Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] 1 out of 2 proteins in asymmetric unit does not fit density

[Jürgen Bosch]

What’s your spacegroup ? RWork / RFree?

Twinning by any chance?

Jürgen 

__
Jürgen Bosch, Ph.D.
Division of Pediatric Pulmonology and Allergy/Immunology
Case Western Reserve University
2109 Adelbert Rd, BRB 835
Cleveland, OH 44106
Phone: 216.368.7565
Fax: 216.368.4223
https://www.linkedin.com/in/jubosch/

CEO & Co-Founder at InterRayBio, LLC

Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology

> On Dec 16, 2019, at 1:50 PM, Jessica Besaw  wrote:
> 
> I am crystallizing this membrane protein in a medium (bicelles) that forms 
> lamella like sheets that stack on top of each other. 
> 
> The layer packing is shown below. Is this structure unreasonable?
> 
> 
> 
> On Mon, 16 Dec 2019 at 13:38, Reza Khayat  <mailto:rkha...@ccny.cuny.edu>> wrote:
> ​​Hi Jessica,
> 
> 
> 
> The gap between the two proteins is a bit troubling. Perhaps it's the image, 
> but why would a crystal form if there is no crystal contact between the two 
> proteins?
> 
> 
> 
> Reza
> 
> 
> 
> Reza Khayat, PhD
> Assistant Professor 
> City College of New York
> Department of Chemistry
> New York, NY 10031
> From: CCP4 bulletin board  <mailto:CCP4BB@JISCMAIL.AC.UK>> on behalf of Ashish Kumar 
> mailto:mail2ashish...@gmail.com>>
> Sent: Monday, December 16, 2019 1:24 PM
> To: CCP4BB@JISCMAIL.AC.UK <mailto:CCP4BB@JISCMAIL.AC.UK>
> Subject: [EXTERNAL] Re: [ccp4bb] 1 out of 2 proteins in asymmetric unit does 
> not fit density
>  
> Hi Jessica,
> 
> It may be possible because of wrong MR solution as well. How were your stats 
> after MR. 
> Also it is correct that it could be possible because of wrong space group.
> Try changing the Space group and repeat MR.
> 
> Best Regards
> Ashish
> 
> On 16 Dec 2019 22:56, "Jessica Besaw"  <mailto:jbesaw1...@gmail.com>> wrote:
> Dear community, 
> 
> I am having a lot of trouble solving a protein structure. I think my problem 
> may caused by incorrectly placed proteins in molecular replacement. I have 
> two proteins in my asymmetric unit. It appears that one protein fits 
> perfectly, and the other one has many errors. (See snapshots below). I have 
> tried deleting the parts of the protein (and even the whole protein) to try 
> and rebuild it in COOT, but it was a bit too difficult for me to solve. 
> 
> I would appreciate any and all suggestions for potential strategies moving 
> forward. 
> 
> Other information: 
> (1) 2.4 Angstrom
> (2) 99% complete
> (3) "Translational NCS may be present at a level that may complicate 
> refinement"
> 
> Cheers!
> 
> Jessica 
> 
> 
> 
> 
> 
> 
> 
> 
> 
> 
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Re: [ccp4bb] need someone officially settle a pdb dispute for a publication

[Jürgen Bosch]

I guess I should just reply with “B.” since you replied with A. :-)

More seriously, 

Both A & B will need to have real hard proof of their claims as this is a real 
mess in which they got themselves into.

It seems as A despite not been hired by B succeeded in getting another 
position, which is good. B has taken advantage of his/her power position by not 
hiring A. He/she will find excuses why A was not a good fit blabla. From a 
moral perspective B sucks.
Just imagine what A in B’s lab could have achieved by having someone in the 
project already knowing everything about the dubious protein with a four letter 
PDB code. Must be one of those Ego’s out there without true team leadership 
skills.

Regarding the IP, that’s more difficult and again will require written evidence 
unless a patent has been filed and then that should be relatively easy and have 
the lawyers go after it.

All of these actions require extra attention and mental resources - is it worth 
for A? If you just want to be right, then read on to the bottom of this email.

Is the publication in C formally correct? Other than that A claims B took 
his/her coordinates to solve the structure. All that really counts is that the 
science around PDB  is correct.

From my own experience during my postdoc time, we had a paper under review for 
nine months. I know who one of the reviewers was because he copy-protected his 
comments in the pdf - unfortunately for him on a Mac your login user account is 
added per default to the pdf as creator, and that was just his plain name. 
The day after his paper was accepted at a different journal, ours was finally 
accepted as well. It is remarkable how some figures in his paper just adopt the 
same orientation and show the same things as ours. I never followed up on this 
as it was not worth my time. I will not reveal the authors names but I’m sure 
smart pople like you can write a little perl script to query the PDB in a 
meaningful manner.

This was more than 2 cents I wanted to add to this discussion and hopefully A 
does see the value in my last paragraph.

Jürgen 


______
Jürgen Bosch, Ph.D.
Division of Pediatric Pulmonology and Allergy/Immunology
Case Western Reserve University
2109 Adelbert Rd, BRB 835
Cleveland, OH 44106
Phone: 216.368.7565
Fax: 216.368.4223
https://www.linkedin.com/in/jubosch/

CEO & Co-Founder at InterRayBio, LLC

Johns Hopkins University
Bloomberg School of Public Health
Department of Biochemistry & Molecular Biology

> On Aug 21, 2019, at 4:49 AM, Anastassis Perrakis  wrote:
> 
> Something is unclear to me in the original question. What does “has used his 
> pdb for a publication” mean? Somebody used an entry already in the PDB? 
> Somebody used a “.pdb” coordinates file for publication (without “.mtz”)? 
> What was and is the relationship between A and B? 
> 
> In any case, assuming that A and B are not in talking terms (have you tried 
> through a mediator?), it is the director or designated ombudsperson of the 
> institute of A, that should review the case internally, and officially 
> contact the corresponding person of the institute of B. I can’t see what the 
> journal has to do with it, without a settlement between institutes. I also do 
> not consider a direct contact if A to the director of B appropriate. There 
> should be procedures for these cases. 
> 
> A. 
> 
> Sent from my iPhone
> 
> On 21 Aug 2019, at 10:12, Mark J van Raaij  <mailto:mjvanra...@cnb.csic.es>> wrote:
> 
>> Dear Flemming,
>> 
>> As I understand it (I may be wrong), the final responsible institutions are 
>> those where the authors work. But as you say, they sometimes don't even 
>> reply - or they just may be very slow because they want to be really sure 
>> before committing to any answer.
>> 
>> But the journal has a responsibility also, to retract the paper if there is 
>> a serious suspicion the data were not obtained ethically. Of course, it may 
>> be difficult to prove ownership of a pdb file, if both authors claim 
>> ownership there is not really a way the journal can decide who is right. In 
>> my opinion, the journal should officially contact the institutions where the 
>> authors work to try and resolve this. The institutions may take the journal 
>> more seriously than a single researcher.
>> 
>> A generally respected institution that may advise on authorship disputes is 
>> COPE, Committee on Publication Ethics: https://publicationethics.org/ 
>> <https://publicationethics.org/>
>> May also take a while though...
>> They have a database with anonymised examples of previously resolved 
>> disputes that may be helpful - you may find a similar situation on which 
>> they have "ruled". These are of course not

Re: [ccp4bb] need someone officially settle a pdb dispute for a publication

[Jürgen Bosch]

Can’t help you with this, however, I have a couple of PDB deposits that I did 
not publish on and if somebody took them and used my coordinates that’s fine as 
long as they cite the DOI affiliated with the deposition.

Now the question for you, were A and B collaborating on this? If so then 
there’s a different problem and indeed misconduct or at least not good practice 
by B.

Communication is a skill, that some people definitely need to learn.

Jürgen 
 

> On Aug 20, 2019, at 11:45 AM, Flemming Goery  
> wrote:
> 
> Dear All, 
> 
> A and B belong to 2 different institutes. A claimed B has used his pdb for a 
> publication in Journal C. Journal C did not give the retraction, but permit 
> complain related to the journal publication author issue, with the 
> prerequisite journal C did not have the authority on authorship dispute. Then 
> A has e-mailed to the institute head of B with academic misconduct by B as 
> claim, the institute head of B did not give reply.
> 
> In this situation, can A have the journal  authorship  dispute settled by a 
> neutral reviewer (Journal C view: you (A) need to reach out to the 
> institutions that have authority to adjudicate on such matters, as 
> investigation and adjudication on authorship claims falls outside the remit 
> of journal editors. )? Who are qualified as the neutral reviewer so that the 
> review decision can be submitted to Journal C?
> 
> If you believe you are qualified, or you know somebody or some organization 
> qualified, please let me know and I will introduce the issue to you by 
> separate e-mail (it is best not disseminated, am I right?)
> 
> Best regards.
> 
> Flemming
> 
> 
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Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Better Beamline suggestion!

[Jürgen Bosch]

Hi Chandra,

If I remember correctly, the SSRL beam lines 12-2 and 14-1  allow kappa offset 
plus you can swing out the detector.
You might have to come up with a special collection strategy, that you collect 
in resolution bins to avoid overlaps.

Jürgen

P.S. my previous messages got blocked, let’s see if this one passes.

> On Aug 20, 2019, at 5:18 AM, colin.n...@diamond.ac.uk 
>  wrote:
> 
> I agree with Ed that focusing on the detector might be better than on the 
> crystal. An alternative is to reduce the beam divergence which might be 
> easier on some beamlines. 
> 
> The idea is to ensure the instrument parameters (e.g. beam size, beam 
> divergence, detector distance) and data collection parameters (e.g. rotation 
> increments) do not degrade the diffraction pattern. This should ensure all 
> spots which are intrinsically separated are properly resolved. I don't really 
> see the need for a multi circle goniometer if this is done. If the spots are 
> naturally smeared due to crystal imperfections then they overlap anyway. In 
> this case, as Ed says, one has to ensure the profiles are adequately sampled 
> so that any deconvolution procedure has a chance of succeeding.
> 
> Colin
> 
> 
> -Original Message-
> From: CCP4 bulletin board  On Behalf Of Edward A. Berry
> Sent: 19 August 2019 17:08
> To: ccp4bb 
> Subject: Re: [ccp4bb] [EXTERNAL] Re: [ccp4bb] Better Beamline suggestion!
> 
>  I would respectfully suggest that higher pixel resolution does not generally 
> help much in these situations. If an average spot is 10 or more pixels wide 
> then the profile is defined pretty well. But if the spots overlap, they still 
> overlap with higher pixel density. It may make profile fitting more accurate, 
> allowing more accurate "deconvolution" of the compound spot into its 
> components, but it will not improve the overlap. Smaller or better-focused 
> (on the detector, not the crystal?) beam, and longer camera length can help.
>   It is analogous to chromatography- If two peaks coming off the column 
> overlap, collecting smaller fractions will not help to resolve them. It may 
> allow a better-informed decision on the cut-off points when you pool the 
> fractions, but it won't separate the overlap.
>   On the other hand a multi-circle goniometer is very useful. I remember in 
> one of our last trips at SSRL (2007-8?) we used a (Huber 4-circle?) and it 
> was very easy to have the long axis in the plane of the image throughout the 
> rotation.  In the absence of such you can resort to carefully bending the 
> loop or bending the pin (Jim Holton made a nifty device for bending the pin) 
> while keeping the xtal bathed in the cold stream.
> 
> 
> On 08/19/2019 11:12 AM, graeme.win...@diamond.ac.uk wrote:
>> Chandra
>> 
>> What you are looking for here is a beamline with a detector with many pixels 
>> (so you can resolve the long axis) and a multi-axis goniometer - probably a 
>> SmarGon / kappa and an Eiger 16M would make a good combination for this. 
>> Searching on
>> 
>> https://urldefense.proofpoint.com/v2/url?u=http-3A__biosync.sbkb.org_=DwIFAg=ogn2iPkgF7TkVSicOVBfKg=cFgyH4s-peZ6Pfyh0zB379rxK2XG5oHu7VblrALfYPA=VcmIp54F7yM1JdiEMdBdR0y7xinGb-nsn2-3LI_BHto=5VFyqMBHljO-AEcXr3-pqjF8xFyEejXetVFOxOXLp_Y=
>> 
>> Should allow you to make up a short list
>> 
>> Best wishes Graeme
>> 
>> On 19 Aug 2019, at 16:08, Chandramohan Kattamuri 
>> <1c5b7cb6c764-dmarc-requ...@jiscmail.ac.uk>
>>  wrote:
>> 
>> 
>> Dear All
>> We recently collected a data set at APS, Chicago with unit cell dimensions 
>> of 68.4; 68.4 and 991.6 A. Our diffraction data extends to 3A with the APS 
>> set up, however, the long axis has been problematic, resulting in streaking 
>> of the diffraction data and requires a very specific orientation of the 
>> crystal for usable diffraction. Can anyone recommend beamlines that can give 
>> us higher resolution, or a source with a better goniometer allowing for more 
>> angle manipulation after looping?
>> Thank a lot
>> Chandra K
>> 
>> 
>> 
>> 
>> 
>> 
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>> 
>> 
> 
> 
> 
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Re: [ccp4bb] raw data deposition

[Jürgen Bosch]

Which trps protein check the MSGPP or SGPP website they might have what you are 
looking for.

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/

On Oct 28, 2011, at 8:19, Vellieux Frederic frederic.velli...@ibs.fr wrote:

 I must say that there were some emails exchanged between me and Gerard 
 later, in which I pointed out that I wasn't against deposition of images 
 (data frames). In fact, if SR sources kept user's data there would be 
 one more structure from here in the PDB: HDD failure here, the data on a 
 mirror HDD but the company in charge of maintenance erased the data 
 frames and data processing statistics by accident. For a trypanosomal 
 enzyme there is no chance that I can ever get funding now to replicate 
 the work (protein production and purification, crystallisation, data 
 collection) so that Table 1 could be produced for a manuscript.
 
 However, my email to the bb was provocative - I admit I was doing this 
 willingly - to write that in such harsh funding times someone could 
 start a career, get some small grant, enough to clone produce purify 
 crystallize and collect a first data set. And then find him or herself 
 without funding for X years (success rate = less than 10% these days). 
 If this person then gets scooped by whoever, end of a promising career. 
 Unfortunately, such a prospect doesn't seem to be science fiction any 
 more nowadays. I hope this clears things. I wanted to be provocative and 
 point out the difficulties we are all facing wrt funding so that we 
 shouldn't set up a system that may result in killing careers. Our 
 politicians do not need any help from us on that I think.
 
 Fred.
 
 Gerard Bricogne wrote:
 Dear Remy,
 
 You are right, and I was about to send a message confessing that I had
 been rash in my response to Fred's. Another person e-mailed me off-list to
 point out that sometimes a structure can be quickly solved, but that doing
 all the rest of the work involved in wrapping that structure into a good
 biological story for publication can take a very long time, and that it
 would be wrong for a SR source's forced disclosure policy to start imposing
 deadlines on that process. I entirely agree with both of you and admit that
 I reacted too quickly and with insufficient thought to Fred's message.
 
 However, as you point out yourself, this issue is related to a
 different question (SR sources' disclosure policy towards all data collected
 on their beamlines) from the original one that started this thread
 (deposition of raw images with the pdb entries they led to). The two topics
 became entangled through the idea of prototyping an approach to the latter
 by tweaking the storage and access features involved in the former. 
 
 Many thanks to you and to the other correspondent for picking up and
 correcting my error. This however leaves the main topic of this thread
 untouched.
 
 
 With best wishes,
 
  Gerard.
 
 --
 On Fri, Oct 28, 2011 at 01:38:29PM +0200, Remy Loris wrote:
 
 Dear Gerard,
 
 I cannot agree. Last year my group published a paper in Cell which 
 contained a structure for which the native data were collected at a 
 synchrotron around 1997. Various reasons contributed to the long lag period 
 for solving this structure, but basically it all came down to money needed 
 to do the work. Equally I am sure there are other cases for which a first 
 good native data set is a breakthrough you wish to protect rather than hand 
 it out to anyone who might potentially scoop you after you have put lots of 
 money and effort into the project.
 
 Therefore: Images corresponding to structures I deposit in the PDB: No 
 problem. That is what we do with processed data as well. But images of 
 unsolved structures, I don't see why that should be enforced or done 
 automatically by synchrotrons. Nobody deposits processed data without an 
 accompanying structure either.
 
 I do agree that one could be given the option to deposit interesting data 
 with which he/se will not continue for whatever reason. But this should be 
 optional, and a clear consensus should emerge within the community as how 
 the original producers of the data have to be acknowledged if these data 
 are used and the results published by another team, especially if the use 
 of that particular dataset is crucial for the publication.
 
 Remy Loris
 Vrije Universiteit Brussel and VIB
 

Re: [ccp4bb] Off-topic: DSF thermo cycler low temp limit

[Jürgen Bosch]

CD spec with Pelletier is an option too

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/

On Oct 26, 2011, at 19:08, Reginald McNulty rmcnu...@uci.edu wrote:

 We are trying to determine Tm value of rather unstable proteins with a Tm in
 the mid 20 C range using DSF/thermofluor.  Our Stratagene thermocycler has a
 low temp limit of 25 C (Peltier).  I called the company and they said it's a
 'hardware limit' that cannot be changed.
 
 1) Has anyone been able to 'hotwire' the MX3000/3005 to go below this limit?
 
 2) Are there other thermofluor machines that allow lower starting
 temperatures (say 4, 10 or 15 C)?
 
 Best regards,
 -Reggie
  
 

Re: [ccp4bb] UV imaging of crystals

[Jürgen Bosch]

I once tested such a commercial system in Seattle about 4 years ago. It did not 
impress me. In particular the discrimination between salt and protein did not 
work for about 10 different proteins from which we already had collected data. 
sure those were small between 10 and 100 micrometer. Excuse was to few 
tryptophans
So in theory it is nice but a cheaper variant might be to add Gfp to your 
protein and screen for something green.
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/

On Sep 15, 2011, at 16:03, Frank von Delft frank.vonde...@sgc.ox.ac.uk wrote:

 A while ago I was trying to be cheap, so we played around with it quite 
 a bit in the lab.  After rediscovering some of the basics of 
 signal-to-noise and microscope transmission efficiency and that sort of 
 rot, I realised that the commercial systems may not be all that 
 ridiculously overpriced after all.  Not if one wants to be able to say 
 something useful about really really small crystals -- the only ones 
 that really matter in the grand scheme of things (big ones are quick to 
 test; little ones must first be optimized = money+time).
 
 But maybe I was just being incompetent.  Happens.
 phx.
 
 
 
 
 On 15/09/2011 20:50, Andrew Purkiss-Trew wrote:
 Quoting Harman, Christinechristine.har...@fda.hhs.gov:
 
 Hi All,
 I was curious if any of you have tried or even know if it is
 possible to adapt a stereoscope (in my case an Olympus SZX10 model)
 so as to view protein crystals with UV illumination. Basically, I
 want a cheap manual version of what a Rock UV Imager does.  I know
 this is probably a crazy dream.  However, I would greatly appreciate
 any comments, advice or experience any of you may have.
 
 Molecular Dimension do such an adaptor which fits to existing microscopes.
 
 See
 http://www.moleculardimensions.com/shopdisplayproducts.asp?id=121cat=X%2DtaLight%3Csup%3E%99%3C%2Fsup%3E+100+%2D+UV+for+Microscope+
 
 
 
 This message was sent using IMP, the Internet Messaging Program.

Re: [ccp4bb] Mac OSX 10.7 Lion

[Jürgen Bosch]

One really nice feature though comes with what Bill's wife hates. No worries 
about saving an edited file in many programs. The background versioning is 
great no more needs of having my_manuscript_###1.doc :-) one file to handle 
them all. And you can go back to older versions of course. The only disturbing 
feature about this integrated auto backup is sometimes your Mac goes idle 
before you can typebecause it is saving another version. This is particularly 
annoying when you edit large Keynote files.

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/

On Sep 10, 2011, at 5:26, harry powell ha...@mrc-lmb.cam.ac.uk wrote:

 Hi
 
 My two ha'porth.
 
 If you are thinking of upgrading your sole Mac software development  
 box to Lion I'd say don't do it unless you like a lot of pain.  
 Anything built on Snow Leopard should run okay on Lion (my Tiger  
 builds seem okay on 10.4, 10.5, 10.6...), so unless you really have  
 an over-riding need to move to 10.7, I'd hang fire.
 
 If you're only running applications and not developing, you can  
 always shout at the developers if things go wrong.
 
 My plan is to install Lion on a spare bootable disk and see what  
 happens - if all else fails, at least I can ignore the upgrade until  
 Apple release 10.7.1, 10.7.2, etc and fix most of their screw-ups.
 
 On 10 Sep 2011, at 08:03, Jacques-Philippe Colletier wrote:
 
 Hi,
 
 Overall, the transition from 10.6 is seemingless,  
 crystallographically-wise.
 
 Of course you need to have the new XCode 4.1 installed, and you  
 should also download new, 10.7-dedicated 64bits gcc/gfortran/g77  
 bundles from http://hpc.sourceforge.net/
 And then, CNS, Phenix, CCP4, etc... will just run perfect.
 I had to reinstall Coot -- but that's minor.
 (Mac)Pymol also works fine, yet (for some reason) uses a lot of  
 resources even when idle.
 As per the Upsalla Soft. Factory programs, you'll have to recompile  
 then using the above mentioned bundles, or get already compiled  
 binaries from Mark Harris.
 You'll also need to recompile Gromacs (and fftw3) if you're using  
 it -- but it then works great.
 
