In the last couple of months, CCP4 started outputting PDB files that appeared > in WORDPAD as one continuous line, i.e. no line feed. This was an > INTERMITTENT behaviour. I could not reproduce it to order. These files are > readable by NOTEPAD, despite my dislike of this editor. I have not changed > anything in my set up or in the CCP4 or Coot installation. So I could blame > it on some system updates in WINDOWS that are invisible to me, but then again > it could be a fault of something else, perhaps like changing the > combination already mentioned. The files are usually readable in COOT, which > reads anything coming out of CCP4, but again output from COOT is occasionally > unreadable in CCP4. NOTEPAD readable files are usually OK. Unfortunately, I > don't have a recipe for correct action. > > Having said all that, at the moment output PDB files from CCP4 and COOT > appear to be all well behaved in terms of readability, both in the Windows > editors and in CCP4. I would like to think this problem has disappeared just > as it had appeared suddenly. I shall continue to look out for symptoms of > trouble. > > Pierre > *** > Dr Pierre Rizkallah, Senior Lecturer in Structural Biology, > Wales Heart Research Institute, Heath Campus, Cardiff CF14 4XN > email: rizkall...@cardiff.ac.uk phone + 44 29 2074 2248 > > -CCP4 bulletin board wrote: - > To: CCP4BB@JISCMAIL.AC.UK > From: Robbie Joosten > Sent by: CCP4 bulletin board > Date: 27-09-2011 05:22 > Subject: Re: [ccp4bb] Superpose, SSM > > One would assume that Windows software would read DOS/Windows type text > files... > Open the file in Wordpad. Unlike Notepad, it is able to work with Windows and > Unix type text files. If you edit something and save the file, it will be in > Windows style. If Superpose stops on that, it should really be updated. I'm > sure that there are Windows versions op the programs Unix2dos and Dos2unix > which were the programs to use to convert one type to the other. You can also > use Word to search and replace the linefeeds. > > Good luck with this very retro problem. > Cheers, > Robbie > >> Date: Mon, 26 Sep 2011 17:07:50 -0600 >> From: xtald...@gmail.com >> Subject: Re: [ccp4bb] Superpose, SSM >> To: CCP4BB@JISCMAIL.AC.UK >> >> I think something in your workflow is inserting dos line feeds (\n\r or >> \r\n, I can't remember which). >> >> If I have guessed correctly, you want to remove those "\r"s before >> proceeding (or never let them get in there in the first place). >> >> You claim to open it with MS something, which would insert dos line feeds as >> part of Operation Vendor Lock. Did you happen to save it, perhaps by habit? >> That would do the trick. It might even do something insidious and insert >> those linefeeds without your purposefully saving the document. >> >> Your best bet to fix the file after corruption is vim (used to be that >> "crystallographers" could use real text editors). >> >> The command in vim is: >> >> :%s/\r//g >> >> You might find some third party utility that fixes linefeeds for $30.00 >> somewhere, if vim is too "retro". >> >> Otherwise, you may want to start over, skip checking it out in MS something, >> and go straight to superpose. >> >> James >> >> >> >> On Sep 26, 2011, at 2:51 PM, Matthias Zebisch wrote: >> >> > Hi again, >> > >> > Thanks for your quick replies but I think I made myself not clear. here is >> > what I'm doing: >> > >> > 1) superpose proteinA.pdb onto proteinB.pdb : works, but gives out >> > proteinA_lsq1.pdb with extra empty lines (not the anisou lines ;o) ) &g
[ccp4bb] Database of Known Medically-Relevant Mutations
Dear Crystallographers, does anyone have a favorite site which lays out in a clear way all of the known medically-relevant or phenotype-changing mutations of a given protein and their phenotypes with references? Some of the existing sites are very busy graphically and somewhat difficult to interpret, making what would seem to be a simple task take much longer... Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] detect dsDNA
I actually looked at an EtBr MSDS a while ago, and was shocked at how benign it was. I also heard from someone that they used to feed it to Argentinian cows routinely a few years back... JPK On Sat, Oct 1, 2011 at 1:56 AM, James Stroud wrote: > If you can reproduce the crystals and have the material > > 1. Harvest several large crystals. > 2. Make several transfers to fresh mother liquor to wash. > 3. Dissolve in DNA loading dye without SDS > 4. Run on a native gel (e.g. 6% polyacrylamide, 0.5XTBE, etc.). > 5. Include positive control lanes for protein, DNA, and complex. > 6. Stain with ETBr. Take a picture. > 7. Wash out the ethidium in accordance with state, local, federal, UN, laws, > filling out the proper documentation ad nauseum. Take a safety class just to > be sure you didn't miss something. Hug a bureaucrat. > 8. Stain with coomassie. Take a picture. > > That should tell you more than you need to know. > > James > > On Sep 30, 2011, at 9:36 PM, zq deng wrote: > >> Hi all, >> . >> recently,I got a crystal of protein-DNA crystal.i used silver stainto prove >> that it is a protein crystal.Does anyone have method to detect if there is >> DNA in the crystal. >> any suggestion will be appreciated. >> >> Regards, >> deng > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] detect dsDNA
>> There exists a less toxic chemical than EtBr to stain DNA: SYBR safe >> DNA stain (a fluorescence dye sold by a certain vendor). > > SYBR Safe is about 10X less sensitive though. Can you do the toothbrush test with SYBR Safe? JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Bicarb at low pH
Dear Crystallographers, I would like to soak my crystals in bicarbonate (a possible substrate), but the crystals have grown--and only grow--in pH 5.2-6.0, so the bicarb/CO2 will just keep evolving out of the solution and reliquishing its hydroxyls until the pH is elevated sufficiently out of range. Does anyone have a clever way of getting bicarb into these crystals? Grow them under CO2? Transfer them to higher pH, and hope for the best? Jacob Keller
Re: [ccp4bb] Anomalous patterson not consistent with systematic extinctions
Why not just go to P1, then build up the symmetry? Is completeness low? JPK On Mon, Oct 3, 2011 at 10:14 AM, Eleanor Dodson wrote: > Further Qs. > > Do you have a noncryst translation parallel to the b axis (ctruncate will > list any such translation..) > > If the b shift is 0.5 then the 0k0 "absences" will be present whether the > spacegroup is P2 or P21. > > How many Xe sites do you expect? If there is only one then phasing is more > difficult in monoclinic SGs - you have to break the centrosymmetry of the > heavy atom distribution. > > Do you have native data without Xe? > > I always check for peaks which are consistent in the isomorphous and > anomalous difference pattersons. > > The NCS symmetry will doubtless make the exptl phasing more complicated, but > it should help you with averaging later! > Eleanor > > > > On 10/03/2011 03:33 PM, Marta Ferraroni wrote: >> >> Dear all, >> >> We collected some Xenon derivatives of a protein (an heterotetramer >> α2β2)that seems to crystallize in P21 since the 0k0 reflections with >> k=2n+1 are not present. However in the anomalous Patterson we found >> strong peaks in the section v=0 yet none in the Harker section v=1/2. >> Furthermore we weren't able to solve the structure both in P2 and P21 >> using these derivatives with the most commonly used programs. >> >> The cell is 89 125 90 90 102 90 so a is approximately equal to c that >> could permit pseudomerohedral twinning albeit the tests (Padilla-Yeates >> and Britton) estimate a fraction of twinning around 0.05. >> >> Data cannot be scaled as C orthorhombic even if the data reduction >> programs (XDS and imosflm) assign a higher score to the related >> orthorhombic cell with dimensions 112 139 124 90.00 90.00 90.00. In the >> native Patterson map there are not strong peaks. According to the >> Matthews coefficient the asymmetric unit contains 2 heterotetramers and >> the self rotation function may indicate a 222 non crystallographic >> symmetry with one two-fold axis perpendicular to the crystallographic >> one (see figure attached). >> >> Our questions are: >> >> 1) why the anomalous Patterson is not consistent with the space group? >> >> 2) Is there the possibility that the NCS could hamper the determination >> of the correct space group and eventually determine a lower estimate of >> the twinning fraction? >> >> thanks in advance for your help >> >> Regards, >> >> >> Marta Ferraroni >> Dept. of Chemistry >> University of Florence >> Italy >> > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Finding a sequence motif with BLAST
Dear Crystallographers, I cannot get BLAST to find all proteins with the motif cxcxcxc or at least cxcxc. It seems to think of "x" as an actual amino acid rather than a wildcard. There must be some easy way to do this? Ordinarily to find a short motif, I would just paste the sequence and get the answer, but here the C's are an absolute requirement and there is no constraint on the x's except that they be only one residue. JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Finding a sequence motif with BLAST
Thanks everybody, I tried using --toolkit tuebingen mpi --Scanprosite I think my regex syntax was different from the Tuebingen site's, but scanprosite worked well and found many hits, although without really hitting paydirt. I think both of these programs would do the job well, though. Thanks very much for your speedy help (this BB is truly amazing!), Jacob On Tue, Oct 4, 2011 at 3:47 PM, David Briggs wrote: > Hi Jacob, > SCAN PROSITE > http://prosite.expasy.org/scanprosite/ > will do precisely what you want. > C-X-C-X-C-X-C > or > C-X-C-X-C > would be the pattern using Prosite syntax. > Cheers, > Dave > > David C. Briggs PhD > Father, Structural Biologist and Sceptic > > University of Manchester E-mail: > david.c.bri...@manchester.ac.uk > > http://manchester.academia.edu/DavidBriggs (v.sensible) > http://xtaldave.wordpress.com/ (sensible) > http://xtaldave.posterous.com/ (less sensible) > Twitter: @xtaldave > Skype: DocDCB > > > > On 4 October 2011 21:34, Jacob Keller > wrote: >> >> Dear Crystallographers, >> >> I cannot get BLAST to find all proteins with the motif cxcxcxc or at >> least cxcxc. It seems to think of "x" as an actual amino acid rather >> than a wildcard. There must be some easy way to do this? Ordinarily to >> find a short motif, I would just paste the sequence and get the >> answer, but here the C's are an absolute requirement and there is no >> constraint on the x's except that they be only one residue. >> >> JPK >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> cel: 773.608.9185 >> email: j-kell...@northwestern.edu >> *** > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Finding a sequence motif with BLAST
Just for kicks, check out this sequence I found in the process (conjecture: maybe when the virus causes its synthesis, it uses up all the cysteines/methionines!): >sp|Q69566|U88_HHV6U Uncharacterized protein U88 OS=Human herpesvirus 6A >(strain Uganda-1102) GN=U88 PE=4 SV=1 MYVSVSVHVSVHVSVRVSVRVSVCVSVRVSVHVSVRVSVSVRVSVRVSVSVRVSVRVSVSVHVSVRVSVRVSVSVRVSVCARVCARVCVCARVCVCARVCVCARVCVCARVCARVCVCACVCVCACLCVCACLCVCACLCVCACLCVCACLCVCACLCVCACLCVCACLCVCVCVCLCVCVCLCVCVCLCVCVCLCVCVCLCVCVCLCVCVCLCVCVCLCVCVCLCVCVCLCVCVCVCVCVCVCVCVCVCVCVCVCLCVCVCLCVCLCVCLCVCVCVCVCLCVCLCVCLCVCVCVCVCLLCMSLCMCMCMCMCMCMCMCMCMSLCMSLCMCMCMCMCMCMCICMCMCICICMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCMCIIEGNK Maybe it's just a sequencing glitch? JPK On Tue, Oct 4, 2011 at 4:05 PM, Jacob Keller wrote: > Thanks everybody, I tried using > > --toolkit tuebingen mpi > --Scanprosite > > I think my regex syntax was different from the Tuebingen site's, but > scanprosite worked well and found many hits, although without really > hitting paydirt. I think both of these programs would do the job well, > though. > > Thanks very much for your speedy help (this BB is truly amazing!), > > Jacob > > > > On Tue, Oct 4, 2011 at 3:47 PM, David Briggs wrote: >> Hi Jacob, >> SCAN PROSITE >> http://prosite.expasy.org/scanprosite/ >> will do precisely what you want. >> C-X-C-X-C-X-C >> or >> C-X-C-X-C >> would be the pattern using Prosite syntax. >> Cheers, >> Dave >> >> David C. Briggs PhD >> Father, Structural Biologist and Sceptic >> >> University of Manchester E-mail: >> david.c.bri...@manchester.ac.uk >> >> http://manchester.academia.edu/DavidBriggs (v.sensible) >> http://xtaldave.wordpress.com/ (sensible) >> http://xtaldave.posterous.com/ (less sensible) >> Twitter: @xtaldave >> Skype: DocDCB >> >> >> >> On 4 October 2011 21:34, Jacob Keller >> wrote: >>> >>> Dear Crystallographers, >>> >>> I cannot get BLAST to find all proteins with the motif cxcxcxc or at >>> least cxcxc. It seems to think of "x" as an actual amino acid rather >>> than a wildcard. There must be some easy way to do this? Ordinarily to >>> find a short motif, I would just paste the sequence and get the >>> answer, but here the C's are an absolute requirement and there is no >>> constraint on the x's except that they be only one residue. >>> >>> JPK >>> >>> -- >>> *** >>> Jacob Pearson Keller >>> Northwestern University >>> Medical Scientist Training Program >>> cel: 773.608.9185 >>> email: j-kell...@northwestern.edu >>> *** >> >> > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Finding a sequence motif with BLAST
> Maybe it's just a sequencing glitch? Not so--BLAST showed there are a whole cadre of these things in various genomes. Go figure. JPK > > JPK > > > On Tue, Oct 4, 2011 at 4:05 PM, Jacob Keller > wrote: >> Thanks everybody, I tried using >> >> --toolkit tuebingen mpi >> --Scanprosite >> >> I think my regex syntax was different from the Tuebingen site's, but >> scanprosite worked well and found many hits, although without really >> hitting paydirt. I think both of these programs would do the job well, >> though. >> >> Thanks very much for your speedy help (this BB is truly amazing!), >> >> Jacob >> >> >> >> On Tue, Oct 4, 2011 at 3:47 PM, David Briggs >> wrote: >>> Hi Jacob, >>> SCAN PROSITE >>> http://prosite.expasy.org/scanprosite/ >>> will do precisely what you want. >>> C-X-C-X-C-X-C >>> or >>> C-X-C-X-C >>> would be the pattern using Prosite syntax. >>> Cheers, >>> Dave >>> >>> David C. Briggs PhD >>> Father, Structural Biologist and Sceptic >>> >>> University of Manchester E-mail: >>> david.c.bri...@manchester.ac.uk >>> ======== >>> http://manchester.academia.edu/DavidBriggs (v.sensible) >>> http://xtaldave.wordpress.com/ (sensible) >>> http://xtaldave.posterous.com/ (less sensible) >>> Twitter: @xtaldave >>> Skype: DocDCB >>> >>> >>> >>> On 4 October 2011 21:34, Jacob Keller >>> wrote: >>>> >>>> Dear Crystallographers, >>>> >>>> I cannot get BLAST to find all proteins with the motif cxcxcxc or at >>>> least cxcxc. It seems to think of "x" as an actual amino acid rather >>>> than a wildcard. There must be some easy way to do this? Ordinarily to >>>> find a short motif, I would just paste the sequence and get the >>>> answer, but here the C's are an absolute requirement and there is no >>>> constraint on the x's except that they be only one residue. >>>> >>>> JPK >>>> >>>> -- >>>> *** >>>> Jacob Pearson Keller >>>> Northwestern University >>>> Medical Scientist Training Program >>>> cel: 773.608.9185 >>>> email: j-kell...@northwestern.edu >>>> *** >>> >>> >> >> >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> cel: 773.608.9185 >> email: j-kell...@northwestern.edu >> *** >> > > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > cel: 773.608.9185 > email: j-kell...@northwestern.edu > *** > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program cel: 773.608.9185 email: j-kell...@northwestern.edu ***
[ccp4bb] Cell Params as a Function of Temperature
Dear Crystallographers, is anyone aware of references studying variation in cell params systematically as a function of temperature, with many points on the curve (not just RT and 100K?) Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] WaterTidy fails in windows ccp4i
