Re: [ccp4bb] X-Ray films
Wasnt it the tramp whom they beat to death - and the book was R James.. That movie gave the cold shivers.. eleanor Philip Leonard wrote: I have a vague recollection of a student carrying books about crystallography getting beaten up at the start of Clockwork Orange. This might only be in the book though... This opens a whole new can of worms! How many films would contain references to crystallography if only the screenwriter hadn't decide to omit the reference in favour of something else more fun. Phil. -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of harry powell Sent: Thursday, April 15, 2010 5:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] X-Ray films Hi Not a question about films for recording X-rays on, but a question about films about X-rays, Crystallography and related subjects! I was wondering what ccp4bbers favourite movies involving real science, especially crystallography might be? If they're from Hollywood, though, I'd guess it should be favorite... I'm a little tired, but the only one I can think of at the moment is actually based on results from fibre diffraction - Life Story, with Jeff Goldblum. There must be others, though. Harry -- Dr Harry Powell, MRC Laboratory of Molecular Biology, Hills Road, Cambridge, CB2 0QH -- DISCLAIMER: This e-mail and any attachments are confidential. They may contain privileged information and are intended for the named addressee(s) only. If you are not the named addressee do not disseminate, distribute, copy or retain this e-mail, or any part of it, in any manner. Please notify the sender immediately if you have received this e-mail by mistake and delete it from your system. The sender does not accept any liability for any errors or omissions in the contents of this message, which arise as a result of e-mail transmission. Unless expressly stated, opinions in this e-mail are those of the individual sender, and not of Galapagos NV or any of its subsidiaries including BioFocus. Galapagos NV and its subsidiaries including BioFocus reserve the right to monitor all e-mail communications through its network. Although this e-mail has been scanned for all known viruses, the sender does not guarantee that this message is virus-free and disclaims any liability for viruses that may be transmitted with this message.
Re: [ccp4bb] Proportion of MR in PDB
Here are my counts of the various methods used for PDBs as of a few days ago: 37851 MR 7802 MAD/SAD NULL 669 OTHER 53 N/A 993 MIR 352 SIRAS 316 MIRAS 188 AB-INITIO 88 SIR 6 RIP 2 UNCONVENTIONAL 1 UNCONVENTIANAL 1 FIBER-DIFFRACTION 56171 TOTAL The other 8605 don't have REM200 entries. Note that creating this list involves some heuristics on my part. For example PDB files that don't say how they were solved, but mention another PDB entry as the source of phases are put into the MR bin. I should also note that the 5:1 ratio of MR:MAD/SAD is consistent with the 5:1 ratio of native to anomalous datasets I see collected at my beamline (Advanced Light Source 8.3.1). This is despite the fact that an average of 10 MAD/SAD datasets are collected for every one that gets solved. -James Holton MAD Scientist Nicholas Keep wrote: Thanks to several people for helpful comments to my question on the Proportion of MR in the PDB. I got two very detailed responses one from the OCA team at the Weizmann which went to the Bulletin Board This is what OCA has: From un total of 64,623 PDB structure files, 30,784 have 'MOLECULAR REPLACEMENT' as Method for Structure Determination. However, you must remember that we have a large number of false negatives Several reasons: - Only 47,557 structure files from the total of 64,623 report which method was used for structure determination. For example, 1CRY whose title reports using the MR method does not include the info in the proper REMARK. - Users are allowed to write almost anything as the METHOD USED TO DETERMINE THE STRUCTURE, making it difficult an accurate report. OCA found PDB italian speaking structure files reporting 'MOLECULARE REPLACEMENT' ... This and other problems are being reported to RCSB. and one from U of Virginia Based on information from Remark200 lines, 31761 structures were solved using MR, what comprise for 56.8% of all X-RAY structures (55843). Considering structures which were determined using 'primitive MR' the number grows to 34949 (62.6%). There are also some structures determined using combination of MR with SAD, MAD, SIR and MIR. If we would add them, the number will increase to 35258 (63.1%). Thanks again Nick
Re: [ccp4bb] Mysterious Crystals?
Perhaps this is a case of a 'phantom crystal' that was discussed on the CCP4bb awhile back: http://bit.ly/b6Rgw7 All the Best, Sean
[ccp4bb] XVII BCA/CCP4 Summer School in Macromolecular Crystallography: closing date for pre-registration 1st May 2010
Any applicants for this Summer School who have not already done so should pre-register by 1st May 2010, on which date the WWW site will close. As stated on the pre-registration form, applicants will not be considered for a place on the School unless a supporting letter from their supervisor has been sent to elspeth.gar...@bioch.ox.ac.uk by midnight on 1st May. We currently only have supporting letters for about 60% of our 80 pre-registrants. All supervisors whose support letters have been received so far should have had acknowledgement e-mails from Elspeth Garman. Best wishes Elspeth BCA/CCP4 Summer School XVII Biochemistry Department and St. Edmund Hall, University of Oxford. 5th-10th September 2010. Scope. The BCA Summer School is a combined taught and practical course intended primarily for students and researchers new to crystallography. Its aim is to provide comprehensive training in the theory and practise of crystallography, and to promote the exchange of experience and best practise within the British crystallographic community. The meeting covers the gamut of modern crystallographic theory and practice, from lattices, through phasing, to maximum likelihood and refinement. The focus is very much on relating theory to practise. The practical aspect of the workshop takes the form of intensively supervised computer tutorials. Although CCP4 programs will primarily be used in these tutorials, the aim will be to approach crystallographic questions in a software-agnostic manner. Eligibility. The course is eligible to any crystallographic researcher in a British university or research institute, although a small number of overseas students may be accepted. Applicants should have started their research degree before 2010, and must provide a supporting letter or e-mail from their supervisor. Cost. The cost of the Summer School, including full board, will be only 120 GBP, thanks to substantial resources allocated to bursaries, including