 I agree with W. Scott on the fact that the new OS is really greedy  
 in terms of resources.
 I surely wont upgrade my other, older mac computers that run just  
 fine on 10.6.
 But if you have a new Mac, Lion is is really neat.
 
 Best
 Jacques
 
 
 Le Sep 10, 2011 à 2:09 AM, William Scott a écrit :
 
 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/
 
 Harry
 --
 Dr Harry Powell,
 MRC Laboratory of Molecular Biology,
 Hills Road,
 Cambridge,
 CB2 0QH

Re: [ccp4bb] PyMol plugin

[Jürgen Bosch]

Symexp is not sufficient for hour purpose ?
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/

On Aug 14, 2011, at 18:15, Yuri Pompeu yuri.pom...@ufl.edu wrote:

 I installed the plugin superSym  but when I triesd to run it, ended up with 
 the following message:
  File 
 /home/local/UFAD/yuri.pompeu/Desktop/PHENIX_installation/phenix-installer-dev-833/phenix-dev-833/pymol/modules/pmg_tk/PMGApp.py,
 line 156, in initialize_plugins
__builtin__.__import__(mod_name)
  File 
 /home/local/UFAD/yuri.pompeu/Desktop/PHENIX_installation/phenix-installer-dev-833/phenix-dev-833/pymol/modules/pmg_tk/startup/SuperSymPlugin12.py,
 line 18, in ?
 Color: map_colour defined as [ 0.000, 0.500, 1.000 ].
 Color: iso_diff_map_colour defined as [ 0.000, 1.000, 0.250 ].
quit(Oops! SuperSym requires cctbx and numeric python to function. Please 
 install these.)
 
 I then installed cctbx+Python bundle from http://cci.lbl.gov/cctbx_build/ and 
 sourced it.
 I still get the same result in PyMol though..
 Does anyone know what to do?
 Thank you for your time
 Best,
 Yuri

Re: [ccp4bb] Apochromatic and Achromatic

[Jürgen Bosch]

What type of camera were you thinking to add to the microscope ? Faveon based 
chip ? 30+ megapixel regular CMOS based chip ? If the answer to both is no, 
then go with the cheaper lense (achromatic). After all it is not about the 
beauty of the crystals, if they diffract or not does not depend on apo- or 
achromatic lenses.

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/

On Jul 30, 2011, at 5:46, Ramanathan Natesh r.nat...@mail.cryst.bbk.ac.uk 
wrote:

 Dear All,
In crystal examination stereo zoom microscopes, how important is 
 it if one chooses a Achromatic objective lens compared to Apochromatic 
 lens.
   Achromatic can give higher working distance ~ 70 mm or more as 
 compared to Apochromatic, which may be important for picking crystals / 
 manipulating before flash freezing. 
   Is it good to compromise on working distance to get Apochromatic 
 objective?
 
  An input from the community will be of great help to me.
 
 Many thanks in advance,
 Natesh.
 
 --- 
 Ramanathan Natesh 
 Now at IISER-TVM (India).
 
 Live Simply and do Serious Things .. 
 - Dorothy Mary Crowfoot Hodgkin OM, FRS 
 
 In Science truth always wins
 - Max Ferdinand Perutz OM FRS
 ---

Re: [ccp4bb] ARP/wARP install on 6.2.0 RHEL 6?

[Jürgen Bosch]

Hi Jonathan,
seems to be a UW centered day today on the BB (Eric, Jan, you, me).
Have the permissions changed ? I assume you are installing as root ?
Wouldn't be surprised if Ethan replies soon :-)

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/

On Jul 27, 2011, at 20:31, Jonathan Kay jp...@u.washington.edu wrote:

 Hi all,
 
 I have a RHEL 6 x86_64 machine I recently installed CCP4-6.2.0 onto; the 
 install went through fine, but when I went to install the ARP/wARP GUI (via 
 System Administration - Install/uninstall task), I received the following 
 error in the shell window I started ccp4i from:
 
 UnpackTaskArchive: uncompress failed to create 
 /tmp/user/install_ARP_wARP_CCP4I6/ARP_wARP_CCP4I6.tar
 ExamineTaskArchive: failed to unpack temporary copy of 
 /usr/local/arp_warp_7.1/ARP_wARP_CCP4I6.tar.gz
 
 /tmp is not at all full and has plenty of inodes left.
 (running the install.sh from the arp_warp_7.1 directory doesn't install it 
 either)
 
 I have searched around for some solutions, but haven't found anything really 
 relevant.
 The odd thing I have another x86_64 machine running RHEL 5 that I can do the 
 exact same install method and it works (and using the install.sh from 
 arp_warp_7.1/ works too), so I wonder if something changed with RHEL6 that 
 might be causing problems?
 
 Anyone have any suggestions?
 
 Thanks!
 Jonathan

Re: [ccp4bb] hello

[Jürgen Bosch]

In addition to Fred's suggestion, are you certain about your space group ? I 
assume you tried phaser with different space groups turned on ?
Another thing to look at maybe the model you are using for MR has some 
extensions that lead to clashes in your crystal packing and therefore the 
second molecule can not be placed. You can trim your model to a core and try 
searching with that. 
Or if your one molecule is correctly placed do some rigid body refinement, then 
use this solution as fixed input model for molrep and try placing the second 
one.
How does your selfrotation function look like ?
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/

On Jun 22, 2011, at 5:08, Afshan Begum afshan...@yahoo.com wrote:

 Dear all,
 
  Could any one help me regarding my serious problem actually i have collected 
 data at 3.0 and cut off 3.1 where the data statics showed the good  values 
 for the further processing.
  According to the methew coefficient there would be two molecule in the 
 asymmetric unit but after running the molrep its provide only one monomer 
 instead of two, for this reason R values is very high 50%. Actually 
 homologous model having  P6322 space group where as my one is F4132.  I had 
 tried to run phaser as well  phenix but both were failed to process further. 
 i really do not know how can i get the second chain in my structure. Please 
 if you have some ideas i will appreciate and would be many thankful to you.
 
 Hope to hearing you soon
 
 Best Regards
 
 AFSHAN
 
 

Re: [ccp4bb] citric acid + 40%MPD (pHI -H1 condition)

[Jürgen Bosch]

It will freeze out of the drop. So that's good.
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/

On Jun 20, 2011, at 19:09, Jayashankar s.jayashan...@gmail.com wrote:

 Dear friends,
 
 Should I be happy if i get my crystals with one of the condition with a 
 commercial kit
 containing citric acid and 40%MPD+ protein in(hepes+MgCl2)+ADP+VO3
 
 any insights ...
 
 
 S.Jayashankar 
 Research Student 
 Institute for Biophysical Chemistry
 Hannover Medical School 
 Germany.

Re: [ccp4bb] Help! low resolution protein-DNA complex

[Jürgen Bosch]

Another thing you should bookmark and check out is the Molprobity page (google 
for it), upload your pdb file and let it evaluate what you have done. The chart 
it generates will guide you through your structure and you will be able to do a 
good job.
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/

On Jun 17, 2011, at 8:30, Robbie Joosten robbie_joos...@hotmail.com wrote:

 Dear Xun,
  
   I have a 3.2A dataset for a protein-DNA complex. The protein is
   a homodimer, and the DNA is almost palindromic (except one base pair
   in the middle and two or three base pairs at both two ends). It is my
   first time solving structures, and unfortunately the resolution is
   low. No body in our lab has used ccp4 or phenix, so I am really
   frustrated as a second year student. 
  Your frustration is understandable. It is somewhat of an expectation in
  academia that your advisor will either help you directly or if she/he is
  not familiar with the methodology you are forced to use, will find
  someone to help you. The questions you ask surely may be answered by
  someone in your department. IMHO, a second year student should not be
  left alone to battle his first structure which happens to be 3.2A
  protein/DNA complex.
 Indeed, this is just asking for problems. It's a good call that you asked for 
 help. Perhaps your supervisor can arrage for you to be embedded in a 
 crystallography lab for a while. That should give you easy access to people 
 with experience.
  
   I mainly used ccp4. So far, the best R/Rfree I got is 0.27/0.34.
  and that is not bad given the resolution
 You are heading the right way. You should be able to close the R/R-free gap a 
 bit more.
 
   I went to the crystallography meeting, and people suggested me to
   rely more on geometry. I remember I got a DNA restraints file and a
   refmac script from someone on this mailing list, and that really
   helped (otherwise the DNA base pairing will be weird). Can someone
   tell me how to restraint the protein (helix)?
  one way of doing it would be to restrain the hydrogen bonds that
  stabilize the helix. It is not advisable at higher resolution, but
  sounds alright at 3.2A. I once used a restraint file to keep DNA sane
  by forcing Watson-Crick pairing, the helical restraints would work
  pretty mnuch in the same way. Look at the structure of the restraint
  file that you have and modify it to include the helix-stabilizing
  hydrogen bonds.
 I like real-space refining everything in Coot with tight helical restraints. 
 You may need to chainge the default restraint weight matrix (lower numbers 
 give tighter restraints). The options are under the R/RC button.
  
   People also suggested me to include NCS and TLS in the
   refinement, but I don't know how to. For NCS, I should define a region
   that are the same in both monomers? Should I use tight or loose
   restraints? For TLS, I don't have a clue.
  Yes and tight (at least at first). For TLS you may want to take a look
  at the TLSMD server. (Also, consider tighter restraints on B-factors).
  Otherwise, just define TLS for the whole thing, then protein and DNA
  separately, then individual monomers and whatever pieces of DNA common
  sense suggests would move together. Keep whatever combination gives you
  the lowest Rfree.
 In Refmac you can use local NCS which takes away the need to mess with NCS 
 selections (which can be really difficult). Although it is not needed for 
 Refmac, you should make sure that the same residues in different monomers 
 have the same residue number. Be conservative with TLS (in the beginning). 
 One group per chain sounds right. In the case of your DNA you can consider 
 putting both chains in one group. Tight B-factor weights may be needed, you 
 could also trying one overall B factor. I personally only do that when TLS 
 works well. Oh, and always use riding hydrogens in refinement. It helps a lot 
 at low resolution, because of the VDW restraints. For that same reason you 
 should not be too conservative with the sidechains (at least not for the ones 
 in the core of the protein).
 
 Since you have only started building you should probably go through the 
 entire structure a few times. After that, use structure validation tools 
 frequently. WHAT_CHECK and Molprobity are must-use tools for that. Coot also 
 has many usefull features for validation. Good luck.
  
 Cheers,
 Robbie Joosten

Re: [ccp4bb] Problems in refinement

[Jürgen Bosch]

60% occupied Zn site perhaps ?

Q2 do you have leftover atoms from a previous dual conformation refinement ?
Try deleting the corresponding residues in a texteditor and not coot to ensure 
they are really gone, then rebuild the section into the new diff- map.

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/

On May 26, 2011, at 6:11, Petr Kolenko kole...@imc.cas.cz wrote:

 Dear colleagues,
 
 I have two problems in two structure refinements using REFMAC5.
 
 1) 1.8A resolution, zinc in the active site. Refinement using work
 reflections - ADP for Zinc was about 14. Final refinement including
 all reflections increase ADP to 20 or even higher values - followed by
 very high positive difference density in position of the zinc. I have
 tried also PHENIX, the same thing. I changed ADP manually to 14 and
 only calculated maps (no refinement) look good. May I deposit the
 structure using manually fixed ADP according to the best agreement
 to the observed and difference electron density? By the way, it is
 clear that this is zinc.
 
 2) 1.9A resolution, about 600AA, all of them OK in electron density.
 But, somehow, about ten atoms give very strong positive electron
 density suggesting they are not taken into account in refinement. On
 the other hand, ADPs are reasonable and seem to be refined. All of
 these atoms are fully occupied. I tried to omit whole residues and
 build them again, but the maxima appeared again. Using of PHENIX
 resulted in no difference electron density for these atoms. I have
 also tried to take PHENIX output to REFMAC, but the maxima are there
 again. It is always one or two atoms from the same residues -
 sometimes Calpha, sometimes Cbeta, sometimes C, sometimes NH1, but
 still on the same five residues. Does anyone have any idea how to
 solve this problem?
 
 Many thanks for any response.
 
 Petr
 
 
 -- 
 Petr Kolenko
 petr.kole...@biochemtech.uni-halle.de
 http://kolda.webz.cz

Re: [ccp4bb] Tev Cleavage issue !!

[Jürgen Bosch]

I'd say since you obtained crystals with your tag it is not a disturbing factor 
and either disordered or making contacts. So removing the tag you might end up 
not getting crystals in the worst case. Now to the question why they don't 
diffract. Did you test the old fashioned way at RT in capillary ? Maybe your 
freezing is the problem. 
The inability to purify TEVed protein suggests that you have at least a dimer 
perhaps or higher multimer. Since you observe three bands on the gel, 
uncleaved, cleaved and TEV itself.  Any idea about DLS or migration behavior on 
a SEC column for your uncleaved protein ?
Since you have crystals what I would do is crush them up and rescreen whatever 
commercial screens you have available using some of the crushed crystals as 
seed.
A second option would be to take your current condition and modify it with say 
the most frequent 12 cryo solutions added  in maybe 5-10% effective 
concentration and see if you still obtain crystals. Before freezing them I 
would then freeze directly from the drop and a second crystal would be soaked 
into whatever concentration of the cryo is required to properly freeze. I bet 
you will find something that works.
Of course you don't stop there. Once your crystals are on the gonio and 
diffract or don't diffract you will flash anneal them once or multiple times 
and report back in a nice table which condition resulted in the 2.3A dataset.
So you have roughly 146 experiments to do alone from cryo- optimization.
Good luck !
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/

On Apr 7, 2011, at 22:37, anita p crystals...@gmail.com wrote:

 Hi Crystallographers,
  I am working of 23 Kda protein with a Nterminal  His tag and a TEV cleavage 
 site.
  I am getting crystals with the his tag and tev site intact, but they dont 
 diffract.
  Is it probable that they dont diffract because of the extra his tag and the 
 tev site?
 
  I am trying to get rid of this tag but the reaction is optimum at 10:1 
 protein to TEV ratio in micrograms overnight incubation without shaking.
  I tried to run it on histrap column after this reaction but I am not able to 
 purify  cleaved protein from TEV and uncleaved.
  I have tried several times but I get 3 bands ie., the TEV, uncleaved and 
 Cleaved.
  I have also tried to use the Nibeads instead of the histrap column, but no 
 difference is seen.
  Is there a possible way to approach this problem?
 
  Suggestions awaited
  Anita

Re: [ccp4bb] Crystallographic Breakthrough - DarkMatter Version 1.0

[Jürgen Bosch]

I assume TLS is supported or do we have to wait for version 1.1 ? When will you 
have a 10.7 (lion) standalone version compiled ? 

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/

On Apr 1, 2011, at 2:06, Ethan Merritt merr...@u.washington.edu wrote:

 Hi to all on ccp4bb:
 
 What better day to announce the availability of a breakthrough technique
 in macromolecular crystallography?
 
 Given recent discussion and in particular James Holton's suggestion that
 the problem of disordered sidechains is a problem akin to the difficulty
 of describing dark matter and dark energy...
 
 I am happy to announce a new crystallographic tool that can improve your
 model by accounting for an often-neglected physical property. A detailed
 explanation, references, and a preliminary implementation of the program
 can be downloaded from
 
http://skuld.bmsc.washington.edu/DarkMatter
 
 -- 
 Ethan A Merritt
 Karmic Diffraction Project
 Fine crystallography since April 1, 2011
 What goes around, comes around - usually as a symmetry equivalent

Re: [ccp4bb] (off-topic) Measurement of channel pore dimensions

[Jürgen Bosch]

Try the program Hollow. Or visit USF and find the cavity script. There's also 
Carver out there but I never got it really to work for my purposes, but it's 
been written for channels.

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/

On Mar 23, 2011, at 9:02, Van Den Berg, Bert 
lambertus.vandenb...@umassmed.edu wrote:

 Hello all,
 
 Does anyone know how to get values for pore sizes of membrane channels? I’m 
 not interested in A x B angstrom values measured between atom centers and 
 assuming a regular pore shape, but a “real-life” value of either surface area 
 at the narrowest point or the volume of a block centered on the narrowest 
 point of the pore.
 
 Thanks, Bert

Re: [ccp4bb] while on the subject of stereo

[Jürgen Bosch]

I never thought I would agree with Tassos :-)
But same experience 3D and wiggling with a snaked ligand makes live so much 
easier.
It helps to have a fast graphics card though.
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/

On Mar 23, 2011, at 4:21, Anastassis Perrakis a.perra...@nki.nl wrote:

 To add my two cents, I am not at all a fun of stereo for routine use, but I 
 appreciate it if I have to look at a long ligand bent 90 deg in the middle to 
 bury in my protein, and all that at 3.0-3.5 A resolution ... So, I must have 
 both types of brain damage that Jan refers to. No surprises here I guess.
 
   A.
 
 On Mar 23, 2011, at 3:16, Phoebe Rice wrote:
 
 My 2 cents worth on the stereo-dependent:
 
 1) They have carpal tunnel syndrome that makes it painful to keep the 
 molecule in motion while rebuilding it (NOTE: enough constant mouse-wiggling 
 and you will get carpal tunnel problems if you don't have them yet!)
 
 2) They work on big, low-resolution structures where you need to see a 
 bigger-picture view.  I've had people tell me that can fit 3-3.5A maps just 
 fine without stereo, but having viewed their work, I beg to differ.
 
  Phoebe
 
  Original message 
 Date: Tue, 1 Mar 2011 22:30:54 +
 From: CCP4 bulletin board CCP4BB@JISCMAIL.AC.UK (on behalf of Jan Löwe 
 j...@mrc-lmb.cam.ac.uk)
 Subject: Re: [ccp4bb] while on the subject of stereo  
 To: CCP4BB@JISCMAIL.AC.UK
 
 Ah! The question of to stereo or not to stereo! There has to be a 
 scientific reason why this question is more popular than asking for what 
 Linux distro is more fashionable this spring or why an Rmerge of 0.90 in 
 the outermost shell is good for you and your structure.
 
 I am offering my two (conflicting) theories (and apologies that both 
 seem to imply some problem):
 
 A) people who do use stereo have a problem with their brain because they 
 cannot produce three dimensional vision from depth cues alone.
 
 B) people who do not use stereo have a problem with their brain because 
 they cannot see properly in three dimensions and rely on depth cues alone.
 
 I personally prefer people with A) when I am their passenger in a car 
 since they do not need to rotate by 90° to see how far the braking 
 lights of the car in front are away :-)
 
 jan
 
 
 
 On 01/03/2011 21:35, Jim Pflugrath wrote:
 I will offer my view.
 
 I hate stereo glasses and hate stereo in general.
 
 One should be able to see 3D from the depth-cueing and by keeping the view
 in motion.  For fitting, I like to flip the view by 90 degrees.  I know I 
 am
 going to move in displayX and displayY, but never in displayZ.  I then
 rotate the view around the vertical axis so thatn the old displayZ becomes
 displayX.
 
 Furthermore, I don't waste too much time fitting.  I know the software can
 fit the map better than me, so I let it do its job.  I only need to get the
 coordinates within the radius of convergence of the refinement program.  I
 also know that 9 times out of 10, the displayed electron density is 
 probably
 suspect, so I believe in stereochemistry more than I believe in the map.
 
 The main trick is to realize that as a human being, you really are not that
 good at fitting the map or that it is unnecessary to waste your time since
 the software is really so much better than you.  Refinement is quick enough
 that you can try various hypotheses as in:  If I move this here, then
 refinement will do the trick and Well, that didn't work, so I will move
 that over there and see if refinement will do the trick.
 
 As for stereo figures, you should be able to convey what you want to say
 from a good figure with depth-cueing, shadows, etc.  Don't ever use stereo
 glasses in a public seminar.  Maybe my opinion will change with better
 stereo technology.
 
 OK, I know quite a lot of people will disagree with me. :)
 
 Jim
 
 -Original Message-
 From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David
 Roberts
 Sent: Tuesday, March 01, 2011 10:29 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] while on the subject of stereo
 
 Hi again,
 
 I'd like to ask a question about the pedagogy of stereo.  That is, using
 stereo with students in the classroom.
 
 Do you all find that, after setting up these elaborate stereo devices,
 students really use the stereo or do they tend not to?
 
 I am a huge fan of stereo - and frankly here we have quite a few options 
 for
 doing stereo - from the active Nvidia systems that people have recently 
 been
 discussing to passive zalmans. ...
 
 As I mentioned, I like stereo a lot, but really projecting on a nice bright
 lcd monitor also has it's advantages, and with the ease of moving things
 using

Re: [ccp4bb] while on the subject of stereo

[Jürgen Bosch]

I think the weight of the shutter glasses puts them off. Compared to the 30g or 
less of the Zalmans the shutter glasses  feel like bricks. I would estimate 
them to at least 270g. After one hour wearing them you feel them on your nose.
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/

On Mar 23, 2011, at 9:40, Christoph Parthier cparth...@googlemail.com wrote:

 Hi Dave,
 
 We recently equipped  a pool of 25 computers with Nvidia 3D shutter 
 glasses, they're used in structural biology courses for undergraduate 
 students of biochemistry . We teach mainly PyMOL, but (in an advanced 
 course) also model building in COOT. Of course, we let the students 
 decide whether they want to use hardware stereo or not. They all try. 
 After several courses now I realized no more than 30% of the students 
 keep using them in visualization and model building, while the majority 
 of students put the glasses aside... Some of the 30% said, it helps, but 
 could also do without. Haven't assessed this properly yet... ;-)
 
 Christoph
 
 
 David Roberts wrote:
 Thanks for the comments, I do appreciate them.  I guess we went off in 
 a direction I wasn't thinking of - related to your personal like or 
 dislike of stereo.  What I am really looking for is an answer to a 
 simple question in that is stereo a nice thing from a pedagogy 
 standpoint for showing students complex biomolecules.
 
