Re: [ccp4bb] Crystallizing a membrane associated protein

[Van Den Berg, Bert]

Hi Daniel,

Whether or not I would introduce detergents would depend on the behavior of the 
protein during purification and crystallization. Does it behave well during 
purification in the absence of detergents? In your crystallization screens, do 
most of the drops have heavy precipitation? If the answer to the first question 
is "yes" and the answer to the second question "no" I don't think you would 
need to complicate screening and add detergents. Of course it is possible that 
association with the membrane induces a conformation that is more emenable to 
crystallization, but again I wouldn't expect the protein to behave well in the 
absence of detergents.

Good luck, Bert


On 8/1/10 2:13 PM, "Daniel Bonsor"  wrote:

I have a 30kDa protein which I have been trying to crystallize. I have tried in 
the conventional way using Hampton screens but no luck so far. CD of the 
protein shows it is folded. However reading the literature I found out it is 
strongly associated with the inner membrane of H. pylori. This was done by 
Western blots of the supernatant  and membranes. Triton X-100 and N-lauroyl 
sarcosine were found to release the protein into the supernatant.

My question is do I change tactics and try and crystallize it as a membrane 
protein or try adding a small amount of detergents to the protein and treat it 
as a soluble protein? Or something else?


Thanks in advance for all of your suggestions.


Daniel Bonsor

Re: [ccp4bb] protein turns brown

[Van Den Berg, Bert]

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"  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
 

Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?

[Van Den Berg, Bert]

I find this interesting as well, mainly because I have never seen this myself 
and I have looked at plenty of badly diffracting crystals. In my hands, 
synchrotron data at most end up being ~1.5 angstrom better in terms of 
resolution than the same crystals on our home source. I'm wondering if this 
phenomenon is real, or whether it is just a matter of people screening 
more/better crystals at the synchrotron compared to at home? Or a lousy home 
source?

Bert


On 9/28/10 1:27 PM, "Francis E Reyes"  wrote:

Hi all

I'm interested in the scenario where crystals were screened at home
and gave lousy (say < 8-10A) but when illuminated with synchrotron
radiation gave reasonable diffraction ( > 3A) ? Why the discrepancy?

Thanks

F

-
Francis E. Reyes M.Sc.
215 UCB
University of Colorado at Boulder

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D

Re: [ccp4bb] How to detect the concentration of detergent?

[Van Den Berg, Bert]

Hi YB,

For membrane protein crystallization it is common practice (although not always 
necessary) to dialyze the protein after the final concentration step (against 
GF buffer). The problem with DDM is that dialysis is slow due to the low cmc, 
and in general it is advisable to finish the prep and set up drops as quickly 
as possible. I would not dialyze more than 1 x O/N, although if your protein is 
really stable you could try longer. You could also try 100 kDa cutoff 
concentrators, as these may allow passage of empty DDM micelles. As for 
measuring the detergent concentration, in the case of DDM and other 
sugar-containing detergents you could do a sugar (Fehling's) kind of assay. I'm 
not sure if anything has been published, but it should be fairly easy to do. 
You could also try TLC, but this may be less accurate.

Also, 10 mg/ml is not high at all (although its a good starting point), and you 
should try much higher if most drops are clear: try 50 mg/ml and see what 
happens.

Good luck, Bert


On 10/4/10 10:28 AM, "yybbll"  wrote:

Dear all,

I want to crystallize a symport transporter, which contains 12 transmembrane 
alpha-helices. We used Ni-resin column firstly, and then size exclusion. After 
size exclusion, only one peak, it is very nice. the final condition is 10 mM 
mes, 100 mM NaCl, 10 mM sucrose, 1 mM DTT, and 0.02% DDM. The CMC of DDM is 
about 0.008%. However, when we concentrate protein using a concentrator with 50 
kDa cutoff, detergent all was concentrated. So final the concentration of 
detergent should be very high (10 times more than CMC). We don't know how to 
detect the concentration of detergent. We used these samples to grow crystal. 
We found almost drops are clear, and the final concentration of protein is 
about 10 mg/ml. For membrane protein, I think this concentration is high. But 
for us, we can obtain so high concentration easily.

Could anybody tell me how to detect the concentration of detergent?

And how to  dilute detergent?

Thanks all.

Y.B. Lin

2010-10-04

yybbll

Re: [ccp4bb] Help with Optimizing Crystals

[Van Den Berg, Bert]

Hi Matt,

You'll probably get many different answers to a question like this, but what I 
would do is go back to your protein and make different constructs; chop off 
termini, surface mutations etc, maybe cleave off the tag. Of course more 
screening and optimization might work, but my sense is that since you get many 
hits pretty easily that however don't diffract, there may be something on the 
protein level that needs correcting.

Good luck, Bert


On 10/26/10 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] Additional band on gel due to his-tag: any references?

[Van Den Berg, Bert]

Hi Seb,

I'm not aware of the notion (and neither are your reviewers apparently) that a 
His tag often results in two bands on a lane in SDS page. Why would that be? 
Extra SDS binding to the positive patch?
Just wondering if there's any truth to your statement.

Also, since in this case there seems to be consensus (!) among reviewers 
regarding the double band, they may have a legitimate point..my 2 cents.

Regards, Bert


On 10/28/10 7:16 AM, "Sebastiaan Werten"  
wrote:

Dear all,

we have a his-tagged protein that shows a minor accompanying band in
SDS-PAGE, just above the main band. According to all other methods
available to us the material is homogeneous, the protein has the
correct
mass in MALDI-TOF, epitopes are recognized, etc. etc.

I know that the additional band is a very common artifact with
his-tagged proteins, but I was wondering if anyone is aware of a paper
that formally describes the phenomenon, as we need to appease a couple
of rather bloody-minded referees.

Thanks very much for any suggestions, Seb.

--
Dr. Sebastiaan Werten
Institut für Biochemie
Universität Greifswald
Felix-Hausdorff-Str. 4
D-17489 Greifswald
Germany
Tel: +49 38 34 86 44 61
E-mail: sebastiaan.wer...@uni-greifswald.de

Re: [ccp4bb] Strange spots

[Van Den Berg, Bert]

Autoindexing in the truest sense of the word? ;-)


On 10/29/10 12:08 PM, "David Goldstone"  wrote:

Dear All,

Does anyone have any insight into what the circles around the spots
might be?

cheers

Dave
--
David Goldstone, PhD
National Institute for Medical Research
Molecular Structure
The Ridgeway
Mill Hill
London NW7 1AA

Re: [ccp4bb] DLS

[Van Den Berg, Bert]

Hi Shukuri,

If you're on a tight budget and you only want to use DLS to verify sample 
homogeneity I would save my money or use it for something else (a 
crystallization robot, for example). You definitely do NOT need DLS to solve 
crystal structures, including those of membrane proteins. Many monodisperse 
samples do not crystallize and I can tell you from experience that polydisperse 
samples can crystallize perfectly well. Gel filtration chromatography is a good 
and much cheaper alternative to DLS, and you'll have the columns already anyway.
Admittedly, DLS will give you somewhat more info than gel filtration, but 
again, if money is an issue I would not get a DLS instrument. For me its 
comparable to a UV microscope for verification of crystals; nice for sure, but 
not worth the $$$.

Cheers, Bert


On 10/30/10 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] Citations in supplementary material

[Van Den Berg, Bert]

?? I don't know if I understand the question, but don't most journals have 
references that do include the article titles?
Science, Nature, Cell, NSMB, PNAS, JMB, Structure all have references 
titlesas they should.

Bert


On 11/22/10 9:35 AM, "John R Helliwell"  wrote:

Dear Jacob,
Additional content, like article titles, whether print or online, need
to be checked properly for accuracy.
Article titles (if supplied by authors) can often need heavy checking,
and online systems to check
bibliographic information are not always reliable or comprehensive.
This I think explains the paucity of
article titles coverage in references lists of many/most Journals.
Best wishes,
John



On Mon, Nov 22, 2010 at 12:16 PM, Jacob Keller
 wrote:
> It seems to me that this problem is really a hold-over from compromise
> with the exigencies of hard-copy publishing, i.e. a way to save
> physical space. Further, there seem to be many aspects of
> e-publication that have not adapted to the strengths/weaknesses of the
> new medium, which could and should be remedied. One aspect is citation
> format--why not capitalize on the luxury of having plenty of space?
> Maybe even the abstracts could be included (why not?). Another thing I
> mentioned in a previous email: why not remove length limitations? Let
> the authors have the space they need to say what needs to be said! I
> think EMBO actually espouses this idea, although I am not sure how far
> they would go (I am pretty sure they do not limit number of
> references, for example.) Anyway, it seems to be an interesting and
> historical time in the publishing world.
>
> JPK
>
> On Sun, Nov 21, 2010 at 8:07 PM, Francois Berenger  wrote:
>> Hello,
>>
>> For me the citation format is also a major problem.
>>
>> When the title of the paper is not shown, it really hinders the work
>> of trying to find which references are worthwhile reading.
>> I think it may even have a negative impact on the number of citations
>> a paper get.
>>
>> I don't know if it has been solved in recent issues of IUCr journals,
>> so please forgive me if this is an old and dead topic.
>>
>> Regards,
>> Francois.
>>
>



--
Professor John R Helliwell DSc

Re: [ccp4bb] Citations in supplementary material

[Van Den Berg, Bert]

OK I get it, its about the supplements...apologies.

Bert


On 11/22/10 9:35 AM, "John R Helliwell"  wrote:

Dear Jacob,
Additional content, like article titles, whether print or online, need
to be checked properly for accuracy.
Article titles (if supplied by authors) can often need heavy checking,
and online systems to check
bibliographic information are not always reliable or comprehensive.
This I think explains the paucity of
article titles coverage in references lists of many/most Journals.
Best wishes,
John



On Mon, Nov 22, 2010 at 12:16 PM, Jacob Keller
 wrote:
> It seems to me that this problem is really a hold-over from compromise
> with the exigencies of hard-copy publishing, i.e. a way to save
> physical space. Further, there seem to be many aspects of
> e-publication that have not adapted to the strengths/weaknesses of the
> new medium, which could and should be remedied. One aspect is citation
> format--why not capitalize on the luxury of having plenty of space?
> Maybe even the abstracts could be included (why not?). Another thing I
> mentioned in a previous email: why not remove length limitations? Let
> the authors have the space they need to say what needs to be said! I
> think EMBO actually espouses this idea, although I am not sure how far
> they would go (I am pretty sure they do not limit number of
> references, for example.) Anyway, it seems to be an interesting and
> historical time in the publishing world.
>
> JPK
>
> On Sun, Nov 21, 2010 at 8:07 PM, Francois Berenger  wrote:
>> Hello,
>>
>> For me the citation format is also a major problem.
>>
>> When the title of the paper is not shown, it really hinders the work
>> of trying to find which references are worthwhile reading.
>> I think it may even have a negative impact on the number of citations
>> a paper get.
>>
>> I don't know if it has been solved in recent issues of IUCr journals,
>> so please forgive me if this is an old and dead topic.
>>
>> Regards,
>> Francois.
>>
>



--
Professor John R Helliwell DSc

Re: [ccp4bb] TEV protease

[Van Den Berg, Bert]

The way to go is make-your-own, especially since it is a pretty lousy enzyme 
(we often use 1:5 to 1:10 molar ratio of TEV:protein). You can get expression 
vectors here:

http://mcl1.ncifcrf.gov/waugh_tev.html

Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gloria Borgstahl 
[gborgst...@gmail.com]
Sent: Friday, January 14, 2011 9:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] TEV protease

We are using TEV protease to make a big batch of protein for
structural studies.  We usually use thrombin, so this is our first
time using this enzyme on a large scale.  Boy is it expensive!  Does
anyone know of a bulk source for this enzyme and what ratios of use do
you recommend.  Imidazole inhibits?
Many thanks. Gloria

Re: [ccp4bb] Merging data to increase multiplicity

[Van Den Berg, Bert]

I have heard this before. I'm wondering though, does anybody know of a 
systematic study where different data processing programs are compared with 
real-life, non-lysozyme data?

Bert


On 1/28/11 7:58 AM, "Bosch, Juergen"  wrote:

I was a bit reductive with my statement (iPhone)
The equation below is suppose to read:
If you have bad data, then you need to process with XDS in order to get the 
maximum out of your data.

Thanks Tim,

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 7:44 AM, Tim Gruene wrote:

Dear Jürgen,

is this an assignment operator or an equal sign? For if it's the latter it could
read that the result of processing data with XDS are bad data, which is rather
rude and probably not what you meant.

Tim

On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote:
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  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] Merging data to increase multiplicity

[Van Den Berg, Bert]

Interesting! But when will it be published? :-)


On 1/28/11 8:45 AM, "Bosch, Juergen"  wrote:

Yes.
But we have not published this.
Mark Robien and I did a "systematic" study on about 30 data sets while we were 
at SGPP. The easy cases can be processed with anything the difficult cases 
worked only with XDS.
This was mostly SeMet data or HA data, so de novo phasing no MR stuff.
If you compare the signal of the SeMet peaks between the different processing 
options you got the strongest peaks via XDS.
Success was assessed using ShelxD for finding HA sites.