Dear CCP4ers, I am trying to run watertidy from the windows gui to add waters, but get the error message below. Since the gui is so short, I don't think I am missing anything, so what I am doing wrong? Is there an alternative water-picker in the gui? I used to use watpick, but I believe that was in XPLOR... Jacob #CCP4I VERSION CCP4Interface 2.1.0#CCP4I SCRIPT LOG watertidy#CCP4I DATE 19 Oct 2011 12:30:14#CCP4I USER 'UNKNOWN'#CCP4I PROJECT Tues2bot#CCP4I JOB_ID 18#CCP4I SCRATCH C:/Ccp4Temp#CCP4I HOSTNAME chloe#CCP4I PID 9820 ### ### ### ### CCP4 6.2: DISTANG version 6.2 : 02/04/08## ### User: Jacob Run date: 19/10/2011 Run time: 12:30:15 Please reference: Collaborative Computational Project, Number 4. 1994. "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Logical name: XYZIN File name: C:/Users/Jacob/Desktop/Dallos_Lab/3MGL_Crystals/APS_10_10_2011/jacob/2botnr3/TuesProcessing/Tues2bot_4.1_buccaneer2_refmac1.pdb PDB file is being opened on unit 1 for INPUT. MATRICES DERIVED FROM CRYST1 CARD IN COORDINATE FILE RF RO 0.012 -0.000 -0.000 -0.000 82.115 0.000 0.000 -0.000 -0.000 0.012 -0.000 0.000 0.000 82.115 0.000 0.000 0.000 -0.000 0.006 -0.000 0.000 0.000 159.320 -0.000 -0.000 0.000 -0.000 1.000 -0.000 0.000 -0.000 1.000 ARSE after CALL RBSPGRP(SPGRP)P 43 21 2 Data line--- title [No title given] Data line--- distance vdw intra Data line--- symmetry P43212 Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib Data line--- radii CA 1.5 C 1.5 N 1.5 O 1.5 SG 1.5 OW 1.5 Data line--- from atom 1 to 20 Data line--- to atom to DISTANG: *** Two limits required***Times: User: 0.0s System: 0.0s Elapsed: 0:00 Information from CCP4Interface script***The program run with command: distang XYZIN "C:/Users/Jacob/Desktop/Dallos_Lab/3MGL_Crystals/APS_10_10_2011/jacob/2botnr3/TuesProcessing/Tues2bot_4.1_buccaneer2_refmac1.pdb" DISTOUT "C:/Ccp4Temp/Tues2bot_18_1_log_1.tmp" has failed with error message DISTANG: *** Two limits required** #CCP4I TERMINATION STATUS 0 " DISTANG: *** Two limits required***"#CCP4I TERMINATION TIME 19 Oct 2011 12:30:15#CCP4I MESSAGE Task failed -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] WaterTidy fails in windows ccp4i
Well, I see that it fixes/edits waters, but it seems to add them too, according to the documentation "Add/Tidy Waters - Watertidy This task rationalises waters at the end of refinement. See program documentation: Watertidy, Distang." Is there a program in the gui that adds waters, if not this one? There is of course arp/warp, but not in windows... Jacob On Wed, Oct 19, 2011 at 1:32 PM, Ed Pozharski wrote: > On Wed, 2011-10-19 at 12:45 -0500, Jacob Keller wrote: >> Is there an >> alternative water-picker in the gui? > > watertidy is not a water-picker > > -- > "I'd jump in myself, if I weren't so good at whistling." > Julian, King of Lemurs > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] IUCr committees, depositing images
Is anyone seriously questioning whether we should archive the images used for published structures? That amount of space is trivial, could be implemented just as another link in the PDB website, and would be really helpful in some cases. One person could set it up in a day! You could just make it a policy: no images, no PDB submission, no publishing! Jacob On Wed, Oct 26, 2011 at 11:15 AM, Gloria Borgstahl wrote: > I just want to jump in to state that I am ALL FOR the notion of > depositing the images that go with the structure factors and the > refined structure. > > Through the years, I have been interviewing folks about the strange > satellite diffraction they saw, but ignored, > used the mains that they could integrate and deposited that structure, > does not help me to > justify the existance of modulated protein crystals to reviewers. > > But if I could go and retrieve those images, and reanalyze with new methods. > Dream come true. Reviewers convinced. > > On Wed, Oct 26, 2011 at 10:59 AM, Patrick Shaw Stewart > wrote: >> >> Could you perhaps use the principle of "capture storage" that is used by >> wild-life photographers with high-speed cameras? >> The principle is that the movie is written to the same area of memory, >> jumping back to the beginning when it is full (this part is not essential, >> but it makes the principle clear). Then, when the photographer takes his >> finger off the trigger, the last x seconds is permanently stored. So you >> keep your wits about you, and press the metaphorical "store" button just >> after you have got the movie in the can so to speak >> >> Just a thought >> Patrick >> >> On Wed, Oct 26, 2011 at 2:18 PM, John R Helliwell >> wrote: >>> >>> Dear Frank, >>> re 'who will write the grant?'. >>> >>> This is not as easy as it sounds, would that it were! >>> >>> There are two possible business plans:- >>> Option 1. Specifically for MX is the PDB as the first and foremost >>> candidate to seek such additional funds for full diffraction data >>> deposition for each future PDB deposiition entry. This business plan >>> possibility is best answered by PDB/EBI (eg Gerard Kleywegt has >>> answered this in the negative thus far at the CCP4 January 2010). >>> >>> Option 2 The Journals that host the publications could add the cost to >>> the subscriber and/or the author according to their funding model. As >>> an example and as a start a draft business plan has been written by >>> one of us [JRH] for IUCr Acta Cryst E; this seemed attractive because >>> of its simpler 'author pays' financing. This proposed business plan is >>> now with IUCr Journals to digest and hopefully refine. Initial >>> indications are that Acta Cryst C would be perceived by IUCr Journals >>> as a better place to start considering this in detail, as it involves >>> fewer crystal structures than Acta E and would thus be more >>> manageable. The overall advantage of the responsibility being with >>> Journals as we see it is that it encourages such 'archiving of data >>> with literature' across all crystallography related techniques (single >>> crystal, SAXS, SANS, Electron crystallography etc) and fields >>> (Biology, Chemistry, Materials, Condensed Matter Physics etc) ie not >>> just one technique and field, although obviously biology is dear to >>> our hearts here in the CCP4bb. >>> >>> Yours sincerely, >>> John and Tom >>> John Helliwell and Tom Terwilliger >>> >>> On Wed, Oct 26, 2011 at 9:21 AM, Frank von Delft >>> wrote: >>> > Since when has the cost of any project been limited by the cost of >>> > hardware? Someone has to implement this -- and make a career out of it; >>> > thunderingly absent from this thread has been the chorus of volunteers >>> > who >>> > will write the grant. >>> > phx >>> > >>> > >>> > On 25/10/2011 21:10, Herbert J. Bernstein wrote: >>> > >>> > To be fair to those concerned about cost, a more conservative estimate >>> > from the NSF RDLM workshop last summer in Princeton is $1,000 to $3,000 >>> > per terabyte per year for long term storage allowing for overhead in >>> > moderate-sized institutions such as the PDB. Larger entities, such >>> > as Google are able to do it for much lower annual costs in the range of >>> > $100 to $300 per terabyte per year. Indeed, if this becomes a serious >>> > effort, one might wish to consider involving the large storage farm >>> > businesses such as Google and Amazon. They might be willing to help >>> > support science partially in exchange for eyeballs going to their sites. >>> > >>> > Regards, >>> > H. J. Bernstein >>> > >>> > At 1:56 PM -0600 10/25/11, James Stroud wrote: >>> > >>> > On Oct 24, 2011, at 3:56 PM, James Holton wrote: >>> > >>> > The PDB only gets about 8000 depositions per year >>> > >>> > Just to put this into dollars. If each dataset is about 17 GB in >>> > size, then that's about 14 TB of storage that needs to come online >>> > every year to store the raw data for every structure. A two second >>> > sear
Re: [ccp4bb] IUCr committees, depositing images
Touche! But alas, I have no access to the PDB's server, so... JPK On Wed, Oct 26, 2011 at 11:54 AM, Frank von Delft wrote: > Cool - we've found our volunteer!! > > On 26/10/2011 17:28, Jacob Keller wrote: >> >> Is anyone seriously questioning whether we should archive the images >> used for published structures? That amount of space is trivial, could >> be implemented just as another link in the PDB website, and would be >> really helpful in some cases. One person could set it up in a day! You >> could just make it a policy: no images, no PDB submission, no >> publishing! >> >> Jacob >> >> >> On Wed, Oct 26, 2011 at 11:15 AM, Gloria Borgstahl >> wrote: >>> >>> I just want to jump in to state that I am ALL FOR the notion of >>> depositing the images that go with the structure factors and the >>> refined structure. >>> >>> Through the years, I have been interviewing folks about the strange >>> satellite diffraction they saw, but ignored, >>> used the mains that they could integrate and deposited that structure, >>> does not help me to >>> justify the existance of modulated protein crystals to reviewers. >>> >>> But if I could go and retrieve those images, and reanalyze with new >>> methods. >>> Dream come true. Reviewers convinced. >>> >>> On Wed, Oct 26, 2011 at 10:59 AM, Patrick Shaw Stewart >>> wrote: >>>> >>>> Could you perhaps use the principle of "capture storage" that is used by >>>> wild-life photographers with high-speed cameras? >>>> The principle is that the movie is written to the same area of memory, >>>> jumping back to the beginning when it is full (this part is not >>>> essential, >>>> but it makes the principle clear). Then, when the photographer takes >>>> his >>>> finger off the trigger, the last x seconds is permanently stored. So >>>> you >>>> keep your wits about you, and press the metaphorical "store" button just >>>> after you have got the movie in the can so to speak >>>> >>>> Just a thought >>>> Patrick >>>> >>>> On Wed, Oct 26, 2011 at 2:18 PM, John R Helliwell >>>> wrote: >>>>> >>>>> Dear Frank, >>>>> re 'who will write the grant?'. >>>>> >>>>> This is not as easy as it sounds, would that it were! >>>>> >>>>> There are two possible business plans:- >>>>> Option 1. Specifically for MX is the PDB as the first and foremost >>>>> candidate to seek such additional funds for full diffraction data >>>>> deposition for each future PDB deposiition entry. This business plan >>>>> possibility is best answered by PDB/EBI (eg Gerard Kleywegt has >>>>> answered this in the negative thus far at the CCP4 January 2010). >>>>> >>>>> Option 2 The Journals that host the publications could add the cost to >>>>> the subscriber and/or the author according to their funding model. As >>>>> an example and as a start a draft business plan has been written by >>>>> one of us [JRH] for IUCr Acta Cryst E; this seemed attractive because >>>>> of its simpler 'author pays' financing. This proposed business plan is >>>>> now with IUCr Journals to digest and hopefully refine. Initial >>>>> indications are that Acta Cryst C would be perceived by IUCr Journals >>>>> as a better place to start considering this in detail, as it involves >>>>> fewer crystal structures than Acta E and would thus be more >>>>> manageable. The overall advantage of the responsibility being with >>>>> Journals as we see it is that it encourages such 'archiving of data >>>>> with literature' across all crystallography related techniques (single >>>>> crystal, SAXS, SANS, Electron crystallography etc) and fields >>>>> (Biology, Chemistry, Materials, Condensed Matter Physics etc) ie not >>>>> just one technique and field, although obviously biology is dear to >>>>> our hearts here in the CCP4bb. >>>>> >>>>> Yours sincerely, >>>>> John and Tom >>>>> John Helliwell and Tom Terwilliger >>>>> >>>>> On Wed, Oct 26, 2011 at 9:21 AM, Frank von Delft >>>>> wrote: >>>>>> >>
Re: [ccp4bb] raw data deposition
One thing that the poll is useful for is something I find surprising: ~40% when I checked found storing images a waste of time. So, I guess this might be useful for finding the "silent [significant] minority." Why not have those folks chime in about why they think this is useless, even to store images of solved datasets? JPK On Thu, Oct 27, 2011 at 11:25 AM, Gerard Bricogne wrote: > Dear Ed, > > I am really puzzled by this initiative. It assumes that there is a > pre-formed "collective opinion" out there, independent from and unaffected > by the exchanges of views that have taken place on this BB, that would be > worth more than the conclusions we might reach by pursuing these exchanges. > > The thread you are obviously deciding to dissociate yourself from was > initiated in response to a suggestion that views on this topic would > usefully be aired publicly on this BB rather than posted off-list to Tom > Terwilliger, who immediately agreed that this was a good idea and has been > very supportive of this discussion. > > Shouldn't we continue to try and put our heads together to reach a > consensus, rather than collect opinions that may be little more than prior > prejudices? > > What shall we gain by such a vote? I may be misunderstanding what you > have in mind, of course :-) . > > > With best wishes, > > Gerard. > > -- > On Thu, Oct 27, 2011 at 12:08:24PM -0400, Ed Pozharski wrote: >> I am curious as to what the collective opinion on the raw data >> deposition actually is across the cross-section of the macromolecular >> crystallography community subscribed to the bb. So, if you have a >> second and a formed opinion on the idea of the depositions of the raw >> data, please vote here >> >> http://tinyurl.com/3qlwwsh >> >> I'll post the results as soon as they look settled. >> >> Cheers, >> >> Ed. >> >> -- >> "Hurry up before we all come back to our senses!" >> Julian, King of Lemurs > > -- > > === > * * > * Gerard Bricogne g...@globalphasing.com * > * * > * Global Phasing Ltd. * > * Sheraton House, Castle Park Tel: +44-(0)1223-353033 * > * Cambridge CB3 0AX, UK Fax: +44-(0)1223-366889 * > * * > === > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] raw data deposition
In medical school, I found out that there could be a large population in a class which was completely lost or completely disagreed with what was being said, but there was only silence. When the lecturer would pose a question, it would take a painful silence before anyone in the 100+ student class would hazard an answer (one can only speculate why this was, but so be it.) Even a yes-no show of hands would yield only ~10% participation. Too much commitment? Anyway, the solution for many lecturers was to pass out "clickers," which allowed the vote anonymously for a given answer in a multiple choice question. Here we have Ed's version of clickers for the ccp4bb. The problem is that clickers are not very articulate--they just click! I fully agree that those who disagree should articulate their opinions--maybe subscribe anonymously if necessary? I don't like it that people are so stymied, but this seems to be the way things are, so the question is how to work with it. Jacob On Thu, Oct 27, 2011 at 11:48 AM, Gerard Bricogne wrote: > Dear Jacob, > > I agree, of course, with the goal of giving everyone a voice, but > knowing that 40% of the voters find storing images a waste of time falls > short of knowing why they think so and taking their arguments into account. > Disagreeing without saying why when a topic is being actively discussed is a > position that does not contribute anything very constructive. > > I think there should be an extra category of answer that would be > "I don't care", so that people who have no opinion do not get confused with > those who have an articulate position against the proposal, and wh should > then articulate it! > > > With best wishes, > > Gerard. > > -- > On Thu, Oct 27, 2011 at 11:30:20AM -0500, Jacob Keller wrote: >> One thing that the poll is useful for is something I find surprising: >> ~40% when I checked found storing images a waste of time. So, I guess >> this might be useful for finding the "silent [significant] minority." >> Why not have those folks chime in about why they think this is >> useless, even to store images of solved datasets? >> >> JPK >> >> On Thu, Oct 27, 2011 at 11:25 AM, Gerard Bricogne >> wrote: >> > Dear Ed, >> > >> > I am really puzzled by this initiative. It assumes that there is a >> > pre-formed "collective opinion" out there, independent from and unaffected >> > by the exchanges of views that have taken place on this BB, that would be >> > worth more than the conclusions we might reach by pursuing these exchanges. >> > >> > The thread you are obviously deciding to dissociate yourself from was >> > initiated in response to a suggestion that views on this topic would >> > usefully be aired publicly on this BB rather than posted off-list to Tom >> > Terwilliger, who immediately agreed that this was a good idea and has been >> > very supportive of this discussion. >> > >> > Shouldn't we continue to try and put our heads together to reach a >> > consensus, rather than collect opinions that may be little more than prior >> > prejudices? >> > >> > What shall we gain by such a vote? I may be misunderstanding what you >> > have in mind, of course :-) . >> > >> > >> > With best wishes, >> > >> > Gerard. >> > >> > -- >> > On Thu, Oct 27, 2011 at 12:08:24PM -0400, Ed Pozharski wrote: >> >> I am curious as to what the collective opinion on the raw data >> >> deposition actually is across the cross-section of the macromolecular >> >> crystallography community subscribed to the bb. So, if you have a >> >> second and a formed opinion on the idea of the depositions of the raw >> >> data, please vote here >> >> >> >> http://tinyurl.com/3qlwwsh >> >> >> >> I'll post the results as soon as they look settled. >> >> >> >> Cheers, >> >> >> >> Ed. >> >> >> >> -- >> >> "Hurry up before we all come back to our senses!" >> >> Julian, King of Lemurs >> > >> > -- >> > >> > === >> > * * >> > * Gerard Bricogne g...@globalphasing.com * >> > * * >> > * Global Phasing Ltd.