 I am in a chemistry department - undergraduate only.  We focus on 
 3-dimensional shape and the importance of shape of chemical 
 function/reactivity/etc...  With small molecules (PF5, etc...), it's 
 easy to see how shape works by simply rotating the molecule.  The 
 molecules are small enough, the concept of 3D can be visualized easily 
 in these systems.  Furthermore, they can make a simple model using 
 your standard organic or inorganic model kit, no worries.
 
 Now, bring in a huge protein, or a protein-protein complex.  The issue 
 of 3Dness becomes fuzzier.  It's not so easy to see which hydrogen 
 will get plucked off during a chemical reaction, even with careful 
 zooming and mouse manipulation.  So my question still is, how many of 
 you feel stereo is important from a pedagogy standpoint (not looking 
 at maps, just structures that are huge and complex).  Is it something 
 that we need to try to bring to the classroom, or is it just a cool 
 toy like the 3D TV that hopefully is going nowhere and will soon fade 
 out like the viewmaster of old.  I know a large percentage of people 
 cannot see stereo (at least the way we present it), and so it isn't 
 for everybody.  But, does it help, and if so, does it help when done 
 in a huge classroom or when put on an individual screen.  Has anybody 
 tried to assess this (there's a horrible word for you).
 
 That's what I was wondering about.  Presenting the stereo is a 
 different issue (how is that done), but I think there are lots of 
 avenues for that depending on your particular situation.
 
 Thanks again
 
 Dave

Re: [ccp4bb] how to use mac to solve structures

[Jürgen Bosch]

Just google for crystallography on os x and you will find Bill Scott's 
excellent guide through the galaxy.
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/

On Feb 5, 2011, at 0:33, LISA science...@gmail.com wrote:

 Hi all, 
  I just start to install crystallography software on my new mac. My os X 
 version is 10.6. Can some one show me how to install phenix, cns , ccp4 and 
 so on? Thank you.
 
 Lisa
 

Re: [ccp4bb] Merging data to increase multiplicity

[Jürgen Bosch]

Bad data = processing with XDS

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/

On Jan 28, 2011, at 6:46, José Trincão trin...@dq.fct.unl.pt wrote:

 Hello all,
 I have been trying to squeeze the most out of a bad data set (P1, 
 anisotropic, crystals not reproducible). I had very incomplete data due to 
 high mosaicity and lots of overlaps. The completeness was about 80% overall 
 to ~3A. Yesterday I noticed that I could process the data much better fixing 
 the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but with 
 a multiplicity of 1.7. I tried to integrate the same data fixing the 
 mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in 
 completeness, Rmerge and multiplicity.
 Now, is there any reason why I should not just merge all these together and 
 feed them to scala in order to increase multiplicity?
 Am I missing something?
 
 Thanks for any comments!
 
 Jose
 
 
 José Trincão, PhDCQFB@FCT-UNL
 2829-516 Caparica, Portugal
 
 It's very hard to make predictions... especially about the future - Niels 
 Bohr

Re: [ccp4bb] How to use XDS programme to process data collected at Q315r detector

[Jürgen Bosch]

I don't see the job card in your script.
So it will assume job= all and then it is more than normal that xds will 
complain about the indexing being inaccurate. Look at the idxref.lp file and 
pick your space group then rerun xds adding job=devpix
See what happens
Am on my phone will send you my script later
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/

On Dec 8, 2010, at 9:53, wu donghui wdh0...@gmail.com wrote:

 Dear all,
 
 Thank you very much for your valuable inputs. This is an update based from 
 the suggestions.
 
 Following Pierre's suggestion, I installed xdsme 
 (http://code.google.com/p/xdsme/) and run it. xdsme extracts image header as 
 the below:
 
  Image format:  adsc
  Detector type: ADSC 315
  Detector distance:   480.00 mm
  X-ray wavelength:1.1397 A
  Oscillation range:   0.5000 degree
   Beam coordinate X:   1535.3 pixel
 Y:   1535.3 pixel
 
 As the detector type should be ADSC 315r, the indexing shows warning 
 information that  !!! WARNING in IDXREF. Solution is inaccurate.
 
 Based on Miles's suggestion, I run the program adxv ( 
 http://www.scripps.edu/~arvai/adxv.html) to extract image header as the below:
 
 Distance 480.000 mm
 Pixel Size 0.10259 mm
 Wavelength 1.13967 A
 Beam Center X: 1535 pixels
 Y: 1536 pixels
 OSC_RANGE 0.5000 degree
 
 However the beam center pixel seems to indicate that ccd detector is Q315 but 
 not Q315r based on the provided template script from XDS website as below.
 
 *
 ! Example file XDS.INP for the ADSC Q105 CCD-detector 
 ! Characters in a line to the right of an exclamation mark are comment.
 !*
 !NOTE: XDS can handle only SMV images of TYPE=unsigned_short.
 !  Images are expected to be already corrected for spatial distortions.
 !
 !Standard settings for the ADSC Q105 that rarely need to be changed
 
  DETECTOR=ADSC  MINIMUM_VALID_PIXEL_VALUE=1  OVERLOAD= 65000
  DIRECTION_OF_DETECTOR_X-AXIS= 1.0 0.0 0.0
  DIRECTION_OF_DETECTOR_Y-AXIS= 0.0 1.0 0.0
  TRUSTED_REGION=0.0 1.05 !Relative radii limiting trusted detector region
 !UNTRUSTED_RECTANGLE= 570 1920  1469 2048  ! rectangle: X1 X2   Y1 Y2  
 !UNTRUSTED_ELLIPSE= 910 11101010 1210  ! ellipse enclosed by X1 X2 Y1 Y2
 !File name, access, format of dark-current (non-Xray background) image
 !DARK_CURRENT_IMAGE=../images/blank.tif!hardly ever used
 
 !MAXIMUM_NUMBER_OF_JOBS=4  !Speeds-up COLSPOT  INTEGRATE on a Linux-cluster
 !MAXIMUM_NUMBER_OF_PROCESSORS=4!33;ignored by single cpu version of xds
 !MINUTE=0   !Maximum number of minutes to wait until data image must appears
 !TEST=1 !Test flag. 1,2 additional diagnostics and images
 
 !NX=number of fast pixels (along X); QX=length of a X-pixel (mm)
 !NY=number of slow pixels (along Y); QY=length of a Y-pixel (mm)
 !For the ADSC, the number of detector pixels and their sizes are
 !obtained from the image header. It is therefore unnecessary to
 !specify values for NX, NY, QX, QY.
 !NX=2304 NY=2304 QX=0.0816 QY=0.0816   !ADSC Q4
 !NX=2048 NY=2048 QX=0.0500 QY=0.0500   !ADSC Q105
 !NX=2048 NY=2048 QX=0.1024 QY=0.1024   !ADSC Q210 at ESRF ID-29
 !NX=4096 NY=4096 QX=0.051  QY=0.051!ADSC Q210r
 !NX=3072 NY=3072 QX=0.10259 QY=0.10259 !ADSC Q315 at SSRL 9-2
 !NX=6144 NY=6144 QX=0.0513 QY=0.0513   !ADSC Q315r at ESRF ID-29
 
 
 In summary, I can not index my data collected at Q315r Taiwan 13B1 beamline 
 using XDS program. It seems that XDS process data from Q315 is more efficient 
 that from Q315r.
 
 Any suggestions will be much appreciated. 
 
 Best regards,
 
 Donghui
 
 
 
 
 
 On Wed, Dec 8, 2010 at 11:03 AM, wu donghui wdh0...@gmail.com wrote:
 Dear all,
 
 Recently I collected several data sets at 13B1 Taiwan beamline with Q315r 
 detector. It's no problem to index these datasets using mosflm, but Rms 
 residual and weighted residual is high. Here I want to try XDS to play my 
 data. I downloaded a template example as below.
 
 !*
 ! Example file XDS.INP for the ADSC Q105 CCD-detector 
 ! Characters in a line to the right of an exclamation mark are comment.
 !*
 !NOTE: XDS can handle only SMV images of TYPE=unsigned_short.
 !  Images are expected to be already corrected for spatial distortions.
 !
 !Standard settings for the ADSC Q105 that rarely need to be changed
 
  DETECTOR=ADSC  MINIMUM_VALID_PIXEL_VALUE=1  OVERLOAD

Re: [ccp4bb] How to use XDS programme to process data collected at Q315r detector

[Jürgen Bosch]

Look into the header of your image file via more or run mosflm on one image and 
look at the output in the terminal window.

You'll get the pixelsize and you'll need to convert the beamcenter into pixels 
from mosflm. Keep in mind that beam X = orgy in xds and beam y is orgx
Plus the usual stuff lambda distance 2theta

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/

On Dec 7, 2010, at 22:03, wu donghui wdh0...@gmail.com wrote:

 Dear all,
 
 Recently I collected several data sets at 13B1 Taiwan beamline with Q315r 
 detector. It's no problem to index these datasets using mosflm, but Rms 
 residual and weighted residual is high. Here I want to try XDS to play my 
 data. I downloaded a template example as below.
 
 !*
 ! Example file XDS.INP for the ADSC Q105 CCD-detector 
 ! Characters in a line to the right of an exclamation mark are comment.
 !*
 !NOTE: XDS can handle only SMV images of TYPE=unsigned_short.
 !  Images are expected to be already corrected for spatial distortions.
 !
 !Standard settings for the ADSC Q105 that rarely need to be changed
 
  DETECTOR=ADSC  MINIMUM_VALID_PIXEL_VALUE=1  OVERLOAD= 65000
  DIRECTION_OF_DETECTOR_X-AXIS= 1.0 0.0 0.0
  DIRECTION_OF_DETECTOR_Y-AXIS= 0.0 1.0 0.0
  TRUSTED_REGION=0.0 1.05 !Relative radii limiting trusted detector region
 !UNTRUSTED_RECTANGLE= 570 1920  1469 2048  ! rectangle: X1 X2   Y1 Y2  
 !UNTRUSTED_ELLIPSE= 910 11101010 1210  ! ellipse enclosed by X1 X2 Y1 Y2
 !File name, access, format of dark-current (non-Xray background) image
 !DARK_CURRENT_IMAGE=../images/blank.tif!hardly ever used
 
 !MAXIMUM_NUMBER_OF_JOBS=4  !Speeds-up COLSPOT  INTEGRATE on a Linux-cluster
 !MAXIMUM_NUMBER_OF_PROCESSORS=4!33;ignored by single cpu version of xds
 !MINUTE=0   !Maximum number of minutes to wait until data image must appears
 !TEST=1 !Test flag. 1,2 additional diagnostics and images
 
 !NX=number of fast pixels (along X); QX=length of a X-pixel (mm)
 !NY=number of slow pixels (along Y); QY=length of a Y-pixel (mm)
 !For the ADSC, the number of detector pixels and their sizes are
 !obtained from the image header. It is therefore unnecessary to
 !specify values for NX, NY, QX, QY.
 !NX=2304 NY=2304 QX=0.0816 QY=0.0816   !ADSC Q4
 !NX=2048 NY=2048 QX=0.0500 QY=0.0500   !ADSC Q105
 !NX=2048 NY=2048 QX=0.1024 QY=0.1024   !ADSC Q210 at ESRF ID-29
 !NX=4096 NY=4096 QX=0.051  QY=0.051!ADSC Q210r
 !NX=3072 NY=3072 QX=0.10259 QY=0.10259 !ADSC Q315 at SSRL 9-2
 !NX=6144 NY=6144 QX=0.0513 QY=0.0513   !ADSC Q315r at ESRF ID-29
 
 !AIR=0.001 !Air absorption coefficient of x-rays is computed by XDS by default
 
 !== JOB CONTROL PARAMETERS ===
 !JOB= XYCORR INIT COLSPOT IDXREF DEFPIX XPLAN INTEGRATE CORRECT
 
 !== GEOMETRICAL PARAMETERS ===
 !ORGX and ORGY are often close to the image center, i.e. ORGX=NX/2, ORGY=NY/2
  ORGX=3072.0  ORGY=3072   !Detector origin (pixels). ORGX=NX/2; ORGY=NY/2
  DETECTOR_DISTANCE= 480  !(mm)
 
  ROTATION_AXIS= 1.0  0.0 0.0
  OSCILLATION_RANGE=0.5!degrees (0)
 
  X-RAY_WAVELENGTH=1.14   !Angstroem
  INCIDENT_BEAM_DIRECTION=0.0 0.0 1.0
  FRACTION_OF_POLARIZATION=0.90 !default=0.5 for unpolarized beam
  POLARIZATION_PLANE_NORMAL= 0.0 1.0 0.0
 
 !=== CRYSTAL PARAMETERS =
  SPACE_GROUP_NUMBER=0  !0 for unknown crystals; cell constants are ignored.
  UNIT_CELL_CONSTANTS= 139.53   249.90   119.83  90.000 112.511 90.000
 
 ! You may specify here the x,y,z components for the unit cell vectors if
 ! known from a previous run using the same crystal in the same orientation
 !UNIT_CELL_A-AXIS=
 !UNIT_CELL_B-AXIS=
 !UNIT_CELL_C-AXIS=
 
 !Optional reindexing transformation to apply on reflection indices
 !REIDX=   0  0 -1  0  0 -1  0  0 -1  0  0  0
 
 !FRIEDEL'S_LAW=FALSE !Default is TRUE.
 
 
 
 
 However indexed cell parameter for a b and c is small and error information 
 indicates that cell solution is not accurate.  I suspect that I need to 
 change some input parameters besides selection of ORGX=3072.0  ORGY=3072 when 
 using XDS as the template is for ADSC Q105.
 
 Here I want to know if anyone has experience to use XDS to process data 
 collected at Q315r and what error I made for using this script. 
 
 Thank you very much for any suggestion.
 
 Best regards,
 
 Donghui

Re: [ccp4bb] Tough 'shell' on disturbed drop

[Jürgen Bosch]

Often you can also avoid this skin formation by adding a bit of your reservoir 
solution first to make it a larger droplet.
Then the microtools as mentioned earlier or just two loops
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/

On Nov 25, 2010, at 6:45, Rick mbp08...@sheffield.ac.uk wrote:

 Dear CCP4
 
 I looped a v.thin rod emerging from a cluster of v.thin rods that grew in 
 29%PEG1500 and 0.1M SPG buffer at pH7.5 (succinic acid, sodium dihydrogen 
 orthophospate and glycine). The loop i used had been washed more than 10 
 times with deionised water (so assumed as 'clean'). The crystals had grown at 
 17degreesC, and looped out probably just below room temperature (~20-23 
 degreesC). When transferred to 5% glycerol cryo-buffer the crystal 
 disintegrated (maybe due to glycerol being an unfavourable addition to the 
 mother-liquor). When i looked back at the original cluster-containing drop, a 
 very tough shell had formed over the surface of the drop, from which chunks 
 could be dug out...the nearest analogy is maybe like when you blow-torch 
 sugar on top of creme brulee, and have to crack it with your spoon. The 
 crystals within had also disintegrated. Any clues to what might have caused 
 this very tough shell to form, and maybe how to deal with it? 
 
 Much appreciated
 
 Rick Salmon

Re: [ccp4bb] Question on calculation of RMSD

[Jürgen Bosch]

Take any protein where a helix moves as a rigid body and look at your results.
Then fix the alpha helix and superimpose the rest of the structure on that 
moving helix and compare both results.
If you want to give it a shot try 2pc4 and the unliganded structure.

You see the difference in both methods ?

From a structural/functional perspective  one method will highlight the real 
differences in terms of movements.

For an initial understanding I recommend SSM and a morphing movie, that helps 
dissecting what might be going on.

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/

On Nov 16, 2010, at 19:58, Clement Angkawidjaja 
clem...@bio.mls.eng.osaka-u.ac.jp wrote:

 The lowest rmsd might not be the biological relevant one
 True, however the least square fit using the right residues (thus producing 
 the lowest rmsd possible) can really tell you the most significant 
 differences (or not signifcant ones) that cause biological changes. Human 
 eyes are often not good in doing this. Using automated lsq fit programs can 
 give you precise (initial) information using the lowest human effort and low 
 chance of human error.
  
 Clement
  
 As confucius would say, don't trust the output of a program if you have not 
 programmed it yourself or know what it's doing.
 
 Last words of wisdom for tonight.
 
 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/
  
 On Nov 14, 2010, at 8:32 PM, Clement Angkawidjaja wrote:
 
 DALI server (http://ekhidna.biocenter.helsinki.fi/dali_server/). Choose the 
 pairwise alignment function. It is all automatic and will give you the 
 lowest rmsd. Note, however, that it will omit parts that are very different 
 for calculation.
 
 Within CCP4, SUPERPOSE or COOT can also do that. You need to specify the 
 residues you want to use for calculation for the lsq fit function.
 
 Regards,
 Clement Angkawidjaja, PhD
 Specially Appointed CMP Assistant Professor
 Graduate School of Engineering
 Osaka University
 2-1 Yamadaoka GSE Commoon East 8F
 Suita-shi, Osaka 565-0871, Japan
 Tel/Fax +81-6-6879-4580
 http://www.mls.eng.osaka-u.ac.jp/~bio_ext/mlsbe123/clement.html
 ///
 G30 Chemistry/Biology Combined Major Program
 http://cmp.sci.osaka-u.ac.jp/CMP/
 
 -Original Message- 
 From: E rajakumar
 Sent: Monday, November 15, 2010 6:52 AM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Question on calculation of RMSD
 
 Dear All
 I have two structures of homo-dimeric protein complex with different DNA.
 I want to calculate RMS deviation between second monomer from these two 
 complexes by fixing superposed firstmonomer.
 
 This I require to know what is the effect of DNA on relative orientation of 
 two monomers in the dimer.
 
 Previously I was using MOLEMAN2 to do this calculation.
 
 Please can you suggest me any other program to do this calculation.
 
 Thanking you
 Raj
 
 
 E. Rajakumara
 Postdoctoral Fellow  Strcutural Biology Program  Memorial Sloan-Kettering 
 Cancer Center  New York-10021  NY  001 212 639 7986 (Lab)  001 917 674 6266 
 (Mobile)
 
  

Re: [ccp4bb] Crystal gel band

[Jürgen Bosch]

The other thing you can try to do is the-cheap-men-silver-stain

scan your gel and bump up the contrast, you'd be surprised what you can detect 
even from a regular stained gel.
Best results are if you make the gel first as a greyscale image.

Google for Neuhoff stain and bump up the phosphoric acid to 10%.

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/

On Nov 2, 2010, at 10:19 AM, Jim Pflugrath wrote:

 It reads like you need to run a lane or two with a positive control of some 
 kind.  Can you grow lysozyme, glucose isomerase, hemoglobin or other crystals 
 of a protein around the same expected molecular weight and try run on the gel 
 lanes with about the same amount of crystalline volume as your putative 
 protein crystals?
 
 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of 
 xaravich ivan
 Sent: Monday, November 01, 2010 9:51 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] Crystal gel band
 
 Hi everyone,
 I have grown some crystals after micro-seeding starting from thin-small 
 needles from needle-clusters. These crystals are larger in size than the 
 needles but are comparable to the shape and don't look like salt crystals. 
 But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a 
 home source,handy and would like to send these to the synchrotron.
 
 Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say, 
 the amount of protein is  1uG? 
 Has anyone experienced such a thing (no band in gel, but crystal diffracts)? 
 It would be nice if I get observations/suggestions.
 
 ivan

Re: [ccp4bb] Lysis of Pichia pastoris

[Jürgen Bosch]

You shouldn't have clogging up problems in the first place. We don't use Pichia 
but have the same system and I've used it before at the MPI in Martinsried. The 
system is designed that there are no parts which break, you only have to 
maintain it on a regular basis and clean it properly.
Do you have a manual or pressure driven controller ? With the manual one, 
people tend to over tighten the valve and thereby ruining the system as a metal 
spike hits a metal outlet (in the end you grind a hole into the base and can 
break the whole system). That's really the only part that can be broken. With 
the pressure controlled system this user error is eliminated. Take it apart, 
clean it and reassemble it is my suggestion - did you ever took it apart to 
clean it up or add the vacuum lubricant ? By the way the Hampton heavy duty 
vacuum grease works very well for that purpose, needs to be replaced every 
three to four months.

The only other alternative would be a glass beater and regularly autoclave and 
clean the glass beads.

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/

On Nov 2, 2010, at 5:33 PM, Cory Brooks wrote:

 Hello all;
 
 I have successfully expressed a membrane protein in Pichia pastoris,
 however I am having a difficult time with cell lysis.
 I have used a Avestin emulsiflex to lyse them, however I have had many
 difficulties with the system clogging up, and parts wearing out with the
 high pressures.
 
 So I am wondering what other people out there use to lyse their Pichia?
 In particular we have been considering
 a microfluidizer
 a Retsch mixer mill
 a TS-series cell disruptor (Constans systems)
 
 Any thoughts on these options, or other systems would be much appreciated!
 