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/ <http://web.me.com/bosch_lab/>

On Jan 28, 2011, at 8:37 AM, Van Den Berg, Bert wrote:

I have heard this before. I'm wondering though, does anybody know of a 
systematic study where different data processing programs are compared with 
real-life, non-lysozyme data?

Bert


On 1/28/11 7:58 AM, "Bosch, Juergen"  > wrote:

I was a bit reductive with my statement (iPhone)
The equation below is suppose to read:
If you have bad data, then you need to process with XDS in order to get the 
maximum out of your data.

Thanks Tim,

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/ <http://web.me.com/bosch_lab/>

On Jan 28, 2011, at 7:44 AM, Tim Gruene wrote:

Dear Jürgen,

is this an assignment operator or an equal sign? For if it's the latter it could
read that the result of processing data with XDS are bad data, which is rather
rude and probably not what you meant.

Tim

On Fri, Jan 28, 2011 at 06:55:43AM -0500, Jürgen Bosch wrote:
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  > 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] I/sigmaI of >3.0 rule

[Van Den Berg, Bert]

There seem to be quite a few "rule" followers out there regarding resolution 
cutoffs. One that I have encountered several times is reviewers objecting to 
high Rsym values (say 60-80% in the last shell), which may be even worse than 
using some fixed value of I/sigI.


On 3/3/11 9:55 AM, "Ed Pozharski"  wrote:

On Thu, 2011-03-03 at 12:29 +0100, Roberto Battistutta wrote:
> Does anyone know the origin or the theoretical basis of this "I/sigmaI
> >3.0" rule for an appropriate resolution?

There is none.  Did editor ask you to follow this "suggestion"?  I
wonder if there is anyone among the subscribers of this bb who would
come forward and support this "I/sigmaI >3.0" claim.

What was your I/sigma, by the way?  I almost always collect data to
I/sigma=1, which has the downside of generating somewhat higher
R-values.  Shall I, according to this reviewer, retract/amend every
single one of them?  What a mess.

Cheers,

Ed.

--
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] I/sigmaI of >3.0 rule

[Van Den Berg, Bert]

Does the position of this "inflection point" depend on the redundancy? Maybe it 
does not; for high-redundancy data one would simply get a much higher 
corresponding Rsym.


On 3/3/11 11:13 AM, "Ed Pozharski"  wrote:

On Thu, 2011-03-03 at 16:02 +0100, Vellieux Frederic wrote:
> For myself, I decide on the high resolution cutoff by looking at the
> Rsym vs resolution curve. The curve rises, and for all data sets I
> have
> processed (so far) there is a break in the curve and the curve shoots
> up. To near vertical. This "inflexion point" is where I decide to
> place
> the high resolution cutoff, I never look at the I/sigma(I) values nor
> at
> the Rsym in the high resolution shell.
>

Fred,

while your procedure is definitely more sophisticated than what I do,
let me point out that the Rsym is genuinely a bad measure for this, as
it depends strongly on redundancy.  Does more robust measures (e.g.
Rpim) show similar "inflexion"?  I suspect it will at least shift
towards higher resolution.

Cheers,

Ed.

--
"I'd jump in myself, if I weren't so good at whistling."
   Julian, King of Lemurs

Re: [ccp4bb] I/sigmaI of >3.0 rule

[Van Den Berg, Bert]

We should compile this discussion and send it as compulsive reading to journal 
editors...;-)

Bert


On 3/3/11 12:07 PM, "Simon Phillips"  wrote:



I take the point about a tendency in those days to apply sigma cutoffs to get 
lower R values, which were erroneously expected to indicate better structures.  
I wonder how many of us remember this paper by Arnberg et al (1979) Acta Cryst 
A35, 497-499, where it is shown for (small molecule) structures that had been 
refined with only reflections I>3*sigma(I) that the models were degraded by 
leaving out weak data (although the R factors looked better of course).

Arnberg et al took published structures and showed the refined models got 
better when the weak data were included.  The best bit, I think, was when they 
went on to demonstrate successful refinement of a structure using ONLY the weak 
data where I<3*sigma(I) and ignoring all the strong ones.  This shows, as was 
alluded to earlier in the discussion, that a weak reflection puts a powerful 
constraint on a refinement, especially if there are other stronger reflections 
in the same resolution range.

---
| Simon E.V. Phillips |
---
| Director, Research Complex at Harwell (RCaH)|
| Rutherford Appleton Laboratory  |
| Harwell Science and Innovation Campus   |
| Didcot  |
| Oxon OX11 0FA   |
| United Kingdom  |
| Email: simon.phill...@rc-harwell.ac.uk  |
| Tel:   +44 (0)1235 567701   |
|+44 (0)1235 567700 (sec) |
|+44 (0)7884 436011 (mobile)  |
| www.rc-harwell.ac.uk|
---
| Astbury Centre for Structural Molecular Biology |
| Institute of Molecular and Cellular Biology |
| University of LEEDS |
| LEEDS LS2 9JT   |
| United Kingdom  |
| Email: s.e.v.phill...@leeds.ac.uk   |
| Tel:   +44 (0)113 343 3027  |
| WWW:   http://www.astbury.leeds.ac.uk/People/staffpage.php?StaffID=SEVP 
  |
---

Re: [ccp4bb] off topic: GPCR membane insertion/orientation

[Van Den Berg, Bert]

Hi Justin,

I'm not sure if there are papers regarding this for GPCRs, but the phenomenon 
you're referring to is the "positive inside rule". This basically means that 
the SecY translocon (in a way that is only partially clear) mediates membrane 
protein insertion in such a way that the (net) positively charged side of the 
first TM segment stays inside the cytosol. The orientation of the first TM 
dictates that of the subsequent ones (up, down, up etc). People have played 
with this successfully. It's generally valid for membrane proteins. A recent 
reference to get you started is this:

Orientation of small multidrug resistance transporter subunits in the membrane: 
correlation with the positive-inside rule. 


Kolbusz MA, ter Horst R, Slotboom DJ, Lolkema JS.

J Mol Biol. 2010 Sep 10;402(1):127-38.


Good luck, Bert


On 3/4/11 11:31 AM, "Justin Hall"  wrote:

Dear Community,

In trying to trouble shoot an experiment I have become interested in
the cellular process that regulates the insertion and proper
orientation of membrane proteins. I am looking for references for how
a GPCR is correctly oriented during expression (i.e. the extra
cellular domain ends up extra cellularly oriented instead of a 50/50
mix in and out), my intuition is that there must be an N-terminal
sequence that directs this process, but I am having no luck finding
information on what this sequence is for GPCRs, what players are
involved or how orientation is thought to be controlled. Any
suggestions?

This is all spurred by my wanting to use phage display with a protein
that binds to the intracellular side of a GPCR, but of course that is
the hard side to present to the outside of a cell so I need to figure
out how to flip these guys around. I have thought about adding a new
TM helix before TM1 (or removing TM1) to flip these guys, but was
hoping there might be another way around that doesn't involve such
massive architectural rearrangement such as simply clipping the
N-terminal sequence responsible for proper orientation (if such a
thing exists). Cheers~

~Justin

Re: [ccp4bb] off-topic replay: glycerol in protein stock solutions

[Van Den Berg, Bert]

Hi Tom,

Adding glycerol to (crystallization) buffers is a very common practice when 
working with membrane proteins. Many membrane proteins have been crystallized 
(perhaps even the majority) with glycerol, even up to 30% v/v. So, at least for 
membrane proteins, there is no problem.

Bert


On 3/4/11 8:25 PM, "Brett, Thomas"  wrote:

Hi guys:
I know this has been asked before, but I want to get (current) opinions and 
observations once again. Every once in a while, you work with a protein that 
needs to have a little something added to the buffer to keep it soluble. The 
most common trick is addition of glycerol (usually 5-10%). I'm looking for 
general observations on this. I was of the opinion that this was usually a bad 
thing to do with protein stock that you intend to cyrstallize because glycerol 
will coat the protein in a non-homogeneous manner and make your homogeneous 
protein prep heterogeneous (in a way). Or do people usually have good luck 
crystallizing proteins that have to be stored in some glycerol? Or is there a 
better additive? Also, when having to use glycerol, do you put it on your 
sizing columns, etc? I am concerned with putting glycerol on my columns I may 
never be able to completely wash it away. What are your thoughts, community?
thanks in advance,
-tom

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

[Van Den Berg, Bert]

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] protein aggregation

[Van Den Berg, Bert]

Try different detergents.
Try 10%  or more glycerol.
Try adding ligands (if present/known).
Try varying ionic strength and/or pH.
Try giving more specifics so people on the board may be able to help you better.

Bert


On 3/23/11 1:51 PM, "gauri misra"  wrote:

The protein purifies nicely there is no problem in that. Just at the last step 
when it is concentrated it starts precipitating beyond a concentration of 
1mg/ml.
Already the purification buffers have the detergent.

On Wed, Mar 23, 2011 at 1:44 PM, Kornelius Zeth 
 wrote:

2 M urea and detergents

On Wed, 23 Mar 2011 13:41:55 -0400
 gauri misra  wrote:
> Hi,
> What are the different methods to prevent protein aggregation while
> concentrating so as to increase the concentration of the protein?
> I have some idea of adding EDTA and charged amino acids like L-Arg and
> L-Glu.
> I would appreciate if the readers share their experiences.
>
> Thanks!
>
> Gauri

 --
 Kornelius Zeth
 Max Planck Institute for Developmental Biology
 Dept. Protein Evolution
 Spemannstr. 35
 72076 Tuebingen, Germany
 kornelius.z...@tuebingen.mpg.de
 Tel -49 7071 601 323
 Fax -49 7071 601 349

[ccp4bb] Structures of crosslinked complexes

[Van Den Berg, Bert]

Dear All,

Does anybody know of examples where labile protein complexes have been 
crystallized after using non-specific crosslinkers (so excluding complexes with 
engineered SS bonds, for example)?

Thanks in advance for any examples,

Bert


On 3/23/11 10:19 AM, "Parthasarathy Sampathkumar"  wrote:

Dear Bert,

You  could try the program Hole from
http://www.ncbi.nlm.nih.gov/pubmed/9195488

J Mol Graph. <http://www.ncbi.nlm.nih.gov/pubmed/9195488>  1996 
Dec;14(6):354-60, 376.
HOLE: a program for the analysis of the pore dimensions of ion channel 
structural models.
Smart OS <http://www.ncbi.nlm.nih.gov/pubmed?term=%22Smart%20OS%22%5BAuthor%5D> 
, Neduvelil JG 
<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Neduvelil%20JG%22%5BAuthor%5D> , 
Wang X <http://www.ncbi.nlm.nih.gov/pubmed?term=%22Wang%20X%22%5BAuthor%5D> , 
Wallace BA 
<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Wallace%20BA%22%5BAuthor%5D> , 
Sansom MS 
<http://www.ncbi.nlm.nih.gov/pubmed?term=%22Sansom%20MS%22%5BAuthor%5D> .



-Partha



On Wed, Mar 23, 2011 at 9:02 AM, Van Den Berg, Bert 
 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] where to buy stereo glasses for making stereo figures?

[Van Den Berg, Bert]

http://hamptonresearch.com/product_detail.aspx?cid=26&sid=145&pid=439

These work pretty well and are cheap.

Bert


On 3/30/11 10:24 PM, "Jiamu Du"  wrote:

For side by side stereo figures.
Thanks.

On Wed, Mar 30, 2011 at 9:42 PM, Ingrid Attinost  
wrote:
Dear Jiamu,


What kind of stereo are you refering to? Do you mean anaglyph stereo (like the 
kind of stereo used nowadays when watching 3D movines at the theater) or side 
by side stereo for your figures?

Ingrid


Date: Wed, 30 Mar 2011 20:59:43 -0400
From: jiam...@gmail.com
Subject: [ccp4bb] where to buy stereo glasses for making stereo figures?

To: CCP4BB@JISCMAIL.AC.UK

Dear All,
I want to buy a stereo glasses for making stereo figures, especially for the 
labeling. Is there a company sell it?

Thanks and best wishes.

Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

[Van Den Berg, Bert]

You may have a fairly long cell edge (or two if you are dealing with P3 or P6), 
but you also seem to have high mosaicity (pic spot 1). Try the useful 
strategies suggested here. It may also be worthwhile to shoot a few roomtemp 
crystals to see if your cryo is at fault for your high mosaicity.

Bert


On 4/5/11 9:10 AM, "dengzq1987"  wrote:

hello Jürgen_Bosch,

because i have collected the data,but can't index.in some direction,the spot is 
separated .but the others are set close together(picture spot1 and spot 2).so  
we think there  is one  long unit cell axes.