particular support for BBSRC funded researchers. Registration Preliminary applications should be made online at http://www.bioch.ox.ac.uk/bca2010 *The closing date for applications is 1st May 2010. Numbers are strictly limited to 45, and historically the course has been significantly oversubscribed. Applications will be collected for a closed field selection procedure in which we will try to allocate places on the grounds of: # Geographical distribution (departments are encouraged to coordinate applications and indicate priorities if they support more than one applicant: it is unlikely that two students from a single department will be accepted). # Anticipated benefit to the student # First come first served All applicants will be notified immediately after the selection meeting (scheduled for mid-May) Venue. Lectures will take place in the New Biochemistry Department of Oxford University, with tutorials in the Medical Sciences Teaching Centre, Sir William Dunn School of Pathology. Accommodation. The course is a residential one, with accommodation provided at the nearby St. Edmund Hall, in the heart of Oxford University. Social Programme. We recognise that the social programme plays a regrettably large part in the perceived success of the course. There will be a conference dinner on the night of the 9th September, and a number of other events, including a boat trip and a tour of the Diamond synchrotron have been arranged. Sponsors This meeting would not be possible without generous financial contributions from the following (confirmed as at 2/1/10) # BBSRC # BSG of the BCA # Bruker # CCP4 # Molecular Dimensions # Oxford Cryosystems # Oxford Diffraction # Rigaku Contact details. Further details can be obtained by contacting the organisers: # elspeth.gar...@bioch.ox.ac.uk # martin.no...@bioch.ox.ac.uk -- Professor Elspeth F. Garman, President, British Crystallographic Association Postal address: Laboratory of Molecular Biophysics, Department of Biochemistry, University of Oxford, Tel: (44)-1865-613297 South Parks Road, FAX: (44)-1865-613201 OXFORD, OX1 3QU, U.K. E-mail: elspeth.gar...@bioch.ox.ac.uk http://www.biop.ox.ac.uk/www/garman/gindex.html -
[ccp4bb] Automated beta-sheet definition for PDB-deposition ?
Dear depositors, does exist there any program that is able to handle the beta-sheets of complicated structures like beta-helices, bifurcated sheets ... to produce the input for PDB-deposition. The program included in the PDB-deposition procedure failes in our case and there are lots of strands and sheets. Production of beta sheet records by hand is such a boring task. Thanks a lot, Juergen
Re: [ccp4bb] merging Pymol sessions ? Non-CCP4 question
Jürgen, PyMOL sessions are essentially pickled Python dictionaries. At this point I'm not aware of a way to easily merge the contents of two sessions. This would be a nice new feature; I've added it to my list of features enhancement requests. Thanks, -- Jason On Sun, Apr 18, 2010 at 6:36 PM, Jürgen Bosch jubo...@jhsph.edu wrote: Dear CCP4bb, I'm running into some difficulties with Pymol, in particular I want to visualize via APBS the electrostatic surface of the protein, a protein peptide and an inhibitor molecule. I can render the electrostatic surfaces for each of them separately but I fail to show individual surfaces for each of the items in question. Since the .pse files are in binary format I was unable to find a way how to merge the individual .pse files in a meaningful manner (only the first file will be interpreted when all files are cated) If anybody has some suggestions I would be very glad to try them out. Thanks, Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ -- Jason Vertrees, PhD PyMOL Product Manager Schrodinger, LLC (e) jason.vertr...@schrodinger.com (o) +1 (603) 374-7120
Re: [ccp4bb] unable to fly? - Pittsburgh
I would like to extend this to Pittsburgh as well. We are in Eastern Pittsburgh, available by bus or taxi to Pittsburgh International Airport. We have wireless internet but not much apartment space. If you are stuck in Pittsburgh, please send me an email to make arrangements. Regina - Original Message From: Frances C. Bernstein f...@bernstein-plus-sons.com To: CCP4BB@JISCMAIL.AC.UK Sent: Sat, April 17, 2010 6:56:46 PM Subject: [ccp4bb] unable to fly? If you are stuck in the NYC area and unable to get home to Europe because of the volcano, we might be willing to host you for a reasonable time. We are located about 1.5 hours east of JFK and you can get close to us by train. Note that we have two dogs that are friendly. We also have wireless internet access. Please contact us by e-mail to see if arrangements can be made. I hope that other people on this list will make similar offers. Frances and Herbert Bernstein = Bernstein + Sons * * Information Systems Consultants 5 Brewster Lane, Bellport, NY 11713-2803 * * *** *Frances C. Bernstein * *** f...@bernstein-plus-sons.com *** * * *** 1-631-286-1339FAX: 1-631-286-1999 =
Re: [ccp4bb] Proportion of MR in PDB
Quite a lot of structures in the PDB involve minor variations on structures that have already been solved (different ligands, mutants, high res), so the solution involves refining the previous structure against the new data, perhaps starting with rigid body refinement to correct minor variations in cell parameters. Would you include this in molecular replacement? Ed Nicholas Keep wrote: Thanks to several people for helpful comments to my question on the Proportion of MR in the PDB. I got two very detailed responses one from the OCA team at the Weizmann which went to the Bulletin Board This is what OCA has: From un total of 64,623 PDB structure files, 30,784 have 'MOLECULAR REPLACEMENT' as Method for Structure Determination. However, you must remember that we have a large number of false negatives Several reasons: - Only 47,557 structure files from the total of 64,623 report which method was used for structure determination. For example, 1CRY whose title reports using the MR method does not include the info in the proper REMARK. - Users are allowed to write almost anything as the METHOD USED TO DETERMINE THE STRUCTURE, making it difficult an accurate report. OCA found PDB italian speaking structure files reporting 'MOLECULARE REPLACEMENT' ... This and other problems are being reported to RCSB. and one from U of Virginia Based on information from Remark200 lines, 31761 structures were solved using MR, what comprise for 56.8% of all X-RAY structures (55843). Considering structures which were determined using 'primitive MR' the number grows to 34949 (62.6%). There are also some structures determined using combination of MR with SAD, MAD, SIR and MIR. If we would add them, the number will increase to 35258 (63.1%). Thanks again Nick
Re: [ccp4bb] Mysterious Crystals?