Re: [ccp4bb] Weird blob appears
Part of what is bothering me is that the density showed up at one exact point in the refinement, and I am currently testing what exactly it was that changed things. To me, the blob almost looks like a mask of the molecule, and there is very little 2Fo density, so that's weird. I am also really bothered that the R value is so low, and yet there's this huge blob. Something seems buggy to me. Also a clarification: the blob runs into a two-fold axis in the middle, so things get dodgy there too. I will let you know what I find out. JPK On Thu, Oct 27, 2011 at 2:53 PM, David Briggs wrote: > I agree with Rafael, > > From those pictures it looks like a sugar chain - maybe 2-3 > saccharides in a row. > > HTH > > D > > David C. Briggs PhD > Father, Structural Biologist and Sceptic > > University of Manchester E-mail: > david.c.bri...@manchester.ac.uk > > http://manchester.academia.edu/DavidBriggs (v.sensible) > http://xtaldave.wordpress.com/ (sensible) > http://xtaldave.posterous.com/ (less sensible) > Twitter: @xtaldave > Skype: DocDCB > ==== > > > > On 27 October 2011 17:27, Jacob Keller wrote: >> Dear Crystallographers, >> >> In the course of a reasonably smooth refinement, all of a sudden there >> is a huge worm-hole-type blob in the electron density (see pics). Has >> anyone seen this before? Is it some effect of the refinement >> over-fitting or something? For some background, resolution is 2.2Ang >> and there are 4mol/ASU, but one of them is markedly worse in terms of >> density than the rest, and appears to be in a non-crucial part of the >> lattice, as if just sitting in one of the interstices. R/Rfree is >> ~19.5/24.5. >> >> Jacob >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> email: j-kell...@northwestern.edu >> *** >> > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] raw data deposition
Since this hasn't been brought up--there is the consideration that in 10 or more years maybe x-ray crystallography will be completely a thing of the past, with some kind of massively-superior modality taking over. Of course there is no way to bank on this, but I am wondering whether this is something to consider or not. Do we really think people will still be crystallizing proteins in 50 years, or far less, looking up structures determined in 2011? Has anybody recently used the original myoglobin structure? JPK On Thu, Oct 27, 2011 at 4:55 PM, Michel Fodje wrote: > We store raw data for two main reasons: > a) We currently use only a fraction of the information actually contained in > raw images and extraction of that fraction can be improved. Destroying the > data means > - we lose the extra information, and make future research in some areas > either impossible or more costly > - we make it more difficult to improve current data reduction methods > b) Raw data is the best way to independently validate a published structure > and prevent fraud. > > The majority of crystallographers already recognize these truths. That is why > almost all of them do keep backups of their data even after structures have > been published. > > To those still against making data public I would ask a simple question: > Would you object to providing the raw data from a published structure if such > data were available and you did not have to bear an unreasonable > inconvenience in the process? My guess is that most crystallographers are > reasonable scientists and such a "Poll" will probably result in ~100% "Yes" > and ~0% "No". I'm I wrong? > > The real issue then is how do we make the data available in such a way that > the inconvenience (if any) to all the stake-holders is reasonable. Some > great ideas have already been advanced. > > In the short-term, we could start by using the fact that synchrotron > facilities already store raw data for a period. However, a lot of data is > collected which is not published. Given the limited disk space, it may be > useful to know exactly which datasets result in a publication and should be > kept for an extended period. If a unique ID (such as the DOI suggestion) is > provided to every dataset and required during deposition/publication, then > synchrotron facilities can preserve only those datasets which have been > published after a given "grace" period. Combined with a central Meta-data > server similar to TARDIS, such a system could be developed in a relatively > short period of time, while longer term central storage ideas are worked out. > > Again the best solution is going to be one which requires the least amount of > effort from crystallographers. In fact, I can see a system in which the > experiment metadata for a PDB entry/dataset comes directly from the > synchrotron facility during deposition so that users simply provide a unique > dataset ID and the experimental details are pre-filled for them. > > Of course the above completely ignores home sources. > > > /Michel >> -Original Message- >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of D >> Bonsor >> Sent: October-27-11 3:10 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] raw data deposition >> >> Why should we store images? >> >> From most of the posts it seems to aid in software development. If that is >> the case, there should be a Failed Protein Databank (FPDB) where people >> could upload datasets which they cannot solve. This would aid software >> development and allow someone else to have ago at solving the structure. >> >> If it is for historical reasons, how can someone decide whether their >> structure is historical? I would propose that images should be uploaded for a >> protein or protein-complex that has never be solved before. That way the >> images are there if that structure does become historical. >> >> The question is not whether or not images should be uploaded but who >> would use the images that were uploaded. >> >> For example, people who use crystallography as a tool to aid in >> characterization of their protein, would probably not look at images for >> 99.5% >> of other protein datasets, and they probably would not look at images for a >> protein that is related to their own protein. They are more interested in the >> final structure. I too would probably not be interested in reprocessing and >> solving a structure again when I can easily access the final product already. > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Weird blob appears
Blob is gone--something funny happened, I guess. I went back to using the original mtz from scala, removed and replaced a bunch of waters, and no more worm! I can't really figure it out, and wish I knew exactly what happened, but I think I am just going to non-chalantly move along. Jacob On Thu, Oct 27, 2011 at 8:41 PM, Yuri Pompeu wrote: > Jacob, > By simply looking at the figures you show, it does look like you have some > type of long, maybe polymeric, molecule bound. > With that being said: > 1- It is in the symmetry axis so maybe be a little noisy there > 2-If you are in doubt about it being real or not check the density and how it > fits into your protein and (symm. related neighbours of course). If the > desity appears to be in position for some real interactions- i.e, there are > some peaks at H-bond distances of polar groups etc...- it may be real. If its > all random and through your protein atoms, probably not > Keep in mind alternate conformers > HTH > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] raw data deposition
What about a case in which two investigators have differences about what cutoff to apply to the data, for example, A thinks that Rsym of 50 should be used regardless of I/sig, and B thinks that I/sig of 2 and Rpim should be used. Usually A would cut off the data at a lower resolution than B, especially with high multiplicity, so B would love to have the images to see what extra info could be gleaned from a higher-res cutoff. Or the converse, A is skeptical of B's cutoff, and wants to see whether the data according to A's cutoff justify B's conclusion. Don't these types of things happen a lot, and wouldn't images be helpful for this? JPK
Re: [ccp4bb] raw data deposition
I don't think anyone is considering archiving all images *as a first step*! I think the obvious first step is to try to get depositors of new structures to the PDB to deposit images at the same time, which to me seems trivially easy and has a reasonably high benefit:cost ratio. Let's just do it, maybe even making it optional at first. I don't even think that in the beginning a common image format would be required--most programs can use multiple formats. Anyway, that could be addressed down the line. Jacob 2011/10/28 Boaz Shaanan : > Hi Jacob, > > There is (very) BIG difference between depositing images for deposited > structures and depositing all images ever recorded by any crystallographer on > the planet. In the case you presented, A and B can settle the issue by > looking at each other's images whether through the database or by exchanging > data on their own initiative or even by writing a note to a journal that they > completely disagree with one another and start a debate (in case one of them > is not willing to exchange images). Besides, I thought that by now there are > some standards on how data should be processed (this has been discussed on > this BB once every few months, if I'm not mistaken). Isn't that part of the > validation process that so many good people have established? Also, to the > best of my knowledge (and experience) referees (at least of some journals) > are instructed to look into those issues these days and comment about them, > aren't they? > > Cheers, > > Boaz > > > Boaz Shaanan, Ph.D. > Dept. of Life Sciences > Ben-Gurion University of the Negev > Beer-Sheva 84105 > Israel > > E-mail: bshaa...@bgu.ac.il > Phone: 972-8-647-2220 Skype: boaz.shaanan > Fax: 972-8-647-2992 or 972-8-646-1710 > > > > > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jacob Keller > [j-kell...@fsm.northwestern.edu] > Sent: Friday, October 28, 2011 5:05 PM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] raw data deposition > > What about a case in which two investigators have differences about > what cutoff to apply to the data, for example, A thinks that Rsym of > 50 should be used regardless of I/sig, and B thinks that I/sig of 2 > and Rpim should be used. Usually A would cut off the data at a lower > resolution than B, especially with high multiplicity, so B would love > to have the images to see what extra info could be gleaned from a > higher-res cutoff. Or the converse, A is skeptical of B's cutoff, and > wants to see whether the data according to A's cutoff justify B's > conclusion. Don't these types of things happen a lot, and wouldn't > images be helpful for this? > > JPK > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Archiving Images for PDB Depositions
Dear Crystallographers, I am sending this to try to start a thread which addresses only the specific issue of whether to archive, at least as a start, images corresponding to PDB-deposited structures. I believe there could be a real consensus about the low cost and usefulness of this degree of archiving, but the discussion keeps swinging around to all levels of archiving, obfuscating who's for what and for what reason. What about this level, alone? All of the accompanying info is already entered into the PDB, so there would be no additional costs on that score. There could just be a simple link, added to the "download files" pulldown, which could say "go to image archive," or something along those lines. Images would be pre-zipped, maybe even tarred, and people could just download from there. What's so bad? The benefits are that sometimes there are structures in which resolution cutoffs might be unreasonable, or perhaps there is some potential radiation damage in the later frames that might be deleterious to interpretations, or perhaps there are ugly features in the images which are invisible or obscure in the statistics. In any case, it seems to me that this step would be pretty painless, as it is merely an extension of the current system--just add a link to the pulldown menu! Best Regards, Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Archiving Images for PDB Depositions
Pilot phase, opt-in--eventually, mandatory? Like structure factors? Jacob On Mon, Oct 31, 2011 at 11:29 AM, Adrian Goldman wrote: > I have no problem with this idea as an opt-in. However I loathe being forced > to do things - for my own good or anyone else's. But unless I read the tenor > of this discussion completely wrongly, opt-in is precisely what is not being > proposed. > > Adrian Goldman > > Sent from my iPhone > > On 31 Oct 2011, at 18:02, Jacob Keller wrote: > >> Dear Crystallographers, >> >> I am sending this to try to start a thread which addresses only the >> specific issue of whether to archive, at least as a start, images >> corresponding to PDB-deposited structures. I believe there could be a >> real consensus about the low cost and usefulness of this degree of >> archiving, but the discussion keeps swinging around to all levels of >> archiving, obfuscating who's for what and for what reason. What about >> this level, alone? All of the accompanying info is already entered >> into the PDB, so there would be no additional costs on that score. >> There could just be a simple link, added to the "download files" >> pulldown, which could say "go to image archive," or something along >> those lines. Images would be pre-zipped, maybe even tarred, and people >> could just download from there. What's so bad? >> >> The benefits are that sometimes there are structures in which >> resolution cutoffs might be unreasonable, or perhaps there is some >> potential radiation damage in the later frames that might be >> deleterious to interpretations, or perhaps there are ugly features in >> the images which are invisible or obscure in the statistics. >> >> In any case, it seems to me that this step would be pretty painless, >> as it is merely an extension of the current system--just add a link to >> the pulldown menu! >> >> Best Regards, >> >> Jacob Keller >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> email: j-kell...@northwestern.edu >> *** >> > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Off topic: Kendrew Model
Maybe you could refine it using our new-fangled methods to improve the model? (Couldn't resist such irony!) Jacob On Tue, Nov 1, 2011 at 9:34 AM, Katherine Sippel wrote: > Hi all, > > I'm going to interject into the middle of this rousing though protracted > debate to pick your brains. I am in possession of a rather large and intact > brass scale Kendrew model (sans mirrors). Due to facility restructuring we > no longer have room for it. I have approached the local health science and > natural science museums but have gotten nothing but the run around. This > amazing model is in need of a forever home and I'm stumped as far as > alternative ideas. I am seriously considering suspending a Mars bars in the > sugar binding cleft, calling it MBP, and trying to spin it to the art museum > as a modernist piece commenting on the diets in Western civilization. Either > that or putting it in my dining room if I can get it in the door. Any > suggestions would be appreciated. > > Cheers, > > Katherine > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Best route from x-files to scala
Dear Crystallographers, I would like to transfer data from x-files (HKL2000) into scala, and think that the best way to do it is to tell HKL "no merge original index." Is that right, or is "no merge include partials" better? Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Installation of CCP4 under Windows 7
Hmm, works fine for me. Maybe disable user account control? Jacob On Thu, Nov 3, 2011 at 7:54 AM, Jan Dohnalek wrote: > does not seem to create anything "runnable" - please any experience here? > > I downloaded the latest Windows package "all users" - type. Installed under > admin (as it would not let me otherwise). > Now as a user I cannot start the interface no matter what I try. There is no > ccp4.setup file ... > > Jan > > > -- > Jan Dohnalek, Ph.D > Institute of Macromolecular Chemistry > Academy of Sciences of the Czech Republic > Heyrovskeho nam. 2 > 16206 Praha 6 > Czech Republic > > Tel: +420 296 809 390 > Fax: +420 296 809 410 > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Can I combine selenoMet data and MR model to solve the phase problem?
How about Phaser with partial MR structure? JPK On Tue, Nov 15, 2011 at 3:56 PM, Feng Guo wrote: > Hi, there, > > Maybe someone asked this question before, but I couldn't find it in the > archive. > > We use the native data to do molecular replacement before, but only part of > the model fit the density. After collect a new set of selenoMet data, we try > to use it to solve the phase, it solve some of the phase problem other than > the MR, but still not complete. Is there anyway that I can somehow combine > the two phases together? Thank you. > > Best, > > Feng > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Distinguish NH4 from Na?