2011-04-05

dengzq1987

发件人： Jürgen_Bosch
发送时间： 2011-04-05  20:29:18
收件人： dengzq1987
抄送： CCP4BB@JISCMAIL.AC.UK
主题： Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

What do you consider long ? 200, 300 ? 600 A ? Before shooting try to run 
strategy or xplan. Move the detector back to first reliably be able to 
determine your cell. Then double your estimated mosaicity and see what strategy 
suggests. If you don't get many overlaps (<5%) then try a closer distance. 
Don't rotate 1degrees but take 1/2 of the mosaicity. Obviously you want to make 
good use of the detector area so adjust the edges to where your crystal really 
diffracts. And if that resolution leads to too many overlaps then limit your 
resolution and get first a good datasets home. You then can play with 2theta 
for a higher resolution dataset.
Another obvious thing to do and you don't mention what reduction program you 
use is to let XDS sort your problem out. Unless you collected to high 
resolution without being cautious XDS could help. If not, well then you had 
your experience and now should know better.
SSRL has options to collect 450 A cells to 3A without much hassle. That was my 
largest cell so far.
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 5, 2011, at 1:05, dengzq1987  wrote:



hello all,

does anyone have the experience of   Collecting Data from Long Unit Cell Axes ? 
I  have a crystal that diffracts to about 4 A. in some direction   the spots 
overlap. we can't use the data to index .we think it is because that  there is 
a long unit cell axes. so  is there any method to solve this  problem?



best wishes.



2011-04-05



dengzq1987

Re: [ccp4bb] Lyophilized protein sample (purchased)

[Van Den Berg, Bert]

I think that's impossible to say. Some proteins lyophilize fine, some don't. 
Generally your chances are best if the protein is sturdy. Is also depends how 
it was lyophilized (any salts, buffer etc). Getting the protein in solution may 
be tricky; in my limited experience plain water works best (not buffered).

Good luck, Bert


On 5/6/11 4:01 PM, "REX PALMER"  wrote:

We have purchased a 5mg commercial sample of a protein we want to crystallize.
On arrival it transpired that the sample had been lyophilized.
Does anyone know if this is likely to give problems and if so what can/should 
be done about it?
Thanks in advance

Rex Palmer
Birkbeck College

[ccp4bb] postdoctoral positions available

[Van Den Berg, Bert]

Two postdoctoral positions are available immediately to study 
structure-function relationships of integral membrane proteins. One position 
(funded by NIH) will focus on the transport mechanism of hydrophobic compounds 
across the bacterial outer membrane as mediated by FadL-family proteins. The 
second position (funded by a PEW scholar grant) concerns prokaryotic and 
eukaryotic divalent metal transporters belonging to the ZIP (Zrt/IRT-like 
protein) family. Work will involve overexpression and purification of membrane 
proteins in E. coli and Pichia pastoris, membrane protein crystallization and 
structure determination, and development of in vitro substrate binding and 
transport assays. Applicants should have a strong background in molecular 
biology and protein chemistry. Experience in membrane protein purification and 
X-ray crystallography would be desirable, but is not required. Interested and 
highly motivated candidates should state their project of interest and e-mail 
their CV and the contact details of three references to:

 
Bert van den Berg
 
e-mail: [EMAIL PROTECTED]

University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm

Re: [ccp4bb] Solvent content of membrane protein crystals

[Van Den Berg, Bert]

The problem is that micelle shape/size depends on many factors such as ionic 
strength, pH, temperature etc (this also depends on the particular detergent). 
So, even if one would take the ROUGH estimate of the amount of detergent 
associated with the protein from gel filtration/light scattering experiments, 
more likely than not this would not correspond to the amount of detergent 
associated with the protein in the crystal. So a "micelle-corrected" Vm value 
would still have a large degree of uncertainty and would not really help 
narrowing down the number of molecules/AU.

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Savvas Savvides
Sent: Sun 9/23/2007 4:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Solvent content of membrane protein crystals
 
Indeed, but wouldn't consideration of micelle size affect our  
estimation of the number of molecules in the asu, in some cases  
significantly?
The crystal packing of some membrane proteins shows that they tend to  
pack as "potatoes in space" with relatively few protein-protein  
contacts and with detergent micelles presumably providing the rest of  
the crystal packing interactions. That also explains the often  
significant diffraction anisotropy observed in such crystals. One  
classic example is the prototypical potassium channel structure (KCSA)  
(PDB entry 1bl8).

Savvas


Quoting Edward Berry <[EMAIL PROTECTED]>:

> I would use a very general definition for "solvent",
> including disordered detergent and lipids.
> As you know in many cases ordered detergents and lipids
> have been modeled in the coordinates, so they are part of
> the model not the solvent. In some cases I think waters
> should be included in the model not solvent- say for
> structural waters buried in the protein at least.
>
> Ed
>
> Savvas Savvides wrote:
>
>> Dear colleagues,
>>
>> in estimating the solvent content of membrane protein crystals it
>> would only seem reasonable that micelle size should also be taken   
>> into account. Depending on the aggregation number and MW of a given  
>>   detergent, the concentation of detergent used, and the buffer
>> conditions, one may have micelles on the order of 15-25 kDa or even  
>>   35-50 kDa for detergents with alkyl chains of more than 10 carbons.
>>
>> However, when I took a look in a handful of papers reporting   
>> Matthews' numbers for membrane protein crystals, it became apparent  
>>  that only  the protein MW is used in such estimates. I am  
>> beginning  to wonder if  one should even bother reporting a  
>> Matthews number  for a membrane  protein crystal given the  
>> uncertainties surrounding  size and role of  micelles in crystal  
>> packing.
>>
>> Any thoughts on this?
>>
>> best wishes
>> Savvas

Re: [ccp4bb] Protein-detergent micelle sizes

[Van Den Berg, Bert]

Jacob,
 
Whether the calbiochem/anatrace catalogues will work for you depends on what 
you want to know. They do have useful info on micelle sizes (aggregation #s, 
cmc's etc), but these are values for micelles alone, either in water or 0.1 M 
salt or something like that. If your question is how much detergent is actually 
associated with a particular protein you'll unfortunately have to measure that 
yourself (see Michaels message), since this will be protein and condition 
dependent (ie a large membrane protein will have more detergent associated with 
it than a small one). There was a thread on this subject on this board a couple 
weeks back i think.
 
Cheers, Bert
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Savvas Savvides
Sent: Tue 11/20/2007 3:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Protein-detergent micelle sizes



Hi Jacob,
the Calbiochem and Anatrace product catalogues will do the trick for
you.
best wishes
Savvas


Savvas N. Savvides
L-ProBE, Unit for Structural Biology
Ghent University
K.L. Ledeganckstraat 35
9000 Ghent, BELGIUM
Phone: +32-(0)9-264.51.24 ; +32-(0)472-92.85.19
Email: [EMAIL PROTECTED]
http://www.eiwitbiochemie.ugent.be/units_en/structbio_en.html



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Jacob Keller
Sent: Tuesday, November 20, 2007 12:25 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein-detergent micelle sizes


Dear Crystallographers,

I am trying to gather literature values on the size of the
detergent/micelle
belts on solublized membrane proteins. Does anybody know of a good
repository of this info, whether as a database or as a review paper, or
otherwise? The rough values of 15-25kD to 35-50kD were given in one
recent
BB posting, but I was looking for a more systematic tabulation...

Thanks,

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.467.4049
cel: 773.608.9185
email: [EMAIL PROTECTED]
***

Re: [ccp4bb] detergent concentration used for membrane protein purification

[Van Den Berg, Bert]

Hi Rongjin,
 
the concentration (and total amount) of detergent during purification depends a 
lot on what stage of purification you're at. For membrane extraction (1st 
step), people typically use anything from 1-5%. This depends on your budget, on 
the cmc of the detergent, and of course on the amount of cells you have. b-OG, 
having a high cmc (~0.7%) is not very effective at 1%, so you have to use a 
higher concentration. 1% is often enough for things like DDM and LDAO. My lab 
uses typically 100 ml of 1% detergent to extract membrane proteins from 12 l 
Ecoli culture (so 1 g detergent total).
 
Later on in purification, when the amounts of stabilizing endogenous lipids 
decrease, it makes sense to lower the detergent concentration as too high 
concentrations are often denaturing. I would not go lower than ~ 2-fold cmc (it 
is hard to know what the actual cmc is in your buffer). In the case of DDM, we 
do our 1st purification step in 0.2% DDM (Ni-column) and subsequent steps in 
0.03-0.05% DDM (approx. 2x cmc). 2% DDM for purification seems excessive and 
will very likely not allow crystallization (if you're attempting that).
 
Purification in one detergent followed by exchange later is perfectly fine. 
However, it seems your protein is not very happy in DDM, so I think it would 
make sense to start off (extraction) from a different detergent.
 
Cheers, Bert
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Rongjin Guan
Sent: Fri 12/21/2007 2:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] detergent concentration used for membrane protein purification


Dear All
 
sorry for a non-ccp4 related question. 
 
what is the typical concentration of detergents used in membrane protein 
purification? 
Is it usually within 1-10 times of CMC? 
 
My collaborator was using 10% DDM in purifying a membrane protein. I suggested
to reduce it and now he is working with 2% DDM. He said lower than that the 
protein 
is not soluble. 
 
so another question is, can we purify the membrane protein first at high 
concentration
of one detergent (say, 2% DDM), and then screen different detergents later to 
find a 
better one? Or it is better to screen the detergents first before purification? 
 
 
any suggestions will be appreciated. 
 
Have a nice holiday!
 
Rongjin Guan 
 
 

Re: [ccp4bb] crystallization robot

[Van Den Berg, Bert]

Hi Lisa,
 
is the Phoenix capable of setting up hanging drops? For me that is one of the 
big plusses of the Mosquito. Are there any other robots out there capable of 
doing hanging drops?
 
Cheers, Bert
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Lisa A Nagy
Sent: Wed 1/9/2008 11:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization robot



We chose the Phoenix crystallization robot because:

It has no expensive consumables (tips) intrinsic to the machine. This
was also a big item for us because we worry about being able to run the
machine for more than 3 years. Would the tips for our 2008 machine be
available in 2014?

It is easy to program (BIG ITEM) for different tray configurations, and
various dispensing methods- even on the same tray. Right now you may not
think you'll have to vary drop sizes or add additional components
(ligand? detergent?) to your drops, but you probably will.

It can draw from 2ml block plates. Reformatting from block plates to
your trays is a pain.

The nitinol tips won't break (Compared to ~$700 apiece for the
incredibly breakable ceramic tips on some other machines).

It has cooling blocks for your samples. This is more important than you
think.

It's fast.

It is easily integrated into a fully automated lab system. Right now,
though, our humans (including me) are cheaper than rails and robots.

It's incredibly accurate, even with 30% PEG 4000 (we tested this
ourselves).

You can use it for other low volume dispensing applications.


--
Lisa Nagy
University of Alabama-Birmingham
[EMAIL PROTECTED] 

Re: [ccp4bb] crystallization robot

[Van Den Berg, Bert]

The mosquito has special (albeit fairly pricey at $13 each) plastic sheets that 
allow setup of hanging drops in a 96-well format. It can also do multiple drops 
per well. As far as I know this is a capability unique to the Mosquito but I 
may be wrong.
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Lisa A Nagy
Sent: Wed 1/9/2008 11:53 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization robot



Looking at the mosquito, it doesn't have any cover-slip handling robotics, 
either. So it's the same thing- rearrange the dispense location and flip the 
cover- which is either a glass plate or mylar or  a tape seal.

--
Lisa Nagy
University of Alabama-Birmingham
[EMAIL PROTECTED] 

[ccp4bb] low-res cutoff in refinement

[Van Den Berg, Bert]

Hi all,

during refinement of our (membrane protein) structures, basically in all cases 
the R/Rfree values depend a lot on the low resolution cutoff. Putting the 
cutoff at lower res (20-50 A) results in substantially higher R/Rfree values 
(sometimes few percent). For this reason we mostly refine the data from the 
high-res limit down to 10A or so. I have noticed that this occurs fairly often 
in the literature, but I don't know if this is a membrane protein related issue 
or not.

Could it be that the bulk solvent model used in CNS (we refine exclusively with 
CNS) does not model the situation with membrane proteins, due to the presence 
of detergents? Or is it related to data collection issues (low-res spots 
overloaded etc)? Anything else? What could be done to overcome the problem, and 
to use all the data in refinement?

Thanks, Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115

Re: [ccp4bb] low-res cutoff in refinement

[Van Den Berg, Bert]

No, we have been using version 1.1 so far. Thanks for the suggestion, we'll use 
version 1.2 from now on and try Phenix as well.

Bert



-Original Message-
From: Axel Brunger [mailto:[EMAIL PROTECTED]
Sent: Thu 1/24/2008 7:35 PM
To: Van Den Berg, Bert
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] low-res cutoff in refinement
 
Are you using CNS 1.2?  This version has a robust bulk solvent model and 
anisotropic correction
that is much improved compared to CNS 1.1.  It is similarly robust as 
that of Phenix (although
different in detail). 