Well, the good news is that they don't seem to be salt crystals. But have you really ruled that out? With small molecule crystals the diffraction pattern is so sparse, and at such high resolution, that you are likely to miss it altogether in a one-degree oscillation and looking for spots around the beamstop. Next time you have one in the beam, put the detector up close and take a 180-degree oscillation (one picture, exposure say 10 sec or however fast the phi-axis drive can spin). Now if there are still no spots you can begin work on extending the diffraction out from behind the beamstop. (Don't forget to set delta-phi back to a reasonable value, or the next shot on a protein crystal is going to be very disappointing!) Ed tat cheung cheng wrote: Hi all, I have got some crystals, the purified protein was in Tris buffer with 300mM NaCl for crystallization. they grew in light weight PEG, PEG400 or monomethyl ethyl PEG500, they were needle shaped, could be long (~0.2mm) but very thin all the time and sometimes grew into sea-urchin like needle cluster. What interesting is, when i gridded crystallization conditions against pH or PEG amount, the crystals sizes and shapes varied, and the crystals were fragile so i believed they were protein crystals in nature. But upon X-ray diffraction, they gave no reflection at all, not even a faint spot. I wonder, beside silly mistakes like misalignment of the crystal to the beam, not enough exposure time, what could be the reason for this mysterious crystals? Are they protein or PEG or what? Thanks very much. Tc
Re: [ccp4bb] sigma cutoff for fitting waters in model
I second Tim's opinion. In the days of CNS/O, there was a popular rule to place waters in 3 sigma peaks that make chemical sense, then re-refine and keep those waters that produce more than 1 sigma in 2fo-fc map. (With Coot the default cutoff is 5). There could be a bizarre probabilistic argument for a particular choice of sigma cutoff - with 3 sigmas you have ~0.3% chance of a particular peak to be simply a random spike. Which means that if the map is on, say, 0.5A grid, there is a decent chance to have one such peak per 3.5x3.5x3.5A volume. With 5 sigmas the size of the cube goes up to ~60x60x60A, so 5 sigma peaks are almost guaranteed not to be flukes. On Sat, 2010-04-17 at 22:46 +0200, Tim Gruene wrote: Hello Sudhir Kumar, most of all the waters in your structure should make chemical sense. When the density around the water is weak it may just mean that the water is not fully occupied. Tim On Sat, Apr 17, 2010 at 09:47:35PM +0900, Sudhir Kumar wrote: hi all sorry for such a basic query, i'ld like to know what is the acceptable sigma cut off for waters to be kept in a model if data is of about 1.6 A. thanks in advance Sudhir Kumar Research Scholar Structural Biology Laboratory SLS, JNU, New Delhi-110067 -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
Re: [ccp4bb] Proportion of MR in PDB
I would say it should still be classed as MR: the distinguishing feature of MR is surely that it uses an known structure as a starting model, not that it does a rotation/translation search. In any case the distinction between a rigid-body search and RB refinement is moot. At Astex our automated scripts routinely do a limited MR search for all our protein-ligand structures, even though it may not be necessary in all cases. This is because we have found from experience that RB refinement alone is often not sufficient: presumably the degree of non-isomorphism induced by soaking and/or freezing the crystals (e.g. we often see 5-10% changes in cell parameters) takes it beyond the radius of convergence of the refinement. We use a limited search MR to avoid issues of finding symmetry-related or origin-shifted solutions. Cheers -- Ian On Mon, Apr 19, 2010 at 3:06 PM, Edward A. Berry ber...@upstate.edu wrote: Quite a lot of structures in the PDB involve minor variations on structures that have already been solved (different ligands, mutants, high res), so the solution involves refining the previous structure against the new data, perhaps starting with rigid body refinement to correct minor variations in cell parameters. Would you include this in molecular replacement? Ed Nicholas Keep wrote: Thanks to several people for helpful comments to my question on the Proportion of MR in the PDB. I got two very detailed responses one from the OCA team at the Weizmann which went to the Bulletin Board This is what OCA has: From un total of 64,623 PDB structure files, 30,784 have 'MOLECULAR REPLACEMENT' as Method for Structure Determination. However, you must remember that we have a large number of false negatives Several reasons: - Only 47,557 structure files from the total of 64,623 report which method was used for structure determination. For example, 1CRY whose title reports using the MR method does not include the info in the proper REMARK. - Users are allowed to write almost anything as the METHOD USED TO DETERMINE THE STRUCTURE, making it difficult an accurate report. OCA found PDB italian speaking structure files reporting 'MOLECULARE REPLACEMENT' ... This and other problems are being reported to RCSB. and one from U of Virginia Based on information from Remark200 lines, 31761 structures were solved using MR, what comprise for 56.8% of all X-RAY structures (55843). Considering structures which were determined using 'primitive MR' the number grows to 34949 (62.6%). There are also some structures determined using combination of MR with SAD, MAD, SIR and MIR. If we would add them, the number will increase to 35258 (63.1%). Thanks again Nick
Re: [ccp4bb] Re： [ccp4bb] Mysterious Crystals?