 Best regards,
 Cory
 
 Cory Brooks, Ph.D.
 Postdoctoral Fellow
 University of Alberta

Re: [ccp4bb] Lysis of Pichia pastoris

[Jürgen Bosch]

Yes that's how it's conventionally done if you are working with plants.
Instead of a drill (what did safety inspection said to that, did you have 
proper precautions in place etc.) they use a mortar cooled in LN2 and also 
containing LN2 within the mortar and a suitable grinder plus cryo gloves (for 
you).

Then you keep grinding until you have a nice pesto. Thinking about this, we 
should do that for smaller amounts instead of diluting the cells so that they 
can be run through the Emulsiflex ... Need to go to my freezer and pull out a 
pellet and grind them up.

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/

On Nov 2, 2010, at 6:19 PM, Jacob Keller wrote:

 I know this seems a bit crazy, but I used to lyse s. cerevisiae by
 pelleting the cells into a 50mL conical, freezing in liquid nitrogen,
 and using a power drill with a drill bit fitting the caliber of the
 tube, all at liquid nitrogen temp through periodic (or constant)
 re-immersion in nitrogen. It seemed to work really well, and it was
 nice how no proteolysis could take place because of the temp. It
 yielded a talc-like powder which, once all nitrogen was gone, was
 resuspended in whatever buffer. This can also be done for e coli
 pellets, and might be advisable for really proteolysis-sensitive
 proteins.
 
 JPK
 
 On Tue, Nov 2, 2010 at 4:33 PM, Cory Brooks cbro...@uvic.ca wrote:
 Hello all;
 
 I have successfully expressed a membrane protein in Pichia pastoris,
 however I am having a difficult time with cell lysis.
 I have used a Avestin emulsiflex to lyse them, however I have had many
 difficulties with the system clogging up, and parts wearing out with the
 high pressures.
 
 So I am wondering what other people out there use to lyse their Pichia?
 In particular we have been considering
 a microfluidizer
 a Retsch mixer mill
 a TS-series cell disruptor (Constans systems)
 
 Any thoughts on these options, or other systems would be much appreciated!
 
 Best regards,
 Cory
 
 Cory Brooks, Ph.D.
 Postdoctoral Fellow
 University of Alberta
 

Re: [ccp4bb] How to get PDB of small molecule which is not available in data base..??

[Jürgen Bosch]

Pubchem ?
Hicup ?
Prodrg ?

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/

On Oct 31, 2010, at 2:13, Hussain Bhukyagps hsn...@yahoo.com wrote:

 Hi all..,
 
 How to get a PDB of small molecule which is not available in data base..??
 
 is there any server that provides a area where in we can do a chemdraw and 
 get the 3D picture of it and also PDB format
 
 plz help.
 
 Thank you, 
 
 Regards,
 Hussey
 

Re: [ccp4bb] DLS

[Jürgen Bosch]

instead of DLS you could also look into buying a fancy PCR machine ~30K where 
you can perform
a) regular PCR
b) quantitative PCR
c) thermal stability tests for your protein

Ericsson et al. Thermofluor-based high-throughput stability optimization of 
proteins for structural studies. Anal Biochem (2006) vol. 357 (2) pp. 289-98

Crowther et al. Buffer Optimization of Thermal Melt Assays of Plasmodium 
Proteins for Detection of Small-Molecule Ligands. Journal of biomolecular 
screening : the official journal of the Society for Biomolecular Screening 
(2009) pp. 

Sure you are missing the quantification for how homogeneous your sample is and 
an estimation of the molecular weight of the particles in solution but the 
correlation between crystallizability and DLS is not so good compared to 
thermal shifts as pointed out in Ericsson 2006.

You will have a high end purification system so you could get a sense of your 
sample homogeneity via SEC instead

Just a thought,

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/

On Oct 30, 2010, at 12:02 AM, Mohd Shukuri Mohamad Ali wrote:

 Hi there
 
 Sorry for the off ccp4 topics. I am planning to get a DLS to set up my
 crystallography lab. I got a tight budget. Around USD 8 perhaps. I'm
 quite new to this field.
 
 Thanks in advance for your suggestions
 
 Regards
 Shukuri

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

[Jürgen Bosch]

Regarding the riding hydrogens:
They are obviously not visible, but your protein in solution is also not 
visible but still has those weird riding hydrogens:-)
Use them, they are there. And ther was a recent compendium of neutron 
scattering in one of our favorite journals if you really want to see them.

Another rule to add:
You are 98% done with refinement of your structure, does it really matter - I 
mean from a functional/ biological perspective ?
Is it wrong to stop at some point ?
This of course implies you've already passed rule 3-5 from Robbie.

Just some Espresso-thoughts in the morning

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/

On Oct 27, 2010, at 1:29, Robbie Joosten robbie_joos...@hotmail.com wrote:

 Dear Anthony,
 
 That is an excellent question! I believe there are quite a lot of 'rules of 
 thumb' going around. Some of them seem to lead to very dogmatic thinking and 
 have caused (refereeing) trouble for good structures and lack of trouble for 
 bad structures. A lot of them were discussed at the CCP4BB so it may be nice 
 to try to list them all.
 
 
 Rule 1: If Rwork  20%, you are done.
 Rule 2: If R-free - Rwork  5%, your structure is wrong.
 Rule 3: At resolution X, the bond length rmsd should be  than Y (What is the 
 rmsd thing people keep talking about?)
 Rule 4: If your resolution is lower than X, you should not 
 use_anisotropic_Bs/riding_hydrogens
 Rule 5: You should not build waters/alternates at resolutions lower than X
 Rule 6: You should do the final refinement with ALL reflections
 Rule 7: No one cares about getting the carbohydrates right  
 
 
 Obviously, this list is not complete. I may also have overstated some of the 
 rules to get the discussion going. Any addidtions are welcome.
 
 Cheers,
 Robbie Joosten
 Netherlands Cancer Institute
 
 Apologies if I have missed a recent relevant thread, but are lists of
 rules of thumb for model building and refinement?
 
 
 
 
 
 Anthony
 
 
 
 Anthony Duff Telephone: 02 9717 3493 Mob: 043 189 1076
 
 
 

Re: [ccp4bb] Help with Optimizing Crystals

[Jürgen Bosch]

You did check on a gel that they are indeed your protein ?

If you have sufficient amounts available try digesting it with various 
proteases and see if you can identify a stable fragment.

A less radical approach, which might not be accessible to you, you could screen 
your protein for alternative buffer conditions using DSF and then pick a 
condition under which it seems to be very stable according to its melting 
temperature in the buffer.

You've spared us the details of your purification procedure, maybe a polishing 
step at the end with a SEC might do wonders.

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/

On Oct 26, 2010, at 4:23 PM, Matthew Bratkowski wrote:

 Hello.
 
 I have obtained disk shaped crystals of a protein that I am working on.  I 
 got hits in about 10 different conditions, with a few common precipitants and 
 pHs, and I have optimized two conditions so far.  In the optimized 
 conditions, the crystals appear overnight, usually surrounded by or hiding 
 under heavy precipitant. Under the best conditions, I get what I would 
 describe as single disks, some of which are of decent size and very round, 
 that rotate light very well.  Sub-optimal conditions can give small to large 
 crystal clusters.  I shot the large disk crystals grown from one conditions 
 at the synchrotron. but they do not diffract.
 
 I was wondering if anyone had any advice about optimizing these crystals in 
 order to get them to diffract better?  As mentioned before, I have only tried 
 optimizing a few of the hit conditions (varying precipitant conc., pH, etc.), 
 but crystals from all of the hits look the same: always round disks or disk 
 clusters.  This leads me to believe that optimized conditions of the other 
 hits will produce similar results as before.  Would it be worthwhile to try 
 optimizing these conditions as well?  I have also tried seeding, which just 
 produces a lot of clusters, and an additive screen.  Some of the additives 
 help to produce larger crystals, but again I always get single or disk 
 clusters.
 
 Any advice would be helpful.
 
 Thanks,
 Matt
 

Re: [ccp4bb] Help with Optimizing Crystals

[Jürgen Bosch]

 
 Hi.
 
 Here is some additional information.
 
 1.  The purification method that I used included Ni, tag cleavage, and SEC as 
 a final step.  I have tried samples from three different purification batches 
 that range in purity, and even the batch with the worst purity seems to 
 produce crystals.
Resource Q ? two or more species perhaps ? Does it run as a monomer dimer 
multimer on your SEC ?

 
 2. The protein is a proteolyzed fragment since the full length version did 
 not crystallize.  Mutagenesis and methylation, however, may be techniques to 
 consider since the protein contains quite a few lysines.
 
 3. There are not any detergents in the buffer, so these are not detergent 
 crystals.  The protein buffer just contains Tris at pH 8, NaCl, and DTT.
 
 4. Some experiments that I have done thus far seem to suggest that the 
 crystals are protein.  Izit dye soaks well into the crystals, and the few 
 crystals that I shot previously did not produce any diffraction pattern 
 whatsoever.  However, I have had difficulty seeming them on a gel and they 
 are a bit tough to break.
Do they float or do they sink quickly when you try to mount them ?
 
 5.  I tried seeding previously as follows: I broke some crystals, made a seed 
 stock, dipped in a hair, and did serial streak seeding.  After seeding, I 
 usually saw small disks or clusters along the path of the hair but nothing 
 larger or better looking.
 
 I also had one more question.  Has anyone had an instance where changing the 
 precipitation condition or including an additive improved diffraction but did 
 not drastically change the shape of the protein?  If so, I may just try 
 further optimization with the current conditions and shoot some more crystals.
 

The additive screen from Hampton is not bad and can make a big difference.


A different topic is it a direct cryo what you are using as a condition ? If 
not what do you use a s a cryo ? Have you tried the old-fashioned way of 
shooting at crystals at room temperature using capillaries (WTHIT ?)

You might be killing your crystal by trying to cryo it is what I'm trying to 
say here.

Jürgen


 Thanks for all the helpful advice thus far,
 Matt
 
 

Re: [ccp4bb] Hardware question

[Jürgen Bosch]

Hi Ed,

I have four of those 
http://www.newegg.com/Product/Product.aspx?Item=N82E16822136514
and would now buy these
http://www.newegg.com/Product/Product.aspx?Item=N82E16822136764

DELLete it, I mean the quote you have and shop somewhere else.

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/

On Oct 26, 2010, at 9:52 PM, Edward A. Berry wrote:

 Another question about computer hardware- If I configure a computer at the
 Dell site, it costs about $700 to add a 2TB SATA drive.
 On amazon.com or Staples or such, a 2TB drive costs ~$110. to $200
 depending on brand.
 
 Are the Dell-installed drives much faster, or more reliable, or have
 a better warranty?  After all, RAID is supposed to stand for redundant
 array of inexpensive disks, and we could afford a lot more redundancy
 at the Amazon.com price.
 
 And, are there any brands or models that should be avoided due to known
 reliability issues?
 
 Thanks,
 eab

Re: [ccp4bb] **Possible spam**cryoprotetant for 35% Dioxane

[Jürgen Bosch]

And if you are not so into reading papers, you can use this database
http://idb.exst.jaxa.jp/db_data/protein/search-e.php?

Jürgen

P.S. Who wants to write an App for that, wouldn't this be very handy at the 
beamline ? I take 5% of the income for the idea :-)
-
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/

On Oct 21, 2010, at 1:07 PM, Edward Snell wrote:

 Not to forget
 
 J. Appl. Cryst. (1996). 29, 584-587[ doi:10.1107/S0021889896004190 ]
 Glycerol concentrations required for cryoprotection of 50 typical protein 
 crystallization solutions
 E. F. Garman and E. P. Mitchell
 
 Which prompted the McFerrin and Snell work.
 
 Also worth checking out (and I apologize if I missed others)
 
 Acta Cryst. (2008). D64, 287-301[ doi:10.1107/S0907444907067613 ]
 Glycerol concentrations required for the successful vitrification of cocktail 
 conditions in a high-throughput crystallization screen
 R. Kempkes, E. Stofko, K. Lam and E. H. Snell
 
 J. Appl. Cryst. (2006). 39, 244-251[ doi:10.1107/S0021889806004717 ]
 Effects of cryoprotectant concentration and cooling rate on vitrification of 
 aqueous solutions
 V. Berejnov, N. S. Husseini, O. A. Alsaied and R. E. Thorne
 
 
 Cheers,
 
 Eddie
 
 Edward Snell Ph.D.
 Assistant Prof. Department of Structural Biology, SUNY Buffalo,
 Senior Scientist, Hauptman-Woodward Medical Research Institute
 700 Ellicott Street, Buffalo, NY 14203-1102
 Phone: (716) 898 8631 Fax: (716) 898 8660 
 Skype:  eddie.snell Email: esn...@hwi.buffalo.edu  
 Telepathy: 42.2 GHz
 
 Heisenberg was probably here!
 
 
 -Original Message-
 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Eric 
 Larson
 Sent: Thursday, October 21, 2010 12:14 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] **Possible spam**cryoprotetant for 35% Dioxane
 
 Hi Jerry,
 
 A great reference for initial cryocondition searches for many standard 
 crystallization solutions is the tables in:
 
 J. Appl. Cryst. (2002). 35, 538-545  [ doi:10.1107/S0021889802009238 ]
 The development and application of a method to quantify the quality of 
 cryoprotectant solutions using standard area-detector X-ray images
 M. B. McFerrin and E. H. Snell
 
 On the bottom of page 542 it says for 35% dioxane as the precipitant in 
 Hampton Crystal Screen II condition # 4:
 
 25% glycerol, 25% PEG 400, 20% ethylene glycol, 15% propylene glycol 
 (1,2-propanediol)
 
 good luck,
 
 Eric
 
 __
 Eric Larson, PhD
 Biomolecular Structure Center
 Department of Biochemistry
 Box 357742
 University of Washington
 Seattle, WA 98195
 
 
 
 On Wed, 20 Oct 2010, Jerry McCully wrote:
 
 | Dear All;
 | 
 | We just got some crystals from 35% (v/v) Dioxane. We are going to 
 collect some data soon.
 | 
 | Does anyone have the experience with the cryoprotectant in this 
 condition?
 | 
 | Thanks a lot,
 | 
 | Jerry McCully
 | 
 | 
 | 
 |

Re: [ccp4bb] JAC (2010), 43,1242-1249

[Jürgen Bosch]

Thank you all for not sharing the pdf.

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/

On Oct 19, 2010, at 11:33 AM, Michel Fodje wrote:

 It is available online at
  
 http://www.stat.ucla.edu/history/essay.pdf
  
  
 
 Michel Fodje
 Staff Scientist
 Canadian Macromolecular Crystallography Facility
 Canadian Light Source
 Tel: 306 657 3758
  
  
  
 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Jürgen 
 Bosch
 Sent: October-18-10 8:34 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] JAC (2010), 43,1242-1249
  
 Lesenswert !
 Worth reading !
 http://journals.iucr.org/j/issues/2010/05/02/issconts.html
  
 Could somebody send me the pdf from Bayes 1763 ?
  
 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/
  

Re: [ccp4bb] Refinement

[Jürgen Bosch]

1. How does the MR map look like ? Do you see features resembling a real 
solution or just noise ?

2. If the map looks decent, then try running a rigid body refinement using 
Refmac. Does the Rwork/Rfree dro ?

3. At this point you should look at the difference density map in Coot and be 
able to identify wrong amino acids e.g. if your MR model had an Ala where 
there is truly a His or Phe, you should see a huge blob of green density 
indicating the missing atoms in your current model.

4. Fix the sequence of your MR model to your true sequence

5. WARNING don't continue to read the next option if you want to learn and have 
fun by yourself !

6. at 1.9 Å ARP/wARP will be able to build most of your structure automatically

Jürgen

P.S. It's likely you have the correct space group simply by gaussian 
distribution over P222 pointgroups, but maybe that's what the Rwork/Rfree are 
indicating  Did you run Phaser in P222 + all other possible space groups or did 
you feed it with P212121 and only ran it in this space group ?
-
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/

On Oct 18, 2010, at 7:39 PM, Jyotica Batra wrote:

 Hi All
 
 I have a dataset at 1.9A, spacegroup-P212121 (unit cell: 37.7, 39.52, 231.72, 
 90, 90, 90), 
 I used MR phaser and got a structure solution with LLG= 320 (1copy/a.u) .  
 During refinement, the R-free (50%) and R-factors (42%) never go down.
 I have tried refining in phenix and refmac both, but still the high R-free 
 problem persists.  At this point I'm seeking help to know the possible 
 reasons for this high R-free.
 
 Thanks in advance!
 
 Jyotica
 
 

[ccp4bb] JAC (2010), 43,1242-1249

[Jürgen Bosch]

Lesenswert !
Worth reading !
http://journals.iucr.org/j/issues/2010/05/02/issconts.html

Could somebody send me the pdf from Bayes 1763 ?

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/

Re: [ccp4bb] quantum diffraction

[Jürgen Bosch]

Can't find your reference unless you are referring to this one:
JOURNAL OF MODERN OPTICS, 1994, VOL .41, NO .12, 2413-2423

In case I'm wrong, could you post the direct link for download of Dale's paper ?

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/

On Oct 16, 2010, at 11:36 AM, Bernhard Rupp wrote:

 The wave function doesn't collapse to a single outcome 
 until the detector measures something 
 
 Reference:
 Tronrud D, Entanglement-phasing in Quantumcryptocrystallography,
 Nature epub, doi:0101010.
 
 
 

Re: [ccp4bb] Too many microcrystals.......

[Jürgen Bosch]

Sure it's not the peptide that crystallizes ?
You could try adding glycerol, exchanging the PEG, lowering the PEG
playing with the pH or changing to a different buffer system.
You might have done all these already, then you could also try a larger drop 
size.
You could also mix your protein with your reservoir and spin it down, then 
setup the tray but not adding additional reservoir to it.
How about temperature ? Is your protein more soluble at 4˚C or 37˚C ?

Just some thought before the sun rises,

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/

On Oct 10, 2010, at 2:15 AM, Dilip Badgujar wrote:

 hi guys 
 
 I am trying to crystallize protein complexed with peptide(14mer).
 In initial screening i got microcrystals in following condition
 - 0.2M MgCL2
 0.1M Tris pH 8.5
 28 % PEG 4000
 Ptotein concentration - 12.5mg/ml and peptide conc.-5mg/ml
 Complex ratio- (Protein-Peptide)(1:1.5)
 Incubation temp.-22 degree 
 Method - Sitting drop vapor diffusion method 
 
 within four hours of setting trials I can see too many microcrystals.
 Though i tried reduction of protein and precipitant conc. as well as using 
 mineral oil to decrease evaporation rate but still it is not working.
 
 I am waiting for your valuable suggestions.
 

Re: [ccp4bb] The color of GFP crystals?

[Jürgen Bosch]

What GFP did you use, your pH is 4.6 or something around there according to 
your picture. If the pH is true GFP will be colorless. You certainly have some 
more GFP lying around (without your protein fused to it), just titrate it and 
see what pH dependency you have.

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/

On Sep 29, 2010, at 4:41 AM, Tri Ngo wrote:

 Dear CCP4 community,
 
 I wonder if anybody has experience about the color of GFP crystals. Is there 
 any chance that the GFP crystal will become colorless? We are working on one 
 membrane protein fused with GFP. Since we got some crystals which didn't show 
 any color (Condition 01), I am not sure if I should go for optimization.
 
 Please check the link below for the image details: 
 http://www.docstoc.com/docs/55950590/GFP-crystals
 
 Because of the limited amount of sample, we used Topaz system (screening 
 chip) to do the screening and there is no way to confirm these crystals are 
 actually protein crystals. I noticed that the crystals are bigger day by day. 
 Is it a good sign to confirm the protein crystals? 
 
 Thank you very much for your helpful information!
 
 Best wishes,
 
 TriNgo
 
 Structural Biology Lab
 School of Medicine - Sungkyunkwan University
 Phone: 031-299-6150
 

Re: [ccp4bb] Micromatrix seeding using the mosquito

[Jürgen Bosch]

Not with membrane proteins, however we use our Mosquito with 20 nl seeding 
standard.

Strip 1 Protein, Strip 2 Seed stock solution
We first copy the plate by either picking up protein, then reservoir and 
dispensing together 200-400 nl or we multidispense protein and add the 
reservoir later (larger drops, in particular if we can't concentrate the 
protein well then we add 600 nl and hope that enough evaporates before we come 
with the reservoir solution).
As last step we add the 20 nl seed solution.
We've also done seed titrations then we use three strips 1)Protein 2) seed 
buffer solution 3) seed. In that case the combination of 2+3 is always 100nl 
and we start out with 5 nl Seed stock.
In the IntelliPlates (3well) we play the protein/reservoir ratio game + seed
Reproducibility is pretty good. We keep our seed stock where we grew the 
crystals e.g. 20˚C or 4˚C.

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/

On Sep 29, 2010, at 5:23 AM, Patrick Shaw Stewart wrote:

 Hi Jeroen
  
 I think that part of the point of the method is that it’s incredibly quick 
 and easy to do – you can make seeds and run an experiment in 20 mins.  But 
 I’m sure it’s important to use the seed stock quickly and freeze it.
  
  
 Maria
  
 I think most people use 100 nl seed stock, 200 nl reservoir, 300 nl protein 
 as D’Arcy and co originally suggested.  Maybe more seed stock would be better 
 as Roberto says, to increase the additive effect.  The main reasons not to 
 put seed stock into the reservoir are (1) it’s more work – compared to your 
 robot picking up the seed stock from a PCR tube - and (2) sometimes your seed 
 stock is very valuable, for example if you only have one crystal and you make 
 up say 5 ul of seed stock.
  