Axel Brunger



Van Den Berg, Bert wrote:
>
> Hi all,
>
> during refinement of our (membrane protein) structures, basically in 
> all cases the R/Rfree values depend a lot on the low resolution 
> cutoff. Putting the cutoff at lower res (20-50 A) results in 
> substantially higher R/Rfree values (sometimes few percent). For this 
> reason we mostly refine the data from the high-res limit down to 10A 
> or so. I have noticed that this occurs fairly often in the literature, 
> but I don't know if this is a membrane protein related issue or not.
>
> Could it be that the bulk solvent model used in CNS (we refine 
> exclusively with CNS) does not model the situation with membrane 
> proteins, due to the presence of detergents? Or is it related to data 
> collection issues (low-res spots overloaded etc)? Anything else? What 
> could be done to overcome the problem, and to use all the data in 
> refinement?
>
> Thanks, Bert
>
> Bert van den Berg
> University of Massachusetts Medical School
> Program in Molecular Medicine
> Biotech II, 373 Plantation Street, Suite 115
>
>

-- 
Axel T. Brunger
Investigator,  Howard Hughes Medical Institute
Professor of Molecular and Cellular Physiology
Stanford University

Web:http://atb.slac.stanford.edu
Email:  [EMAIL PROTECTED]  
Phone:  +1 650-736-1031
Fax:+1 650-745-1463

Re: [ccp4bb] Low resol structure

[Van Den Berg, Bert]

We had a similar issue recently where we tried to draw conclusions about a 
domain having the same conformation in wildtype and mutant proteins. One 
reviewer got back saying the resolution of the structures was too low (3-3.5 A) 
to say anything meaningful. Of course everything will depend not only on 
resolution, but also on how small the changes are that you want to draw 
conclusions about. 
 
So how does the uncertainty of atom positons depend on the resolution? Can one 
just take the Luzatti coordinate error or is it more complicated than that?
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Jim Naismith
Sent: Fri 4/11/2008 6:04 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Low resol structure



Dear All,
I have an interesting problem, we have a 3.45A structure of
a membrane protein. We have just been told that the structure is "too low
resolution to be considered as the uncertainty is too high". We use the
structure to identify helices which have moved.

Is there a blanket ban on low res structure operating at the moment?

The structure was refined extremely tightly, MolPROB 98th centile. (I will
happily send the data and structure to anyone who wishes to validate.) The
editors simply ignored everything but the res limit (I/sI in the last shell
was 1.8 with a redundancy of 4)

Of course we will begin the usual journal shopping. However, does anyone
know how to convince editors and non-xtallographers that 3.45A is valid?

Best
Jim


James H. Naismith FRSE   |Research mailto:[EMAIL PROTECTED]
Professor of Chemical Biology|Teaching mailto:[EMAIL PROTECTED]
Centre for Biomolecular Sciences |Office: 1334-463792
The North Haugh  |Fax : 1334-467229
The University   |Lab : 1334-467245
St. Andrews  |In UK add 0 to start of number
Fife Scotland, U.K., KY16 9ST|http://www.st-and.ac.uk/~strucbio

The University of St Andrews is a charity registered in Scotland : No
SC013532

Re: [ccp4bb] Optimization of needle crystals?

[Van Den Berg, Bert]

Hi Ngo,
 
your needles actually look like very thin plates. They seem promising to me. 
From appearance its impossible to tell whether the crystals are detergent or 
not. DDM is apparently known to form crystals, but I've never really gotten any 
(DDM is very soluble). What temperature have you used? 4C would favor forming 
DDM crystals. The round crystals are often seen in membrane protein 
crystallization. They generally don't diffract, contain a lot of detergent 
(maybe all detergent) and are very hard to get better (ie get sharp edges 
and/or diffraction).
What I would do at this point is trying to get the needle/plate crystals better 
(by using techniques/tricks that are used for soluble proteins, such as vary 
Mg2+ salt conc and identity, temperature, volume/reservoir ratio, protein conc. 
and even trying different detergents and/or mixtures including DDM). For now I 
would go with the assumption that the crystals are protein. 
 
Good luck!
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Ngo Duc Tri
Sent: Wed 4/16/2008 9:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Optimization of needle crystals?


Dear experts,




You didn't mention which detergent in the conditions. From the picture 
it looks
like crystals of detergent. 



The initial buffer contains  50mM Tris 7.7, 100mM NaCl and 0.015% DDM. 

I got two forms of crystal: needle and round shape. 
The needle crystals grow from conditions containing Mg2+ salt and high pH.
The round crystal grow from conditions containing Mg2+, high pH and small PEG.

I already fail to optimize the round shape (by changing the PEG concentration 
as well as pH or protein concentration). So that's the reason why I think about 
optimization of the needle form. 

Here I attach more pictures to show you the needle and the round crystal. I 
never applied powder diffraction before so maybe it's hard for me to confirm 
it's the crystal of detergent or not.

Thank you very much for all of your helpful suggestion!

My best regards,
TriNgo
PhD Student- Sungkyunkwan University, Korea

Re: [ccp4bb] SUMARY: Optimization of needle crystals

[Van Den Berg, Bert]

Another possible, and easy, way to optimize initial hits is to take your hit 
condition in the reservoir and add small volumes (say 10% or so) of other 
screen solutions. In this way you get small deviations from the initial hit 
condition that may lead to better crystals or at least gives you hints as what 
to try next. The only thing to watch out for is incompatibilities that could 
lead to salt crystals.


Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Ngo Duc Tri
Sent: Thu 4/17/2008 4:09 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] SUMARY: Optimization of needle crystals
 
Dear ccp4 Users,

I would like to say thank you to all of your valuable advices to optimize
the needle crystal and related problems when dealing with membrane protein.
Here I report the summary of your advices to help other students who got the
same problem:

1. Additive screening from Hampton Research maybe useful in some cases.
2. Can apply techniques/tricks that are used for soluble proteins, such as
vary Mg2+ salt conc and identity, temperature, volume/reservoir ratio,
protein conc. and even trying different detergents and/or mixtures including
DDM
3. Round shape crystal is maybe detergent crystal, but other opinion shows
that DDM cannot create crystal in aqueous solution. I am still confused
about this problem but it's hard to optimize these kind of crystal.
4. Try to collect data on the micro-diffraction beamline (but the facilities
is only available in US and Europe).
5. Powder diffraction also can be used to check the protein crystal or
detergent crystal and even solve the structure.

Thank you very much for your advices and suggestion!

My best regards,
TriNgo
PhD Student- Sungkyunkwan University, Korea

[ccp4bb] discontinuous data wedges in XDS

[Van Den Berg, Bert]

Hi all,

is it possible to input discontinuous data wedges into XDS (obtained from for 
example inverse beam sweeps)? (So wedge se1 goes from 0-90 deg (image 1-90), 
se2 from 180-270 (image 1-90), etc). Or do I have to rename everything so that 
I get one data file in which the rotation ranges are continuous?

Thanks, Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm

Re: [ccp4bb] mutation to cysteines

[Van Den Berg, Bert]

Try the program disulfide by design: http://www.ehscenter.org/dbd/
Its easy to install (at least on windows) and to run. You put in a PDB file and 
the program will look in the structure where disulphide bonds are possible. It 
also ranks the candidates in energies. Finally, it can generate a PDB file with 
a particular disulphide. Quite a nice program.


Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of amit sharma
Sent: Thu 5/29/2008 11:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] mutation to cysteines
 
Dear All,

Sorry for a non-CCP4 question. I intend to mutate a couple of residues to
cysteines(so that they form a disulphide linkage) in a certain region of my
protein. Could I please be directed to program(s) that would reliably let me
do that, prior to primer designing?

Thanks in advance,
Amit

[ccp4bb] build/refine individual methyl/metylene groups in membrane protein structures

[Van Den Berg, Bert]

Hi all,

a common issue with membrane protein strustures is that at the end of 
refinemnt, there are (almost) always blobs of unassigned density covering the 
hydrophobic region that can't be built (because they are too small for a 
complete detergent molecule for example). Would it be feasible/allowed to build 
individual methyl/methylene groups in those blobs, much like is done for 
waters? Within CNS, what topology/parameter files would have to be used?

I seem to recollect a fairly recent membrane protein structure paper that 
introduced something like this, but for the life of me I can't remember the 
reference.

Cheers, Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm

Re: [ccp4bb] Re: [ccp4bb] Advice for phasing

[Van Den Berg, Bert]

For the SeMet dataset, which wavelength did you collect first? If it is the 
peak, you could try doing SAD with just the peak wavelength. Maybe combine the 
Se peak data with the Hg dataset (provided they are isomorphous) and do MIRAS 
as Jacob suggested.
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Thu 7/17/2008 11:23 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject:  Re: [ccp4bb] Advice for phasing


Wouldn't it make sense to do MIRAS with both the Se and Hg derivatives, and add 
in some multi-crystal averaging from the native set? You seem like you are 
almost there, though. I assume you have already tried DM and NCS averaging (if 
there is any)?
 
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: [EMAIL PROTECTED]
***


- Original Message - 
From: Junyu Xiao   
To: CCP4BB@JISCMAIL.AC.UK 
Sent: Thursday, July 17, 2008 10:13 AM
Subject: [ccp4bb] Advice for phasing

Hi, 

I have a very good native data set. However, the selenomethionyl 
crystal has a different space group and always suffers from radiation damage. A 
three-wavelength data set was still collected, and after phasing in SHARP, some 
features can be seen but the map is not good enough for tracing.  Then I did 
some heavy atom soaking with the native crystals and collected data at home, 
however, the crystal was again converted into the same space group as the 
selenomethionyl crystal. At lease 4 Hg sites can be found both by isomorphous 
difference Fourier calculated using the partial Se-MAD phase and by isomorphous 
difference Patterson, suggesting they should be real. Now with these 
information, what's the best way to do the phasing in SHARP, or in some other 
programs?

I hope I have made myself clear; and I would like to supply more 
details.  Any suggestion will be highly appreciated.

Best regards,
Junyu




==
Junyu Xiao
Department of Biological Chemistry,
University of Michigan

Lab address:
3163 Life Sciences Institute
University of Michigan
210 Washtenaw Avenue
Ann Arbor, MI, 48109-2216
Phone: 734-615-2078
==

Re: [ccp4bb] Cryo with succinate

[Van Den Berg, Bert]

It should be pretty straightforward to figure that out by freezing and shooting 
loops with mother liquor only. You can easily fine-tune by including any 
additional buffer components you may have.
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Tjaard Pijning
Sent: Tue 7/22/2008 8:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Cryo with succinate


I have crystals grown from 1 M sodium succinate pH 7 and have been searching 
for suitable cryo conditions.
Is succinate in itself already a cryoprotectant (maybe at somewhat higher 
concentration) or should I
add e.g. glycerol ? Any experience or ideas are welcome.
 
Thanks in advance,
  

 

Ing. Tjaard Pijning

Protein Crystallography Group
University of Groningen
Nijenborgh 4
9747 AG Groningen
The Netherlands
Tel. (31)(0)50 363 4385

 

<><>

[ccp4bb] space group stats

[Van Den Berg, Bert]

Regarding Phil's comment about the space group, check the PBD stats and you'll 
see that c222 is pretty rare. It occurs only in 0.24% of all cases, versus 5.1% 
of C2221. So i guess you could say that without doing any analysis, there's a 
95% chance that a centered orthorhombic cell is c2221 rather than c222.
 
In terms of stats, its also pretty interesting that 58% of all structures are 
in only 5 (low-symmetry) space groups: p212121, p21, c2, p21212 and c2221.
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Phil Evans
Sent: Fri 7/25/2008 9:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] MR problem --molrep



Perhaps obvious - are you sure the space group is C222 not C2221?
Phil

On 25 Jul 2008, at 14:19, Roger Rowlett wrote:

> Carl Soja wrote:
>> Dear all
>> I tried to solve one structure by ccp4i molrep(resolution at 3.0 A, 
>> space group C222, sequence ID 30%). I can get a good Rfactor 0.528  
>> at first  translation function. However, the second translation 
>> function Rfac is 0.526, the third is 0.525, the fourth  is 0.525.  
>> All of the  translation function Rfacs are too closed. I changed 
>> the model and minimum resolution for search, the Rfactor closed no 
>> any improved. My structure estmates  four molecules in the 
>> aymmetric unit. This is my first time found the closed Rfac by 
>> molrep.  From the low translation function Rfac, it seems that it 
>> is correct solution. I checked the solution and found some clashes 
>> in the structure.I am not sure why the Rfactor too closed in next 
>> the molecule search? I know this is unusual solution by molrep. 
>> Does it mean the diffraction data has problem?
>> Any suggestions are welcome.Thank you in advance!
>> Carl soja
> To be honest, I would try both Phaser and EPMR (now Open-EPMR) first 
> to do MR. Both especially excel at finding good MR solutions for 
> multimers, and are very fast as well. These programs can find 
> solutions that are very difficult to find using other rotation-
> translation programs, including MOLREP. In my experience (using 
> Phaser and EPMR) reasonable MR solutions almost always have an R-
> factor under 50%.
>
> Cheers,
>
>
> --
> 
> Roger S. Rowlett
> Professor
> Colgate University Presidential Scholar
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: [EMAIL PROTECTED]

Re: [ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)

[Van Den Berg, Bert]

By R-fac you mean Rsym? If it is, a value of 0.3 seems way too high. How many 
rejections do you have with scaling?
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: [EMAIL PROTECTED]
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of JINJIN ZHANG
Sent: Tue 7/29/2008 5:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)


Dear All,


Here are updated details for the high R problem of my 1.75A structure. 