Couldn't they simply be too thin? After all, unit cell dimensions are routinely about 0.01um, so if these needles are only fraction of a micron thick, there is simply not enough material for diffraction. Nice looking but non-diffracting protein crystals are too disordered (i.e. while packing is present, there is no long-range spatial correlation among individual unit cells). On Mon, 2010-04-19 at 11:35 +0800, tat cheung cheng wrote: Thank you. Forget to mention, no diffraction observed no matter with or without cyro cooling. __ 寄件人﹕ tom.p...@csiro.au tom.p...@csiro.au 收件人﹕ theif...@yahoo.com.hk 傳送日期﹕ 2010/4/19 (一) 11:29:38 AM 主題： RE: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? Hello Tc, It isn’t that unusual to get protein crystals that don’t diffract. This happens probably 50% of the time. One can try dehydration of the crystals, crystal annealing and additive screens to see if any of these things will give you some diffraction. In addition, you didn’t mention whether you froze these crystals- one should also try putting a crystal in the beam without cryo-cooling, as cryo-cooling can often be detrimental to diffraction. Cheers, tom __ From:CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of tat cheung cheng Sent: Monday, 19 April 2010 1:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? Yes, I have just done that. They are protein. But if they are protein, why no diffraction? That's intriguing. __ 寄件人﹕ Jürgen Bosch jubo...@jhsph.edu 收件人﹕ccp...@jiscmail.ac.uk 傳送日期﹕ 2010/4/19 (一) 10:57:40 AM 主題： Re: [ccp4bb] Mysterious Crystals? Fish and wash some crystals then run them on a SDS-gel, then you will know for sure if it's protein or not. J僡gen On Apr 18, 2010, at 10:46 PM, tat cheung cheng wrote: Hi all, I have got some crystals, the purified protein was in Tris buffer with 300mM NaCl for crystallization. they grew in light weight PEG, PEG400 or monomethyl ethyl PEG500, they were needle shaped, could be long (~0.2mm) but very thin all the time and sometimes grew into sea-urchin like needle cluster. What interesting is, when i gridded crystallization conditions against pH or PEG amount, the crystals sizes and shapes varied, and the crystals were fragile so i believed they were protein crystals in nature. But upon X-ray diffraction, they gave no reflection at all, not even a faint spot. I wonder, beside silly mistakes like misalignment of the crystal to the beam, not enough exposure time, what could be the reason for this mysterious crystals? Are they protein or PEG or what? Thanks very much. Tc - J僡gen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street , W8708 Baltimore , MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse /
[ccp4bb] Calculation of electron density map for a twinned dataset
Hi All, I have a dataset that shows about 50 % twinning. I was curious what will be the best way for the refinement and calculation of electron density maps, including the composite omit map. Thanks a lot. Mariah -- Mariah Jones Department of Biochemistry University of Florida
Re: [ccp4bb] sigma cutoff for fitting waters in model
Hello - The sigma issue a bit more complicated. What we call usually sigma is the root mean square deviation (rmsd) of the map. Lets first recall, that the variation within the protein region is quite large, while the solvent is rather flat. Now, lets take an 'extreme' example, of a protein with 80% solvent. The rmsd for that will be quite low, since most of the AU is flat. Thus, I would argue that you might want to consider waters in relatively 'low sigma'levels. Of course 80% solvent will also mean that most likely this protein will only diffract to low resolution, so you should maybe not be putting any waters. The inverse case argument also applies. Similar issues, maybe more severe, come up for atom removal. Admittedly, we had this discussion with Victor back at the EMBL- Hamburg library, back to what will soon be two decades ago, and I do not think we have good answers, although we try new things every couple of years. A. If anyone is interested thats the ARP/wARP code for removal atom sigma, I am not sure when we came up with that, but it did make some sense at the time. Cant even recall if its my or Victor or both putting that in ... basically it leaves waters in if they are above 1.0 rmsd (if resolution is better than 2.0 A), or above 0.6 rmsd if resolution is less than 2.8 A (I can hear the screams already ...), and uses a value in between for resolutions between 0.6-1.0 A. The rmsd for adding atoms is 3.4, since it does not really matter what to add, if you remove what should not be there ... (what a silly assumption ...? It looked logical at the time though) On Apr 19, 2010, at 16:38, Ed Pozharski wrote: I second Tim's opinion. In the days of CNS/O, there was a popular rule to place waters in 3 sigma peaks that make chemical sense, then re-refine and keep those waters that produce more than 1 sigma in 2fo-fc map. (With Coot the default cutoff is 5). There could be a bizarre probabilistic argument for a particular choice of sigma cutoff - with 3 sigmas you have ~0.3% chance of a particular peak to be simply a random spike. Which means that if the map is on, say, 0.5A grid, there is a decent chance to have one such peak per 3.5x3.5x3.5A volume. With 5 sigmas the size of the cube goes up to ~60x60x60A, so 5 sigma peaks are almost guaranteed not to be flukes. On Sat, 2010-04-17 at 22:46 +0200, Tim Gruene wrote: Hello Sudhir Kumar, most of all the waters in your structure should make chemical sense. When the density around the water is weak it may just mean that the water is not fully occupied. Tim On Sat, Apr 17, 2010 at 09:47:35PM +0900, Sudhir Kumar wrote: hi all sorry for such a basic query, i'ld like to know what is the acceptable sigma cut off for waters to be kept in a model if data is of about 1.6 A. thanks in advance Sudhir Kumar Research Scholar Structural Biology Laboratory SLS, JNU, New Delhi-110067 -- Edwin Pozharski, PhD, Assistant Professor University of Maryland, Baltimore -- When the Way is forgotten duty and justice appear; Then knowledge and wisdom are born along with hypocrisy. When harmonious relationships dissolve then respect and devotion arise; When a nation falls to chaos then loyalty and patriotism are born. -- / Lao Tse / P please don't print this e-mail unless you really need to Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791
Re: [ccp4bb] Calculation of electron density map for a twinned dataset
The newest refmac will deal with this and generate maps with twinning corrected.. Eleanor protein.chemist protein.chemist wrote: Hi All, I have a dataset that shows about 50 % twinning. I was curious what will be the best way for the refinement and calculation of electron density maps, including the composite omit map. Thanks a lot. Mariah
Re: [ccp4bb] sigma cutoff for fitting waters in model