 Has anyone had success with MMS microseeding with membrane proteins?  I 
 wonder if membrane protein crystals dissolve more easily.
  
 Patrick
  
  
  
 --
 For information and discussion about protein crystallization and automation, 
 please join
 our bulletin board at http://groups-beta.google.com/group/oryx_group?hl=en
  
  patr...@douglas.co.ukDouglas Instruments Ltd.
  DouglasHouse, EastGarston, Hungerford, Berkshire, RG177HD, UK
  Directors: Peter Baldock, Patrick Shaw Stewart
  http://www.douglas.co.uk/
  Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
  Regd. England 2177994, VAT Reg. GB 480 7371 36
  
 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of mesters
 Sent: 28 September 2010 07:26
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Micromatrix seeding using the mosquito
  
 Hello Maria,
 
 the success of this method depends on a few things.
 First, your seed stock-solution. Most people prepare it by smashing up seeds 
 in mother liquor (i.e. reservoir solution). Looking at the phase diagram, you 
 will quickly understand that this might not be the best way to go as you will 
 dissolve your seeds fairly quickly over time. That is why people prepare 
 really fresh stocks, keep them on ice and freeze them as soon as possible in 
 order to stabilize the seeds. Keeping on ice will only help if your protein 
 is less soluble at lower temperatures, which true for roughly 45% of proteins 
 (45% will react retrograde, 10% do not show a clear preference). Looking at 
 the phase diagram again, you will also understand that seeding only works for 
 near equilibrated drops that are metastable-supersaturated. If 
 undersaturated, seeding will not work as the seeds will dissolve over time.
 
 Keeping the above considerations in mind, best is to seed after the drops 
 have equilibrated. Next best option is to add the seeds or the protein as the 
 last component to the drop...
 
 Jeroen.
 
 
 
 
 On 20.09.10 09:53, Maria Håkansson wrote:
 Dear all,
  
 Anyone who have had success using the mosquito for micromatrix seeding?
 Which way is the optimal way of adding seed solution to sitting drops on an 
 MRC plate?
  
 Is it better to add 100 nl protein + 10 nl seed solution + 100 nl reservoir 
 solution or
 to add concentrated seed solution (1 microliter) directly to the reservoir 
 solution and then pipett
 100 nl protein + 100 nl reservoir solution.
  
 Best regards and thanks in advance,
  
 Maria Håkansson
  
  
 
 __
 Maria Håkansson, Ph.D.
 Senior Scientist, Max-lab, Lund University
 Phone: +46 (0) 76 8585 706
 Fax: +46 (0) 46 222 47 10
 Ole Römers väg 1 (P.O. Box 188)  
 SE-221 00 Lund, Sweden   
  
 Web address: www.maxlab.lu.se  
 Email: maria.hakans...@maxlab.lu.se
 __
  
  
  
  
  
 
 --
 
 image002.jpg--
 Dr. Jeroen R. Mesters
  
 Gruppenleiter Strukturelle Neurobiologie und Kristallogenese
 Institut für

Re: [ccp4bb] Regarding quality of protein for crystallization

[Jürgen Bosch]

How about incomplete TEV cleavage ?
Your protein is a dimer/multimer in solution and some of it is cleaved and some 
not and they stick together and are separated on the SDS gel ?

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/

On Sep 27, 2010, at 9:09 AM, vikrant saa wrote:

 Dear all
 Thanks for yours valuable suggestion.
 Just some addition information to make the query clear.
  
 1)My protein has 14 cysteine residues.
 2)It is not a metal binding protein.
 3)I have added the protease inhibitor cocktail + PMSF during sonication and 
 cleavage. I saw only one band of protein bind on beads. Two band appears 
 after cleavage.
 4) I used TEV protease for cleavage. I express it at 24 degree for 16 hrs in 
 Rosetta 2DE3
 5) I have to do MALDI and MS MS to know exact molecular weight and sequence 
 that  differ  in both the proteins. Most of the  peaks in peptide mass 
 fingerprint match exactly while some are at different position and size. 
 Amino acids sequence similarity  in both the cases are upto 1-405 amino acids 
 (as per limitation of peptide mass fingerprint it show some region upto 1-405 
 matching in both the proteins).
  
  
 I  have certain doubt/solution(s) on the basis of yours feedback and above 
 information.
  
 1) How come PTM can play role when protein expressed in bacteria.
 2) TEV is a very specific protease hence non specific cleavage less likely 
 occur.
 3) Cysteine residues or temperature dependent expression may be one of the 
 reason of two band of protein.
  
 Further suggestion to get rid of this problem will be highly appreciated.
  
  
  With Regards
  
 Vikrant
  
 
 

Re: [ccp4bb] Regarding quality of protein for crystallization

[Jürgen Bosch]

What are the minor differences in peptides ?
Any PTM's perhaps ?

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/

On Sep 26, 2010, at 3:43 PM, vikrant saa wrote:

 
 I am working on 45 kd protein (pI 5.3) for crystallization . It is in pGEX 
 vector.I do the expression (induction 24 degree 16 hrs) and purification from 
  Rosetta 2 DE3 cells (has additional rare codon).when I run the affinity 
 chromatography purified protein (cleaved protein after removal of tag) on SDS 
 PAGE I do find 2 bands (one of 45 kd and other 2-3 kd less). The same pattern 
 being retained after FPLC.  Intensity of both the bands remain same even 
 after one or two week of storage at 4 degree. Peptide mass fingerprinting 
 (after trypsin digestion) suggest both are my proteins except minor 
 difference in some peptide peaks.  Is it  because of rare codon/degradation  
 of protein of interest or any other possibilities? Can I use this mixure for 
 crystallization?
  
  
 With Regards
  
 Vikrant
 
  
  
  
 
 

Re: [ccp4bb] protein turns brown

[Jürgen Bosch]

[FeS] clluster ?

Or some metal bound to your protein ?
Is it dark brown or yellowish brown ? What's your protein concentration ?  25 
mg/ml ?

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/

On Sep 24, 2010, at 9:03 AM, Van Den Berg, Bert wrote:

 Maybe you should give us a hint about the identity of your protein (if you 
 dare;-)). I’m sure there are folks around who may be able to say whether 
 or not your protein is supposed to be brown. You can’t expect too much help 
 if you don’t provide (m)any details.
 
 Cheers, Bert
 
 
 On 9/24/10 4:34 AM, sandeep toskgu...@rediffmail.com wrote:
 
 Dear all,
 
 I have purified protein from E.coli. expression system. the protein has been 
 purified with three independant columns. Now during concentration step using 
 amicon, the protein shows brown colour. what could be the reason.
 
 best regards and Thanks,
 sandy 
  
 http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline@middle?
  

Re: [ccp4bb] How to define NCS in REFMAC

[Jürgen Bosch]

Take a look at this part of a script, I added your NCS operators in two options.
So you are working on GroEL ?

Good luck,

Jürgen


#!/bin/csh -f
set prevVer = 01
set prevRun = 00
set currVer = 02
set currRun = 04nolig

set currData = my_latest_structure
# 
set xyzin =  omitted_ligands.pdb
set xyzot = v{$currVer}r{$currRun}_{$currData}.pdb
#

#XDS processed data
set hklin = v02r02_Pf_Cmp24.mtz

set hklot = v{$currVer}r{$currRun}_{$currData}.mtz
#
set log   = v{$currVer}r{$currRun}_{$currData}.log
#

refmac5 \
HKLIN $hklin HKLOUT $hklot \
#   LIBIN Cmp24.cif \
# TLSIN tls_def.tlsin TLSOUT tls.out \
XYZIN tmp.pdb XYZOUT $xyzot \
EOF  $log
MAKE HYDRogens ALL  
MAKE CHECK 0
MAKE CISP N BUILD Y
LABI FP=FP SIGFP=SIGFP FREE=FreeR_flag

REFI TYPE RESTrained RESOlution 25 1.7
#option # 1 over the whole chain
#NCSRestraints NCHAins 7 CHAIns  A B C D E F G 

#or option #2 two domains per chain
#for definitions of restrain codes
# RTFM
# http://www.ccp4.ac.uk/html/refmac5/keywords/restraints.html#ncsr

NCSRestraints NCHAins 7 CHAIns  A B C D E F G NSPANS 2 2 135 1 136 190 1
REFI RESI MLKF
#BFACtor SET_to 90
#REFI TLSC 10
REFI BREF ISOT  ! Refine overall B-values
WEIG MATR 0.1
DAMP 0.5 0.5
SCALe TYPE BULK
SCALe LSSCale 
SCALe LSSCale ANISotropic
SCAL MLSC 
NCYC 10
TEMP 1.0 4.0 6.0 6.0 10.0
MONI MANY DIST 4 TORS 4 ANGL 4 CHIR 4 VDWR 3 NCSR 4 PLAN 4 NCSR 4 BFAC 4
BINS 10
EOF

-
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/

On Sep 9, 2010, at 8:29 PM, Hailiang Zhang wrote:

 Hi there:
 
 The REFMAC manual give me a hard time to define the NCS during refinement.
 Can anybody give a first time user a sample script based on the following
 PDB header (NCS part only is ok, but please include how todefine tight
 restrant only for both positional and B ref, for both NCS groups)? Thanks
 a lot!
 
 Best Regards, Hailiang
 
 REMARK   3  NCS RESTRAINTS STATISTICS
 REMARK   3   NUMBER OF DIFFERENT NCS GROUPS : 2
 REMARK   3
 REMARK   3  NCS GROUP NUMBER   : 1
 REMARK   3 CHAIN NAMES: A B C D E F G
 REMARK   3 NUMBER OF COMPONENTS NCS GROUP : 2
 REMARK   3   COMPONENT C  SSSEQI  TO  C   SSSEQI   CODE
 REMARK   3   1 A  2   A 135  1
 REMARK   3   1 B  2   B 135  1
 REMARK   3   GROUP CHAINCOUNT   RMS WEIGHT
 REMARK   3   TIGHT POSITIONAL   1A(A):   1806 ; 0.092 ; 0.050
 REMARK   3   TIGHT POSITIONAL   1B(A):   1806 ; 0.131 ; 0.050
 REMARK   3   TIGHT THERMAL  1A (A**2):   1806 ; 0.192 ; 0.500
 REMARK   3   TIGHT THERMAL  1B (A**2):   1806 ; 0.228 ; 0.500
 
 REMARK   3
 REMARK   3  NCS GROUP NUMBER   : 2
 REMARK   3 CHAIN NAMES: A B C D E F G
 REMARK   3 NUMBER OF COMPONENTS NCS GROUP : 2
 REMARK   3   COMPONENT C  SSSEQI  TO  C   SSSEQI   CODE
 REMARK   3   1 A136   A 190  1
 REMARK   3   1 B136   B 190  1
 REMARK   3   GROUP CHAINCOUNT   RMS WEIGHT
 REMARK   3   TIGHT POSITIONAL   1A(A):   1806 ; 0.092 ; 0.050
 REMARK   3   TIGHT POSITIONAL   1B(A):   1806 ; 0.131 ; 0.050
 REMARK   3   TIGHT THERMAL  1A (A**2):   1806 ; 0.192 ; 0.500
 REMARK   3   TIGHT THERMAL  1B (A**2):   1806 ; 0.228 ; 0.500

Re: [ccp4bb] Fab purification and crystallization

[Jürgen Bosch]

I assume you separate your complex over a size exclusion before setting up 
trays ?
If not try that and see if you get better crystals.

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/

On Sep 9, 2010, at 10:59 PM, xaravich ivan wrote:

 Hi CCP4bb,
 
 I have two questions regarding Fab purification and Fab-antigen complex 
 crystallization and would really appreciate any input from the experienced 
 board.
 
 1) I have got some hits for Fab-antigen complex (150 kD) but they are all 
 needle clusters. Whatever fine screen I formulate, it always gives me these 
 needle clusters. Are there some better common ways to change needles to 
 single crystals?
 
 2) I have certain IgGs from which I purify the Fab by papain digestion (resin 
 from ThermoSci). One of the first steps is to dialyze the IgG with the 
 digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I 
 always get 30-60% of the IgG precipitated during this overnight dialysis. I 
 tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone 
 experienced such problem? Is there any thing that could be tried to stop this 
 precipitation.
 
 thanks in advance.
 
 ivan
 

Re: [ccp4bb] why I can't reproduce R based on the same program?

[Jürgen Bosch]

I see in your script you used Weight Auto - play a bit with that value and you 
will find a better fit.
Have you used the same TLS groups ? I don't see that you are using TLS 
refinement in your script.

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/

On Sep 10, 2010, at 1:33 AM, Hailiang Zhang wrote:

 Hi there:
 
 I want to reproduce the R factor provided by PDB file. The structure was
 refined by REFMAC, and so I think if I try a REFMAC refinement based on
 the pdb file and reflection data, the initial R factor given by REFMAC
 should be it.
 
 The pdb file provides residual B factors with TLS given by the header. I
 therefore generated the PDB with the total anisotropic B, based on which I
 tried REFMAC. However, the initial R_free was higher than provide by PDB
 (0.226 vs 0.216).
 
 Not sure why I can't reproduce R based on the same program. Thanks for any
 advice.
 
 Best Regards, Hailiang

Re: [ccp4bb] Reverse Translatase

[Jürgen Bosch]

A large amount of hypotheticals can be found in Plasmodium :-)

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/

On Sep 8, 2010, at 9:29 PM, Ed Pozharski wrote:

 David is absolutely right.  There is no design, Jacob, we just
 instinctively look for it everywhere because seeking purpose instead of
 understanding mechanism conveys advantage to our species.  Your
 rationale is flawed - just because it is imaginable (with caveats) does
 not mean that it must exist on this particular planet.  Complementary,
 not every feature observed has functional significance (in part because
 biomacromolecules are structurally redundant).
 
 On Wed, 2010-09-08 at 09:04 -0400, David Schuller wrote:
 Ah, so many possibilities! And as I said before, considering that
 it would be so useful, and that the genius of macromolecular design
 observed
 in nature is apparently so unlimited, shouldn't it be out there
 somewhere?
 Design? I think there are more appropriate descriptions for life as
 it 
 has been observed. The complexity of life can be explained fairly
 well 
 by Darwinian evolution, i.e. replication with variation coupled with 
 selection. This works through modification of existing entities. The 
 relatedness of many molecules and the theme of modification of 
 pre-existing parts ought to be apparent to someone who has learned
 about 
 replication and sources of genetic novelty, and spent any time
 studying 
 protein structure.
 
 

Re: [ccp4bb] Anybody using PVM by any chance ?

[Jürgen Bosch]

Fixed it, details are available if somebody is interested in them.

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/

On Sep 3, 2010, at 5:15 PM, Jürgen Bosch wrote:

 Dear BB,
 
 I'm stuck with PVM (Parallel Virtual Machine) right now, trying to tell the 
 master to use a remote host as slave.
 The individual machines run fine with PVM but they don't seem to communicate 
 with each other and I don't even get an error message, which makes it hard to 
 troubleshoot.
 
 If there is somebody on the BB with PVM experience, please contact me off the 
 board.
 
 Thanks in advance,
 
 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/
 

[ccp4bb] Anybody using PVM by any chance ?

[Jürgen Bosch]

Dear BB,

I'm stuck with PVM (Parallel Virtual Machine) right now, trying to tell the 
master to use a remote host as slave.
The individual machines run fine with PVM but they don't seem to communicate 
with each other and I don't even get an error message, which makes it hard to 
troubleshoot.

If there is somebody on the BB with PVM experience, please contact me off the 
board.

Thanks in advance,

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/

Re: [ccp4bb] Problem with getting protein-inhibitor complex structure

[Jürgen Bosch]

What concentration/ratio of your inhibitor did you use ?

Also check this out: (short soaks, see time course experiment)
Bosch et al. Using fragment cocktail crystallography to assist inhibitor design 
of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med Chem (2006) 
vol. 49 (20) pp. 5939-46

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/

On Aug 28, 2010, at 1:40 AM, 胡小鹏 wrote:

 Dear all,
We are working on protein-inhibitor complex crystal structures, although 
 we did get nice crystals and soaked them with inhibitor for several days, all 
 structures we obtained were empty!!There are nothing at the active site. For 
 several crystals. their color turned very clearly from colorless to yellow 
 the color of the inhibitors, nothing found either.
These inhibitors work very well in enzyme assay. We, especially our 
 students, are very depressed...
Any suggestions will be really appreciated!
 
 
 
 
 Xiaopeng Hu, D.Sc.,
 School of Pharmaceutical Sciences
 Sun Yat-sen University,
 Higher Education Mega Center
 Guangzhou, Guangdong, China 510006
 Tel. +86 020 39943032 

Re: [ccp4bb] Your Ccp4 question

[Jürgen Bosch]

-
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/

On Aug 28, 2010, at 8:11 AM, 胡小鹏 wrote:
 
 
 4. Do you have inhibitor in cryo/mounting solution?
 
 No, I just sock the crystal very quickly (1S) in 30% glycerol.
 
 

Bad idea, even if your ligands are not very soluble have them around in your 
cryo. Perhaps the solubility is even higher in Glycerol and you are loosing 
your nicely bound inhibitor even faster. See my previous postin even one second 
is enough to soak in a ligand so why shouldn't it be enough to loose it in your 
cryo step.

Try optimizing your conditions in such a way that you can directly freeze them 
out of the drop.

Jürgen

Re: [ccp4bb] Problem with getting protein-inhibitor complex structure

[Jürgen Bosch]

If the solubility is a problem, you can start out with a low concentrated but 
soluble solution of your inhibitor in a diluted protein sample. Then 
concentrate up your protein to the desired amount in the presence of the 
inhibitor, set up trays and be happy. Additionally you could add more inhibitor 
into your precipitant solution when setting up the trays. Since you are not 
screening for new conditions you could prepare a mixture of 
[protein-inhibitor]+inhibitor+reservoir solution and directly dispense this for 
crystallization over non-inhibitor containing reservoir solution.

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/

On Aug 28, 2010, at 8:16 AM, 胡小鹏 wrote:

 Some inhibitors have bad solubility but low Ki (less then 1 uM), we keep  
 inhibitors solid in the soaking drops and don't know the exact concentration.
 For inhibitors with good solubility, we keep the ration at 10 or higher, 
 unfortunately, Ki of these inhibitors are not good, for example, around 30 uM.
 
 
 - 原始邮件 -
 发件人: Jürgen Bosch jubo...@jhsph.edu
 收件人: 胡小鹏 huxp...@mail.sysu.edu.cn
 抄送: CCP4BB@JISCMAIL.AC.UK
 发送时间: 星期六, 2010年 8 月 28日 下午 8:00:46
 主题: Re: [ccp4bb] Problem with getting protein-inhibitor complex structure
 
 What concentration/ratio of your inhibitor did you use ?
 
 
 Also check this out: (short soaks, see time course experiment)
 Bosch et al. Using fragment cocktail crystallography to assist inhibitor
 design of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med
 Chem (2006) vol. 49 (20) pp. 5939-46
 
 
 
 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/
 
 
 On Aug 28, 2010, at 1:40 AM, 胡小鹏 wrote:
 
 
 
 Dear all,
 We are working on protein-inhibitor complex crystal structures, although
 we did get nice crystals and soaked them with inhibitor for several
 days, all structures we obtained were empty!!There are nothing at the
 active site. For several crystals. their color turned very clearly from
 colorless to yellow the color of the inhibitors, nothing found either.
 These inhibitors work very well in enzyme assay. We, especially our
 students, are very depressed...
 Any suggestions will be really appreciated!
 
 
 
 
 Xiaopeng Hu, D.Sc.,
 School of Pharmaceutical Sciences
 Sun Yat-sen University,
 Higher Education Mega Center
 Guangzhou, Guangdong, China 510006
 Tel. +86 020 39943032
 
 -- 
 -- 
 胡小鹏   理学博士
 教授
 中山大学药学院结构生物学实验室
 广东省 广州市 番禺区 大学城 510006
 Tel: 020-39943032
 
 
 Xiaopeng Hu, D.Sc.,
 Professor
 School of Pharmaceutical Sciences
 Sun Yat-sen University,
 Higher Education Mega Center
 Guangzhou, Guangdong, China 510006
 Tel. +86 020 39943032 

Re: [ccp4bb] DM NCS averaging question

[Jürgen Bosch]

Regarding your script:
change a couple of things:
 
MODE HIST SOLV MULT AVER
COMBINE PERT 
SCHEME RES FROM 3.0 (or from where you have FOM 70%, as your low res phases 
will be more reliable)
NCYCLE 50 (since you have 12 molecules you should get a significant benefit 
from averaging.

Then the first Aver card does not require the REFI card as it should not be 
refined. For all the others I would change them to AVER REFI EVERY 3 to update 
the matrices while performing the averaging.

You could also update the solvent mask with the command SOLMASK UPDATE 20 if 
you want.