1. Protein 100 aa, peptide 6 aa, co-crystalized
2. Space group: P43212. Overall R-fac: 0.3. Redundancy: >5 for 98% of 
reflections. B-factor:55
3. CNS refinement
4. Phenix xtriage checked, no twinning is suspected.
5. Water molecules added


Thanks a lot for all your comments and suggestions.


Best,
Jinjin Zhang 

Re: [ccp4bb] Progresss with Stereo 3D under Mac OS X Leopard

[Van Den Berg, Bert]

On Sep 16, 2008, at 5:26 PM, Warren DeLano wrote:

> Also, do crystallographers still consider stereo 3D to be a
> high-priority or must-have feature in a graphics workstation?


Yes, I do. Really, how can you do without?
As for LCD stereo: yes, please!

Re: [ccp4bb] foam dewar usage ?

[Van Den Berg, Bert]

Funny indeed. Our experience has been as well that icing of the foam dewars 
seems to be reduced compared to the "classical" ones.

Bert



-Original Message-
From: CCP4 bulletin board on behalf of Schubert, Carsten [PRDUS]
Sent: Thu 10/9/2008 9:15 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] foam dewar usage ?
 
Hmmm, that's funny. Our experience was quite the opposite. The first time we 
used one of the darker small dewars we ended up with a lot of burr in the LN2. 
Those got picked up by the cryo and we ended up with lilac particles embedded 
inside the cryo. You can imagine the reaction when these particles showed up in 
the mounting camera 
We also noticed that the LN2 in these dewars actually ices up more rapidly as 
compared to the glass dewars. We attribute this to the lower insulating 
capacity of the foam dewars, which causes a vigorous boiling action and the 
higher turbulence seems to suck in more ambient air. Needless to say the dewars 
did not make it past the first use...

Carsten



-Original Message-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] Behalf Of
[EMAIL PROTECTED]
Sent: Thursday, October 09, 2008 8:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] foam dewar usage ?


Yes, we use them all the time and they're great. Stuff does not slip on
the bottom like it does in the glass Dewars and the formation of ice is
greatly reduced. Plus, they're much easier to dry.

Artem

> Does anyone in the biocrystallogaphy community use foam dewars for
> handly liquid nitrogen and freezing/manipulating frozen protein
> crystals ?

Re: [ccp4bb] buster-tnt on OSX ?

[Van Den Berg, Bert]

>On a side note we also looked at HKL, Mosflm, XDS processing of data, 
>we observed differences in the ability of e.g. Shelx to locate SeMet 
>sites depending on the processing program you used. Of course perfect 
>data was undistinguishable, but some datasets which were more tricky 
>to process showed significant differences in locating the SeMet 
>positions. In those cases XDS was better. This is of course running 
>all programs with their default parameters and not tweaking them.

Has this (or anything like this) been published? It may be interesting to look 
at this in a systematic way. You often hear that certain data processing 
packages are better than others for problematic datasets, but I have never 
found anything concrete about this.
 
Bert

Re: [ccp4bb] offtopic__which crystals to harvest

[Van Den Berg, Bert]

True. It also appears that most people will be looking for less expensive ways 
to distinguish between protein and salt crystals
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine



From: CCP4 bulletin board on behalf of V. Nagarajan
Sent: Wed 1/14/2009 2:49 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] offtopic__which crystals to harvest


Actually, UV fluorescence imaging appears to be a pretty reliable means of
discriminating protein crystals, provided your protein has Trp residue(s).

V. Nagarajan
JAN Scientific, Inc.
http://www.janscientific.com  

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Artem
Evdokimov
Sent: Tuesday, January 13, 2009 8:10 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] offtopic__which crystals to harvest

Hi,



A few simple hints:



(Please note that I am aware of the inexact language in the statements below
but I don't have the time to write this up exactly - conversational English
would have to do. Caveat emptor.)



Most protein crystals will break or deform when poked with a steel needle.
Most inorganic salts/minerals won't deform from a simple poke, but will
break (often with an audible crack) when pushed hard against something solid
(like the surface of the glass slip etc.). Many, but not all protein
crystals can survive gentle prodding with a thin cat whisker. Nylon loops
are a bit trickier because they can exhert different forces depending on
their geometry, age of the loop, and user's manual aptitude.



To make matters more complicated - crystals of organic materials (i.e. not
salt but also not protein) can display properties similar to either (but
will more often than not tend to behave like salts).



Salt crystals sink very rapidly in most well solutions. Protein crystals
often take their time (lower density). Salt crystals often display Newton
rings (Newton rainbows) when viewed through a polarizer-analyzer pair (not
to be confused with relatively simple gradients of birefringence colors that
are also common to protein crystals!). I have seen a few crystals of
proteins that had distinct Newton rings and they were all exceptionally good
diffractors. Don't be confused by rainbow-like coloring that's often
associated with spherolites - the latter aren't likely to diffract X-rays in
a useful manner :-)



If in doubt - stick your crystals into an X-ray beam. Pretty much the best
way to resolve this ambiguity! The next best choice is to show the crystals
to an experienced crystallographer - oftentimes it's possible to guess just
by eyeballing the drops but it takes experience to learn the traits and
habits. Membrane crystals (or for that matter any crystals grown in the
presence of detergent) can be extremely tricky to identify correctly due to
the inherently soft and nasty nature of detergent crystals and the tendency
of the latter to form various quasi-crystalline artefacts.



Good luck,



Artem



From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
deliang
Sent: Tuesday, January 13, 2009 10:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] offtopic__which crystals to harvest



Hi there,



Since a lot of different forms of crystals shows, I am using a quick/simple
strategy to choose crystals by applying a force on the crystal against the
wall, with the nylon loop. 



Some can never break apart, so they are salt crystals? The others can not
survive the force and lose their intact shape and sharp surface. It seems
these are protein crystals, but are they "bad" crystals?  I just came to
this field, and welcome all your suggestions and experience.



Thanks a lot.



Deliang

Re: [ccp4bb] cryoloops for X-ray data collection from protein crystals at room temperature

[Van Den Berg, Bert]

Hi Cedric,
 
I haven't read this paper, but there's already a system available for roomtemp 
data collection that works quite well. Check out 
http://www.mitegen.com/products/micrort/micrort.shtml
Instead of a capillary they use thin polyester tubing that you slide over 
(special) bases so that everything is reasonably airtight. It apparently has 
also less background scatter compared to capillaries.
Compared to the old ways of cutting capillaries etc this works like a breeze.
 
Cheers, Bert (not affiliated to mitegen in any way, except as a customer...).
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of cedric bauvois
Sent: Fri 1/16/2009 9:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryoloops for X-ray data collection from protein crystals at 
room temperature


Dear CCP4ers,

in their paper entitled " Using cryoloops for X-ray data collection from 
protein crystals at room temperature: A simple applicable method" ( Journal of 
Crystal Growth  
Volume 281, Issues 2-4 

 , 1 August 2005, Pages 592-595.), the authors present a way to mount crystals 
using "a cryoloop accompanied by a glass capillary cap" (see abstract below).
Do you know if any commercial version of such system are now available ?


Abstract: Although cryoloops are now routinely used for X-ray data collection 
from protein crystals in cryocooling condition, it is still necessary to 
collect X-ray diffraction data from protein crystals at room temperature under 
such circumstances as to find resolution limit and/or to avoid damage of 
protein crystals at cryogenic temperature (e.g. 100 K). Here, we show that a 
cryoloop, which is accompanied by a glass capillary cap to maintain humid 
environment of crystal in the cryoloop, can be used not only to examine protein 
or non-protein crystals but also to collect X-ray diffraction data for 
structural analysis from protein crystals at room temperature. The size of 
cryoloop should be carefully chosen so that the crystal does not move in the 
cryoloop. This crystal mounting method can be time-saving compared to the 
traditional method to mount a crystal in a glass capillary tube.


Many thanks 


-- 
Dr. Cedric Bauvois
Cristallographie des protéines
Institut de Recherches Microbiologiques JM Wiame -IRMW
Av E. Gryzon 1, 1070 Brussels (Belgium)  
tél: +32 (0)2 5273634
fax: +32 (0)2 5267273

Re: [ccp4bb] Se oxidation

[Van Den Berg, Bert]

Hi,
 
I wouldn't worry about Se oxidation. In principle having a mix of  
oxidized/reduced seleniums is unfavorable, as you'll have less signal at the 
edge (broadening). However, all-oxidized Se apparently makes things better 
(sharper and more intense peak; I forgot the reference, i think it may have 
been an Acta Cryst. D paper). We never bother with adding EDTA and/or reducing 
agents and haven't had problems determining structures by SAD. You should be 
fine.
 
Cheers, Bert
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of aka akaka
Sent: Mon 2/9/2009 1:27 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Se oxidation


Dear All 

I would like to know whether oxidation of Se entails any problem for SAD or MAD 
experiments and/ or how to resolve it. Cannot use DTT or reducing agents in my 
protein (extracellular and disulphide bonds are important).
Thanks

Dr. R.Depetris
Weill Cornell Medical College





Get 5 GB of storage with Windows Live Hotmail. Sign up today. 

  

[ccp4bb] Questions about (possibly) twinned data

[Van Den Berg, Bert]

Hello all,
 
we have a dataset collected from multiple (2 or 3) parts of  the same crystal 
with a microbeam (20 micron). The merged data scales OK (not great) in 
monoclinic (1-3% rejections). The resolution is 3.2-3.3 A, so the data is not 
fantastic. This is the cell (similar for other datasets):
 
Cell: 70.012   126.449   107.98890.00089.94690.000 p21

Processing in orthorhombic makes the scaling a lot worse, so I'm assuming its 
monoclinic for now. Running xtriage gives the following summary:

---
Twinning and intensity statistics summary (acentric data):

Statistics independent of twin laws
  - /^2 : 1.877
  - ^2/ : 0.834
  - <|E^2-1|>   : 0.663
  - <|L|>, : 0.411, 0.235
   Multivariate Z score L-test: 6.737
   The multivariate Z score is a quality measure of the given
   spread in intensities. Good to reasonable data are expected
   to have a Z score lower than 3.5.
   Large values can indicate twinning, but small values do not
   necessarily exclude it.


Statistics depending on twin laws
-
| Operator | type | R obs. | Britton alpha | H alpha | ML alpha |
-
| h,-k,-l  |  PM  | 0.167  | 0.367 | 0.339   | 0.152|
-

Patterson analyses
  - Largest peak height   : 5.962
   (corresponding p value : 0.72096)


The largest off-origin peak in the Patterson function is 5.96% of the
height of the origin peak. No significant pseudotranslation is detected.

So, I'm assuming that these crystals are monoclinic and that they are 
pseudo-merohedrally twinned. Is this a reasonable assumption? I get a decent 
solution for the P21 data from molecular replacement with a 50% identical model 
(LLG 900, with the rotation Z-scores low (4-5), but the corresponding 
translation Z-scores high (8-20)).

My questions are: what would be the best way to refine? More specifically, what 
twin fraction should be used as the different tests give different fractions. 
Is the twin fraction automatically determined in phenix.refine or does this 
need to be specified? Finally, can twinning be responsible for the fact that 
the data do not scale well (using data collected on different parts of the same 
crystal)?

Any hints appreciated!

Cheers, Bert

 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm

 

Re: [ccp4bb] Importance of low order reflections?

[Van Den Berg, Bert]

Regarding the merging of "regular" datasets and low-res ones, what is the 
proper thing to do? Merging both complete datasets no matter what or cutting 
out the low-res data of the "regular" dataset? I.e. if the low-res data is not 
correctly measured in one dataset, would merging all data not be detrimental?

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of George M. Sheldrick
Sent: Fri 2/20/2009 5:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Importance of low order reflections?
 
I agree with Pavel and would add that we have frequently found that
collecting low resolution data on a home source and then merging it
with the higher resolution synchrotron data collected subsequently
from the same crystal can work wonders. The home source data tend 
to be missing fewer reflections (especially if the home system has
a kappa or three-circle goniometer) and may well be measured more 
precisely at low resolution, as well as suffering much less from
radiation damage.