... or you could just use the RMSD of the difference map (i.e. that using 2(mFo-DFc) coefficients for acentric reflns), which is a reasonable approximation of the uncertainty provided most of the structure is accounted for, as the uncertainty of the Fourier map (i.e. that using 2mFo-DFc for acentrics). Note that for this to work you must use the correct 'minimally biased' coefficients, i.e. 2(mFo-DFc) for acentric reflns, and (mFo-DFc) for centrics; I suspect some programs are still not doing this correctly. Similarly the correct 'minimally biased' coefficient for the Fourier map is 2mFo-DFc for acentrics and mFo for centrics (see Randy Read's original 1986 paper for the proof, which actually if I recall correctly depends on an earlier proof by Peter Main). For example, picking a difference map at random, I see from its header that it has an uncertainty in the electron density (i.e. number density of electrons!!) estimated as 0.0662 A^-3. This isn't actually the RMSD, which happens to be 0.0687 A^-3. The extends program has excluded significant peaks and used just the flat regions of the a.u. of the map output by the fft program to estimate the uncertainty. Now looking at the Fourier map computed from the same data, I see it has an RMSD = 0.1903 A^-3, but really it has the uncertainty of the difference Fourier above, i.e. 0.0662 A^-3. Now 0.1903/0.0662 ~ 2.9 so I should multiply any given RMSD level of the Fourier map by 2.9 to get the true sigma level. Note that I don't change the sigma value in the header of the Fourier map to its correct value because that would cause total confusion! - but strictly that is what one should do. Cheers -- Ian On Mon, Apr 19, 2010 at 4:44 PM, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote: Has anybody ever explored contouring maps using a sigma (i.e. rmsd) derived only from what is clearly the solvent region? Obviously that's not relevant during early phasing, but in the later stages of refinement, that would be relatively clear. And it would be fairly comparable from map to map. (I think...) phx. On 19/04/2010 16:31, Anastassis Perrakis wrote: Hello - The sigma issue a bit more complicated. What we call usually sigma is the root mean square deviation (rmsd) of the map. Lets first recall, that the variation within the protein region is quite large, while the solvent is rather flat. Now, lets take an 'extreme' example, of a protein with 80% solvent. The rmsd for that will be quite low, since most of the AU is flat. Thus, I would argue that you might want to consider waters in relatively 'low sigma'levels. Of course 80% solvent will also mean that most likely this protein will only diffract to low resolution, so you should maybe not be putting any waters. The inverse case argument also applies. Similar issues, maybe more severe, come up for atom removal. Admittedly, we had this discussion with Victor back at the EMBL-Hamburg library, back to what will soon be two decades ago, and I do not think we have good answers, although we try new things every couple of years. A. If anyone is interested thats the ARP/wARP code for removal atom sigma, I am not sure when we came up with that, but it did make some sense at the time. Cant even recall if its my or Victor or both putting that in ... basically it leaves waters in if they are above 1.0 rmsd (if resolution is better than 2.0 A), or above 0.6 rmsd if resolution is less than 2.8 A (I can hear the screams already ...), and uses a value in between for resolutions between 0.6-1.0 A. The rmsd for adding atoms is 3.4, since it does not really matter what to add, if you remove what should not be there ... (what a silly assumption ...? It looked logical at the time though) On Apr 19, 2010, at 16:38, Ed Pozharski wrote: I second Tim's opinion. In the days of CNS/O, there was a popular rule to place waters in 3 sigma peaks that make chemical sense, then re-refine and keep those waters that produce more than 1 sigma in 2fo-fc map. (With Coot the default cutoff is 5). There could be a bizarre probabilistic argument for a particular choice of sigma cutoff - with 3 sigmas you have ~0.3% chance of a particular peak to be simply a random spike. Which means that if the map is on, say, 0.5A grid, there is a decent chance to have one such peak per 3.5x3.5x3.5A volume. With 5 sigmas the size of the cube goes up to ~60x60x60A, so 5 sigma peaks are almost guaranteed not to be flukes. On Sat, 2010-04-17 at 22:46 +0200, Tim Gruene wrote: Hello Sudhir Kumar, most of all the waters in your structure should make chemical sense. When the density around the water is weak it may just mean that the water is not fully occupied. Tim On Sat, Apr 17, 2010 at 09:47:35PM +0900, Sudhir Kumar wrote: hi all sorry for such a basic query, i'ld like to know what is the acceptable sigma cut off for waters to be kept in a model if data is of about 1.6 A. thanks in
Re: [ccp4bb] (slightly more on) sigma cutoff for fitting waters in model
Hello: some more on water picking... After quite a bit of experimenting, I found this working the way I like (implemented in phenix.refine): 1) peak at mFo-DFc map is higher than ~3sigma, and 2) peak center is within a hydrogen bond to another atom (water or macromolecule), and 3) peak has approximately the same shape as a water molecule would have at this resolution and local environment, and 4) peak at 2mFo-DFc is higher than ~1.5 sigma, 5) refine B-factor (and occupancy, resolution permitting) of water only, 6) apply criteria 3-4 to waters, after a round of coordinate and B-factor refinement (whole structure), check criteria (2-3-4) are still ok, and 7) apply B-factor filtering criteria to all waters, 8) keep cycling 1-8 until convergence (number of defined macro-cycles). Of course I don't mention various technicalities related to partial occupancies, alternative conformations, Hydrogen atoms on waters in case of refinement against neutron data or ultra-high resolution X-ray data. These are special cases that get special treatment. There are a few other technical tricks to make this process robust and efficient at high resolution, higher than ~1.2A or so. All the parameters of the above protocol are available to adjust so one can customize the procedure. Also, the whole process of water picking can be relatively safely enabled at earlier stages of refinement than it is typically done, IF fix_rotamers=true option is used. In this case there is no danger that a water will end up in an unoccupied yet side chain density, since when the side chains move during refinement the water is treated as just a density peak, making possible for a side chain run into water's atom. I'm still playing with this and some summary is here (for those who is really interested -:) ): http://cci.lbl.gov/~afonine/rsr.pdf Regarding sigma cutoff: one of more ideas to try out is to lower sigma cutoff criteria as refinement progresses and the structure improves. Pavel. On 4/17/10 5:47 AM, Sudhir Kumar wrote: hi all sorry for such a basic query, i'ld like to know what is the acceptable sigma cut off for waters to be kept in a model if data is of about 1.6 A. thanks in advance Sudhir Kumar Research Scholar Structural Biology Laboratory SLS, JNU, New Delhi-110067
Re: [ccp4bb] sigma cutoff for fitting waters in model
Hi Ian, ... or you could just use the RMSD of the difference map (i.e. that using 2(mFo-DFc) coefficients for acentric reflns), which is a reasonable approximation of the uncertainty provided most of the structure is accounted for, as the uncertainty of the Fourier map (i.e. that using 2mFo-DFc for acentrics). Note that for this to work you must use the correct 'minimally biased' coefficients, i.e. 2(mFo-DFc) for acentric reflns, and (mFo-DFc) for centrics; I suspect some programs are still not doing this correctly. thanks for shedding some light on this subject! I coded this way of map calculation in phenix.refine without actually understanding why it has to be this way. Did anybody discuss this in the literature (or it's too obvious to be worth of discussion -:) )? Pavel.