Good luck,

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/

On Aug 28, 2010, at 10:05 PM, Hailiang Zhang wrote:

 Hi,
 
 I am using the following DM script to perform a NCS averaging. I have a
 fundemental question: after NCS averaging, are the density distrubitions
 of different NCS unit being averaged supposed to be the same? I found they
 are different by checking FCDM/PHICDM, and maybe I am wrong somewhere...
 
 
 dm NCSIN ${PDB}.msk HKLIN ${PDBALL}.mtz HKLOUT ${PDBALL}-dm.mtz \
 dmtest
   mode AVER
   ncycle 1
   combine PERT
   scheme ALL
   solc 0.6213
 #Identical
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0
  TRAN 0.0 0.0 0.0
 #AC
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX -0.22748  0.97259  0.04813 -0.97372 -0.22662 -0.02273
 -0.01120 -0.05203  0.99858
  TRAN  101.4683781.74413 2.89341
 #AD
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX -0.90337  0.42792  0.02831 -0.42883 -0.90066 -0.07011
 -0.00451 -0.07547  0.99714
  TRAN   158.0331736.91842 3.25853
 #AF
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX -0.21272 -0.97702  0.01352 0.97675 -0.21300 -0.02424 0.02657
 0.00805  0.99961
  TRAN   100.69797   -81.01860-1.71365
 #AG
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX 0.61704 -0.78687 -0.01005 0.78590  0.61553  0.05899 -0.04023
 -0.04429  0.99821
  TRAN   32.50667   -65.76570 7.05504
 #AH
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX -0.85814 -0.51116 -0.04813 -0.51290  0.85771  0.03558
 0.02310  0.05522 -0.99821
  TRAN   156.1498142.2387348.93406
 #AI
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX -0.13703 -0.98975 -0.04032 -0.99048  0.13635  0.01902
 -0.01332  0.04254 -0.99901
  TRAN  95.9763082.3094852.82510
 #AJ
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX 0.69662 -0.71695 -0.02645 -0.71716 -0.69691  0.00230
 -0.02008  0.01737 -0.99965
  TRAN   25.8458859.7628653.68224
 #AK
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX 0.99467  0.10258 -0.01072 0.10259 -0.99472  0.00088 -0.01057
 -0.00197 -0.4
  TRAN0.47215-8.8608252.78315
 #AL
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX 0.55782  0.82946  0.02875 0.82987 -0.55793 -0.00466 0.01218 
 0.02646 -0.99958
  TRAN   36.68436   -68.6383349.21587
 #AM
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX -0.30892  0.95102 -0.01113 0.95109  0.30890 -0.00370
 -0.8 -0.01173 -0.3
  TRAN   109.48987   -79.0555052.39334
 #AN
  AVER REFI
  NCSMASK NMER 1
  ROTA MATRIX -0.93676  0.34855 -0.03147 0.34600  0.93589  0.06627 0.05255
 0.05119 -0.99731
  TRAN   162.32979   -29.2680045.41564
   LABIN FP = FWT PHIO = PHIC  FOMO = WCMB
   LABOUT  FDM=FDM PHIDM=PHIDM FOMDM=FOMDM FCDM=FCDM PHICDM=PHICDM
   END
 dmtest

Re: [ccp4bb] database-assisted data archive

[Jürgen Bosch]

Do you want the frames to be accessible too ?
If not, then a.wiki would be an easy solution.
Alternatively a Filemaker database would do the trick too.

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/

On Aug 18, 2010, at 5:52, Andreas Förster docandr...@gmail.com wrote:

 Dear all,
 
 going through some previous lab member's data and trying to make sense 
 of it, I was wondering what kind of solutions exist to simply the 
 archiving and retrieval process.
 
 In particular, what I have in mind is a web interface that allows a user 
 who has just returned from the synchrotron or the in-house detector to 
 fill in a few boxes (user, name of protein, mutant, light source, 
 quality of data, number of frames, status of project, etc) and then 
 upload his data from the USB stick, portable hard drive or remote storage.
 
 The database application would put the data in a safe place (some file 
 server that's periodically backed up) and let users browse through all 
 the collected data of the lab with minimal effort later.
 
 I doesn't seem too hard to implement this, which is why I'm asking if 
 anyone has done so already.
 
 Thanks.
 
 
 Andreas
 
 -- 
 Andreas Förster, Research Associate
 Paul Freemont  Xiaodong Zhang Labs
 Department of Biochemistry, Imperial College London
 http://www.msf.bio.ic.ac.uk

Re: [ccp4bb] I-TASSER predicts NADPH binding, need to confirm with experiment

[Jürgen Bosch]

Check the thermal stability with and without your ligand. You could do this via 
CD or with the help of Sypro Orange in a RT-PCR machine.
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/

On Aug 4, 2010, at 4:10, Xuan Yang pattisy...@gmail.com wrote:

 Dear All,
  
 3D structure modeling server I-TASSER predicts a binding site for NADPH and I 
 want to test this prediction. What would be the nice quick way to tell 
 whether this protein bind NADPH or not, when I have a lot of recombinant 
 protein? 
  
 Sincerely,
  
 Xuan Yang

Re: [ccp4bb] Data processing problem with mosflm

[Jürgen Bosch]

Have you picked from multiple images and do you have more than 1000 reflections 
to index from?
You might also want to limit the resolution manually to perhaps 3 first.
Before processing your data you should refine your cell by using multiple  
wedges of 10 degrees.
Check out the manual for additional keywords.

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/

On Jul 20, 2010, at 10:03 PM, Sampath Natarajan wrote:

 
 Dear All,
 
 I have the problem in processing the data with imosflm. The data was 
 collected with 2.5A resolution about 300 images.  Auto index found the space 
 group P222 with unit cell a= 40.2; b= 79.2; c= 194.7 and α=b=γ = 90.0. For 
 this cell, the penalty is 7. I feel this penalty is good enough to select the 
 cell based from the list. When I start to refine the cell, the new popup 
 window is opening with some suggestions as below.
 
 “The index process has failed. It might be worthwhile trying again with”
 
 1. A larger or smaller largest cell edge
 2. Using more or fewer reflections (200-1000 is best)
 3. Using more and / or different images
 4. Checking your direct beam position carefully
  
 Could anyone help me to solve these problems?
 
 Thanks,
 
  Sincerely
 
 Sampath N
 

Re: [ccp4bb] A strange case of MR

[Jürgen Bosch]

Your beta and gamma angle in P1 are awfully close to 90 degrees. Are you sure 
about P1 ?
Could it be C2 with beta of 104 ˚ ? I'm assuming the CRYST card below belongs 
to your structure right ?

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/

On Jul 19, 2010, at 10:54 PM, 孙庆祥 wrote:

 Hi, Collins,
  
 Thanks for the valuable comments.
  
 I tried refineing using 2.0A cutoff, but the problem persists. (No Molrep 
 solution is found with R less than 0.7, ). 
 Could you please show me how can I indentify and eliminate the catastrophic 
 error that I'm having with the data?
  
 Regards,
 
 At 2010-07-19 19:48:35，Collins, Edward J edward_coll...@med.unc.edu wrote:
 Right.  So, you have a good map (not perfect since your CC in fo-fc map is in 
 80% range- should be in 90%s, but lousy overall statistics. What do the R 
 factors/Rfree look like as a function of resolution?  Later in this output 
 file, there are those statistics after each cycle. I just want to know if you 
 have a good R factor and Rfree at low resolution but really bad at high 
 resolution?  It is possible, based on the postrefine statistics that you 
 chose to use all of your data, but there was really only good data to about 
 2.0 angstroms.  
 
 Depending on how bad the statistics are at high resolution, you could get 
 this bad of an Rfree, but that would be strange.  At least, I have never seen 
 the Rfactor at 40% in such a case.  high 30% would be more likely.  The one 
 bin showed in this file is for the highest resolution (50%) which is pretty 
 bad.  If you make your resolution cutoff 20-2.0 angstroms and run for a few 
 cycles, you would see if this is a function of resolution.
 
 If those statistics look similar (that is, the low resolution R factor and 
 Rfree are bad too), you have a catastrophic error in your data somewhere. It 
 could be pseudo symmetry/wrong space group identification.  It could be 
 misindexing in some weird way.  It could be a second crystal that 
 unfortunately for you had a series of overlaps -especially on high intensity 
 reflections.
 Good luck,
 -ed
 
 On Jul 19, 2010, at 3:41 AM, 孙庆祥 wrote:
 
 Dear Collins and all,
  
 Thanks for the reply. Please find the following pdb header. For your 
 information, this is after refmac5 with twin-refinement option selected. 
 This is the best R values that I'm getting. Cutting resolution does not help 
 much in getting the Rs down...
  
  
 HEADERSWISS-MODEL SERVER (pa-JAN-g)      

 COMPNDSS-MODEL   

 REMARK   3
 REMARK   3 REFINEMENT.
 REMARK   3   PROGRAM : REFMAC 5.5.0109 
 REMARK   3   AUTHORS : MURSHUDOV,VAGIN,DODSON
 REMARK   3
 REMARK   3REFINEMENT TARGET : MAXIMUM LIKELIHOOD
 REMARK   3
 REMARK   3  DATA USED IN REFINEMENT.
 REMARK   3   RESOLUTION RANGE HIGH (ANGSTROMS) :   1.52
 REMARK   3   RESOLUTION RANGE LOW  (ANGSTROMS) :  74.96
 REMARK   3   DATA CUTOFF(SIGMA(F)) : NONE
 REMARK   3   COMPLETENESS FOR RANGE(%) :  91.56
 REMARK   3   NUMBER OF REFLECTIONS :   27851
 REMARK   3
 REMARK   3  FIT TO DATA USED IN REFINEMENT.
 REMARK   3   CROSS-VALIDATION METHOD  : THROUGHOUT
 REMARK   3   FREE R VALUE TEST SET SELECTION  : RANDOM
 REMARK   3   R VALUE (WORKING + TEST SET) : 0.42077
 REMARK   3   R VALUE(WORKING SET) :  0.42170
 REMARK   3   FREE R VALUE :  0.40609
 REMARK   3   FREE R VALUE TEST SET SIZE   (%) :  5.4
 REMARK   3   FREE R VALUE TEST SET COUNT  :  1584
 REMARK   3
 REMARK   3  FIT IN THE HIGHEST RESOLUTION BIN.
 REMARK   3   TOTAL NUMBER OF BINS USED   :  20
 REMARK   3   BIN RESOLUTION RANGE HIGH   :1.522
 REMARK   3   BIN RESOLUTION RANGE LOW:1.561
 REMARK   3   REFLECTION IN BIN (WORKING SET) : 1804
 REMARK   3   BIN COMPLETENESS (WORKING+TEST) (%) :82.44
 REMARK   3   BIN R VALUE   (WORKING SET) :0.529
 REMARK   3   BIN FREE R VALUE SET COUNT  :  121
 REMARK   3   BIN FREE R VALUE:0.622
 REMARK   3
 REMARK   3  NUMBER OF NON-HYDROGEN ATOMS USED IN REFINEMENT.
 REMARK   3   ALL ATOMS:  984
 REMARK   3
 REMARK   3  B VALUES.
 REMARK   3   FROM WILSON PLOT   (A**2) : NULL
 REMARK   3   MEAN B VALUE  (OVERALL, A**2) :  18.096
 REMARK   3   OVERALL ANISOTROPIC B VALUE.
 REMARK   3B11 (A**2) : 8.45
 REMARK   3B22 (A**2) :   -12.86
 REMARK   3B33 (A**2) : 4.41
 REMARK   3B12 (A**2) : 0.51
 REMARK   3B13 (A**2) :-1.32
 REMARK   3B23 (A**2) :-8.65
 REMARK   3
 REMARK   3  ESTIMATED OVERALL COORDINATE ERROR.
 REMARK

Re: [ccp4bb] Structure Based Sequence Alignment

[Jürgen Bosch]

http://ww2.cs.mu.oz.au/~arun/Site/mustang.html

http://tcoffee.vital-it.ch/cgi-bin/Tcoffee/tcoffee_cgi/index.cgi?stage1=1daction=EXPRESSO(3DCoffee)::Regular

or maybe what you are searching for is Consurf ?
http://consurf.tau.ac.il/

Jürgen

On May 25, 2010, at 2:39 PM, Muhammed bashir Khan wrote:

 Dear All;
 
 Can some body tell me a website for structure based sequence alignment,
 which can also pin point the similar and identical residues in different
 colors.
 
 regards
 
 Bashir
 
 
 
 
 -- 
 Muhammad Bashir Khan
 Department for Biomolecular Structural Chemistry
 Max F. Perutz Laboratories
 University of Vienna
 Campus Vienna Biocenter 5
 A-1030 Vienna
 Austria
 
 Phone: +43(1)427752224
 Fax: +43(1)42779522

-
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/

Re: [ccp4bb] Finding best model for molecular replacement

[Jürgen Bosch]

You've also applied BRAIN 2.0 ?

I mean looked at homologous structures, superimposed them and decided which 
parts are to be removed ?
Never trust programs :-) There could be a flexible alpha helix which if you 
removed it would have given you in all programs a solution.

it's Monday,

Jürgen


On May 24, 2010, at 10:24 AM, Paul Lindblom wrote:

 The last molrep job just finished and it found only an odd solution. So I 
 think I will try to get my phases elsewhere. But I am somewhat astonished 
 that there are still enough cases you can't solve by MR.
 
 Thanks to all who replied. Here is a list of servers/programs to find a MR 
 model:
 
 http://www.ebi.ac.uk/pdbe-srv/view/
 
 http://www.ebi.ac.uk/Tools/fasta33/index.html
 
 http://meta.bioinfo.pl/submit_wizard.pl
 
 XtalPred
 http://ffas.burnham.org/XtalPred-cgi/xtal.pl
 
 Balbes  http://www.ysbl.york.ac.uk/~fei/balbes/
 
 use the OCA browser for FASTA searches of the PDB
 
 Modeller or Rosetta (both also available as web servers)
 
 ensemble of many proteins with Phaser
 
 FFAS server maintained by the Godzik lab
 
 generate some models using the Phyre server   ( 
 http://www.sbg.bio.ic.ac.uk/~phyre/ )  and feed the best .pdbs into Mr Bump.
 
 
 
 
 

-
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/

Re: [ccp4bb] Finding best model for molecular replacement

[Jürgen Bosch]

XtalPred
http://ffas.burnham.org/XtalPred-cgi/xtal.pl

or if you have data already let Balbes do the job.

Jürgen

On May 23, 2010, at 3:34 PM, Miri Hirshberg wrote:

 Sun., May 23rd 2010
 EBI
 
 Paul,
 
 1. yes you can run your sequence against all PDB.
 
 1. http://www.ebi.ac.uk/pdbe-srv/view/
 drop the one letter sequence in the sequence box
 and search
 
 2. http://www.ebi.ac.uk/Tools/fasta33/index.html
 
 From the Databases protein you pick
 Protein structure Sequence
 
 You drop your 1-letter code sequence in the sequence box and search
 You
 
 both 12 run the same search but the layout of the output is a 
 bit different.
 
 3. You can also try http://meta.bioinfo.pl/submit_wizard.pl
 it will predict 3D structure using many methods.
 
 Miri
 
 On Sun, 23 May 2010, Paul Lindblom wrote:
 
 
 Hi everybody,
 
 I just crystallized a new project protein. How can I find a possible model 
 for using molecular replacement? I have
 the sequence of my protein. Is it enough to make a sequence search in the 
 pdb? Or is there another approach I can
 use?
 
 Thanks a lot,
 
 Paul
 
 
 
 
 
 
 Dr Miri Hirshberg
 European Bioinformatics Institute UK
 PDBe - EBI -EMBL
 http://www.ebi.ac.uk/pdbe
 
 Phone: +44 (0) 1223 492647
 FAX:   +44 (0) 1223 494468
 

-
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/

Re: [ccp4bb] Finding best model for molecular replacement

[Jürgen Bosch]

unambiguous space group ?

Jürgen

On May 23, 2010, at 4:52 PM, Paul Lindblom wrote:

 Thank you all for your quick answers. I already tried the phyre server but 
 could not find a appropriate model. Balbes found a structure with 29% 
 identity, but no solution. Now I am running molrep with the model balbes 
 found...
 
 2010/5/23 Nathaniel Echols nathaniel.ech...@gmail.com
 On Sun, May 23, 2010 at 12:57 PM, David Briggs drdavidcbri...@gmail.com 
 wrote:
 I like to generate some models using the Phyre server
 
 ( http://www.sbg.bio.ic.ac.uk/~phyre/ )
 
 Feed the best .pdbs into Mr Bump.
 
 Go and get coffee. Come back and find a solution with post-refmac
 R/Rfree in the mid-30s.
 
 IMHO, Phyre models are often pretty good, and Phyre is worth running
 on any sequence you are messing with - the output is nice and
 informative.
 
 My experience has been exactly the opposite; I've seen many people waste a 
 lot of time this way.  Phyre is a fold-recognition server - it is not 
 intended to generate optimal, physically accurate models.  It's possible that 
 they've improved the output since I last used it, but I've seen plenty of 
 models with overlapping sidechains and completely unrealistic packing.  Use 
 Modeller or Rosetta (both also available as web servers) if you want as 
 accurate a homology model as possible.  Even so, if you can find homologs in 
 the PDB with better than 30% sequence identity, you'll probably have at least 
 as much success using those as search models, after judicious pruning (which 
 I think MrBUMP can do automatically).
 
 This is not to say that homology modelling isn't occasionally useful for MR, 
 but it is not the first thing I'd try unless I had put a great deal of 
 thought into the pipeline and had a large cluster to run it on.  I know the 
 JCSG has had some luck with this approach, but they have full-time 
 programmers and something like 400 CPUs.
 
 -Nat
 

-
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/

Re: [ccp4bb] Help needed in solving a MAD dataset

[Jürgen Bosch]

I don't like the site finding option in autosharp, takes too long in most of my 
cases.
So my approach is locate sites via SHELX, then feed them into Sharp.

Sorry Gerard :-)

Jürgen

On May 20, 2010, at 10:02 PM, Jeremiah Farelli wrote:

 I second autoSHARP/SHARP.  It makes great initial maps, and once you get it 
 running, it is totally worth it. 

-
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/

[ccp4bb] Hope for breakthroughs

[Jürgen Bosch]

http://www.ebi.ac.uk/chemblntd
And the related publication
Thousands of chemical starting points for antimalarial lead identification
http://www.nature.com/nature/journal/v465/n7296/pdf/nature09107.pdf

Good luck with the goldmine !

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/

Re: [ccp4bb] Native Gel Theory and Practice

[Jürgen Bosch]

Not quite correct, look into Blue Native PAGE. There you can seperate  
natively by mass.


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/

On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca wrote:


Dear Jacob, I offer  you my opinion.
Are you talking about electrophoresis? As far as I know it does not  
work

for the mass. The velocity of a protein depends on the charge at a
particular pH, the mass and shape of molecules etc. It's very  
difficult

to take all these things into consideration. Otherwise this would be a
very convenient method, much easier than the analytical centrifugation
or   gel-filtration that are usually used. However, electrophoresis  
does

not work for mass determination. Besides, complex formation hugely
depends on the protein concentration. If you dilute your mixture, your
complexes might dissociate. There is equilibrium constant between
different types of complexes.

Maia


Jacob Keller wrote:

Dear Crystallographers,

I am trying to optimize a native gel experiment of a two-protein
complex, running the smallest-detectable amount of protein  
component A

with varying amounts of component B.

  MWCharge MW/Charge
A   22 -5-4308
B   17-24 -702

This experiment is partly to determine stoichiometry, but also to
determine roughly the strength of the interaction.

B definitely runs much faster than A alone, as predicted, but I am
wondering what to expect with various oligomers. Should ABB run  
faster
or slower than AB? What about AABB? Theoretically, AA should  
certainly

run slower than A, and BB slower than B, simply because the
mass/charge ratio is the same, but the overall mass is greater. But
what happens when you have AAB, for example? There must be an  
equation

relating the mass/charge and mass (and perhaps gel percentage) to the
speed traveled in the gel--but what is the equation?

Thanks for your consideration,

Jacob

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Native Gel Theory and Practice

[Jürgen Bosch]

You don't necessarily need the second dimension.
BN-PAGE gel:
protein A alone| some proteins you know the size as reference|protein B alone| 
your mixture

You will be able to see in the mixture a) one or b) multiple bands, since the 
Coomassie is equally distributed and attached to your protein in a 
non-denaturing way it is directly proportional to the molecular mass of your 
protein complex.

I'm searching for the protocol which was I believe either from the Görg group 
or a group in Berlin, perhaps also Mann demonstrating dissociation of a 
multimeric complex in dependence of reducing agent.

If I find it I will post it tonight.

Jürgen

On May 19, 2010, at 10:01 AM, Nadir T. Mrabet wrote:

 Maia speaks about native PAGE for which protein mobility (migration) 
 depends on 3 different parameters as she states: charge, mass and shape.
 Blue native PAGE, which might be the answer to Jacob's question, is a 2D 
 gel: Native in the first direction, then SDS-PAGE in the second one.
 You actually need both data to infer stoechiometry and subunit composition.
 
 Nadir
 
 Pr. Nadir T. Mrabet
 Structural  Molecular Biochemistry
 Nutrigenex - INSERM U-954
 Nancy University, School of Medicine
 9, Avenue de la Foret de Haye, BP 184
 54505 Vandoeuvre-les-Nancy Cedex
 France
 Phone: +33 (0)3.83.68.32.73
 Fax:   +33 (0)3.83.68.32.79
 E-mail: Nadir.Mrabetat  medecine.uhp-nancy.fr
 
 
 
 On 19/05/2010 13:01, Jürgen Bosch wrote:
 Not quite correct, look into Blue Native PAGE. There you can seperate 
 natively by mass.
 