George  

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582


On Fri, 20 Feb 2009, Pavel Afonine wrote:

> Sent on behalf of Sacha Urzhumtsev:
> 
> Dear Richard,
> 
> I answer to the whole CCP4bb since the question was quite general and the
> answer may
> be interesting for other people.
> 
> As already mentioned by Bruno Klaholz, low-resolution reflections are not
> only "a few reflections more" but they contain information
> complementary to that in high-resolution reflections, in other words,
> that is different to it.
> 
> Low-resolution reflections correspond to 'electron density waves'
> of a large period. A right combination of low-resolution
> structure factors gives the molecular envelope. Inversely, excluding
> low-resolution reflections from the Fourier series calculation
> artificially increases the map values in some large regions and decreases
> it in others eventually destroying the molecular envelope in the maps.
> If the peaks of the Fourier series are relatively low, this loss
> of low-resolution data may completely kill the signal.
> 
> A map improvement by "phase extension for low-resolution" was illustrated
> by Podjarny et al. (1981) Acta Cryst., A37, 662-668, and by Schevitz et al.
> (1981) Acta Cryst., A37, 669-677. Later we studied in purpose the role
> of low-resolution data for the map quality (1991; Acta Cryst., A47, 794-801).
> In particular a practical illustration is given (EFG structure solution)
> where an addition of 29 reflections to about 3000 available drastically
> improved the map. At my knowledge, low-resolution data were crucial also
> for the ribosomal structure solution (J.Cate).
> 
> Also as Bruno noted, the same data are important for refinement of atomic
> models
> (see for example Kostrewa, 1997, CCP4 Newslet., 34; I remember he was
> presenting
> something earlier but did not find the trace of that presentation). For
> teaching
> goals, recently with our students we prepared an example when a rigid-body
> refinement of a protein model fails using 7377 reflections of the resolution
> 2-20 A and gives a perfect answer when 12 (!) reflections only are added
> at the resolution below 20 A.
> 
> Finally, the same low-resolution reflections may significantly
> reinforce molecular-replacement translation searches (see
> 1995; Acta Cryst., D51, 888-895; Fokine & Urzhumtsev, 2002, Acta Cryst, D58,
> 72-74).
> 
> Obviously, getting low-resolution data requires special efforts during
> experiment
> (see a recent discussion in the CCP4bb) but it is worthy to do and is feasible
> in
> practice at most of synchrotrons. Another feature of these data is a very
> strong
> contribution of the bulk solvent that should be taken into account as soon as
> one starts to use an atomic model.
> 
> I hope these few comments and some bibliography answer your questions and
> may help in some studies.
> 
> With best regards,
> 
> Sacha
> 
> sa...@igbmc.fr
> 
> 
> On 2/20/2009 8:22 AM, Richard Gillilan wrote:
> > Several times I have heard that low order (small angle) reflections are more
> > important when solving low-resolution structures. I presume it is more than
> > just a question of obtaining greater number of reflections.
> >
> > Does anyone know why low-order reflections are so important in these cases?
> >
> > Richard Gillilan
> > MacCHESS
> 

[ccp4bb] Post-doc position available

[Van Den Berg, Bert]

A postdoctoral position is available in the laboratory of Bert van den Berg at 
UMass Medical School, Worcester (MA), to work on a NIH-funded project 
investigating the mechanism by which hydrophobic molecules, such as toxic 
xenobiotics, cross the bacterial outer membrane. The focus of the project will 
be on establishing in vivo transport assays for (mono) aromatic hydrocarbons, 
and the purification, crystallization and structure determination of FadL 
homologues from biodegrading bacteria. In addition, channel-substrate complexes 
and site-directed mutants of the toluene channels TodX and TbuX will be 
subjected to structural studies. For more information please see Hearn et al., 
Nature 1 Feb 2009, and Hearn et al., PNAS 105, 8601-8606 (2008), or contact me 
directly.
 
We are looking for a dedicated and enthusiastic person with a strong background 
in molecular biology. Experience with cloning in Pseudomonas species would be 
ideal. Experience in membrane protein biochemistry and X-ray crystallography 
would be advantageous, but is not required. Please send applications and the 
names of two or three references to: bert.vandenb...@umassmed.edu.
 
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



From: CCP4 bulletin board on behalf of Peer Mittl
Sent: Tue 2/24/2009 7:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Open Post-Doc and technician positions



Applications are invited for four positions at various levels at the
University of Zürich (Switzerland). Currently there are four vacant
positions at the postdoctoral and research assistant levels (2 Post-Docs
and 2 research assistants) in the lab of Prof. Markus Grütter in the
Department of Biochemistry. We are focusing on the investigation of the
structure/function relationship of proteins involved in apoptosis,
innate immunity and trans-membrane signaling (PNAS (2008) 105:20251,
Structure (2008) 16:1443, Structure (2007) 15:625, PLoS Biol (2007) 5:e7).

We seek enthusiastic team players with experience in at least one of the
following disciplines: protein X-ray crystallography, protein expression
and purification, and eukaryotic expression systems. Experiences in
membrane protein biochemistry will be an advantage. Good knowledge of
English is mandatory. Candidates applying for the Post-Doc position
should hold a PhD in a life science-related subject. All positions are
time limited for three years. Salary will be according to the
regulations of the Swiss National Science Foundation scheme.

Please submit your electronic application including a CV, a list of
publications (for the Post-Doc positions) and the names of two to three
referees to Mrs. Salome Rittmeyer at the following address:
secgruet...@bioc.uzh.ch

Best regards,
Peer Mittl

Re: [ccp4bb] off-topic detergent hydrolysis?

[Van Den Berg, Bert]

I wouldn't use any stock solution that has been sitting around for a year at 
4C. Why take the risk that something has happened with it/grown in it? Use a 
freshly made solution and do not store it at 4C.

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of deliang
Sent: Wed 2/25/2009 9:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] off-topic detergent hydrolysis?
 
Hi there,

I am using a stock solution of Octyl-Glucoside to crystallize my membrane 
protein, which was made ~1 year before and kept at 4c . I wonder whether OG can 
be hydrolysed under this condition and whether there is a way to test.

Many thanks,

Deliang

[ccp4bb] exclude NCS regions in phenix.refine

[Van Den Berg, Bert]

Hello all,

I'm refining a structure with 4 molecules in the AU. The molecules have 
substantial differences in certain regions, so I want to exclude those regions 
from the NCS restraints calculation and usage. How do i do this? As far as I 
can see, by selecting residues via for example "chain A and (resseq 20:50 or 
resseq 88:299), etc" the NCS restraints are calculated from the specified part 
of the molecules but still applied to the entire molecules, which I don't want.

Thanks for any hints!

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Artem Evdokimov
Sent: Fri 2/27/2009 7:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli
 
Hello,

The short answer is 'yes'. If you can use both methods :) The issue with
limited proteolysis lies in the questionable state of the full-length
protein - if the stuff is nasty and misfolded, then fagments generated by
proteolytic digest aren't going to be meaningful. On the other hand if you
have a small amount of decent quality full-length protein, digest can be
extremely useful. Deuterium exchange is a nice technique if one of your
friends is an altruistic mass-spectroscopist :) 

Purely theoretical methods are limited as well, especially if you're working
with a bunch of unknown domains in a sequence that has low identity with
anything that has associated structures. And in the end, even for known
structures (or very similar ones) truncation can generate surprises - both
positive and negative ones.

Artem

>Hi:
>I am following this with interest. Nice and useful info.
>My question is: how do you "chop the protein into useful hunks"?
>Using some domain identifying software or using limited proteolysis?
>Thanks
>Subbu

Re: [ccp4bb] OT: Crystallisation compatible detergents

[Van Den Berg, Bert]

Hi Darren,

I'm not aware of any (membrane) protein crystal structures solved with tween20. 
It's heterogeneous, and its color suggests it contains impurities and/or 
oxidation products, making it even more heterogenous. It would be better to 
test the behavior of your complex in the presence of well-defined and 
often-used detergents such as octylglucoside, (D)DM, LDAO, C8E4 and the like. 
If this doesn't work only then I'd set up trials with the tween20.

Hope this helps, Bert



Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Darren Hart
Sent: Tue 3/24/2009 11:22 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] OT: Crystallisation compatible detergents
 
Hello,
We can make a nice 1:1 complex between two proteins that then gradually
precipitates during storage at 4 degC. With 0.005% Tween-20 the complex is
stable and does not preciptate noticeably. We have used this in buffers to
measure the kinetics of binding by Biacore so it is clearly compatible with
"functionality".

Is Tween-20 at this concentration compatible with crystallisation? Is it
worth giving a go, or a waste of time? Should we try and remove it, or
reprepare the complex in an alternative detergent?

If we need to try an alternative, what would people recommend?

Thanks in advance for your suggestions,
Darren

-- 
**
Dr. Darren Hart,
Team Leader
High Throughput Protein Lab
Grenoble Outstation
European Molecular Biology Laboratory (EMBL)
**
EMBL webpages:
http://tinyurl.com/6xdltl
http://tinyurl.com/667jrp

Email: h...@embl.fr
Tel: +33 4 76 20 77 68
Fax: +33 4 76 20 71 99
Skype: hartdarren
Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex
9, France
**

Re: [ccp4bb] How to loop up crystal from AmSO4.

[Van Den Berg, Bert]

I also don't understand what you mean by "The crystal always sticks with AmSO4" 
and "the crystal was still surrounded by AmSO4" Is there solid AmSO4 in the 
drop? Judging from the concentration you state, this can't be the case.
Just loop up the crystal and freeze I would say, the MPD + PEG400 should be 
plenty high enough for good cryo.

Bert van den Berg
University of Massachusetts Medical School



-Original Message-
From: CCP4 bulletin board on behalf of James Holton
Sent: Fri 4/3/2009 1:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to loop up crystal from AmSO4.
 
If you want to get rid of mother liquid around crystal, mount it in oil.

Howerver, what is wrong with NH4SO4?  If your crystal can tolerate 
near-saturated NH4SO4, it is a cryoprotectant all by itself.

-James Holton
MAD Scientist


HanJie_HCT Tai wrote:
> Hi,
>  
> I have a protein-DNA complex grown in 30 -40% MPD/0.2M Ammonium 
> sulfate/5% PEG 400/ 0.1M Tris (8.5).
>  
> I believe that 30%MPD above does not require any cryoprotectant but i 
> hv a problem to loop a crystal up. The crystal always sticks with AmSO4.
>  
> I tried to add 2ul up to 6ul reservoir solution into the protein drop 
> (1+1 ul). Although AmSO4 was diluted, the crystal was still surrounded 
> by AmSO4.
>  
> The loop size is 0.1 -0.2mm, the crystal is about 0.1 mm.
>  
> I tried to grow crystal without AMSO4 or 0.1M AmSO4, but no crystals 
> was shown up.
>  
> Any suggestion to loop up the crystal without any AMSO4 attached?
>
> Regards,
> HCT (HanJie)
>
>
>
> 
> Rediscover Hotmail®: Get quick friend updates right in your inbox. 
> Check it out. 
> 

Re: [ccp4bb] How to loop up crystal from AmSO4.

[Van Den Berg, Bert]

How do you know your precipitate is AmSO4? At the low concentration you’re 
using, my guess is that the precipitate is protein.
It doesn’t matter whether AmSo4 is a cryoprotectant or not (at 0.2 M, its not), 
you have more than enough MPD and PEG400 in your solution to do the job.

Have you looked at diffraction patterns yet? The best (and really only) way to 
see if the cryoprotection worked is to see whether there are ice-rings.  Unless 
you’re freezing in the wrong way, the only issue I can think of that would make 
your condition not suitable for direct freezing is if the volume of the drop 
has increased during equilibration, so that the MPD concentration is now much 
less than 30-40% (for example by having glycerol in the sample but not in the 
screen solution).

Hope this helps, Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: HanJie_HCT Tai [mailto:chemtai2...@hotmail.com]
Sent: Fri 4/3/2009 4:10 PM
To: Van Den Berg, Bert; ccp4bb@jiscmail.ac.uk
Subject: RE: [ccp4bb] How to loop up crystal from AmSO4.
 
In the drop, the amSO4 becomes/casuses precipitate (I guess). The crystals were 
grown from the precipitate within a few days. When I looped up the crystal the 
precipitate was also looped up.
 
And I don't know whether AmSO4 is a cryoprotectant or not. When I freeze the 
crystal in LN, the crystal is not in a glassy drop under the microscope.

Rgds,
(HanJie)



  



Date: Fri, 3 Apr 2009 13:45:40 -0400
From: lambertus.vandenb...@umassmed.edu
Subject: Re: [ccp4bb] How to loop up crystal from AmSO4.
To: CCP4BB@JISCMAIL.AC.UK

I also don't understand what you mean by "The crystal always sticks with AmSO4" 
and "the crystal was still surrounded by AmSO4" Is there solid AmSO4 in the 
drop? Judging from the concentration you state, this can't be the case.
Just loop up the crystal and freeze I would say, the MPD + PEG400 should be 
plenty high enough for good cryo.

Bert van den Berg
University of Massachusetts Medical School



-Original Message-
From: CCP4 bulletin board on behalf of James Holton
Sent: Fri 4/3/2009 1:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to loop up crystal from AmSO4.

If you want to get rid of mother liquid around crystal, mount it in oil.

Howerver, what is wrong with NH4SO4?  If your crystal can tolerate
near-saturated NH4SO4, it is a cryoprotectant all by itself.