Dear Crystallographers, I have crystals containing 666mM NH4 and 540mM Na, and there appears to be a "water" which is only about 2.2 Ang from some polar atoms. It is currently reasonably happy as a Na, but is there any reasonable way to decide which cation is there? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] odd arginines
I also have seen similar. I was thinking it was potentially some kind of radiation damage? Is there a good paper which examines what chemistries are seen in rad damage? Jacob On Thu, Nov 17, 2011 at 5:39 AM, Eleanor Dodson wrote: > Yes - I have seen something similar at a lower resolution, but very ugly! I > tried to model it as a solvent molecule - possible but not too convincing.. > > Cl should give an anomalous signal - try a DANO map and see what it shows.. > Eleanor > > > On 11/17/2011 11:09 AM, Dean Derbyshire wrote: >> >> Hi all, >> Has anyone observed 'odd' arginine residues, missing the NH1 atom; and >> possibly related.. very close Chlorine-arginine (NH1 again) distances. >> >> I have a 1.3Å structure and am having trouble getting my head around some >> very odd density features. >> >> I have 2 molecules in the assym. with the equivalent arg residues showing >> the same odd feature. >> Seems to be a chlorine binding site - typical mix of hydrophobic and >> proton rich side chains - but, the NH1 of the particular arginine residue >> concerned (as I say in both NCS copies) seems to have only 50% occupancy and >> the chlorine appears to have a 2nd distinct position closer to the arginine, >> such that if the NH1 atom were present it would be only 2Å away! >> >> ? >> >> Cheers in advance >> >> Dean >> >> >> > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Combining big MTZs
Dear Crystallographers, I am getting an error when I try to merge two mtz's from mosflm, one with 180 and one with 360 frames, each from different but similar crystals--see below. I can't imagine this really exceeds the max number of records, so what am I doing wrong? Additionally but related, what is the optimal procedure in CCP4 for combining data from two similar crystals? JPK #CCP4I VERSION CCP4Interface 2.1.0 #CCP4I SCRIPT LOG sortmtz #CCP4I DATE 18 Nov 2011 09:26:28 #CCP4I USER 'UNKNOWN' #CCP4I PROJECT NatVSulP #CCP4I JOB_ID 26 #CCP4I SCRATCH C:/Ccp4Temp #CCP4I HOSTNAME chloe #CCP4I PID 5396 SORTMTZ ### ### ### ### CCP4 6.2: SORTMTZ version 6.2 : 06/09/05## ### User: Jacob Run date: 18/11/2011 Run time: 09:26:28 Please reference: Collaborative Computational Project, Number 4. 1994. "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. D50, 760-763. as well as any specific reference in the program write-up. Contents Command Input Input File Details Output File Details Header Information for Output MTZ File Command Input ASCEND/DESCEND SORT KEYS Data line--- ASCEND Data line--- H K L M/ISYM BATCH Data line--- "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz" Input File Details OPENED INPUT MTZ FILE Logical Name: C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz Filename: C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz * Title: Untitled * Base dataset: 0 HKL_base HKL_base HKL_base * Number of Datasets = 1 * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength: 1 New New New 82.0600 82.0600 159.2500 90. 90. 90. 0.97856 * Number of Columns = 18 * Number of Reflections = 1526614 * Missing value set to NaN in input mtz file * Number of Batches = 360 * Column Labels : H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH LP MPART FLAG BGPKRATIOS * Column Types : H H H Y B J Q J Q R R R R R R I I R * Associated datasets : 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 82.0600 82.0600 159.2500 90. 90. 90. * Resolution Range : 0.000300.15998 ( 58.026 - 2.500 A ) * Sort Order : 0 0 0 0 0 * Space group = 'P43212' (number 96) Spacegroup information obtained from library file: Logical Name: SYMINFO Filename: C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib 5 sort keys, in columns1 2 3 4 5 Output File Details 1526614 records read from file1 Data line--- "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz" Input File Details OPENED INPUT MTZ FILE Logical Name: C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz Filename: C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_chs_raw_batched_up_to_401.mtz * Title: [No title given] * Base dataset: 0 HKL_base HKL_base HKL_base * Number of Datasets = 1 * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength: 1 New New New 82.3400 82.3400 161.4500 90. 90. 90. 0.97940 * Number of Columns = 18 * Number of Reflections = 536433 * Missing value set to NaN in input mtz file * Number of Batches = 180 * Column Labels : H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH LP MPART FLAG BGPKRATIOS * Column Types : H H H Y B J Q J Q R R R R R R I I R * Associated datasets : 0 0 0 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) 82.3400 82.3400 161.4500 90. 90. 90. * Resolution Range : 0.000150.16000 ( 82.340 - 2.500 A ) * Sort Order : 1 2 3 4 5 * Space group = 'P43212' (number 96) Too many records SORTMTZ failed to release record to sort procedure, status = 1 SORTMTZ: Sorting failed Times: User: 0.0s System:0.0s Elapsed: 0:05 *** * Information from CCP4Interface script *** The program run with command: sortmtz HKLOUT "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_comb_raw.mtz" has failed with error message SORTMTZ: Sorting
Re: [ccp4bb] crystal orientation during data collection
Generally speaking, don't we agree that "planned" or "rational" is better than "random?" (Having trouble understanding the argument for randomness here...) Jacob On Fri, Nov 18, 2011 at 9:40 AM, Sanishvili, Ruslan wrote: > Depending on the crystal shape, “random orientation” is not always random. > Many crystals have tendencies of sitting themselves in one predominant > posture in the mount. Compounding this, many experimenters have tendencies > of rotating the mount into a specific orientation when centering. Then > crystal orientation ends up being not random at all, so understanding it’s > true orientation as my neighbor Frank suggests can be highly beneficial. > > Cheers, > > N. > > > > Ruslan Sanishvili (Nukri), Ph.D. > > GM/CA-CAT > Biosciences Division, ANL > 9700 S. Cass Ave. > Argonne, IL 60439 > > Tel: (630)252-0665 > Fax: (630)252-0667 > rsanishv...@anl.gov > > > > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank > von Delft > Sent: Friday, November 18, 2011 2:27 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] crystal orientation during data collection > > > > I believe you achieve completeness more quickly (fewer crystals) if you just > take random orientations. At least, that's what I learnt from Dave Stuart. > phx > > > > On 18/11/2011 04:20, Frank Murphy wrote: > > Yanwu, > > > > I surmise from your question that you are inquiring how to go about > collecting from many crystals optimally. Merging data ex post facto is a > totally different kettle of fish. > > > > In my opinion, the most robust way to go about this is to use a kappa > goniometer as Jim suggested (I am most familiar with the MK3). Since you > intend to collect from many crystals, align the first and all subsequent > crystals to the same easily attainable (or seemingly so) orientation, and > then collect the sweep suggested by your data collection strategy program of > choice. > > > > To achieve this at NE-CAT, we have a GUI-based system that used STAC for > orientation determination and BEST for strategy generation. As Jim > suggested, more options than STAC exist. > > > > If anyone is unable to get to a kappa goniometer, they can employ Mosflm or > XDS (Xplan) to generate strategies for data collection from a crystal taking > into account previously collected data. This is not nearly as robust a > solution, but is a workable substitute (and also automated at NE-CAT). > > > > I know there are other ways to achieve similar results, but I have suggested > the methods I am most familiar with... > > > > > > Yours, > > Frank Murphy > > > > > > Begin forwarded message: > > From: > > yanwu huo > > Date: > > November 17, 2011 4:00:06 PM CST > > To: > > CCP4BB@JISCMAIL.AC.UK > > Subject: [ccp4bb] crystal orientation during data collection > > Reply-To: > > yanwu huo > > Hi, > I worked on a crystal sensitive to radiation damage, So I need to merge many > crystal to obtain complete dataset, Does anyone know such program that can > tell crystal orientation after first frame exposure. > Thank you in advance. > > > -- > Thank you very much and all the best, > > Yanwu Huo > Postdoctoral Associate > Department of Molecular Biology and Genetics > Cornell University > Ithaca, NY, 14853 > Email:yh...@cornell.edu > > > > > > > > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Combining big MTZs
Okay, it seems there is utility to Pointless--problem solved! Maybe developers should consider upping the limit, though? Thanks very much, Jacob On Fri, Nov 18, 2011 at 9:45 AM, Phil Evans wrote: > There's a limit set in CCP4/lib/src/sorting_main.f > > C NMAX_MEM increased from 1600 to 3200 > PARAMETER (NMAX_MEM = 3200) > > which you could change and recompile (:-)) > > You can combine unmerged files in Pointless, which is usually the best > method, but that reads everything into memory so you may well hit system > limits unless you have a 64-bit system with lots of memory (and a 64-bit > build) > > Phil > > > > On 18 Nov 2011, at 15:36, Jacob Keller wrote: > >> Dear Crystallographers, >> >> I am getting an error when I try to merge two mtz's from mosflm, one >> with 180 and one with 360 frames, each from different but similar >> crystals--see below. I can't imagine this really exceeds the max >> number of records, so what am I doing wrong? Additionally but related, >> what is the optimal procedure in CCP4 for combining data from two >> similar crystals? >> >> JPK >> >> >> >> #CCP4I VERSION CCP4Interface 2.1.0 >> #CCP4I SCRIPT LOG sortmtz >> #CCP4I DATE 18 Nov 2011 09:26:28 >> #CCP4I USER 'UNKNOWN' >> #CCP4I PROJECT NatVSulP >> #CCP4I JOB_ID 26 >> #CCP4I SCRATCH C:/Ccp4Temp >> #CCP4I HOSTNAME chloe >> #CCP4I PID 5396 >> >> >> >> >> SORTMTZ >> >> >> ### >> ### >> ### >> ### CCP4 6.2: SORTMTZ version 6.2 : 06/09/05## >> ### >> User: Jacob Run date: 18/11/2011 Run time: 09:26:28 >> >> >> Please reference: Collaborative Computational Project, Number 4. 1994. >> "The CCP4 Suite: Programs for Protein Crystallography". Acta Cryst. >> D50, 760-763. >> as well as any specific reference in the program write-up. >> >> >> >> Contents >> >> Command Input >> Input File Details >> Output File Details >> Header Information for Output MTZ File >> >> >> >> Command Input >> > href="C:\CCP4-Packages\ccp4-6.2.0\html/sortmtz.html#ascend">ASCEND/DESCEND >> SORT >> KEYS >> >> Data line--- ASCEND >> Data line--- H K L M/ISYM BATCH >> Data line--- >> "C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz" >> >> >> >> Input File Details >> >> >> OPENED INPUT MTZ FILE >> Logical Name: >> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz >> Filename: >> C:/Users/Jacob/Desktop/Dallos_Lab/Analysis/NatVSulP/lo_res_natvsulp_jpk_raw.mtz >> >> * Title: >> >> Untitled >> >> * Base dataset: >> >> 0 HKL_base >> HKL_base >> HKL_base >> >> * Number of Datasets = 1 >> >> * Dataset ID, project/crystal/dataset names, cell dimensions, wavelength: >> >> 1 New >> New >> New >> 82.0600 82.0600 159.2500 90. 90. 90. >> 0.97856 >> >> * Number of Columns = 18 >> >> * Number of Reflections = 1526614 >> >> * Missing value set to NaN in input mtz file >> >> * Number of Batches = 360 >> >> * Column Labels : >> >> H K L M/ISYM BATCH I SIGI IPR SIGIPR FRACTIONCALC XDET YDET ROT WIDTH >> LP MPART FLAG BGPKRATIOS >> >> * Column Types : >> >> H H H Y B J Q J Q R R R R R R I I R >> >> * Associated datasets : >> >> 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 >> >> * Cell Dimensions : (obsolete - refer to dataset cell dimensions above) >> >> 82.0600 82.0600 159.2500 90. 90. 90. >> >> * Resolution Range : >> >> 0.00030 0.15998 ( 58.026 - 2.500 A ) >> >> * Sort Order : >> >> 0 0 0 0 0 >> >> * Space group = 'P43212' (number 96) >> >> >> Spacegroup information obtained from library file: >> Logical Name: SYMINFO Filename: >> C:\CCP4-Packages\ccp4-6.2.0\lib\data\syminfo.lib >> >> 5 sort keys, in columns 1 2 3 4 5 >>
Re: [ccp4bb] Movements of domains
Just to clarify: I think the question is about the mathematical sense of "significance," and not the functional or physiological significance, right? If I understand the question correctly, wouldn't the reasoning be that admittedly each atom in the model has a certain positional error, but all together, it would be very unlikely for all atoms to be skewed in the same direction? Jacob On Mon, Nov 21, 2011 at 4:04 PM, Filip Van Petegem wrote: > Dear crystallographers, > I have a general question concerning the comparison of different > structures. Suppose you have a crystal structure containing a few domains. > You also have another structure of the same, but in a different condition > (with a bound ligand, a mutation, or simply a different crystallization > condition,...). After careful superpositions, you notice that one of the > domains has shifted over a particular distance compared to the other > domains, say 1-1.5 Angstrom. This is a shift of the entire domain. Now > how can you know that this is a 'significant' change? Say the overall > resolution of the structures is lower than the observed distance (2.5A for > example). > Now saying that a 1.5 Angstrom movement of an entire domain is not relevant > at this resolution would seem wrong: we're not talking about some electron > density protruding a bit more in one structure versus another, but all of > the density has moved in a concerted fashion. So this would seem 'real', > and not due to noise. I'm not talking about the fact that this movement > was artificially caused by crystal packing or something similar. Just for > whatever the reason (whether packing, pH, ligand binding, ...), you simply > observe the movement. > So the question is: how you can state that a particular movement was > 'significantly large' compared to the resolution limit? In particular, what > is the theoretical framework that allows you to state that some movement is > signifcant? This type of question of course also applies to other methods > such as cryo-EM. Is a 7A movement of an entire domain 'significant' in a > 10A map? If it is, how do we quantify the significance? > If anybody has a great reference or just an individual opinion, I'd like to > hear about it. > Regards, > Filip Van Petegem > > -- > Filip Van Petegem, PhD > Assistant Professor > The University of British Columbia > Dept. of Biochemistry and Molecular Biology > 2350 Health Sciences Mall - Rm 2.356 > Vancouver, V6T 1Z3 > > phone: +1 604 827 4267 > email: filip.vanpete...@gmail.com > http://crg.ubc.ca/VanPetegem/ > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Movements of domains
I am curious how all of this can be more than splitting hairs, i.e., under what conditions can this 1Ang domain motion mean something biologically significant? Proteins are pretty flexible, after all, especially between domains. JPK On Mon, Nov 21, 2011 at 6:41 PM, James Stroud wrote: > On Nov 21, 2011, at 5:23 PM, James Stroud wrote: >> except that you use Euclid's formula to calculate the distances in higher >> dimensions > > I meant to say "Euclidian distance". "Euclid's formula" has a specific > meaning that is different. > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] dark progression of radiation damage
I understand that absorbed dose increases with presence of heavy atoms, but I don't understand why that should play a role in damaging the crystal, as heavy atoms such as in cacodylate should probably usually not be near enough to protein atoms to cause problems. At 100K, isn't it true that secondary radiation damage plays little role if any? So the only problem I can think of is the case when the cacodylate molecule happens to be within "striking distance" of a protein atom when it turns into a radical (not sure what that distance would be). This should be relatively rare in, say, 55mM cacodylate, when there is only ~1 cacodylate for every 1000 waters, no? Has there been an empirical study comparing similar crystals of the same protein +/- solvent heavy atoms? I guess derivatives are the obvious example--but real derivatives always have ordered, occupied sites. Jacob On Wed, Nov 23, 2011 at 12:28 PM, Elspeth Garman wrote: > Also, cacodylate contains arsenic which is heavy, and thus has a much larger > X-ray absorption cross section than do buffers constituted of lighter atoms. > There is therefore a bigger dose (Joules/kg of crystal) absorbed with > cacodylate in the buffer than there would be without it (and no extra > diffraction strength), so that is another very good reason to avoid it, or to > buffer exchange it out before the diffraction experiment. > > Elspeth > > -Original Message- > From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jim > Pflugrath > Sent: 23 November 2011 18:11 > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] dark progression of radiation damage > > Any cacodylate buffer will cause gas to be produced. One only needs a minute > exposure on a modern home lab source to see this happening. I suggest that > everyone avoid cacodylate in their crystallization drops that end up being > exposed to X-rays. > > Jim > > > From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, > Ruslan [rsanishv...@anl.gov] > Sent: Wednesday, November 23, 2011 11:49 AM > To: CCP4BB@JISCMAIL.AC.UK > Subject: Re: [ccp4bb] dark progression of radiation damage > > I think I need to clarify couple of things in my recent post about > "exploding" crystals during re-mounting by a robot. First, it was a bit > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Anomalous signal for chlorides