 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/
 
 On May 19, 2010, at 1:31, Maia Cherney ch...@ualberta.ca wrote:
 
 Dear Jacob, I offer  you my opinion.
 Are you talking about electrophoresis? As far as I know it does not work
 for the mass. The velocity of a protein depends on the charge at a
 particular pH, the mass and shape of molecules etc. It's very difficult
 to take all these things into consideration. Otherwise this would be a
 very convenient method, much easier than the analytical centrifugation
 or   gel-filtration that are usually used. However, electrophoresis does
 not work for mass determination. Besides, complex formation hugely
 depends on the protein concentration. If you dilute your mixture, your
 complexes might dissociate. There is equilibrium constant between
 different types of complexes.
 
 Maia
 
 
 Jacob Keller wrote:
 Dear Crystallographers,
 
 I am trying to optimize a native gel experiment of a two-protein
 complex, running the smallest-detectable amount of protein component A
 with varying amounts of component B.
 
  MWCharge MW/Charge
 A   22 -5-4308
 B   17-24 -702
 
 This experiment is partly to determine stoichiometry, but also to
 determine roughly the strength of the interaction.
 
 B definitely runs much faster than A alone, as predicted, but I am
 wondering what to expect with various oligomers. Should ABB run faster
 or slower than AB? What about AABB? Theoretically, AA should certainly
 run slower than A, and BB slower than B, simply because the
 mass/charge ratio is the same, but the overall mass is greater. But
 what happens when you have AAB, for example? There must be an equation
 relating the mass/charge and mass (and perhaps gel percentage) to the
 speed traveled in the gel--but what is the equation?
 
 Thanks for your consideration,
 
 Jacob
 
 ***
 Jacob Pearson Keller
 Northwestern University
 Medical Scientist Training Program
 Dallos Laboratory
 F. Searle 1-240
 2240 Campus Drive
 Evanston IL 60208
 lab: 847.491.2438
 cel: 773.608.9185
 email: j-kell...@northwestern.edu
 ***
 
 
 

-
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/

Re: [ccp4bb] Help needed in solving a MAD dataset

[Jürgen Bosch]

CCP4 way:
locate the Se sites with SHELX (if you use the CCP4I gui it's technically a 
ccp4 program :-) )
Try using only your peak data set first. If you can't locate your sites with 
the single wavelength then add remote (DAD) and if that doesn't work go MAD.

non-ccp4 way
run hkl2map as frontend to SHELX, do the same thing.

After 10 minutes go and open a bottle of your favourite sparkling cider* and 
start tracing the structure.

Jürgen

* can be replaced by other sparkling product containing trace amounts of EtOH 
supplemented with flavours

On May 19, 2010, at 12:01 PM, Qing Lu wrote:

 Hi All,
 
 I am new to protein crystallography. I would like to know the steps involved 
 in solving a MAD dataset by using the program in CCP4 where you determine the 
 phases and then obtain the trace. The dataset is collected at 3 different 
 wavelengths (peak, inflection and remote) using Se-Met as the scatterer. The 
 crystals diffracted to resolution of 2 Angstrsoms and has a good anomalous 
 signal. 
 
 Thanks,
 
 Qing Lu

-
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/

Re: [ccp4bb] Translational pseudosymmetry?

[Jürgen Bosch]

Hi Owen,

you should also make the following plot with your data:
y-axis relative intensity of off-origin peak versus x-axis resolution cutoff 
used for calculation (30 Å - 4 Å in 2 Å steps).

You can have multiple cases of shifts and I would start with a perfect hexamer 
first, take some random monomer and apply a perfect sixfold, move it along the 
axis where it should be in your crystal lattice (things get more complicated if 
you have a top/down hexamer, so keep it simple). Now if you shift your hexamer 
to 2/3,1/3,0 your plot should yield a straight line and be independent of 
resolution. Now start rotating the second hexamer relative to the first 
clockwise with your sixfold, I would use increments of 3 degrees, which will 
result in 19 models, then recalculate the off-origin peak heights and see if 
they match up with your data. I should note  here, if your real data does not 
show a strong drop in peak height of the off-origin peak, then you most likely 
don't have a slight rotational translation in your second hexamer.

One other important thing you should look at is the relative orientation of 
your sixfold axis, is it truly perfectly aligned with one of the cell axis ? If 
not fix this in your model, otherwise your calculations will be of academic 
nature. For this particular case the use of GLRF is more helpful than MOLREP 
(sorry Garib, but maybe Garib can come up with a solution to zoom into certain 
peaks like you can do in GLRF).

When the tilt is fixed you should be able to figure out the rotational 
translation in your second hexamer.

Enjoy your puzzle,

Jürgen

P.S. P3 is certain ? Check with pointless or by human brain visual inspection 
(HBVI)

On May 15, 2010, at 11:53 PM, Owen Pornillos wrote:

 Dear ccp4bb –
 
 I have questions with regards to crystal disorder that gives rise to  
 translational pseudosymmetry.
 
 We have a rotationally hexameric protein that crystallized in P3, with  
 one hexamer in the asu.  The local 6-fold axis of the hexamer is non- 
 crystallographic, and is essentially parallel to the crystallographic  
 3-fold, which gave rise to translational pseudosymmetry.  Intensities  
 for the (h,h+/-3n,l) reflections were on average about 8 times  
 stronger than the weak reflections, and the native patterson gave an  
 off-origin peak about 70-80% of origin (depending on the crystal) at  
 fractional coordinates (2/3,1/3,0).  We are hypothesizing that the  
 break in local 6-fold symmetry is caused by small rigid-body  
 displacements of each subunit (as opposed to conformational changes in  
 the protein), and we are trying to estimate the magnitude of the  
 displacements in the crystal.
 
 To do this, a perfectly symmetric hexamer with the local 6-fold axis  
 parallel to the crystallographic 3-fold was generated, and then shifts  
 were introduced to the atomic coordinates.  The direction of the shift  
 was chosen randomly for each atom, and a single magnitude applied to  
 all atoms, which was then changed incrementally.  Structure factors  
 were calculated from these models, and their pattersons were  
 examined.  The magnitude of the off-origin peak could be reproduced  
 with an atomic shift of say, 1 Å.  Because all of these calculations  
 were made with synthetic structure factors, this is not necessarily a  
 reliable estimate.  The questions are, how far off are we, and in what  
 direction (i.e., are these shifts underestimates or overestimates)?   
 Is there a way to obtain a reliable estimate?
 
 Thanks in advance,
 
 Owen

-
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/

Re: [ccp4bb] co-crystallization VS crystal soaking

[Jürgen Bosch]

Hi Rain,

try both is my advice.
In some cases we (www.sgpp.org) were unsuccessful in soaking but successful in 
co-crystallization. I assume your question is directed towards ligands. If you 
are in the comfortable situation of having your own structure at hand, check 
out a) crystal lattice contacts - would they hamper soaking by restricting 
access to your site ? b) do you want to exchange an existing ligand/co-factor 
in your protein, then it's probably more likely to occur in solution c) how's 
your ligand behaving under your crystallization condition, if it crashes out 
try to form a complex in solution and then co-crystallize.
Another tip, use your apo-form crystals to streak seed crystallization attempts 
with new ligands. When you co-crystallize play with the molarity ratio of your 
ligand.

Jürgen

Bosch et al. Using fragment cocktail crystallography to assist inhibitor design 
of Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med Chem (2006) 
vol. 49 (20) pp. 5939-46

@Eric, can you comment on this:
Ojo et al. Toxoplasma gondii calcium-dependent protein kinase 1 is a target for 
selective kinase inhibitors. Nat Struct Mol Biol (2010) vol. 17 (5) pp. 602-7


On May 15, 2010, at 7:47 AM, rainfieldcn wrote:

 Hi, friends:
 Is there any published paper describing the case study of the difference 
 between co-crystallization and crystal soaking?
 I mean has anybody observed different structures by these two methods?
 Thank you!
   
 Rain Fieldcn
 2010-05-15

-
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/

Re: [ccp4bb] Ion exchange protein lost

[Jürgen Bosch]

Hi Megha,

how about your protein is stuck to the top of your column ?
How does your pressure look like before injection and after injection ?
What makes you believe that you have successfully refolded your protein ?

Just some thoughts,

Jürgen

On May 11, 2010, at 10:53 PM, megha goyal wrote:

 Hi all,
  
  
 Our protein is gcsf. We solubilize the inclusion bodies in 8M urea and 0.1M 
 cysteine and then dilute it 1:20 in refodling buffer 0.1% tween 20 pH 8.2. 
 Then we perform concentration using proflux M12 [just concentration and not 
 diafiltration]. Adjust the pH of concentrate to 4.5 and load it to source 15S 
 resin [strong cation exchange]. The problem is we do not recover our protein 
 on performing IEX. HPLC and absorbance reading on concentrate show the 
 presence of our protein. Buffer for loading is 25 mM na acetate pH 4.5 and 
 elution is same buffer with 0.5 M NaCl. No protein is lost in flow thru and 
 even 2M Nacl washing does not show our protein. . Only when we perform NaOH 
 wash we do see some peak but could not analyse it as it is too alkaline and 
 cant run on SDS PAGE or HPLC.
  
 What could be the reason. Where do we lose our protein. Kindly shed some 
 light on this on where shall I be going wrong.
  
 thanks and regards,
  
 meg

-
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/

Re: [ccp4bb] testers needed

[Jürgen Bosch]

Hi Bernhard,

how disturbing is the electrical cord connected to the MagiXsticks ? I could 
imagine that sometimes you might get trapped with it but I guess it's just 
habit of using the device.

Jürgen

On May 10, 2010, at 2:27 PM, Bernhard Rupp wrote:

 Dear All,
 
 I have the parts supply cost now under control to a point where I 
 actually may be able to sell these MagiXsticks for ~300. But before I
 release those, I am looking for a few good men or women (say 2 sites)
 who want to try it out (free, if you like it you keep it). Requirement
 would be to a reasonable amount of harvesting with a reasonably diverse 
 set of pin bases. 
 
 Any takers?
 
 Thx, BR
 
 http://www.ruppweb.org/MagiXstick/default.html
 
 -
 Bernhard Rupp
 001 (925) 209-7429
 +43 (676) 571-0536
 b...@qedlife.com
 bernhardr...@sbcglobal.net 
 http://www.ruppweb.org/ 
 -
 The hard part about playing chicken
 is to know when to flinch
 -

-
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/

Re: [ccp4bb] MacBookPro problems

[Jürgen Bosch]

Yeah, send your Zalman vendor a nice email and let them exchange your DVI 
adapter. Had that problem too - alternatively you can scavenge one DVI adapter 
from another computer and test it. I wouldn't be surprised if it works, that's 
how I figured it out.

Jürgen

On Apr 23, 2010, at 9:37 AM, Joachim Reichelt wrote:

 Dear all,
 
 I just got a MacBookPro.
 I have two questions:
 How to get the keyboard shortcuts in coot or pymol up, e.g. altF to
 open the file dialog.
 
 and
 
 whenever I connect a zalman 220 (stereo ready) monitor to the mac using
 a miniDisplayPort to DVI
 adapter, the monitor is not recognized and so it is completely dark. The
 Mac does not show the monitor in the dialogues but an error message.
 
 -- 
 Joachim

-
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/

[ccp4bb] merging Pymol sessions ? Non-CCP4 question

[Jürgen Bosch]

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/

Re: [ccp4bb] Mysterious Crystals?

[Jürgen Bosch]

Fish and wash some crystals then run them on a SDS-gel, then you will know for 
sure if it's protein or not.

Jürgen
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
 
 
 
 

-
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/

Re: [ccp4bb] Cryo Vs crystal size

[Jürgen Bosch]

There are a couple of additional factors not taken into account here.

1. LN2 versus frozen in strem or propane etc
2. did you try to flash anneal the larger crystal
3. smeary diffraction from the big crystal or not ?
4. how much residual solvent was around your crystal when freezing ?

In general smaller crystals are anyhow better in my hands.

Jürgen

On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote:

 Hi All
 
 I had two crystals grown in same well, one is small and other is 10 times 
 bigger. I treated both crystal in same cryo and same time. The smaller one 
 diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one 
 to diffract high resolution. 
 
 I assume the bigger crystal might have lot of solvent which prevent for high 
 resolution. If it is true what could be the best way to dehydrate crystal 
 without affecting crystal quality?
 
 Thank you
 
 Syed
 
 PS: Taken care of less solvent to be present in the loop
 
 
 

-
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/

Re: [ccp4bb] To model or not to model...

[Jürgen Bosch]

I should add a comment here about density that might or might not be there :-)
Graphent - trying is believing

Jürgen

On Apr 13, 2010, at 9:29 PM, Bernhard Rupp wrote:

 Dear K,
 
 I do think there is much of a hornet's nest to be stirred up by your
 questions.
 I hate to advertise it, but chapter 12 of BMC has a thorough discussion of
 the
 subject.
 
 Ad riding: ADDING riding hydrogens is not exactly the same as MODELLING
 some stuff one has no clue about (like parts with no density). Covalently
 bonded
 hydrogens are not really 'modeled' in a sense of parameterization,
 they are almost certainly there, and add no parameters to a model. They
 just make certain computations of restraints and validation work better when
 
 included. 
 
 Propositions about other non-H parts not visible in density, are an entirely
 different beast. The crystal is made of trillions of molecules, which
 when they have almost identical conformation in almost all the scattering
 molecules
 will show distinct density. So if no density is visible, what are you
 modeling?
 One possible conformation of many? The most probable of the
 non-evidence-bearing ones?
 
 Much better to fess up and leave the residues/atoms out of the model.
 Increased B
 should be in any case a result of refinement and not arbitrarily set (and
 let's not go into
 details of B-restrains which I believe are to this date a challenge to
 properly implement)
 while adjusting occupancy is not an option for a main chain. 
 
 In addition, setting occupancy to 0 for conjured parts is bad because most
 display programs and users will ignore that column. In practice (as theory)
 omitting parts that have no density = high B exponential = negligible
 contribution
 to scattering factor rarely has a significant effect on refinement
 parameters,
 but the model with absent parts is a more truthful, parsimonious model.   
 
 BR   
 
 -Original Message-
 From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
 SIPPEL,KATHERINE H
 Sent: Tuesday, April 13, 2010 5:40 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] To model or not to model...
 
 Dear Crystallographic Community,
 
 Dr. Holton made a comment today that got me thinking on the issue 
 of modeling. This has been a hotly debated topic in our own lab 
 but I would like to hear the current opinions of the community as 
 a whole. It is a question of two parts.
 
 First, what do you think about modeling into regions of poor 
 density? Do you (A) model something in as best you can while 
 conforming to ideal geometry/chemistry with full disclosure about 
 b-factors in the region, (B) reduce the occupancies of the poorly 
 modeled loops/side chains, or (C) truncate your model, removing 
 loops and side chains from the model at the cost of statistical 
 numbers? From a crystallographers point of view we can assess the 
 quality of the model and make informed decisions as to what 
 conclusions to draw, however most of the greater scientific 
 community has no way to judge this. They do not know what a 
 b-factor is or where to look for an occupancy. Are we doing a 
 disservice to science by emphasizing the minimization of Rwork and 
 Rfree over full disclosure of what we can legitimately see?
 
 Secondly, on a similar vein, what is the community's opinion on 
 modeling hydrogens? I have read a lot on the subject and can see 
 both sides of the argument. From a crystallographer's point of 
 view these are very helpful in maintaining geometry and ensuring 
 the model makes chemical sense. I can also see the necessity of 
 submitting them to the pdb so that the statistics can be 
 recreated. On the other hand most biologist has no comprehension 
 of the concept of riding hydrogens. They assume that if the 
 hydrogen is in the pdb, that the crystallographer saw it and use 
 that information to develop experiments.
 
 I realize that I may be kicking a hornet nest here but I would 
 genuinely like to know what people think.
 
 Thanks,
 
 Katherine Sippel
 
 SIPPEL,KATHERINE H
 Ph. D. candidate
 Department of Biochemistry and Molecular Biology
 College of Medicine
 University of Florida

-
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/

Re: [ccp4bb] crystals as carry-on items

[Jürgen Bosch]

You can only carry them with you if they fit in a sealed plastic bag :-)

My last trip with crystals in trays was from Seattle to Berkley couple of years 
ago. I placed the trays simply in a small styrofoam box, well padded and with a 
cryo pad. You will need to open the box but it helps to tell them what they 
will find. The cryo pad might be an issue perhaps.

Jürgen

On Apr 6, 2010, at 2:03 PM, Arthur Glasfeld wrote:

 Sorry for an off-topic, out-of-date concern. Also, I'm almost certain  
 this has been discussed before, but my search of the archives didn't  
 turn up anything.  I am flying to a synchrotron this weekend and   
 would like to hand-transport some crystal plates.  Does anyone have  
 any recent experience with the TSA that might make this an easier  
 task?  Is it even possible any more?
 
 Thanks in advance for any advice that can be provided.
 
 Arthur Glasfeld
 Department of Chemistry
 Reed College
 3203 SE Woodstock Blvd.
 Portland, OR 97202
 USA

-
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/

Re: [ccp4bb] TLS, NCS and refinement

[Jürgen Bosch]

I'd use NCS in the beginning and maybe also DM solvent flattening,  
then build and fix your structure, run refmac without NCS, superimpose  
your chains in Coot and re-define your strict NCS groups accordingly.  
Therafter you can submit your current coordinates to Ethan's TLSMD  
server and continue from there


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/

On Mar 28, 2010, at 18:34, Daniel Bonsor bon...@bbri.org wrote:


Hello again...

I have a 2.7A resolution data set. Spacegroup P212121 as suggested  
by Pointless and best space group when run through Phaser. Unit cell  
is 110.02x160.49x186.55. Mathew's suggest 11 complexes in ASU. Both  
Molrep and Phaser can only find 6. This seems to be a typical  
observation with the complexes that we solve (high solvent content).


Due to the low(ish) resolution I wish to apply TLS and NCS during  
refinement. My question(s) is when to apply TLS and NCS, the order  
in which it should be done, and when to switch off NCS (if at all)  
during the stages of refinement. I have used a homologue complex  
(Protein A -100% identical, Protein B - 80% identical) as a model.


I am open to any and all suggestions on the matter.


Thanks in advance

Dan

Re: [ccp4bb] solve structure by molecular replacement

[Jürgen Bosch]

On Mar 7, 2010, at 4:35 PM, Chunmin L wrote:
  I can not reprocess the data to
 check the systematic absence to determine the space group (I do not
 have the right software to reprocess the raw image at this time). 

What exactly do you mean by that ? The world does not consist of HKL alone, 
there are other options available even CCP4 ones e.g. Mosflm or non-CCP4 e.g. 
XDS. or d*TREK

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/

Re: [ccp4bb] 3D fitting

[Jürgen Bosch]

How about LSQMAN ?
with ATOM_types Side_chain ?

That should do the trick.

Jürgen

P.S. sorry it's not really a CCP4 related program, but I use it very  
frequently in conjunction with the CCP4 suite :-)


On Feb 26, 2010, at 11:22 AM, Ed Pozharski wrote:


On Fri, 2010-02-26 at 16:50 +0100, Gerard DVD Kleywegt wrote:

But it still won't solve Miri's problem. I think what she is asking
for is a
program that detects which atoms should be matched to which,
irrespective of
their names (i.e., not assuming they are called  CA ) and order
(i.e., not
nicely sequential such as amino-acid residues), and then applies the
operator
that follows from that.




How about FM option in molrep?
http://www.msg.ucsf.edu/local/programs/ccp4-6.0.2/html/molrep.html#FM

Other MR programs can be used as well, just generate FC from one model
in a suitable cell and run search with another.

HTH,

Ed.

--
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 /


-
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/

Re: [ccp4bb] Expression of a protein of 43KDa

[Jürgen Bosch]

Well that it's not his protein which he has crystallized but an over  
expressing contaminant :-)
Or the alternative would be although it's supposed to be 29kDa it runs  
funny and appears larger in the SDS gel, in that case it would be OK.  
If we only had some confirmation that the 43kDa band is the right one  
e.g. via Western Blot  Anti-His ...


Jürgen

On Feb 18, 2010, at 4:01 PM, Jon Schuermann wrote:


Maybe I am not getting it, but I don't see your problem or question?

What I understand from your message is that you have a protein that
overexpresses, purifies, and crystallizes... where's the problem? We  
all

wish we had that problem...

Jon




On 02/18/2010 11:49 AM, Armando Albert de la Cruz wrote:

I am trying to overexpress a His-tagged protein of 29KDa in E.coli
(BL21-codon plus) and I end up with a highly expressed product that  
of

43KDa that binds to the Ni-column. I also have nice crystals. Does
anyone have any experience on this.
Armando



--
Jonathan P. Schuermann, Ph. D.
Beamline Scientist
NE-CAT, Building 436E
Advanced Photon Source (APS)
Argonne National Laboratory
9700 South Cass Avenue
Argonne, IL 60439

email: schue...@anl.gov
Tel: (630) 252-0682
Fax: (630) 252-0687


-
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/

Re: [ccp4bb] Crystal rescue

[Jürgen Bosch]

how much %PEG3350 ?
Try different additives such as glycerol, EG, MPD or smaller PEGs 200  
or 400.

Try flash annealing
Try freezing directly in liquid nitrogen if you have previously frozen  
your crystals in the stream
Try liquid annealing by dipping the frozen crystal into a new cryo  
solution and re-freeze it.

The list can be continued quite a bit.
If you should find out that one of the cryo additives helps then try  
to grow your crystals in the presence of those additives.