-James Holton
MAD Scientist


HanJie_HCT Tai wrote:
> Hi,
> 
> I have a protein-DNA complex grown in 30 -40% MPD/0.2M Ammonium
> sulfate/5% PEG 400/ 0.1M Tris (8.5).
> 
> I believe that 30%MPD above does not require any cryoprotectant but i
> hv a problem to loop a crystal up. The crystal always sticks with AmSO4.
> 
> I tried to add 2ul up to 6ul reservoir solution into the protein drop
> (1+1 ul). Although AmSO4 was diluted, the crystal was still surrounded
> by AmSO4.
> 
> The loop size is 0.1 -0.2mm, the crystal is about 0.1 mm.
> 
> I tried to grow crystal without AMSO4 or 0.1M AmSO4, but no crystals
> was shown up.
> 
> Any suggestion to loop up the crystal without any AMSO4 attached?
>
> Regards,
> HCT (HanJie)
>
>
>
> 
> Rediscover Hotmail®: Get quick friend updates right in your inbox.
> Check it out.
> <http://windowslive.com/RediscoverHotmail?ocid=TXT_TAGLM_WL_HM_Rediscover_Updates1_042009>








Quick access to your favorite MSN content and Windows Live with Internet 
Explorer 8. Download FREE now! 
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Re: [ccp4bb] phasing with se-met at low resolution

[Van Den Berg, Bert]

Hi Engin,

first off, i would not consider an overall Rmerge of 6-10% lousy data, but 
quite acceptable for most "real-life", interesting problems (so no lysozyme, 
thaumatin etc). Our structure of the protein translocation channel SecY is an 
example of de novo low-res Se phasing (PDB code 1RHZ). Phases were obtained by 
carefully collected (friedel flipping with 10-20 deg wedges) peak-wavelength 
SAD datasets combined with cross-crystal averaging to get interpretable maps. 
We didn't have NCS. The best resolution was about 3.5 A for the selenium 
datasets. I would say it is possible (but hard) for anything with at least 
orthorhombic symmetry. I have a Se SAD data set myself that will not give 
interpretable maps; the symmetry is P1 however and there are multiple lattices, 
so this seems pretty hopeless...

I would expect there are a bunch of membrane protein structures that fulfill 
your criteria. Off the top of my head is the small mechano-sensitive channel 
MscS solved by the Rees group. I think this is 3.9 A data, with a high degree 
of NCS however.

Good luck, Bert


Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Engin Ozkan
Sent: Sun 5/10/2009 5:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] phasing with se-met at low resolution
 
Hi everyone,

I thought I start a new thread while it is unusually quiet on the bb. I 
am pondering over the practical limitations to MAD and SAD phasing with 
Se-Met at low resolution. What is the lowest resolution at which people 
have solved structures "only" using phases from selenium in a 
"realistic" case? Let me further qualify my question:  My *realistic* 
*low* resolution case is where
1.  Rmerge over all resolution bins is 6-10% (i.e. your crystals are lousy).
2.  Resolution limit is worse than 3.5 Angstroms, where / in 
the last resolution bin is between 1 and 3 (i.e. your crystals are 
really lousy).
3.  Assuming good selenium occupancy (~85%; I work with eukaryotic 
expression systems, so 100% is not usually achieavable),
4.  The number of selenium atoms are enough many that the Crick-Magdoff 
equation would give you *at least* an average 5% change in intensities 
(assuming 6 electrons contributed per selenium, based on both absorptive 
and dispersive differences being at about 6 e- at the absorption edge).
5.  and specifically, no other phases and molecular replacement 
solutions are available.

Obviously, I have a case very similar to what's described above, and 
three years of failure with heavy atom derivatization (I am still 
trying). I would be happy to hear about Se-Met cases, and data 
collection strategies (2wl vs. 3wl MAD vs. SAD, etc.) and phasing 
methods used in these cases, or references of them. Again, no other 
partial phases, and no data cut off at 3.6 A with an I/s of 15 in the 
last resolution bin. Are there any examples out there? Searching the 
RCSB and PubMed did not point out to me many successful cases.

Thanks,

Engin

P.S. I would also appreciate the specific query type for searching the 
PDB on the web for phasing method (MR, MAD, SAD, MIR, etc.).  They seem 
to have everything under the sun searchable, but I cannot find this one.

Re: [ccp4bb] Shredded E coli pellets

[Van Den Berg, Bert]

Hi Jacob,

take some (few ul) of the lysed culture and spread that together with regular 
cells onto an agar plate so that you (would) get a lawn of bacteria after O/N 
incubation. If it is phage you'll get clear plaques in your lawn, very 
distinctive. If it is regular cell lysis induced by the toxicity of your 
protein you won't get plaques.
We have dealt with phage contamination and the best and most reliable way to 
get rid of them is not to grow any cells for a while (at least a week). This is 
a time to do a lot of cloning.
Also, we now have installed large UV lamps in the room where we grow our cells 
so that we can irradiate that room regularly.

Good luck, Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Thu 7/2/2009 2:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Shredded E coli pellets
 
Okay, it seems that the consensus is phage infection. Is there anything to 
seal the diagnosis? Also, does anybody have literature on de-phaging 
glassware? I am assuming that regular autoclaving will not do the trick?

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
***

- Original Message - 
From: "Jacob Keller" 
To: 
Sent: Thursday, July 02, 2009 11:42 AM
Subject: [ccp4bb] Shredded E coli pellets


> Dear crystallographers,
>
> I recently expressed some new constructs, and found after my usual 
> expression protocol that the cell pellets were not compacted at the bottom 
> corner of the bottles us usual, but were instead smeared as a film on the 
> side, and further, were somewhat clumpy, like clots, and with a smaller 
> pellet in the usual location. The centrifugation was exactly as usual. I 
> noticed that there was also a bit more foam in the medium than usual, but 
> I am not convinced that this was the issue, although it might be a 
> symptom. My suspicion is that the constructs are lethal and cause cell 
> lysis, but I am not sure. Has anybody seen this phenomenon before, and 
> gotten to the bottom of it?
>
> Jacob Keller
>
> ***
> 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] Lipid content/removal

[Van Den Berg, Bert]

Hi Jacob,

there are ways, but simple and fast, I don't know. You can do extraction of 
some purified protein with organic solvents and doing TLC. Quantitation may be 
harder. In the case of phospholipids you can do a phosphorous determination 
(with molybdenum, the protocol should be easy to retrieve online) . For 
regular, non-P containing lipids you may have to go back to TLC with standards. 
For removing the lipid, any column that binds your protein would work 
(metal-affinity, ion exchange), just wash with a large volume to gradually push 
the lipid off. Gel filtratiuon is also possible, but as you can imagine less 
efficient.

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Tue 7/7/2009 1:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Lipid content/removal
 
Dear Crystallographers,

does anybody know of a simple, fast way to determine whether a protein 
sample contains lipid, and roughly to determine the quantity? As corollary, 
does anybody know of a quick way to extract/remove the putative lipid? In my 
case, I am working with an extremely robust/stable soluble protein which may 
contain bacterial lipids.

Thanks for your consideration,

Jacob Keller


***
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] detergent crystals

[Van Den Berg, Bert]

Hi Jose,

how do you know that those crystals were detergent and not protein? My 
impression is that it is really hard to crystallize DDM, and even harder for DM 
(solubilities > 20% in water). The easiest (?) way to check this may be to take 
some crystals, wash them well and run them out on a PAGE gel. If you don't see 
anything and you've taken enough crystals, then you're probably dealing with 
pure detergent crystals. As for your second point, you're right. For most 
low-cmc detergents the total detergent concentration will be substantially 
higher than reported, since a substantial amount is always bound to your 
protein. For 1 mM DDM, you would have only ~ 20 uM micelles, assuming an 
aggregation # of 50 (its higher). I don't think people measure the total 
detergent concentration in the end; for maltosides one could in principle do a 
Fehling's based assay to get the concentration.

Cheers, Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Jose Antonio Cuesta Seijo
Sent: Tue 8/4/2009 12:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] detergent crystals
 
Hi Parveen.

DDM crystals can look a bit like that. Like a pencil that has been
sharpened from both ends until there is very little pencil left, but often
the ends are flatter than that. You can get tons of these (also with DM) in
conditions with medium weight PEGs. Of course many proteins will also
crystallize as hexagonal rods with PEGs :-)
I'll take this chance to shre a thought with the community. When we say
"contains x% of detergent y", are we sure of what there is in the protein
stock? Most membrane protein stocks for crystallization will undergo a
concentration step in a filter, and the membrane proteins themselves can be
excellent detergent concentrators. For example, 0.05% DDM is about 1 mM
DDM, hardly enough to make a belt around proteins that are 100 uM or
higher.
Do people measure how much detergent there really is in the final
crystallization stock? It doesn't seem to get published, instead the
concentration in the last purification step is often mentioned.

Jose.

"Parveen Goyal" wrote:
> Hi All,
> 
> I got some hexagonal crystals in one of my crystal condition. The protein
is
> a membrane protein and contains 0.05% DDM. Has anybody seen DDM crysals
and
> if yes, how do they look like?
> 
> thanks in advance
> 
> Parveen Goyal
> 


--
***
Jose Antonio Cuesta-Seijo

Biophysical
Chemistry Group
Department of Chemistry
University of Copenhagen 
Tlf:
+45-35320261
Universitetsparken 5 
DK-2100 Copenhagen,
Denmark
***

Re: [ccp4bb] detergent crystals

[Van Den Berg, Bert]

This has been elaborated before, but you can safely assume they are NOT 
detergent crystals. DDM may be harder to crystallize than your average membrane 
proteinI hope those crystals diffract!

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Parveen Goyal
Sent: Tue 8/4/2009 11:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] detergent crystals
 
Hi All,

I got some hexagonal crystals in one of my crystal condition. The protein is
a membrane protein and contains 0.05% DDM. Has anybody seen DDM crysals and
if yes, how do they look like?

thanks in advance

Parveen Goyal

Re: [ccp4bb] amino acid analysis for SeMet incorporation

[Van Den Berg, Bert]

Hi marija,

you could try mass-spec. It shouldn't be too hard if your protein is a soluble 
protein (membrane proteins are more complicated). As a side note, we have never 
checked Semet incorporation and I'm sure this is true for many others. If you 
use the common protocols (either use an auxotroph or do met biosynthesis in a 
wildtype strain) there's no reason why you wouldn't get good incorporation.

Bert

Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201 (office); 508 856 1211 (lab)
e-mail: bert.vandenb...@umassmed.edu
http://www.umassmed.edu/pmm/faculty/vandenberg.cfm



-Original Message-
From: CCP4 bulletin board on behalf of Marija Backovic
Sent: Mon 9/28/2009 7:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] amino acid analysis for SeMet incorporation
 
Dear all -

Does anyone know of a facility that provides amino acid analysis for  
determination of SeMet incorporation in a recombinant protein?

I contacted centers at Grenoble and Uppsala, but SeMet determination  
is not something they routinely do.

Any suggestions would be highly appreciated!

Best,

Marija


Marija Backovic, Ph.D.

Pasteur Institute
Department of Virology
25 rue du Dr Roux
75015 Paris
France

+33 (0)1 45 68 82 87

Re: [ccp4bb] video that explains, very simply, what Structural Molecular Biology is about

[Van Den Berg, Bert]

I wonder, just as a "side note", whether there will still be a (big) need for 
X-ray crystallography in a couple of decades?

What will be the state of the art then in structure prediction?
How much of structure space will have been covered by then, so that homology 
modeling can do most of the tricks?