Not that Phaser needs my approval, but I recently did exactly what Randy recommended and it really found basically all of the S and Cl sites, with data at resolution 2.2 Ang and wavelength 0.979 Ang, too. I also played a bit with the sigma cutoff for adding new sites so that the stronger sites (Se in my case) are found but the weaker ones not. Also, don't forget to click the "output LLG map coefficients" option to get the right columns in your mtz. Jacob On Sat, Nov 26, 2011 at 6:13 AM, Randy J. Read wrote: > Dear Yuri, > > In addition to the other good options that have been presented, you can use > the log-likelihood gradient maps in Phaser to find anomalous scatterers. We > find this to be very sensitive, and it has the advantage of being iterative > (i.e. when you find some anomalous scatterers, this improves your model and > thus the sensitivity for finding additional sites). > > When you're starting from a refined model, we think it is best to look for > purely imaginary scatterers to add to the real scattering in your model. In > the ccp4i GUI, choose the "SAD with molecular replacement partial structure" > mode, provide the data (with F+ and F-), the wavelength, and the current > model, then turn on LLG-map completion and select the "Complete with purely > anomalous scatterer" option. > > If you want to see the initial LLG map, set the number of completion cycles > to 0 and turn on the option to output log-likelihood-gradient map > coefficients. Open these in coot as a difference map, choosing the columns > FLLG_AX and PHLLG_AX. If you let Phaser complete the sites, then the final > LLG map should be nearly flat and you have to look at the PDB file > containing the sites that it found. > > Best wishes, > > Randy Read > On Nov 26 2011, Yuri Pompeu wrote: > >> Hi Boaz, Yes indeed, the phosphate group of the molecule looks quite >> beautiful at 1.17A and it has a really big peak 18sigma! Is there a utility >> for calculating anomalous maps, or is it simply an option in the refinement >> program? >> > > -- > Randy J. Read > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Superpositions: Deviation by Residue
Dear Crystallographers, is there a ccp4 program--or otherwise--which can compute ca-ca distances of corresponding residues between two superposed structures? Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Superpositions: Deviation by Residue
Let me refine my question (sorry for my lack of clarity): is there a program that will output the distances between the corresponding ca's of a superposition on a residue-by-residue basis, and not just a global RMSD value (doubtless these numbers are part of the superposition algorithm itself)? I want to plot these values as a function of residue number to show which parts of the structures deviate more or less from each other. Jacob
Re: [ccp4bb] Superpositions: Deviation by Residue
Dear Crystallographers, Thanks very much for all who responded to my request. Below is a compiled list of the various ways to skin this crystallographic cat! Jacob -Moleman ca plot distance-CCP4's superpose-distance matrix analysis, e.g. http://www.csb.yale.edu/userguides/datamanip/ddmp/ddmp_descrip.html, or other programs-Lsqkab, with the "deltas" card-If you use the MatchMaker tool in UCSF Chimera to make the superposition, it has an option to show the corresponding sequence alignment. The sequence alignment will have an "RMSD" header running across the top, which is a bar graph of the RMSD values. You can the alignment's Headers->Save... menu item to save the numerical values to a file if you want. OR If you already have the structures superimposed on your own, you can use Chimera's Match->Align tool to create a superposition-based sequence alignment, and do the same thing with its RMSD header.-Coot prints this information to the terminal, so you can start coot and 'tee' its output into a file.-Various fortran/homebrew programs available from individuals-LGA server: http://proteinmodel.org/AS2TS/LGA/lga.html On Tue, Nov 29, 2011 at 3:44 AM, Tim Gruene wrote: > -BEGIN PGP SIGNED MESSAGE- > Hash: SHA1 > > Dear Jacob, > > Coot prints this information to the terminal, so you can start coot and > 'tee' its output into a file. > > Tim > > On 11/28/2011 11:53 PM, Jacob Keller wrote: >> Let me refine my question (sorry for my lack of clarity): is there a >> program that will output the distances between the corresponding ca's >> of a superposition on a residue-by-residue basis, and not just a >> global RMSD value (doubtless these numbers are part of the >> superposition algorithm itself)? I want to plot these values as a >> function of residue number to show which parts of the structures >> deviate more or less from each other. >> >> Jacob >> > > - -- > - -- > Dr Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > GPG Key ID = A46BEE1A > > -BEGIN PGP SIGNATURE- > Version: GnuPG v1.4.10 (GNU/Linux) > Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ > > iD8DBQFO1KmAUxlJ7aRr7hoRAjy0AJ0WepnWXxMHVFfAE1oSX0rQ5BV4zQCggo/N > OQ67cZNE7jMPZyXL4v1zOjE= > =JXFJ > -END PGP SIGNATURE- -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Crystallographic and Physiologic but not Solution Oligomers
Dear Crystallographers, does anyone have any nice examples/references of proteins which form demonstrably physiologically-relevant oligomers in crystals, but which do not appear to do so in solution? I am thinking particularly that domains of membrane proteins which oligomerize primarily through their TM domains would be subject to this, but all comers are certainly welcome... Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Withdrawal of subscription
Just you wait--he'll be back...won't he? On Tue, Nov 29, 2011 at 11:11 PM, James Stroud wrote: > With such a nice parting letter, we find it disheartening to let you go. > > James > > On Nov 29, 2011, at 7:44 PM, debanjan choudhuri wrote: > > To whom it may concern, > I am writing this in request to the cancellation of my subscription to > CCP4BB mail. > It was indeed a very rewarding and a knowledgeable experience to be a part > of this family. > With regards > Debanjan Choudhuri > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Reference for Resolution Cutoffs
Dear Crystallographers, I hate to broach this subject again due to its wildly controversial nature, but I was wondering whether there was any reference which systematically analyses resolution cutoffs as a function of I/sig, Rmerge, Rmeas, Rpim, etc. I strongly dislike Rmerge/Rcryst for determining cutoffs, for obvious reasons--and especially for datasets of higher multiplicity--but nevertheless it is a ubiquitously-reported statistic, and one therefore has to make an argument against using it. Hopefully this could be done by pointing to a definitive reference--or am I stuck with a convention versus the truth? Maybe the ACA or similar could make a public anti-Rmerge proclamation about it, to make it easier for us? Also, more generally, it seems that the refinement programs are now better able to discount lousy high-res data, so why not leave the choice to those programs, and just give them all of the data to the edge of the detector, especially since our computational and data storage capacities are now completely sufficient for that? One could then use some other metric for the goodness of the structure, such as what bin crosses the Rfree = 40% mark or something. One could push this even further and, as has been mentioned on this list before, just give the refinement program all of the intensities of the voxels in the 3D dataset? Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Crystallographic and Physiologic but not Solution Oligomers
Thank you to everyone who responded to this query--here is a list of proteins which oligomerize in crystals and in vivo, but not appreciably in solution: PknB--thanks to Christine Gee and Nat Echols GluR ligand binding domains--thanks to Mark Mayer and Andrew Plested Cadherins--thanks to Rachelle Gaudet EGFR kinase--thanks to Markus Seeliger FokI nuclease--thanks to Artem Evdokimov NKR-P1 receptors--thanks to Jan Dohnalek Insulin--thanks to Eleanor Dodson All the best, Jacob Keller -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Reference for Resolution Cutoffs
Hi Ethan, thanks for pushing me to clarify--see below. >> I hate to broach this subject again due to its wildly controversial >> nature, but I was wondering whether there was any reference which >> systematically analyses resolution cutoffs as a function of I/sig, >> Rmerge, Rmeas, Rpim, etc. I strongly dislike Rmerge/Rcryst for >> determining cutoffs, for obvious reasons--and especially for datasets >> of higher multiplicity--but nevertheless it is a ubiquitously-reported >> statistic, and one therefore has to make an argument against using it. > > What is your question, exactly? The question is: "is there a reference in which Rmerge has been thoroughly, clearly, and authoritatively discredited as a data evaluation metric in the favor of Rmeas, Rpim, etc., and if so, what is that reference?" > I don't follow the logic that because a statistic is reported, one > must therefore argue against it. Let me say it clearer: when there is a conventional, standardized method that one wants to abandon in favor of a better method, in practice one has to make an argument for the new one and against the old one. This is in contrast to continuing to use the conventional method, which, even if apodictically surpassed by the newer method, de facto needs no justification. So, in the current example, if you want to use Rmeas or Rpim and not even report Rsym/merge, it will ruffle feathers, even though the former is certainly superior. Sorry for the confusion, Jacob *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Reference for Resolution Cutoffs
Yes, that's a good point--you can't really discredit Rmerge (it's just a mathematical expression, after all, which must be translated vis a vis redundancy), but you can show that the other R's are pleasanter ways to represent the data. Jacob On Tue, Dec 6, 2011 at 3:02 PM, Anastassis Perrakis wrote: > Also > > Nat Struct Biol. 1997 Apr;4(4):269-75. > Improved R-factors for diffraction data analysis in macromolecular > crystallography. > Diederichs K, Karplus PA. > > But none of these are in any way 'discrediting' Rmerge, they are just > proposing more statistically sound alternatives. That is not the same ... > > A. > > > On 6 Dec 2011, at 21:44, Ed Pozharski wrote: > >> On Tue, 2011-12-06 at 13:43 -0600, Jacob Keller wrote: >>> The question is: "is there a reference in which Rmerge has been >>> thoroughly, clearly, and authoritatively discredited as a data >>> evaluation metric in the favor of Rmeas, Rpim, etc., and if so, what >>> is that reference?" >>> >> >> Aren't these sufficient? >> >> Manfred Weiss & Rolf Hilgenfeld, "On the use of the merging R factor as >> a quality indicator for X-ray data", J.Appl.Cryst. 30, 203-205 (1997) >> >> Manfred Weiss, "Global Indicators of X-ray data quality" J.Appl.Cryst. >> 34, 130-135 (2001) >> >> -- >> Oh, suddenly throwing a giraffe into a volcano to make water is crazy? >> Julian, King of Lemurs -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] artificial tetramer
Can you clarify your reason for doing this? JPK On Mon, Dec 12, 2011 at 3:36 PM, Fred wrote: > Hi James, > In my first post "arbitrary orientation into the cell" only means not > parallel to any crystallographic axis, which would simplify things very > much. I want to apply the 4-fold axis to the protein coordinates. If I have > a cell and therefore an origin, I can take a point at any distance of the > origin, pass a vector/axis through it and take the 3 others molecules by > symmetry. That's trivial, given the point, the orientation and the property > of the rotation. Don't know which program to use. > Regards, > Fred > > > > Em 12-12-2011 19:18, James Stroud escreveu: > >> This is not trivial. Assuming an arbitrary origin, the simplest 4-fold >> symmetry operation (4-fold rotation) has 5 free parameters (translation >> along the symmetry axis is irrelevant). The biggest problem is determining >> the values for these parameters. For example, once you apply the symmetry, >> your molecule may clash with its symmetry mates or not even contact them. >> And even if you solve this latter problem automatically (which is not >> trivial because of irregularity), that leaves a net of 3 parameters >> describing the orientation of the protomer. >> >> James >> >> >> >> On Dec 12, 2011, at 1:34 PM, Fred wrote: >> >>> Hi Tim, >>> Thanks for your message and sorry if I wasn't clear. I don't have neither >>> the axis orientation nor the rotation matrix. I would like to create them >>> but don't know how and which program to use. Theoretically a have to create >>> a axis (vector) at some distance of the molecule into the cell and give it >>> the 4-fold propriety. Quite simple, but don't which program to use. >>> Regards, >>> Fred >>> >>> >>> Em 12-12-2011 18:23, Tim Gruene escreveu: -BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Hello Fred, if you know the rotation matrix, you can use pdbset with its 'rotate' keyword. It is not clear to me whether or not you have the rotation matrix or how you define rotation. Cheers, Tim On 12/12/2011 08:49 PM, Fred wrote: > > Hi List, > I would like to build an artificial tetramer from a monomer PBD file. > All that I have is the coordinates it self with CRYST/CELL information > cards. The artificial 4-fold axis has an arbitrary orientation into the > cell. I mean, its not parallel to any crystallographic axis and have to > be at a certain distance of the molecule. This sounds conceptually > simple, but I would like to do that in batch mode for hundreds of > PDB's. > Could someone, please, tell me the easiest way/program to do that? > Thanks in advance, > Fred > - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.10 (GNU/Linux) Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ iD8DBQFO5mKwUxlJ7aRr7hoRAsyuAKDfnH50sG/EuKiFz7tCzgTUtnZrdQCg3zSn cqs8GHOu5M3JQahA/CofR1k= =tDUj -END PGP SIGNATURE- -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] symmetry for ages 6 and up
Does anyone know where one can acquire some Penrose tiles? I think they'd be great toys as well, and drive you a little bonkers. Maybe a kitchen/bathroom floor made from them? Jacob On Tue, Dec 13, 2011 at 5:03 PM, VAN RAAIJ , MARK JOHAN wrote: > reminds me of these symmetric 2D P3 lizards: > http://www.worldofescher.com/store/Z51.html > I bought the RGB-coloured set/puzzle after visiting an Escher exhibition and > sometimes use them in crystallography/symmetry teaching. > Nice to make the students assemble them and then decide on the symmetry > operator, unit cell and asymmetric unit. > > > Quoting Phoebe Rice: > >> Hi all, >> For those who teach xtallography - we found some plastic turtles that can >> be snapped together in an amazing variety of space groups. Worked well in a >> workshop for our students, so I thought I'd share the shopping tip. They're >> called Reptangles, and we got them from Amazon. >> >> http://www.amazon.com/Fat-Brain-Toys-FA042-1-Reptangles/dp/B00392NSQ4 >> >> Have fun! >> >> Phoebe >> >> = >> Phoebe A. Rice >> Dept. of Biochemistry & Molecular Biology >> The University of Chicago >> phone 773 834 1723 >> >> http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 >> http://www.rsc.org/shop/books/2008/9780854042722.asp >> > > > > Mark J van Raaij > Laboratorio M-4 > Dpto de Estructura de Macromoléculas > Centro Nacional de Biotecnología - CSIC > c/Darwin 3, Campus Cantoblanco > 28049 Madrid > tel. 91 585 4616 > email: mjvanra...@cnb.csic.es -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Pore Dimension Convention
Dear Crystallographers, is there a convention for denoting/measuring pore sizes in protein structures? Maybe inter-atom distances minus van der Waals radii? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] From non-twinned to twinned?