Good luck,

Jürgen

On Jan 26, 2010, at 10:42 AM, Zhiyi Wei wrote:


Dear all,

I got a problem with my crystals. I have two total different proteins
that both can be crystallized in the condition with PEG3350 and  
Tacsimate

(although the concentrations are different) with different shapes. The
crystals look big and beautiful. However, when I test them in  
synchrotron,
both of these two types of crystals showed poor diffractions. As  
showed in
the attached diffraction image, the diffraction is up to ~4 A but  
smear in
one direction while 8 A in the other direction. The interesting  
thing is
that the diffraction pattern is similar for all crystals (from two  
different
proteins) that I tested without exception although they belong to  
different

space groups. So, I wonder whether these kind of pattern is caused by
Tacsimate (I don't know what it is) and how to rescue these  
crystals. Any

suggestions or comments?

Thanks a lot!

Best,
Zhiyi
bad.png


-
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/

Re: [ccp4bb] Zalman LCD availability / suitability

[Jürgen Bosch]

Hi Valerie,

this is a known issue, exchange the cable with any other cable which  
you have in the lab. Some of the cables will only work with PC - I had  
one of them too. I ordered two Zalman's and one was *bad* but changing  
the cable from one to the other made it clear that something was wrong  
with the cable. Since then I ordered another two so in total we have  
four and all are fine.


Are you using the DVI or the VGA cable - try both.

Good luck,

Jürgen

On Jan 15, 2010, at 8:03 AM, Valerie Biou wrote:


Dear Jurgen,

we have many macs but the display says check cable when we plug it
in. Neither of our macs has a graphics card listed on their web site.
Valerie

Le 15 Jan 2010 à 13:09, Jurgen Bosch a écrit :


You are saying you don't have any Macs in your institute?
We're running the Zalmans on Mac mini, MacPro or Macbookpro as
secondary screen.
Puzzeled
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/

On Jan 15, 2010, at 3:08, Valerie Biou b...@lebs.cnrs-gif.fr wrote:


Hello

before you buy a Zalman monitor, check out their list of supported
graphics cards carefully. We bought one for a trial and don't have a
single computer with a suitable graphics card in our institute...
Valerie




On Jan 15, 2010, at 7:49 AM, Takaaki Fukami wrote:


Hi, Francois

We bought it 5 months ago from a supplier in Japan.
(and it has been put back in a box under a desk.)

Zalman has its own web site in Japan.  Model name is the same.
http://www.zalman.com/jpn/product/monitors/ZM-M220W.asp

You can buy it from many shops through the web.
Check the most famous comparison site in Japan, kakaku.com:
http://kakaku.com/item/00854312463/

HTH

Takaaki Fukami


-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf
Of Francois Berenger
Sent: Friday, January 15, 2010 1:31 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Zalman LCD availability

Hello,

By the way, does anyone got this LCD in Japan?

My team is interested to know the model's exact
reference as well as from where you ordered it.

Thanks a lot,
Francois.








-
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/

Re: [ccp4bb] Zalman LCD availability

[Jürgen Bosch]

Yes,
contact mark.gar...@asipartner.com and ask for it.
ASI – Fremont, California
48289 Fremont Blvd.
Fremont, California  94538

Jürgen

On Jan 14, 2010, at 10:07 PM, William G. Scott wrote:


Does anyone know how to get extra glasses?


-
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/

Re: [ccp4bb] Judging a homology model

[Jürgen Bosch]

Hi Raja,

1) how big is your protein ?
2) multiple domains ? What's the ID/sim between domains ?
3) Check Xtalpred in case you've overlooked a closer homolog
http://ffas.burnham.org/XtalPred-cgi/xtal.pl

I would not try to predict an intermediate model and then use that to  
predict the final model.


Jürgen

On Jan 5, 2010, at 12:24 PM, Raja Dey wrote:


Dear Friends,

I have a question about judging a homology  
model. I have three homologous proteins A, B and C of which only A  
has 3D crystal structure available. Their similarities/identities  
are given below.


Pair-wise alignment  similarity/identity (%)

A and B   63/49

A and C   54/39

B and C   60/46

Assuming all the above data are right, which of the following way  
would give the best model for C?


1.   Building homology model of C with A as template.

2.Building homology model of B with A as template and then use B  
as template to build C.


Suppose I used MODELLER for this work where an energy minimization  
step is involved after threading the query sequence on the template.


I need to know which model for C will be better and why?

Thanks for your answer…

Happy New Year to you all,

Raja





The INTERNET now has a personality. YOURS! See your Yahoo! Homepage.


-
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/

Re: [ccp4bb] happy holidays

[Jürgen Bosch]

Now what exactly do you mean by see you next year. Does this imply you  
will be out of work the rest of the year ?

Anyhow enjoy the food  drink wherever you are !

Jürgen

P.S. I'll go to my Lab-Espresso-bar now and enjoy an Amaretti with my  
double Espresso to get into the right mood for the three step  
purification today and have the protein ready to go into trays tomorrow.


On Dec 24, 2009, at 6:43 AM, Vellieux Frederic wrote:


Well, very few people still at work...

So I take this opportunity to wish you all a Happy Holiday season.

And see you next year!

Fred.


-
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/

Re: [ccp4bb] FW: pdb-l: Retraction of 12 Structures...

[Jürgen Bosch]

Not sure if in any of the 112 posts regarding either the retraction  
theme or 'was 12 retraction' theme did mention, that after all despite  
good or as good as possible refinement of the structure, the goal is  
still to describe as accurately as possible the biological system  
using crystallography as a tool.


If you can't confirm your structure by solid biochemical or  
biophysical methods what conclusions should you draw ?
Sure faking data is not the default and looking at the ratio of  
suspected faked structures / deposited structures I do believe we are  
doing much better than other researchers e.g. in medical science.


I agree that validation tools are very helpful and everybody should  
use them prior to deposition of your structure to ensure good  
standards and if you are not happy with the result of those validation  
tools e.g. Procheck, Molprobity well then change something - re-refine  
your structure and fix it prior to deposition.


Regarding paper submissions, I think it would be a good idea to  
include e.g. the Molprobity Ramachandran plot  the summary attached  
to the cover letter when submitting - additional to the table 1 of  
course. Bill Scott once mentioned that he provides a Pymol scene file  
with PDB coordinates  electron density map to reviewers, if one could  
ensure that the coordinates can not be extracted from this file, I  
would be willing to do the same, but it's to easy to extract the  
coordinates and save them for 'other purposes'.


My 2.5 cents,

Jürgen

On Dec 13, 2009, at 1:35 PM, Nicholas M Glykos wrote:


Those Greeks again ...


Finally, validation tools are not there to pass judgement. They are
tools to be used by depositors, referees, and users alike, to help  
them

make a better informed interpretation of crystallographic *models*.
Servers like EDS and PDB_REDO must be seen in that light,


Validation tools are programs written by crystallographers and  
structural

biologists. As it happens, most crystallographers that write programs,
have the neurotic belief that they can treat others' people data  
better

than those other people. And, I share that neurosis ;-))



--


 Dr Nicholas M. Glykos, Department of Molecular Biology
and Genetics, Democritus University of Thrace, University Campus,
 Dragana, 68100 Alexandroupolis, Greece, Tel/Fax (office)  
+302551030620,

   Ext.77620, Tel (lab) +302551030615, http://utopia.duth.gr/~glykos/


-
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/

Re: [ccp4bb] Regarding Open Source

[Jürgen Bosch]

On Nov 15, 2009, at 5:15 AM, George M. Sheldrick wrote:



one DFG attempt about 5 years ago was rejected with the justification
that I was too inexperienced to write crystallographic software.



*lol*
How insightful from the DFG, I guess they must have been right saying  
you are so inexperienced.

I guess it was 5 to 5 when someone read your proposal and had to leave.
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/

Re: [ccp4bb] looking for a computer program called Biomatx

[Jürgen Bosch]

How about Laskowsky et al.
http://nar.oxfordjournals.org/cgi/reprint/33/suppl_2/W89?ijkey=0EJWHewQp1zaEeJkeytype=ref
http://www.ebi.ac.uk/thornton-srv/databases/ProFunc/

Jürgen

On Nov 13, 2009, at 12:39 PM, Vellieux Frederic wrote:


Dear all,

I have heard (really heard, nothing seen in print so far) about a
computer program called Biomatx (I think). Computer program that  
might

be used to search for structural motifs in amino acid sequences (here
might is in between quotes because I haven't seen anything in print,  
and
a search on google, google scholar or Pubmed has not returned  
anything).


I'd happily ask the person I have heard this from. Except, of course,
that I cannot remember who it was nor where it was. Nor when it was.  
And

I might have a need for something similar in the future (there are, of
course, the threading servers but I do not think this is what I will  
need).


Has anyone heard of this computer program? If so, could you kindly  
point

me in the right direction?

Thanks a ton in advance,

Fred.


-
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/

Re: [ccp4bb] sulfur sad phasing

[Jürgen Bosch]

Dear CCP4 community,
(hijacking the thread)
I so far failed to get a sulfur SAD phased structure and I blamed it  
on the low symmetry space group C2 plus weakish diffraction if you  
don't want to overexpose your crystal and be able to collect 20-30x  
redundancy.
What do the expert think about fine slicing versus regular data  
collection for sulfur SAD phasing ?


Thanks,

Jürgen

On Nov 11, 2009, at 8:19 AM, Miguel Ortiz Lombardia wrote:


Le 11 nov. 09 à 13:16, Matthias Zebisch a écrit :


Dear bb!

What is the optimal wavelength for Sulfur SAD phasing?
Is it 1.9A or should one go below that to reduce absorption/damage.

Also, would the same wavelength be appropriate to maximize anomalous
scattering to position chlorides, calcium, sulfate in already  
phased structures?


Thanks in advance,

Matthias




Hi Matthias,

We collected a highly-redundant sulfur-SAD data set at 2.0 A  
wavelength. We managed to solve the structure thanks to two of the  
three sulfur atoms present in the protein, plus one chloride ion  
that happened to be bound to it. Radiation damage was an issue, but  
only after anomalous redundancy was higher than 30. With very, very  
high redundancy it was not possible to solve the structure. Thus, in  
some cases, it may be worth trying with less images.


Of course, as Fred said, the precission of the measurements is  
extremely important for sulfur-SAD phasing.


Best,


-- Miguel

Architecture et Fonction des Macromolécules Biologiques (UMR6098)
CNRS, Universités d'Aix-Marseille I  II
Case 932, 163 Avenue de Luminy, 13288 Marseille cedex 9, France
Tel: +33(0) 491 82 55 93
Fax: +33(0) 491 26 67 20
e-mail: miguel.ortiz-lombar...@afmb.univ-mrs.fr
Web: http://www.pangea.org/mol/spip.php?rubrique2







-
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/

Re: [ccp4bb] Zalman monitor on Linux and Coot

[Jürgen Bosch]

Rubbish, excuse my English :-)
I have four Zalman's running in stereo although I have to confess  
they're all running on various Macs (MacBook Pro, Mac Mini, MacPro)  
under 10.5 or 10.6.

I have no clue if the Zalman runs under linux.
Regarding the stereo effect it is superior than conventional CRT with  
shutter glasses.


My 5 cents,

Jürgen

On Nov 4, 2009, at 1:26 PM, Justin Hall wrote:


Hi Ajit;

One of our CRT monitors broke recently, and in the context of
bemoaning the loss to a friend I was told that LCD monitors will not
work for stereo viewing. I understood the reason to be related to the
difference in refresh rates (?), with LCD's not being fast enough so
that the viewer is left seeing ghosts. The effect, which I have not
seen first hand, was described to me as capable of making most hapless
stereo viewers very ill, very fast. I would encourage you to plumb the
depths of knowledge on this subject further, but that is my simple
understanding.

Best wishes~

~Justin

Quoting Ajit Datta adat...@jhmi.edu:


Hello everyone,
   Sorry for a non-CCP4 related question again. Can anyone let me
know how to make stereo work on linux with Zalman monitor with Coot?
Is it as simple as what we do with CRT monitors? Or do we need
something else? We presently use CRT monitors on a Quadro FX 4600
graphics card. I would like to move to LCDs.

Thanks for all inputs

Ajit B.




-
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/

Re: [ccp4bb] SeMet Peak wavelength and f_prime and f_doubleprime

[Jürgen Bosch]

Hi James,

have a look at the program CROSSEC:
http://www.ccp4.ac.uk/html/crossec.html
should be straightforward to use. However you are at an advantage by  
actually measuring the peak, as a) the beamline could be misaligned  
and b) the chemical environment of your crystal might influence your  
actual peak. In general it's saver to miss the peak on purpose (to  
higher energy) in such a way that you still get some anomalous  
difference for your peak wavelength if you are uncertain where to  
collect.


Jürgen

On Oct 31, 2009, at 10:36 AM, james09 pruza wrote:


Dear All,

Please suggest the Peak wavelength of SeMet and the corresponding  
f_prime and f_doubleprime.


In one of the journal I got it is 0.97905 A and Phenix manual shows  
f_prime and f_doubleprime to be -3 and 4 respectively.


How can one deduce f_prime and f_doubleprime for any HA at a  
particular wavelength.


Thanks.
James.


-
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/

Re: [ccp4bb] small molecule library

[Jürgen Bosch]

Hi Christopher,

have a look at this paper and the references.
Using fragment cocktail crystallography to assist inhibitor design of  
Trypanosoma brucei nucleoside 2-deoxyribosyltransferase. J Med Chem  
(2006) vol. 49 (20) pp. 5939-46


The SEC after soaking is a bad idea, you will loose your fragment as  
it is most likely a low micromolar binder. A better way to approach  
this is to use a filtration device say with 3 kDa cutoff. Concentrate  
your protein + ligands and in a separate tube just the ligands without  
protein. Then run both samples over a mass spec and see if you can  
detect a specific decrease in one or more of your ligands.


Jürgen

P.S. wasn't sure what you mean by biological relevant DeCode has a  
library called Fragments of Life, otherwise Zenobia is not bad.


On Oct 27, 2009, at 10:58 AM, c.weinert wrote:


Dear all.

I am looking for a library of small (biologically relevant)  
compounds to

test binding to a protein. Does somebody know, if there is a company
that sells such a library for fragment based library screening? (to a
reasonable price).

And does anybody has experience with screening such libraries? I  
assume

that one approach would be soaking the protein with the compounds,
followed by SEC and MS analysis. Anybody tried directly soaking  
crystals

followed by X-ray analysis?

Thanks alot already in advance for your help.

Sincerely,
Christopher


-
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/

Re: [ccp4bb] Phase separation to crystals

[Jürgen Bosch]

What do you define as thin and long rods, for some of us this could be  
huge crystals. What's your best guestimate regarding the size ? One  
way of approximating this is taking a loop of know size and focus on  
it, then focus on the crystal and guess.
Small crystals does not necessarily mean they don't diffract. I  
recently got a 2.2 Å dataset with something that was 20x20 x80 µm the  
largest unit cell dimension was 220 Å (needless to say the data was  
collected at [advertisement on] SSRL9-2[advertisement off])


Your MPD condition will freeze no matter what you do with it, so  
that's good (assuming you setup a 1:1 drop, therefore starting at 35%  
MPD). How big are your drops ? If they are fairly large say 1 µl, then  
the following trick might work.
A different option you could try out is the following. Premix your  
protein with the 70% MPD condition in an Eppendorf tube and distribute  
the mix onto a new plate. The reservoir of the plate should then have  
maybe 40-60%MPD, so you are starting off closer to equilibrium but  
then have a smoother gradient to the final concentration. The crystals  
might take longer to appear but they might grow bigger too and you  
might find some conditions without phase separation. Temperature will  
definitely help but then you might need more protein.


Just some thoughts,

Jürgen

On Oct 17, 2009, at 1:44 PM, james09 pruza wrote:


Dear CCP4bbers,

Surprisingly, only At pH 3.6  the needles are appearing after 3 days  
but they are very thin and long rods. I have checked it running SDS- 
PAGE as well as using IZIT dye . Since the proteins has already 50mM  
of NaCl so I added 100mM of NaCl in the crystallization mix. The  
crystal appeared at 70% of MPD. Bunch of florets appears without  
NaCl at the same condition. Will the Additives like Ethanol or 2- 
propanol help in this condition? Should  I not use NaCl in the mix?  
Changing temperature from 4degree to 25 degree will help?

Please advice.

Thanks a lot.
James



-
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/

Re: [ccp4bb] To get the crystal faster...

[Jürgen Bosch]

Hi James,

have you tried limited proteolysis on your protein and see if you can  
identify a stable fragment. Then re-clone and re-crystallize your  
protein. Or a very stupid suggestion, how does your size exclusion  
peak look like ? What you're not running your protein over a SEC to  
polish it ?


Good luck,

Jürgen

On Oct 11, 2009, at 11:27 AM, james09 pruza wrote:


Dear crystallographers,

Sorry for the non-ccp4 query. I am new to this field and need some  
suggestions. My question is, why some protein takes longer time to  
crystallize, say 6-8 months, and it is the only condition to get the  
crystals.? What are the ways to get the crystals faster.


The crystal appears with 60% of 2-Methyl, 2-4 Pentane Diol and only  
at 4 degree with very low concentration of NaCl.  I have got some of  
the sugggestions earlier from the CCP4-discussion board for  
microseeding, but it did not work.


All suggestions from the experts are welcome.

Thanks.
James


-
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/

Re: [ccp4bb] Questions about cryoprotectant

[Jürgen Bosch]

Different suggestion:
I assume you don't have problems obtaining crystals, so I'd setup a 24  
or 96 well plate with your preferred condition then add to each row or  
column a different know cryo at low concentration e.g. 1-10%. Then  
under the conditions in which you still get crystals in the presence  
of the cryo I would soak the crystals in a proper cryo condition for  
this particular additive and flash freeze the crystal in liquid  
nitrogen and not in the stream.


You seem to be very close to cracking your problem, don't give up and  
try a lot of things. As someone mentioned already do flash annealing  
and you can do this also with the liquid nitrogen by lifting your  
crystal out of the bath before plunging it back in - sure you're  
missing a data point there what would the diffraction have been if I  
had not done this. In my hands I ALWAYS try flash annealing with every  
crystal - you can only learn, there's nothing to loose. The question  
is only what do you call a bad diffracting crystal 3Å ? 4 Å 6 Å?


Here's a more recent example July 2009
http://web.mac.com/bosch_lab/jbosch/Labfun.html#4

The crystals are tiny ~20x20x60 (not really visible in the image on  
the web) and before flash annealing diffracted to maybe 5A. I brought  
this data set home with 2.2Å longest cell 220 Å (shot at SSRl 9-2)


Good luck,

Jürgen

On Oct 7, 2009, at 4:54 PM, ycheng wrote:

-
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/

Re: [ccp4bb] Test of cryoconditions without X-rays or cryostream

[Jürgen Bosch]

I think I posted this link maybe two years ago:
http://idb.exst.jaxa.jp/db_data/protein/search-e.php?

Jürgen

On Sep 22, 2009, at 12:48 PM, Elspeth Garman wrote:


or see concentrations given in:
McFerrin and Snell
J.Appl.Cryst (2002) 35, 538
and
Mitchell and Garman,
J.Appl.Cryst. (1996) 29, 584
Good luck
EFG



Paul Leonard wrote:

Hi Claudia

It really is best to test the cryo condition in an X-ray beam.

However, if you don't have access to X-rays in house you could look  
at the
hampton crystallization screen conditions (or another supplier).   
Find a
condition with a similar amount and type of precipitant as you have  
in
your crystallisation condition and look up how much glycerol they  
added as
cryoprotectant.  Then try making up your own crystallisation  
condition
with the amount of glycerol they recommend and freeze your crystals  
in

that solution.

Also try crystal soaks of varying lengths of time (30 seconds - 5  
min) in

the cryo condition as well as trying to soak some crystals where you
gradually increase the %glycerol in the crystal soak prior to  
freezing (in

2% glycerol steps for example).

see
http://hamptonresearch.com/product_detail.aspx?cid=1sid=20pid=3

Good luck.

Paul



Dear List,



Sorry for the probably silly question.



Any suggestions to test cryoconditions without X-rays or cryostream?



I'd need to freeze crystals before going to ESRF and I'm a bit  
anxious. Is
it enough to try to freeze the cryoconditions in liquid nitrogen  
checking

under the microscope (or by eye) or is this still risky?



Thanks,



Claudia





Claudia Scotti Dipartimento di Medicina Sperimentale Sezione di  
Patologia
Generale Universita' di Pavia Piazza Botta, 10 27100 Pavia Italia  
Tel.

0039 0382 986335/8/1 Facs 0039 0382 303673



_
With Windows Live, you can organize, edit, and share your photos.
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Department of Biochemistry
UMDNJ-Robert Wood Johnson Medical School
Center for Advanced Biotechnology and Medicine
679 Hoes Lane
Piscataway, New Jersey 08854-5627

Phone: (732) 235-4206
Email: leon...@cabm.rutgers.edu



--
**NOTE NEW E-MAIL ADDRESS AND PHONE NUMBER

 Elspeth F. Garman,
 President, British Crystallographic Association
 Professor of Molecular Biophysics, University of Oxford
 Visiting Professor in Chemistry, University of Durham
 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
-


-
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/

Re: [ccp4bb] Distinguishing between P6(5)22 and P6(1)22

[Jürgen Bosch]

P6122 or P6522 make sure you get it right, otherwise the  
SpaceGroupPolicePatrol will get you :-)


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/