Re: [ccp4bb] sigma cutoff for fitting waters in model
Hi Pavel AFAIK it's not in the literature, in fact I wasn't even aware it was in phenix, but that's probably only because we don't use phenix (sorry! - being commercial we would have to pay for it!). The problem is always where you put little tidbits like this, unless it's part of a much bigger piece of work. Cheers -- Ian On Mon, Apr 19, 2010 at 7:35 PM, Pavel Afonine pafon...@lbl.gov wrote: Hi Ian, ... or you could just use the RMSD of the difference map (i.e. that using 2(mFo-DFc) coefficients for acentric reflns), which is a reasonable approximation of the uncertainty provided most of the structure is accounted for, as the uncertainty of the Fourier map (i.e. that using 2mFo-DFc for acentrics). Note that for this to work you must use the correct 'minimally biased' coefficients, i.e. 2(mFo-DFc) for acentric reflns, and (mFo-DFc) for centrics; I suspect some programs are still not doing this correctly. thanks for shedding some light on this subject! I coded this way of map calculation in phenix.refine without actually understanding why it has to be this way. Did anybody discuss this in the literature (or it's too obvious to be worth of discussion -:) )? Pavel.
Re: [ccp4bb] Re： [ccp4bb] Mysterious Crystals?
Move the beam stop back? My lab has grown quite a few crystals that only diffract to very low resolution. Phoebe (with sympathy!) Original message Date: Mon, 19 Apr 2010 11:35:11 +0800 From: tat cheung cheng theif...@yahoo.com.hk Subject: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? To: CCP4BB@JISCMAIL.AC.UK Thank you. Forget to mention, no diffraction observed no matter with or without cyro cooling. 寄件人﹕ tom.p...@csiro.au tom.p...@csiro.au 收件人﹕ theif...@yahoo.com.hk 傳送日期﹕ 2010/4/19 (一) 11:29:38 AM 主題： RE: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? Hello Tc, It isn’t that unusual to get protein crystals that don’t diffract. This happens probably 50% of the time. One can try dehydration of the crystals, crystal annealing and additive screens to see if any of these things will give you some diffraction. In addition, you didn’t mention whether you froze these crystals- one should also try putting a crystal in the beam without cryo-cooling, as cryo-cooling can often be detrimental to diffraction. Cheers, tom From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of tat cheung cheng Sent: Monday, 19 April 2010 1:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? Yes, I have just done that. They are protein. But if they are protein, why no diffraction? That's intriguing. 寄件人﹕ Jürgen Bosch jubo...@jhsph.edu 收件人﹕ CCP4BB@JISCMAIL.AC.UK 傳送日期﹕ 2010/4/19 (一) 10:57:40 AM 主題： Re: [ccp4bb] Mysterious Crystals? Fish and wash some crystals then run them on a SDS-gel, then you will know for sure if it's protein or not. J僡gen On Apr 18, 2010, at 10:46 PM, tat cheung cheng wrote: Hi all, I have got some crystals, the purified protein was in Tris buffer with 300mM NaCl for crystallization. they grew in light weight PEG, PEG400 or monomethyl ethyl PEG500, they were needle shaped, could be long (~0.2mm) but very thin all the time and sometimes grew into sea-urchin like needle cluster. What interesting is, when i gridded crystallization conditions against pH or PEG amount, the crystals sizes and shapes varied, and the crystals were fragile so i believed they were protein crystals in nature. But upon X-ray diffraction, they gave no reflection at all, not even a faint spot. I wonder, beside silly mistakes like misalignment of the crystal to the beam, not enough exposure time, what could be the reason for this mysterious crystals? Are they protein or PEG or what? Thanks very much. Tc - J僡gen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street , W8708 Baltimore , MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ Phoebe A. Rice Assoc. Prof., 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 RNA is really nifty DNA is over fifty We have put them both in one book Please do take a really good look http://www.rsc.org/shop/books/2008/9780854042722.asp
[ccp4bb] chiral volumes--losing it
Friends, A question about the definition of chiral volumes: I'm looking for the definition of the SIGN of a chiral volume. The only ccp4 reference I can find (readily) is this: http://www.ysbl.york.ac.uk/~garib/refmac/docs/theory/chiral.html This page gives an algorithm for determining the sign, which I paraphrase here: * Call the central (chiral) carbon a, and its three non-hydrogen substituents b, c, and d. * Arrange things so that as you look from b to c to d, your eye is moving clockwise. * If atom a is below the plane formed by b, c, d, then the chiral volume is positive (otherwise negative) Clear enough. However, when I start to apply this rule to basically every library I have ever used, I get the opposite result. Try it, for example, with the ALA.cif file distributed with ccp4: _chem_comp_chir.comp_id _chem_comp_chir.id _chem_comp_chir.atom_id_centre _chem_comp_chir.atom_id_1 _chem_comp_chir.atom_id_2 _chem_comp_chir.atom_id_3 _chem_comp_chir.volume_sign ALA chir_01 CA N CB C negativ Assigning a as the centre atom and atoms b-d as numbers 1-3, when I apply the rule from the refmac page I get a positive chiral volume, not the negative one found in the cif file. Ditto for every other example that I have tried. Am I mis-reading what is meant by above and below? I'm assuming that if atoms b, c, and d all lie in a vertical plane, and you are facing that plane, then below means on the far side of that plane and above means between you and the plane. When I use the definition V1 * (V2 x V3), where V1 is vector FROM chiral atom to 1st substituent (e.g., CA to N in alanine example above), V2 = vector from chiral atom to 2nd substituent, etc., then I get the expected sign for the chiral volume. So is the refmac page wrong, or am I falling prey to some roaringly obvious stupidity? Having a rough Monday--starting to question my sanity. Thanks, Pat ps It appears that the term _chem_comp_chir.volume_sign is not defined in either the core or mmCIF dictionaries. Can this be right? --- 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