Need more info: how many degrees per frame? Also, on integration, do various stats change over the 'sets? JPK On Wed, Jan 4, 2012 at 7:42 AM, Zhiyi Wei wrote: > Dear all, > > I recently collected a dataset (~2000 frames) from a single crystal. > If merge first 600 frames (sca1) or last 600 frames (sca2), Rmerge > values from scalepack seem to be ok (~10%) though rejection ratios are > high (~5%). But if I merge all frames together, Rmerge value goes up > to ~20% and rejection is extremely high (~20%). Then, I checked sca1 > and sca2 by xtriage in phenix. Surprisingly, the logfiles told me that > sca1 is no twining while sca2 is very likely to be twinned. I never > met this case before. So, I am wondering if it is possible from a > non-twinned structure to a twinned structure just due to radiation > damage. If the answer is yes, does it mean that I should not collect a > large number of frames to amplify anomalous signals by using this > crystal? > > Thanks a lot! > > Best, > Zhiyi -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Molecular Transform Superimposed on a Dataset
Dear Crystallographers, has anyone come across a figure showing a normal diffraction image, and then next to it the equivalent molecular transform, perhaps with one image as phases and one as amplitudes? Seems like it would be a very instructional slide to have to explain how crystallography works (I know about Kevin Cowtan's ducks and cats--I was looking for approximately the same but from protein or NA molecules.) I don't think I have ever seen an actual molecular transform of a protein or NA molecule. All the best, Jacob -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
Actually, as a way to make this type of figure, I think there are programs which output simulated diffraction images, so perhaps I could just input a .pdb file with some really huge (fake) cell parameters (10,000 Ang?), and then the resulting spots would be really close together and approximate the continuous molecular transform. I think this would amount to the same thing as the molecular transform of the model itself--am I right? Does anyone know which software outputs simulated diffraction images? Jacob On Fri, Jan 6, 2012 at 10:25 AM, Jacob Keller wrote: > Dear Crystallographers, > > has anyone come across a figure showing a normal diffraction image, > and then next to it the equivalent molecular transform, perhaps with > one image as phases and one as amplitudes? Seems like it would be a > very instructional slide to have to explain how crystallography works > (I know about Kevin Cowtan's ducks and cats--I was looking for > approximately the same but from protein or NA molecules.) I don't > think I have ever seen an actual molecular transform of a protein or > NA molecule. > > All the best, > > Jacob > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] R-Sym statistics in solved structures
Also R cryst is sometimes used for the same number, I think (of course
there are historical reasons for the different terms, but...).
JPK
On Mon, Jan 9, 2012 at 8:47 AM, Ed Pozharski wrote:
> On Mon, 2012-01-09 at 10:28 +, Guillaume Gotthard wrote:
>> Is there a mean to obtain statistics about R-Sym for deposited
>> structures databases ?
>
> 1. It's actually quite easy to do on your own if you want. This
> one-liner will get you the Rsym
>
> wget http://www.rcsb.org/pdb/files/.pdb?headerOnly=YES -O - -q |
> grep 'R SYM ' | cut -d: -f 2
>
> just replace the with the uppercase pdb id. I don't know what kind
> of statistics you want, but assuming that you are after resolution
> dependence, you can do this
>
> wget http://www.rcsb.org/pdb/files/.pdb?headerOnly=YES -O - -q |
> egrep 'R SYM |RESOLUTION RANGE HIGH \(' | cut -d: -f 2 | tr -d '\n' |
> tr -s ' '; echo
>
> 2. Beware that for probably the majority of the records you will get
> NULL as the answer. Using "R MERGE" instead of "R SYM" would result in
> a better outcome.
>
> 3. In any event, this is, imho, rather pointless. Rsym/Rmerge strongly
> depend on multiplicity/resolution cutoff, thus their values across the
> PDB don't really tell you much. Maybe this is why the PDB report
> generator does not even list it as the option under "Data collection".
>
> Maybe it's time for the PDB to start asking the depositors to provide
> the Rpim instead. Well, maybe this is 10 years overdue.
>
> Cheers,
>
> Ed.
>
> --
> Oh, suddenly throwing a giraffe into a volcano to make water is crazy?
> Julian, King of Lemurs
--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***
Re: [ccp4bb] Sub-angstrom resolution
The word "theory" in this thread/question has to be clarified better. Jacob On Mon, Jan 9, 2012 at 1:27 PM, Tim Gruene wrote: > -BEGIN PGP SIGNED MESSAGE- > Hash: SHA1 > > Hi, > > in my opinion the resolution limit of crystals from large complexes/ > membrane proteins is more likely due to lattice imperfections and > long-range disorder, and cold neither cold temperatures nor stronger > radiation sources would circumvent this problem. > > Tim > > On 01/09/2012 08:02 PM, Cale Dakwar wrote: >> In theory, no: sub-angstrom resolution can be obtained for any and all >> proteins, including membrane proteins, and for large complexes. In >> reality, it becomes technically very difficult to achieve; you would need >> ever-colder temperatures and ever-stronger irradiation sources. >> >> P.S. In theory, the only limit to describing the location of the atoms >> would be described by the heisenberg uncertainty principle. >> >> >> >> On Mon, Jan 9, 2012 at 1:15 PM, Theresa H. Hsu wrote: >> >>> Dear crystallographers >>> >>> A theoretical question - can sub-angstrom resolution structures only be >>> obtained for a limited set of proteins? Is it impossible to achieve for >>> membrane proteins and large complexes? >>> >>> Theresa >>> >> > > - -- > - -- > Dr Tim Gruene > Institut fuer anorganische Chemie > Tammannstr. 4 > D-37077 Goettingen > > GPG Key ID = A46BEE1A > > -BEGIN PGP SIGNATURE- > Version: GnuPG v1.4.10 (GNU/Linux) > Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/ > > iD8DBQFPCz+YUxlJ7aRr7hoRAro2AKD502CdF5N3AK2Bb38hxhAey0nQagCg/GSB > gONh31woZI+cxJqEhSVZHjM= > =4B4D > -END PGP SIGNATURE- -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
I like that animation a lot, as it shows the gradual nature of the lattice effect, but it is not exactly what I am looking for. I am actually just curious what the pattern behind the spots looks like for various molecules, and would like to see an image of that in various orientations. I guess one way to put it is that I would like to see what the 1.5-2 Ang diffraction pattern would be for a single, radiation-damage-impervious protein or RNA/DNA molecule given enough x-rays and time. Would the intensities-based transform image be much less complicated than the phases-based one? Would larger molecules have more complex patterns, corresponding to the amount of information in their structures? JPK On Fri, Jan 6, 2012 at 6:23 PM, James M Holton wrote: > You mean something like the animation at the top of this web page? > > http://bl831.als.lbl.gov/~jamesh/fastBragg/ > > This program is a relative of nearBragg, which Dale already mentioned. > > -James Holton > MAD Scientist > > On Jan 6, 2012, at 5:44 PM, Jacob Keller > wrote: > >> Actually, as a way to make this type of figure, I think there are >> programs which output simulated diffraction images, so perhaps I could >> just input a .pdb file with some really huge (fake) cell parameters >> (10,000 Ang?), and then the resulting spots would be really close >> together and approximate the continuous molecular transform. I think >> this would amount to the same thing as the molecular transform of the >> model itself--am I right? >> >> Does anyone know which software outputs simulated diffraction images? >> >> Jacob >> >> On Fri, Jan 6, 2012 at 10:25 AM, Jacob Keller >> wrote: >>> Dear Crystallographers, >>> >>> has anyone come across a figure showing a normal diffraction image, >>> and then next to it the equivalent molecular transform, perhaps with >>> one image as phases and one as amplitudes? Seems like it would be a >>> very instructional slide to have to explain how crystallography works >>> (I know about Kevin Cowtan's ducks and cats--I was looking for >>> approximately the same but from protein or NA molecules.) I don't >>> think I have ever seen an actual molecular transform of a protein or >>> NA molecule. >>> >>> All the best, >>> >>> Jacob >>> >>> -- >>> *** >>> Jacob Pearson Keller >>> Northwestern University >>> Medical Scientist Training Program >>> email: j-kell...@northwestern.edu >>> *** >> >> >> >> -- >> *** >> Jacob Pearson Keller >> Northwestern University >> Medical Scientist Training Program >> email: j-kell...@northwestern.edu >> *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
The diffraction pattern we see results from the convolution of the crystal lattice with the fourier transform of the electron density, as I understand it. I guess I am interested in seeing the unconvoluted transform of the electron density, just to get a feeling for what the characteristics of those transforms are. As I put it in a previous message, I am curious what the diffraction would look like from a theoretical radiation-damage-impervious single molecule of either protein or nucleic acid. I suspect that for nucleic acids one would see the stacked bases a la Rosalind Franklin's fiber diffraction images, and perhaps other interesting features. Maybe there would be a powder-diffraction-like ring for CC and CN bond lengths? Anyway, I guess the goal would be to see whether one could find any other relationships like phase triplets etc. Jacob On Tue, Jan 10, 2012 at 2:21 AM, Tim Gruene wrote: > -BEGIN PGP SIGNED MESSAGE- > Hash: SHA1 > > Dear Jacob, > > what do you mean by 'molecular transform'? Would you like to visualise > the summed structure factors from the atoms inside the unit cell? > - - What pattern are you talking about/ what pattern do you expect? > - - What benefit do you expect from seeing the phases? What you can > physically observe on the detector are the intensities - the concept of > 'phases' is just a mathematical notion to explain the intensities in > terms of interference from single waves and an atomistic model inside > the crystal. > - - What makes you think the pattern from a larger molecule would have a > more complex pattern? > > Cheers, > Tim > > On 01/10/2012 12:13 AM, Jacob Keller wrote: >> I like that animation a lot, as it shows the gradual nature of the >> lattice effect, but it is not exactly what I am looking for. I am >> actually just curious what the pattern behind the spots looks like for >> various molecules, and would like to see an image of that in various >> orientations. I guess one way to put it is that I would like to see >> what the 1.5-2 Ang diffraction pattern would be for a single, >> radiation-damage-impervious protein or RNA/DNA molecule given enough >> x-rays and time. >> >> Would the intensities-based transform image be much less complicated >> than the phases-based one? >> >> Would larger molecules have more complex patterns, corresponding to >> the amount of information in their structures? >> >> JPK >> >> >> >> On Fri, Jan 6, 2012 at 6:23 PM, James M Holton wrote: >>> You mean something like the animation at the top of this web page? >>> >>> http://bl831.als.lbl.gov/~jamesh/fastBragg/ >>> >>> This program is a relative of nearBragg, which Dale already mentioned. >>> >>> -James Holton >>> MAD Scientist >>> >>> On Jan 6, 2012, at 5:44 PM, Jacob Keller >>> wrote: >>> >>>> Actually, as a way to make this type of figure, I think there are >>>> programs which output simulated diffraction images, so perhaps I could >>>> just input a .pdb file with some really huge (fake) cell parameters >>>> (10,000 Ang?), and then the resulting spots would be really close >>>> together and approximate the continuous molecular transform. I think >>>> this would amount to the same thing as the molecular transform of the >>>> model itself--am I right? >>>> >>>> Does anyone know which software outputs simulated diffraction images? >>>> >>>> Jacob >>>> >>>> On Fri, Jan 6, 2012 at 10:25 AM, Jacob Keller >>>> wrote: >>>>> Dear Crystallographers, >>>>> >>>>> has anyone come across a figure showing a normal diffraction image, >>>>> and then next to it the equivalent molecular transform, perhaps with >>>>> one image as phases and one as amplitudes? Seems like it would be a >>>>> very instructional slide to have to explain how crystallography works >>>>> (I know about Kevin Cowtan's ducks and cats--I was looking for >>>>> approximately the same but from protein or NA molecules.) I don't >>>>> think I have ever seen an actual molecular transform of a protein or >>>>> NA molecule. >>>>> >>>>> All the best, >>>>> >>>>> Jacob >>>>> >>>>> -- >>>>> *** >>>>> Jacob Pearson Keller >>>>> Northwestern University >>>>> Medical Scientist Training Program >>&g
Re: [ccp4bb] Sub-angstrom resolution
I think once you start getting down to such small crystals, the spots are not really important, as the pattern starts getting continuous. Interestingly enough, I guess for single-molecule diffraction, resolution is limited only by radiation damage, and not by any sort of lattice disorder (or even by its disorder wrt itself over time, seeing as these images are collected in the fs range!) Each would seem to be a perfect, unlimited-resolution fourier transform of that particular molecule at that particular moment, and the resolution limits come only when trying to merge the images/particles. It seems, then, that if one somehow picked the images that were taken from the most similar molecules, one could get better and better models... JPK On Tue, Jan 10, 2012 at 10:08 AM, Ed Pozharski wrote: > On Tue, 2012-01-10 at 09:04 +, Colin Nave wrote: >> Yes, I think Ed's analysis is a bit misleading. > > I apologize if I misled anyone. Re-reading my post, I can see that it > lacked precision. Indeed, in a perfect monocrystal all the molecules > are lined up perfectly, so I should have emphasized rather that the > culprit is the decay of correlation between atomic positions. It is > still a bit counterintuitive that a crystal can diffract beyond the > resolution seemingly allowed by possible bragg planes. Shouldn't the > "crystal size" formfactor introduce something akin to sinx/x that will > drive intensity rapidly down past the "bragg limit"? Oh well. > > On a second thought, maybe the unit cell size does not matter directly. > What matters perhaps is how quickly the disorder accumulates over > distance, and that should be more pronounced for larger molecules. Thus > the problem is that larger molecules cannot pack as well as, say, > crambin. > > On empirical side, the largest molecule currently in the PDB with d<1A > is 3ju4, 0.98A and ~75kDa. > > See the distribution of sizes of "subangstrom" structures here. > > http://tinyurl.com/8yhbcvk > > BTW, the 3ju4 is reported on EDS as "unreliable". Shall comment in the > other thread. > > Cheers, > > Ed. > > -- > Oh, suddenly throwing a giraffe into a volcano to make water is crazy? > Julian, King of Lemurs -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Always Modelling Anomalous Signal
Dear Crystallographers, it seems to me that on a certain level we are always throwing away (sort of) about half of our data when we merge Bijvoet pairs--why shouldn't we keep them separate, since we know that they should be a little bit different, especially in light of the higher multiplicities which are more common now? Shouldn't modelling them give better R-values, and wouldn't it just be more true? I guess a sort of proof for this is that sulfurs are almost always detectable on anomalous difference maps, implying that we are actually measuring those differences accurately enough to see them (I don't think these things can arise from model bias, as anomalous differences are not modeled.) At least maybe at the final steps of refinement...? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] DDM crystals
What exactly is your question--I saw tons of "crystals" of DDM and PEGs, I think especially P400, if I recall correctly. JPK On Wed, Jan 11, 2012 at 7:12 AM, Patrick Loll wrote: > Does anyone have any experience with formation of crystals of dodecyl > maltoside in the presence of PEG? > Pat > > --- > > Patrick J. Loll, Ph. D. > > Professor of Biochemistry & Molecular Biology > > Director, Biochemistry Graduate Program > > Drexel University College of Medicine > > Room 10-102 New College Building > > 245 N. 15th St., Mailstop 497 > > Philadelphia, PA 19102-1192 USA > > > (215) 762-7706 > > pat.l...@drexelmed.edu > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
No, I meant the non-lattice-convoluted pattern--the pattern arising from the Fourier-transformed electron density map--which would necessarily become more complicated with larger molecular size, as there is more information to encode. I think this will manifest in what James H called a smaller "grain size." JPK On Fri, Jan 13, 2012 at 11:41 AM, Yuri Pompeu wrote: > to echo Tim's question: > If by pattern you mean the position of the spots on the film, I dont think > they would change based on the complexity of the macromolecule being studied. > As far I know it, the position of the spots are dictated by the reciprocal > lattice points > (therefore the real crystal lattice) (no?) > The intensity will, obviously, vary dramatically... > ps. Very interesting (cool) images James!!! -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Molecular Transform Superimposed on a Dataset
I am trying to think, then, what would the Patterson map of a Fourier-transformed electron density map look like? Would you get the shape/outline of the object, then a sharp drop-off, presumably? Is this used to orient molecules in single-particle FEL diffraction experiments? JPK On Fri, Jan 13, 2012 at 12:33 PM, Dale Tronrud wrote: > > > On 01/13/12 09:53, Jacob Keller wrote: >> No, I meant the non-lattice-convoluted pattern--the pattern arising >> from the Fourier-transformed electron density map--which would >> necessarily become more complicated with larger molecular size, as >> there is more information to encode. I think this will manifest in >> what James H called a smaller "grain size." > > I've been thinking about these matters recently and had a nifty > insight about exactly this matter. (While this idea is new to me > I doubt it is new for others.) > > The lower limit to the size of the features in one of these > "scattergrams" is indicated by the scattergram's highest frequency > Fourier component. Its Fourier transform is the Patterson map. > While we usually think of the Patterson map as describing interatomic > vectors, it is also the frequency space for the diffraction pattern. > For a noncrystalline object the highest frequency component corresponds > to the longest Patterson vector or, in other words, the diameter of > the object! The bigger the object, the higher the highest frequency > of the scattergram, and the smaller its features. > > Dale Tronrud > >> >> JPK >> >> On Fri, Jan 13, 2012 at 11:41 AM, Yuri Pompeu wrote: >>> to echo Tim's question: >>> If by pattern you mean the position of the spots on the film, I dont think >>> they would change based on the complexity of the macromolecule being >>> studied. As far I know it, the position of the spots are dictated by the >>> reciprocal lattice points >>> (therefore the real crystal lattice) (no?) >>> The intensity will, obviously, vary dramatically... >>> ps. Very interesting (cool) images James!!! >> >> >> -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Merging data collected at two different wavelength
> By the way, I wouldn't use "MAD" to describe the mergeing of non-isomorphous > datasets. Strictly speaking, MAD is at least an attempt to measure both > anomalous (f") and dispersive (f') differences, and I don't think it is > appropriate to use the term "MAD" when you know the dispersive signal is > washed out by non-isomorphism. I call such attempts MSAD (mult-SAD), which > I think helps differentiate them from actual MAD data collections where you > at least try not to fry the crystal between measurements that you need to > subtract to get your phasing signal. Unless, of course, you are doing RIP! Isn't it true that we cannot even agree on what MAD stands for? Is the following right? M = Multiple-wavelength. I think everyone agrees to this, although I believe I've seen the occasional (and sometime non-sensical) variant A = Anomalous (I think everyone agrees, although this term should really be changed to "resonant," as there is no anomaly to it anymore...) D = Diffraction, Dispersion, Destruction, Dissolution...? JPK
Re: [ccp4bb] Merging data collected at two different wavelength
That is excellent! You refer obviously to the multiple anomalous discussions on the bb? (Maybe d = disagreement?) JPK On Wed, Jan 18, 2012 at 11:42 AM, D Bonsor wrote: > Isn't it true that we cannot even agree on what MAD stands for? > > Is the following right? > > M = Multiple-wavelength. I think everyone agrees to this, although I > believe I've seen the occasional (and sometime non-sensical) variant > A = Anomalous (I think everyone agrees, although this term should > really be changed to "resonant," as there is no anomaly to it > anymore...) > D = Diffraction, Dispersion, Destruction, Dissolution...? > > JPK > > > D =Discussion? -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Merging data collected at two different wavelength
> Can I be dogmatic about this ? I wish you could, but I don't think so, because even though those sources call it that, others don't. I agree with your thinking, but usage is usage. > a SAD experiment is a single wavelength experiment where you are using the > anomalous/dispersive signals for phasing I think "dispersive" usually refers to differences caused by changes in f'/f" between wavelengths, no? JPK *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Merging data collected at two different wavelength
This begs the question* whether you want the lemmings to understand you. One theory of language, gotten more or less from Strunk and White's Elements of Style, is that the most important feature of language is its transparency to the underlying thoughts. Bad language breaks the transparency, reminds you that you are reading and not simply thinking the thoughts of the author, who should also usually be invisible. Bad writing calls attention to itself and to the author, whereas good writing guides the thoughts of the reader unnoticeably. For Strunk and White, it seems that all rules of writing follow this principle, and it seems to be the right way to think about language. So, conventions, even when somewhat inaccurate, are important in that they are often more transparent, and the reader does not get stuck on them. Anyway, a case in point of lemmings is that once Wayne Hendrickson himself suggested that the term anomalous be decommissioned in favor of "resonant." I don't hear any non-lemmings jumping on that bandwagon... JPK *Is this the right use of "beg the question?" On Wed, Jan 18, 2012 at 1:57 PM, Phoebe Rice wrote: >> >>> Can I be dogmatic about this ? >> >>I wish you could, but I don't think so, because even though those >>sources call it that, others don't. I agree with your thinking, but >>usage is usage. > > And 10,000 lemmings can't be wrong? -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] extra density ??