[ccp4bb] chiral volumes--2nd try
Sorry, the original post looks garbled (mirroring my internal state, no doubt). I'm trying again, sending as plain text: Friends, A question about the definition of chiral volumes: I'm looking for the definition of the SIGN of a chiral volume. The only ccp4 reference I can find (readily) is this: http://www.ysbl.york.ac.uk/~garib/refmac/docs/theory/chiral.html This page gives an algorithm for determining the sign, which I paraphrase here: * Call the central (chiral) carbon a, and its three non-hydrogen substituents b, c, and d. * Arrange things so that as you look from b to c to d, your eye is moving clockwise. * If atom a is below the plane formed by b, c, d, then the chiral volume is positive (otherwise negative) Clear enough. However, when I start to apply this rule to basically every library I have ever used, I get the opposite result. Try it, for example, with the ALA.cif file distributed with ccp4: _chem_comp_chir.comp_id _chem_comp_chir.id _chem_comp_chir.atom_id_centre _chem_comp_chir.atom_id_1 _chem_comp_chir.atom_id_2 _chem_comp_chir.atom_id_3 _chem_comp_chir.volume_sign ALA chir_01 CA N CB C negativ Assigning a as the centre atom and atoms b-d as numbers 1-3, when I apply the rule from the refmac page I get a positive chiral volume, not the negative one found in the cif file. Ditto for every other example that I have tried. Am I mis-reading what is meant by above and below? I'm assuming that if atoms b, c, and d all lie in a vertical plane, and you are facing that plane, then below means on the far side of that plane and above means between you and the plane. When I use the definition V1 * (V2 x V3), where V1 is vector FROM chiral atom to 1st substituent (e.g., CA to N in alanine example above), V2 = vector from chiral atom to 2nd substituent, etc., then I get the expected sign for the chiral volume. So is the refmac page wrong, or am I falling prey to some roaringly obvious stupidity? Having a rough Monday--starting to question my sanity. Thanks, Pat ps It appears that the term _chem_comp_chir.volume_sign is not defined in either the core or mmCIF dictionaries. Can this be right? --- 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
Re: [ccp4bb] chiral volumes--2nd try
The problem of discrete values in restraints can be circumvented by computing a corresponding continuous value such as a chiral volume Vc, which is given by a scalar triple vector product A (B x C) originating at the central atom. With the smallest ligand pointed toward the observer and clockwise assignment of the vectors, the sign of the chiral volume is positive, and computes to about 2.5 Å3. An alternative method of restraining chirality is by restraining the improper torsion (or improper dihedral or zeta-value) between CaNCCb to +34 deg. p 630 ;-) BR -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Patrick Loll Sent: Monday, April 19, 2010 2:40 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] chiral volumes--2nd try Sorry, the original post looks garbled (mirroring my internal state, no doubt). I'm trying again, sending as plain text: Friends, A question about the definition of chiral volumes: I'm looking for the definition of the SIGN of a chiral volume. The only ccp4 reference I can find (readily) is this: http://www.ysbl.york.ac.uk/~garib/refmac/docs/theory/chiral.html This page gives an algorithm for determining the sign, which I paraphrase here: * Call the central (chiral) carbon a, and its three non-hydrogen substituents b, c, and d. * Arrange things so that as you look from b to c to d, your eye is moving clockwise. * If atom a is below the plane formed by b, c, d, then the chiral volume is positive (otherwise negative) Clear enough. However, when I start to apply this rule to basically every library I have ever used, I get the opposite result. Try it, for example, with the ALA.cif file distributed with ccp4: _chem_comp_chir.comp_id _chem_comp_chir.id _chem_comp_chir.atom_id_centre _chem_comp_chir.atom_id_1 _chem_comp_chir.atom_id_2 _chem_comp_chir.atom_id_3 _chem_comp_chir.volume_sign ALA chir_01 CA N CB C negativ Assigning a as the centre atom and atoms b-d as numbers 1-3, when I apply the rule from the refmac page I get a positive chiral volume, not the negative one found in the cif file. Ditto for every other example that I have tried. Am I mis-reading what is meant by above and below? I'm assuming that if atoms b, c, and d all lie in a vertical plane, and you are facing that plane, then below means on the far side of that plane and above means between you and the plane. When I use the definition V1 * (V2 x V3), where V1 is vector FROM chiral atom to 1st substituent (e.g., CA to N in alanine example above), V2 = vector from chiral atom to 2nd substituent, etc., then I get the expected sign for the chiral volume. So is the refmac page wrong, or am I falling prey to some roaringly obvious stupidity? Having a rough Monday--starting to question my sanity. Thanks, Pat ps It appears that the term _chem_comp_chir.volume_sign is not defined in either the core or mmCIF dictionaries. Can this be right? --- 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
Re: [ccp4bb] Re： [ccp4bb] Mysterious Crystals?