Who says this is on a twofold? Also, it would be very helpful to know what was in the crystallization condition. JPK On Thu, Jan 19, 2012 at 12:44 AM, stacy William wrote: > Dear All, > I am working on plant proteins and solved a structure, there is an extra > density which i cannot fix . I am attaching the coot image , can anybody > suggest me what it could be > > THANKS :) -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] native gels
I have actually done this by running a normal PAGE gel without stacking gel and switching the electrodes, which seemed to work swimmingly. JPK On Thu, Jan 19, 2012 at 9:25 AM, Katherine Sippel wrote: > Hi Rashmi, > > In my experience native (even blue native) on proteins around that pI is > sketchy at best. The electrophoretic mobility once it gets past the stacking > gel goes to crap meaning long electrophoresis times and it needs to be done > on a chillable system or in a cold room. If this is a multimer issue I'd > suggest trying analytical ultracentrifugation, analytical size exclusion > (with the caveat that buffer, temperature and protein shape will affect the > output/interpretation), or SAXS first. If native is the only alternative > you'll probably get better results changing up the buffer system from > traditional tris-glycine or those listed in the blue native protocol keeping > in mind that you'll still need to stack the bands. > > Good luck, > > Katherine > > > On Thu, Jan 19, 2012 at 7:03 AM, anita p wrote: >> >> Hi All, >> Has anyone run a native gel for proteins at pI>8 . >> I want to pour my own native gel. Do I run a discontinuous page or a >> continuous one?? Please help with regards to the buffer system to be used, >> and the dye to be used. >> With regards >> Rashmi > > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Protein Fold Motifs- off-topic
A very similar question: how about smaller motifs, such as various turn types, etc.? JPK On Fri, Jan 20, 2012 at 12:37 PM, Jeff Headd wrote: > Hi Yuri, > > I don't know of a cheat-sheet, but I find the "Introduction to Protein > Structure" book by Branden and Tooze to useful for illustrations of > common folds. > > Jeff > > On Fri, Jan 20, 2012 at 10:13 AM, Yuri Pompeu wrote: >> Hello Everyone, >> Does anyone know of a quick (yet somewhat reliable) sort of >> cheat-sheet/quick reference sheet with the more common folds with an >> illustrative example? -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Expression of Viral proteins for crystallography
Inspired by the recent post about "quasispecies:" I have been bothered recently by the following problem: why do species of genetic uniformity exist at all (or do they?)? This first came up when I saw a Nature paper describing live bacteria extracted from a supposedly 250-million-year-old salt crystal whose 16S RNA was 99% identical to marismortui bacteria (ref below). What? Are the bacteria the same now as 250 million years ago? But there is a further question: given the assumptions of evolution, why should there be any bacterium whose genome is the same as any other, assuming that equivalent codons are really equivalent (or at least roughly so), and that even at the protein level, there is such a thing as "neutral drift?" After all, we even see in our lab cultures that they (at least e coli) mutate fairly frequently, so why is there such a thing as "e coli" at all, at least at the nucleotide level? I don't think we usually say that each bacterial species is totally optimized in all its features, do we? Even assuming that every single protein must be just so, shouldn't there be as many species of e coli as there are possible genomes encoding the same protein set, i.e. some extremely large number? Why is there any uniformity at all? Or IS there--maybe the bacteria too are only quasispecies...? And maybe also... JPK Nature 407, 897-900 (19 October 2000) | doi:10.1038/35038060; Received 15 November 1999; Accepted 4 July 2000 Isolation of a 250 million-year-old halotolerant bacterium from a primary salt crystal Russell H. Vreeland1, William D. Rosenzweig1 & Dennis W. Powers2 Department of Biology, West Chester University, West Chester, Pennsylvania 19383 , USA Consulting Geologist, Box 87, Anthony, Texas 79821, USA Correspondence to: Russell H. Vreeland1 Correspondence and requests for materials should be addressed to R.H.V. (e-mail: Email: rvreel...@wcupa.edu). Top of page Bacteria have been found associated with a variety of ancient samples1, however few studies are generally accepted due to questions about sample quality and contamination. When Cano and Borucki2 isolated a strain of Bacillus sphaericus from an extinct bee trapped in 25–30 million-year-old amber, careful sample selection and stringent sterilization techniques were the keys to acceptance. Here we report the isolation and growth of a previously unrecognized spore-forming bacterium (Bacillus species, designated 2-9-3) from a brine inclusion within a 250 million-year-old salt crystal from the Permian Salado Formation. Complete gene sequences of the 16S ribosomal DNA show that the organism is part of the lineage of Bacillus marismortui and Virgibacillus pantothenticus. Delicate crystal structures and sedimentary features indicate the salt has not recrystallized since formation. Samples were rejected if brine inclusions showed physical signs of possible contamination. Surfaces of salt crystal samples were sterilized with strong alkali and acid before extracting brines from inclusions. Sterilization procedures reduce the probability of contamination to less than 1 in 10 9. 2012/1/24 Darren Hart : > I think the explanation is this: > The source is natural viral RNA which is a mixture of naturally mutated > sequences (e.g. flu forms such a quasispecies) > See: > http://www.virology.ws/2009/05/11/the-quasispecies-concept/ > > The pooled RNA has an average sequence that you see when you sequence the > pooled cDNA (individual mutations are hidden by the averaging effect of > having many sequences present). > > But when you clonally separate DNA molecules by transformation (1 plasmid > enters 1 cell to yield 1 colony), you see each individual molecule > represented 100% in the sequencing chromatogram from the plasmid DNA that > you have isolated from colonies. > > This is effect is commonly observed when sequencing influenza virus isolates > from patients. It will have nothing to do with the E. coli strain. You can > avoid it completely by using gene synthesis. > > Darren > > > > 2012/1/24 Rubén Sánchez Eugenia >> >> Dear everyone, >> >> I am trying to clone a viral protein in the E. Coli BSJ strain and i am >> having some problems. >> >> I start from the viral RNA carrying out a reverse transcription and PCR >> (RT-PCR) to obtain the protein cDNA. When I sequence this cDNA to check for >> mutations, there are no mutations. So the RT-PCR works fine. >> >> Then, I digest the cDNA and I ligate it with a pET plasmid to transform >> the E. Coli BSJ strain. I get recombinant colonies (checked by colony-PCR) >> but when I sequence them I get various mutations (aprox. 2 miss-sense) on >> the inserted cDNA. Furthermore, these mutations are different among >> different transformations and even among colonies of the same plate (in the >> same transformation). >> >> Maybe these mutations are produced by the cell (because of the lack of >> mutations in the cDNA) but these E. Coli clonning strains are supposed to be >> "optimized" to prevent the insertio
[ccp4bb] Quasispecies
Whoops--I meant to change the subject line, so if you want to reply, please use this one not to perturb the original post. JPK > Inspired by the recent post about "quasispecies:" > > I have been bothered recently by the following problem: why do species > of genetic uniformity exist at all (or do they?)? This first came up > when I saw a Nature paper describing live bacteria extracted from a > supposedly 250-million-year-old salt crystal whose 16S RNA was 99% > identical to marismortui bacteria (ref below). What? Are the bacteria > the same now as 250 million years ago? But there is a further > question: given the assumptions of evolution, why should there be any > bacterium whose genome is the same as any other, assuming that > equivalent codons are really equivalent (or at least roughly so), and > that even at the protein level, there is such a thing as "neutral > drift?" After all, we even see in our lab cultures that they (at least > e coli) mutate fairly frequently, so why is there such a thing as "e > coli" at all, at least at the nucleotide level? I don't think we > usually say that each bacterial species is totally optimized in all > its features, do we? Even assuming that every single protein must be > just so, shouldn't there be as many species of e coli as there are > possible genomes encoding the same protein set, i.e. some extremely > large number? Why is there any uniformity at all? Or IS there--maybe > the bacteria too are only quasispecies...? And maybe also... > > JPK > > > > > Nature 407, 897-900 (19 October 2000) | doi:10.1038/35038060; Received > 15 November 1999; Accepted 4 July 2000 > > Isolation of a 250 million-year-old halotolerant bacterium from a > primary salt crystal > > Russell H. Vreeland1, William D. Rosenzweig1 & Dennis W. Powers2 > > Department of Biology, West Chester University, West Chester, > Pennsylvania 19383 , USA > Consulting Geologist, Box 87, Anthony, Texas 79821, USA > Correspondence to: Russell H. Vreeland1 Correspondence and requests > for materials should be addressed to R.H.V. (e-mail: Email: > rvreel...@wcupa.edu). > > Top of page > Bacteria have been found associated with a variety of ancient > samples1, however few studies are generally accepted due to questions > about sample quality and contamination. When Cano and Borucki2 > isolated a strain of Bacillus sphaericus from an extinct bee trapped > in 25–30 million-year-old amber, careful sample selection and > stringent sterilization techniques were the keys to acceptance. Here > we report the isolation and growth of a previously unrecognized > spore-forming bacterium (Bacillus species, designated 2-9-3) from a > brine inclusion within a 250 million-year-old salt crystal from the > Permian Salado Formation. Complete gene sequences of the 16S ribosomal > DNA show that the organism is part of the lineage of Bacillus > marismortui and Virgibacillus pantothenticus. Delicate crystal > structures and sedimentary features indicate the salt has not > recrystallized since formation. Samples were rejected if brine > inclusions showed physical signs of possible contamination. Surfaces > of salt crystal samples were sterilized with strong alkali and acid > before extracting brines from inclusions. Sterilization procedures > reduce the probability of contamination to less than 1 in 10 9. > > 2012/1/24 Darren Hart : >> I think the explanation is this: >> The source is natural viral RNA which is a mixture of naturally mutated >> sequences (e.g. flu forms such a quasispecies) >> See: >> http://www.virology.ws/2009/05/11/the-quasispecies-concept/ >> >> The pooled RNA has an average sequence that you see when you sequence the >> pooled cDNA (individual mutations are hidden by the averaging effect of >> having many sequences present). >> >> But when you clonally separate DNA molecules by transformation (1 plasmid >> enters 1 cell to yield 1 colony), you see each individual molecule >> represented 100% in the sequencing chromatogram from the plasmid DNA that >> you have isolated from colonies. >> >> This is effect is commonly observed when sequencing influenza virus isolates >> from patients. It will have nothing to do with the E. coli strain. You can >> avoid it completely by using gene synthesis. >> >> Darren >> >> >> >> 2012/1/24 Rubén Sánchez Eugenia >>> >>> Dear everyone, >>> >>> I am trying to clone a viral protein in the E. Coli BSJ strain and i am >>> having some problems. >>> >>> I start from the viral RNA carrying out a reverse transcription and PCR >>> (RT-PCR) to obtain the protein cDNA. When I sequence this cDNA to check for >>> mutations, there are no mutations. So the RT-PCR works fine. >>> >>> Then, I digest the cDNA and I ligate it with a pET plasmid to transform >>> the E. Coli BSJ strain. I get recombinant colonies (checked by colony-PCR) >>> but when I sequence them I get various mutations (aprox. 2 miss-sense) on >>> the inserted cDNA. Furthermore, these mutations are different among >>> different tr
Re: [ccp4bb] Introducing an ELN
Can't you get a plug-in for that? JPK On Thu, Jan 26, 2012 at 11:35 AM, Dale Tronrud wrote: > Unless you have written on the paper using cursive script. Many schools > in the US have stopped teaching longhand reading/writing so in a generation > or two many paper records will be undecipherable to all but historians. My > wife has some handwritten letters from ancestors written in German around > 1920 that even Germans have great trouble reading today. > > The paper is holding up quite well though. ;-) > > Dale Tronrud > > On 01/26/12 08:30, Phoebe Rice wrote: >> As the proud owner of a carefully organized, highly annotated VMS backup >> tape (reel-to-reel, of course), my main concern is that paper is the only >> format that we'll be able to count on reading a decade (or more) from now. >> >> = >> Phoebe A. Rice >> Dept. of Biochemistry & Molecular Biology >> The University of Chicago >> phone 773 834 1723 >> http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123 >> http://www.rsc.org/shop/books/2008/9780854042722.asp >> >> >> Original message >>> Date: Thu, 26 Jan 2012 16:50:04 +0100 >>> From: CCP4 bulletin board (on behalf of Anastassis >>> Perrakis ) >>> Subject: Re: [ccp4bb] Introducing an ELN >>> To: CCP4BB@JISCMAIL.AC.UK >>> >>> I think that all these points are interesting and >>> valid. >>> On Jan 25, 2012, at 10:37, Chris Morris wrote: >>> >>> Tassos reports: >>> >>> 1. None of the twenty test-users was satisfied >>> with any of the two >>> >>> solutions - and each was annoyed for a different >>> reason. >>> >>> This suggests that the choice of ELN is not the >>> most difficult part of the adoption process. Maybe >>> the test users at the NKI were annoyed by the idea >>> of using an ELN at all. >>> >>> That would surely apply to some users. Some were >>> actually very keen, and thats why they signed up for >>> it. >>> >>> In my experience, the hardest part is ensuring >>> that it provides benefits to the people who have >>> to enter the data, and provides them early. The >>> fact that it will make information retrieval >>> easier in three years is not enough. >>> >>> I suggest focussing on electronic support for >>> housekeeping: booking time on an instrument, >>> finding the files the instrument created, ordering >>> oligos, recording when you use the last of a >>> reagent. Scientists work very independently in >>> most respects, but they do have certain >>> obligations that flow from sharing the lab space. >>> You can make use of these to encourage compliance >>> with the ELN. If you do, then most of the science >>> will get recorded in passing. >>> >>> I think that this was exactly one of the problems. >>> The ELNs we tested had no option for booking >>> instruments, no way to find files from instruments >>> let alone read them (it would support only TIF, >>> JPEG, Doc, XLS, PDF), and would not do stock >>> keeping: all these are thought to be out of the ELN >>> scope. And that makes an ELN inherently less useful. >>> Lack of instrument support is another issue: a >>> machine that would allow us to import real >>> chromatograms to ELN would be cool - alas, the >>> solution that was suggested to us is to save as PDF >>> or XLS and reload ...! (it took 3 weeks to come back >>> with this great plan!) >>> For the rest I have nothing much to say, I basically >>> agree. >>> A. >>> >>> I suggest also ensuring that it includes >>> electronic tools that actually help. Two examples >>> from PiMS are primer design, and automatically >>> uploading and interpreting results from the >>> Caliper GX instrument. >>> >>> It must allow round trips with spreadsheets, i.e. >>> dump ELN data as a spreadsheet, edit it, upload it >>> again. Despite their substantial disadvantages, >>> some scientists will not give them up. It should >>> also allow crossreferencing with paper note books. >>> Some will continue to use a lab notebook. When >>> they discover that the ELN serves as a searchable >>> index to it, they will warm to the ELN. >>> >>> I suggest aiming for "no paper" at your lab >>> progress meetings within say 12 months. When you >>> reach that point, everything important is in the >>> ELN. Before then, the ELN is not giving real >>> value. >>> >>> You will need someone who is keen on the >>> introduction of the ELN, to customise it, provide >>> first line user support, and act as a single point >>> of contact with the supplier. This might be a >>> scientist or an IT person. I have also seen this >>> done well by a technician, Delphine Chesnel when >>> she was at the EMBL Hamburg. If you can't find >>> such a "champion", then introduction will not be >>>
[ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
Dear Crystallographers, I cannot think why any of the various flavors of Rmerge/meas/pim should be used as a data cutoff and not simply I/sigma--can somebody make a good argument or point me to a good reference? My thinking is that signal:noise of >2 is definitely still signal, no matter what the R values are. Am I wrong? I was thinking also possibly the R value cutoff was a historical accident/expedient from when one tried to limit the amount of data in the face of limited computational power--true? So perhaps now, when the computers are so much more powerful, we have the luxury of including more weak data? JPK -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
Clarification: I did not mean I/sigma of 2 per se, I just meant I/sigma is more directly a measure of signal than R values. JPK On Fri, Jan 27, 2012 at 11:47 AM, Jacob Keller wrote: > Dear Crystallographers, > > I cannot think why any of the various flavors of Rmerge/meas/pim > should be used as a data cutoff and not simply I/sigma--can somebody > make a good argument or point me to a good reference? My thinking is > that signal:noise of >2 is definitely still signal, no matter what the > R values are. Am I wrong? I was thinking also possibly the R value > cutoff was a historical accident/expedient from when one tried to > limit the amount of data in the face of limited computational > power--true? So perhaps now, when the computers are so much more > powerful, we have the luxury of including more weak data? > > JPK > > > -- > *** > Jacob Pearson Keller > Northwestern University > Medical Scientist Training Program > email: j-kell...@northwestern.edu > *** -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
he shell averages. Also the average will depend on >> > the width of the resolution bin, so one will get the strange effect >> > that the apparent resolution will depend on how one bins at the data! >> > The assumption being made in taking the bin average is that I/sigma(I) >> > falls off smoothly with d* but that's unlikely to be the reality. >> > >> > It seems to me that a chi-square statistic which takes into account >> > the actual distribution of I/sigma(I) would be a better bet than the >> > bin average, though it's not entirely clear how one would formulate >> > such a metric. One would have to consider subsets of the data as a >> > whole sorted by increasing d* (i.e. not in resolution bins to avoid >> > the 'bin averaging effect' described above), and apply the resolution >> > cut-off where the chi-square statistic has maximum probability. This >> > would automatically take care of incompleteness effects since all >> > unmeasured reflections would be included with I/sigma = 0 just for the >> > purposes of working out the cut-off point. I've skipped the details >> > of implementation and I've no idea how it would work in practice! >> > >> > An obvious question is: do we really need to worry about the exact >> > cut-off anyway, won't our sophisticated maximum likelihood refinement >> > programs handle the weak data correctly? Note that in theory weak >> > intensities should be handled correctly, however the problem may >> > instead lie with incorrectly estimated sigmas: these are obviously >> > much more of an issue for any software which depends critically on >> > accurate estimates of uncertainty! I did some tests where I refined >> > data for a known protein-ligand complex using the original apo model, >> > and looked at the difference density for the ligand, using data cut at >> > 2.5, 2 and 1.5 Ang where the standard metrics strongly suggested there >> > was only data to 2.5 Ang. >> > >> > I have to say that the differences were tiny, well below what I would >> > deem significant (i.e. not only the map resolutions but all the map >> > details were essentially the same), and certainly I would question >> > whether it was worth all the soul-searching on this topic over the >> > years! So it seems that the refinement programs do indeed handle weak >> > data correctly, but I guess this should hardly come as a surprise (but >> > well done to the software developers anyway!). This was actually >> > using Buster: Refmac seems to have more of a problem with scaling & >> > TLS if you include a load of high resolution junk data. However, >> > before anyone acts on this information I would _very_ strongly advise >> > them to repeat the experiment and verify the results for themselves! >> > The bottom line may be that the actual cut-off used only matters for >> > the purpose of quoting the true resolution of the map, but it doesn't >> > significantly affect the appearance of the map itself. >> > >> > Finally an effect which confounds all the quality metrics is data >> > anisotropy: ideally the cut-off surface of significance in reciprocal >> > space should perhaps be an ellipsoid, not a sphere. I know there are >> > several programs for anisotropic scaling, but I'm not aware of any >> > that apply anisotropic resolution cutoffs (or even whether this would >> > be advisable). >> > >> > Cheers >> > >> > -- Ian >> > >> > On 27 January 2012 17:47, Jacob Keller >> > wrote: >> >> Dear Crystallographers, >> >> >> >> I cannot think why any of the various flavors of Rmerge/meas/pim >> >> should be used as a data cutoff and not simply I/sigma--can somebody >> >> make a good argument or point me to a good reference? My thinking is >> >> that signal:noise of >2 is definitely still signal, no matter what the >> >> R values are. Am I wrong? I was thinking also possibly the R value >> >> cutoff was a historical accident/expedient from when one tried to >> >> limit the amount of data in the face of limited computational >> >> power--true? So perhaps now, when the computers are so much more >> >> powerful, we have the luxury of including more weak data? >> >> >> >> JPK >> >> >> >> >> >> -- >> >> *** >> >> Jacob Pearson Keller >> >> Northwestern University >> >> Medical Scientist Training Program >> >> email: j-kell...@northwestern.edu >> >> *** > > > > > -- > > ARKA CHAKRABORTY > CAS in Crystallography and Biophysics > University of Madras > Chennai,India > > > -- > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: + 44 1223 336500 > Wellcome Trust/MRC Building Fax: + 44 1223 336827 > Hills Road E-mail: rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Reasoning for Rmeas or Rpim as Cutoff
ons, not the shell averages. Also the average will depend on >> > the width of the resolution bin, so one will get the strange effect >> > that the apparent resolution will depend on how one bins at the data! >> > The assumption being made in taking the bin average is that I/sigma(I) >> > falls off smoothly with d* but that's unlikely to be the reality. >> > >> > It seems to me that a chi-square statistic which takes into account >> > the actual distribution of I/sigma(I) would be a better bet than the >> > bin average, though it's not entirely clear how one would formulate >> > such a metric. One would have to consider subsets of the data as a >> > whole sorted by increasing d* (i.e. not in resolution bins to avoid >> > the 'bin averaging effect' described above), and apply the resolution >> > cut-off where the chi-square statistic has maximum probability. This >> > would automatically take care of incompleteness effects since all >> > unmeasured reflections would be included with I/sigma = 0 just for the >> > purposes of working out the cut-off point. I've skipped the details >> > of implementation and I've no idea how it would work in practice! >> > >> > An obvious question is: do we really need to worry about the exact >> > cut-off anyway, won't our sophisticated maximum likelihood refinement >> > programs handle the weak data correctly? Note that in theory weak >> > intensities should be handled correctly, however the problem may >> > instead lie with incorrectly estimated sigmas: these are obviously >> > much more of an issue for any software which depends critically on >> > accurate estimates of uncertainty! I did some tests where I refined >> > data for a known protein-ligand complex using the original apo model, >> > and looked at the difference density for the ligand, using data cut at >> > 2.5, 2 and 1.5 Ang where the standard metrics strongly suggested there >> > was only data to 2.5 Ang. >> > >> > I have to say that the differences were tiny, well below what I would >> > deem significant (i.e. not only the map resolutions but all the map >> > details were essentially the same), and certainly I would question >> > whether it was worth all the soul-searching on this topic over the >> > years! So it seems that the refinement programs do indeed handle weak >> > data correctly, but I guess this should hardly come as a surprise (but >> > well done to the software developers anyway!). This was actually >> > using Buster: Refmac seems to have more of a problem with scaling & >> > TLS if you include a load of high resolution junk data. However, >> > before anyone acts on this information I would _very_ strongly advise >> > them to repeat the experiment and verify the results for themselves! >> > The bottom line may be that the actual cut-off used only matters for >> > the purpose of quoting the true resolution of the map, but it doesn't >> > significantly affect the appearance of the map itself. >> > >> > Finally an effect which confounds all the quality metrics is data >> > anisotropy: ideally the cut-off surface of significance in reciprocal >> > space should perhaps be an ellipsoid, not a sphere. I know there are >> > several programs for anisotropic scaling, but I'm not aware of any >> > that apply anisotropic resolution cutoffs (or even whether this would >> > be advisable). >> > >> > Cheers >> > >> > -- Ian >> > >> > On 27 January 2012 17:47, Jacob Keller >> > wrote: >> >> Dear Crystallographers, >> >> >> >> I cannot think why any of the various flavors of Rmerge/meas/pim >> >> should be used as a data cutoff and not simply I/sigma--can somebody >> >> make a good argument or point me to a good reference? My thinking is >> >> that signal:noise of >2 is definitely still signal, no matter what the >> >> R values are. Am I wrong? I was thinking also possibly the R value >> >> cutoff was a historical accident/expedient from when one tried to >> >> limit the amount of data in the face of limited computational >> >> power--true? So perhaps now, when the computers are so much more >> >> powerful, we have the luxury of including more weak data? >> >> >> >> JPK >> >> >> >> >> >> -- >> >> *** >> >> Jacob Pearson Keller >> >> Northwestern University >> >> Medical Scientist Training Program >> >> email: j-kell...@northwestern.edu >> >> *** > > > > > -- > > ARKA CHAKRABORTY > CAS in Crystallography and Biophysics > University of Madras > Chennai,India > > > -- > Randy J. Read > Department of Haematology, University of Cambridge > Cambridge Institute for Medical Research Tel: + 44 1223 336500 > Wellcome Trust/MRC Building Fax: + 44 1223 336827 > Hills Road E-mail: rj...@cam.ac.uk > Cambridge CB2 0XY, U.K. www-structmed.cimr.cam.ac.uk > -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
Re: [ccp4bb] Freezing crystal
One last thing--sometimes crystals can be frozen as is, particularly if you use mitegen mounts and get nearly all of the mother liquor off the crystals by dabbing the loop on the dry surface next to the drop several times. So simple it is always worth a try JPK On Tue, Feb 7, 2012 at 2:37 AM, Mark J van Raaij wrote: > Rationalising it completely may only be possible once you know the nature of > the crystal contacts, i.e. when you have solved the structure. Until then it > is mainly a matter of experimenting. >> >> -Original Message- >> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of >> Theresa H. Hsu >> Sent: Monday, February 06, 2012 11:00 PM >> To: CCP4BB@JISCMAIL.AC.UK >> Subject: Re: [ccp4bb] Freezing crystal >> >> Hi all >> >> Thanks for all the suggestions which I will try soon. >> >> How do the crystallization condition (PEG vs. salts like ammonium >> sulfate) affect the croyprotectant condition? Do factors like presence >> of low concentration of high molecular weight PEG (> 2000) mean PEG is >> better? Do buffers and salts in protein also important? >> >> Trying to rationalize it :) >> >> Theresa -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
[ccp4bb] Invisible Reference on Pubmed?
Dear CCP4BB, this is perhaps my most egregious off-topic post, but can anyone explain why the following reference is not findable in PubMed? I can get it from the ACS website, but not on PubMed or elsewhere. The journal is on PubMed--is it perhaps because it's funded by ExxonMobil? Very strange... Jacob Article A Study of the Separation Principle in Size Exclusion Chromatography AbstractFull Text HTMLHi-Res PDF[140 KB]PDF w/ Links[240 KB]FiguresCiting Articles Thomas Sun* Baytown Polymers Center, ExxonMobil Chemical Company, 5200 Bayway Dr., Baytown, Texas 77520-2101 Ronald R. Chance, William W. Graessley,† and David J. Lohse Corporate Strategic Research, ExxonMobil Research and Engineering, Annandale, New Jersey 08801 Macromolecules, 2004, 37 (11), pp 4304–4312 DOI: 10.1021/ma030586k Publication Date (Web): April 29, 2004 Copyright © 2004 American Chemical Society -- *** Jacob Pearson Keller Northwestern University Medical Scientist Training Program email: j-kell...@northwestern.edu ***