Dear Cheng, You could take your non-diffracting needle urchins and crush them in their growth solution (vortex several minutes and/or try Hampton seed bead). Then make serial dilutions of the seed solution and add them to a whole new screen (the MMS method) or try the optimization method with new drops of fresh protein plus microseeds. Maybe you will get thicker crystals or a different, better-diffracting form. See below; Acta Crystallogr D Biol Crystallogr.javascript:AL_get(this,%20'jour',%20'Acta%20Crystallogr%20D%20Biol%20Crystallogr.');2007 Apr;63(Pt 4):550-4. Epub 2007 Mar 16. An automated microseed matrix-screening method for protein crystallization. D'Arcy Ahttp://www.ncbi.nlm.nih.gov/pubmed?term=%22D%27Arcy%20A%22%5BAuthor%5D, Villard Fhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Villard%20F%22%5BAuthor%5D, Marsh Mhttp://www.ncbi.nlm.nih.gov/pubmed?term=%22Marsh%20M%22%5BAuthor%5D . Novartis Institutes of Biomedical Research, Protease Platform, Klybeckstrasse 144, CH 4002 Basel, Switzerland. allan.da...@novartis.com Abstract A microseed-matrix procedure has been established with the aim of influencing the nucleation event in standard crystallization screens. The method is based on the original description of matrix seeding described by Ireton Stoddard (2004, Acta Cryst. D60, 601-605). Seed stocks are produced using a simple seed-bead method. The protein, reservoir solutions and seed stocks are pipetted simultaneously using a three-bore dispensing tip in drops of 0.6 microl total volume. The number and type of hits produced with the proteins tested in this study has been increased and it is believed that this method could be generally applicable to proteins where little or no nucleation is normally observed. On Mon, Apr 19, 2010 at 3:18 PM, Phoebe Rice pr...@uchicago.edu wrote: Move the beam stop back? My lab has grown quite a few crystals that only diffract to very low resolution. Phoebe (with sympathy!) Original message Date: Mon, 19 Apr 2010 11:35:11 +0800 From: tat cheung cheng theif...@yahoo.com.hk Subject: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? To: CCP4BB@JISCMAIL.AC.UK Thank you. Forget to mention, no diffraction observed no matter with or without cyro cooling. 寄件人﹕ tom.p...@csiro.au tom.p...@csiro.au 收件人﹕ theif...@yahoo.com.hk 傳送日期﹕ 2010/4/19 (一) 11:29:38 AM 主題： RE: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? Hello Tc, It isn’t that unusual to get protein crystals that don’t diffract. This happens probably 50% of the time. One can try dehydration of the crystals, crystal annealing and additive screens to see if any of these things will give you some diffraction. In addition, you didn’t mention whether you froze these crystals- one should also try putting a crystal in the beam without cryo-cooling, as cryo-cooling can often be detrimental to diffraction. Cheers, tom From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of tat cheung cheng Sent: Monday, 19 April 2010 1:26 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Re： [ccp4bb] Mysterious Crystals? Yes, I have just done that. They are protein. But if they are protein, why no diffraction? That's intriguing. 寄件人﹕ Jürgen Bosch jubo...@jhsph.edu 收件人﹕ CCP4BB@JISCMAIL.AC.UK 傳送日期﹕ 2010/4/19 (一) 10:57:40 AM 主題： Re: [ccp4bb] Mysterious Crystals? Fish and wash some crystals then run them on a SDS-gel, then you will know for sure if it's protein or not. J僡gen On Apr 18, 2010, at 10:46 PM, tat cheung cheng wrote: Hi all, I have got some crystals, the purified protein was in Tris buffer with 300mM NaCl for crystallization. they grew in light weight PEG, PEG400 or monomethyl ethyl PEG500, they were needle shaped, could be long (~0.2mm) but very thin all the time and sometimes grew into sea-urchin like needle cluster. What interesting is, when i gridded crystallization conditions against pH or PEG amount, the crystals sizes and shapes varied, and the crystals were fragile so i believed they were protein crystals in nature. But upon X-ray diffraction, they gave no reflection at all, not even a faint spot. I wonder, beside silly mistakes like misalignment of the crystal to the beam, not enough exposure time, what could be the reason for this mysterious crystals? Are they protein or PEG or what? Thanks very much. Tc - J僡gen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street , W8708 Baltimore , MD 21205
[ccp4bb] how to make cholesterol solution
Dear ALL: Sorry for this kind of off-topic question. I am going to co-crystallize one protein with cholesterol. I read some papers saying that their protein can be pre-incubated with 1mM cholesterol in the presence of 5% (v/v) ethanol. TO do so, I first dissolved cholesterol in 100% ethanol at a concentration of 10mM. However, it is very difficult to make the dilution into 5% ethanol either just in water or some buffers. Does anyone have such experience to make cholesterol solution in normal buffers plus some ethanol? Thanks a lot, Jerry McCully _ The New Busy think 9 to 5 is a cute idea. Combine multiple calendars with Hotmail. http://www.windowslive.com/campaign/thenewbusy?tile=multicalendarocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_5