Bert van den Berg
UMass Medical School


-Original Message-
From: CCP4 bulletin board on behalf of George M. Sheldrick
Sent: Sat 11/14/2009 4:08 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] video that explains, very simply, what Structural 
Molecular Biology is about
 

Apologies for the typo, I meant to say 'Bernhard'.
George

Prof. George M. Sheldrick FRS
Dept. Structural Chemistry,
University of Goettingen,
Tammannstr. 4,
D37077 Goettingen, Germany
Tel. +49-551-39-3021 or -3068
Fax. +49-551-39-22582


On Sat, 14 Nov 2009, George M. Sheldrick wrote:

> 
> I also think that it is a very nice video and I will certainly be showing 
> it to my students (and relatives). However the little step between 
> recording the X-ray reflections and getting a final refined map might be
> expanded a little. That is after all what CCP4 etc. is about! Otherwise,
> despite the suggestion in the film that an expert should be consulted, we
> are reinforcing the view held by many biologists and chemists that crystal
> structure determination is a routine analytical method and not suitable
> for an academic career. It worries me that in a couple of decades there 
> will be few people still active who really understand how it works. On
> the other hand, Berhard's impressive new book may solve that problem (if
> people still read books).
> 
> George
> 
> Prof. George M. Sheldrick FRS
> Dept. Structural Chemistry,
> University of Goettingen,
> Tammannstr. 4,
> D37077 Goettingen, Germany
> Tel. +49-551-39-3021 or -3068
> Fax. +49-551-39-22582
> 
> 
> On Fri, 13 Nov 2009, claude sauter wrote:
> 
> > Narayanan Ramasubbu a écrit :
> > > mb1pja wrote:
> > > > Dear Fred
> > > >
> > > > A really nice video that would be great for giving non-crystallographers
> > > > (including colleagues and 1st year students, and perhaps also friends 
> > > > and
> > > > family) an overview of what we do. Thank you for pointing it out - and 
> > > > of
> > > > course very many thanks to Dominique Sauter for making it. I am sure it
> > > > will prove very popular.
> > > >
> > > > bet wishes
> > > > Pete
> > > >
> > > > (Pete Artymiuk)
> > > >
> > > >
> > > >
> > > > On 11 Nov 2009, at 09:44, Vellieux Frederic wrote:
> > > >
> > > >  
> > > > > Dear all,
> > > > >
> > > > > Thought I'd share this with you:
> > > > >
> > > > > I located this through Ms Ines Kahlaoui, from the Beja Higher 
> > > > > Institute
> > > > > of Biotechnology in Tunisia (Ines has to teach and locates videos on 
> > > > > the
> > > > > internet, which she then downloads and uses for teaching). Ines 
> > > > > located
> > > > > this jewel:
> > > > >
> > > > > http://video.google.com/videoplay?docid=7084929825683486794&ei=M3b5SvXqD6em2AK3jY33CQ&q=Plongee+coeur+vivant#
> > > > >  
> > > > >
> > > > > This is the French version (explains everything about Structural
> > > > > Molecular Biology, but for the maths :-( , but also shows what we
> > > > > crystallographers have known for a long time, since the first colour 
> > > > > E&S
> > > > > graphics workstations in fact, that the electron are blue :-) ).
> > > > >
> > > > > Both French and English versions can be downloaded from
> > > > >
> > > > > http://cj.sauter.free.fr/xtal/Film/
> > > > >
> > > > > No rights associated with the movie, and the Strasbourg group intends 
> > > > > to
> > > > > release a higher quality version on DVD soon. Please contact them 
> > > > > about
> > > > > that... I am only sharing what I thought was good for educational
> > > > > purposes. 18 minutes of your life, but worth it I think. So feel free 
> > > > > to
> > > > > share this.
> > > > >
> > > > > Wish you all a nice day,
> > > > >
> > > > > Fred.
> > > > > 
> > > >
> > > >   
> > > Hi:
> > > Could someone point out the name and where to get these crystallization
> > > plates used in the video?
> > > By the way, this is a wonderful video.
> > > Subbu
> > > 
> > 
> > 
> > Dear Subbu and dear xtal lovers,
> > 
> > the fancy plates used in the video are Nextal EasyXtal plates which are now
> > sold by Qiagen.
> > 
> > Concerning the video (thank you Fred for you kind advertisement!), the final
> > version (English/French) will be available in DVD very soon, as well as divx
> > and flash formats, we are working hard to get them ready by Christmas. This
> > material will be released under the Creative Commons licence to make it 
> > easily
> > accessible for all kind of education / teaching purposes.
> > 
> > I'll keep you informed as soon as the final version is ready.
> > 
> > Claude
> > 
> > 
> > -- 
> > Dr Claude Sauter
> > Institut de Biologie Moléculaire et Cellulaire (IBMC-ARN-CNRS)
> > Cristallogenèse & Biologie Structurale  

Re: [ccp4bb] Low resolution refinement

[Van Den Berg, Bert]

Hi Joane,

I'm not sure if I understand your description of the structure (chain A forms 
the dimer? Does it form a dimer with a crystallographically related chain A?) .
But anyway, I don't think you need to worry. Your R/Rfree are excellent for 
this resolution. There's not much you can do about the fact that some molecules 
in the AU are less ordered; this happens fairly often. As you say, you can use 
the best ordered chain as a representative model. Refine everything as well as 
you can and move on I would say.

Bert


On 5/19/11 9:02 AM, "Joane Kathelen Rustiguel"  wrote:

Dear all


I am refining a structure at 3.4 A resolution that contains 3 molecules in the
a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric
functional structure expected for this class of proteins. The other two chains
B and C, which also form the functional dimer, seem to be, somehow, a lot more
flexible than chain A. As a result, whereas the electron density map, b-factor
and geometry for chain A is pretty reasonable for a 3.4 A resolution
structure, the refinement for the other two chains (B and C) does not behave
well. Even playing with different weights for geometry, analysing different
levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map
for the helical regions is ok, but the electron density map for strands and
loops of chains B and C are broken along the main chain, B-factors are really
high, and the geometry keeps being distorted.

Right now, the R-factor and R-free are 24.2 and 28.6, respectively.

Any suggestions in how to proceed the refinement?
And even a more difficult question, how do we report this type of structure?
How do we deposit those coordinates? We can certainly use chain A as a model
to perform interesting studies of structure-function relationship, but we know
that chain B and chain C have problems.

Any help will be greatly appreciated.

Regards

Joane


--
Joane Kathelen Rustiguel Bonalumi
Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP
Laboratório de Cristalografia de Proteínas
Departamento de Física e Química
Fone: +55.16.3602.4193

Re: [ccp4bb] Low resolution refinement

[Van Den Berg, Bert]

One more thing though: have you refined with the NCS restraints off or on? 
Presumably on, seeing that you have a small gap between R and Rfree? It may be 
worth turning the restraints off or modify the NCS selections: if your 3 
molecules are substantially different, applying NCS may make things worse 
rather than better (ie you're averaging things that are different).

Good luck, Bert


On 5/19/11 9:02 AM, "Joane Kathelen Rustiguel"  wrote:

Dear all


I am refining a structure at 3.4 A resolution that contains 3 molecules in the
a.u. The chain A sits on a 2-fold crystallographic axis forming the dimeric
functional structure expected for this class of proteins. The other two chains
B and C, which also form the functional dimer, seem to be, somehow, a lot more
flexible than chain A. As a result, whereas the electron density map, b-factor
and geometry for chain A is pretty reasonable for a 3.4 A resolution
structure, the refinement for the other two chains (B and C) does not behave
well. Even playing with different weights for geometry, analysing different
levels of 2Fo-Fc/Fo-Fc maps, using NCS, TLS, etc..., nothing works. The map
for the helical regions is ok, but the electron density map for strands and
loops of chains B and C are broken along the main chain, B-factors are really
high, and the geometry keeps being distorted.

Right now, the R-factor and R-free are 24.2 and 28.6, respectively.

Any suggestions in how to proceed the refinement?
And even a more difficult question, how do we report this type of structure?
How do we deposit those coordinates? We can certainly use chain A as a model
to perform interesting studies of structure-function relationship, but we know
that chain B and chain C have problems.

Any help will be greatly appreciated.

Regards

Joane


--
Joane Kathelen Rustiguel Bonalumi
Faculdade de Ciências Farmacêuticas de Ribeirão Preto - USP
Laboratório de Cristalografia de Proteínas
Departamento de Física e Química
Fone: +55.16.3602.4193

Re: [ccp4bb] Potential Space Group Issue

[Van Den Berg, Bert]

I'd say its very likely to be orthorhombic. Refinement should tell you.its 
the best way to determine the space group anyway. Why do you doubt its 
orthorhombic? Is Vm reasonable?
It could be monoclinic and merohedrally winned with the beta angle very close 
to 90 degrees, but my money is on orthorhombic. if refinement fails I would try 
monoclinic plus/minus twinning. As for the operators, xx.triage will tell 
you and xx.refine will apply them for you during refinement;-)

Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Raji 
Edayathumangalam [r...@brandeis.edu]
Sent: Friday, July 08, 2011 3:24 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Potential Space Group Issue

Oops sorry for the slippery fingers. I meant h00, 0k0 and 00l in my original 
email and NOT "00h, 00k, 00l". Note the correction especially if you are a 
first-year graduate student trying to learn stuff from these emails :)

Raji



On Thu, Jul 7, 2011 at 11:30 PM, Raji Edayathumangalam 
mailto:r...@brandeis.edu>> wrote:
Hello Everyone,

I have a 3.1 Ang dataset for which I'd like to get to the bottom of what the 
correct space group is.

The current unit cell in p212121 is 98.123   101.095   211.20190.000
90.00090.000
I fed the reflection data into Xtriage to look for twinning and 
pseudotranslational NCS and there is no indication for either issue in the 
Xtriage output. Also, all odd 00h, 00k, 00l reflections are systematically 
absent as they should be for p212121.

However, my colleague who is also working on the same dataset recently 
reprocessed the data in P21. Here's the cell in p21:
98.010  100.940  210.470  90.00  90.04  90.00 p21

I am not sure if BETA=90.04 is significant enough to treat as p21 (0.04% 
deviation of beta angle from ideal lattice for p212121). I don't think so but I 
could be wrong. Could someone please clarify?

Also, what kind of twinning and twinning operators can relate a p212121 cell to 
a p21 cell with almost identical unit cell parameters as that of the p212121 
cell and leave all systematic absences intact?

Thanks much.
Raji


---
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University




--

--
Raji Edayathumangalam
Instructor in Neurology, Harvard Medical School
Research Associate, Brigham and Women's Hospital
Visiting Research Scholar, Brandeis University

Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

[Van Den Berg, Bert]

Just a comment: "positive" results can also be due to poor experimental skills 
and/or lack of attention to detail etc.
Peer review should take care of this, at least to some extent. Negative results 
can be very valuable.

Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Victor 
Bolanos-Garcia [vic...@cryst.bioc.cam.ac.uk]
Sent: Monday, July 11, 2011 7:55 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

Dear David

In addition to what Partha and Tim have just said, what if the negative results 
are simply due to poor experimental skills and/or lack of attention to detail?. 
I guess my question is would a journal that publishes negative results be 
rapidly populated with junk data?.

Kind regards

  Victor




From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Partha 
Chakrabarti
Sent: 11 July 2011 12:06
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

What if the negative results contradict some recent papers in big journals? 
Would the PI risk his / her contacts & connections? Of course for the PhD 
student or postdoc, it matters a lot to get it 'published'..


On Mon, Jul 11, 2011 at 4:27 PM, Bosch, Juergen 
mailto:jubo...@jhsph.edu>> wrote:
http://www.jnrbm.com/

Might this be 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 Jul 11, 2011, at 6:03 AM, F.Xavier Gomis-Rüth wrote:


Dear CCP4ers,
I think this is a very interesting initiative and it could potentially lead to 
a discussion within the board.
Best,
Xavier



 Mensaje original 
Asunto:

Could Biological Negative Results be published?

Fecha:

Sun, 10 Jul 2011 21:39:52 -0500

De:

David Alcantara 


Para:

f...@ibmb.csic.es



 Dear colleague,



As you are well aware it is common in our field that many of our

endeavors do not lead to the results we want or expect. Numerous tests

and experiments have outcomes that we share with our immediate

colleagues during informal meetings, but that we are not considering

material for publication. As a result a wealth of information is never

brought to the attention of the greater public, which is not only

unfortunate, but also has others repeating similar studies to produce

the same negative results. Not only are a lot of resources like time

and money wasted in this way, but it also leads to frustration that

could have been prevented if only the scientists would have been aware

of the negative results of earlier studies.



My name is David Alcantara and, on behalf of our editorial board,

I’d like to invite you to submit your articles to The All Results

Journals: Biology, a new journal that focuses on publishing the grey

literature that has never been published. It is our goal to compile

and publish those experiments that led to negative results or to

outcomes that were not expected and were not before considered for

publication. We of The All Results Journals feel that it is equally

important to publish these results together with interpretations of

the scientists involved and in this way offer a solution to the

problem that publication bias is causing, because of a strong emphasis

on positive results.



The All Results Journals:Biol is a peer-reviewed journal dedicated to

publishing articles with negative results and outcomes that were not

expected and were not before considered for publication in all areas

of Biology (pure and applied). The Journal is TOTAL Open Access (no

fees to publish and read) and is being indexed by well-known

scientific databases such as Web of Knowledge, Scirus, and Pubmed This

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We expect to publish articles within four to six weeks of submission,

and our award-winning OJS Publications Web Editions Platform will

showcase your important findings to the international scientific

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Please check our info for authors to submit your articles at:



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Thank you very much for your time and we look forward to hearing from

you..



With kind regards,









 Alcantara



--

David Alcantara, Ph.D

Managing Editor

alcant...@arjournals.com

Phone: 001 617 575 9152

The All Results Journals:Biol (ISSN: 2172-4784)

http://www.arjournals.com/ojs/index.php

Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

[Van Den Berg, Bert]

Negative results are not necessarily criticisms of colleagues, at least I don't 
think it should be perceived as such. And if it is, folks should just grow 
up...:-)
It may be (too) idealistic, but one could argue that the most important aspect 
of scientific experiments is reproducibility. If no-one is able to reproduce 
those fantastic data, what is the value of those data? I just wonder how many 
"dogma's" are out there that are taken for granted ("its from a great lab"), 
but that no-one has taken the time to verify.
Maybe once we get used to the publication of (properly reviewed) negative data, 
reality will change?

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Bernhard Rupp 
[hofkristall...@gmail.com]
Sent: Monday, July 11, 2011 9:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

Hmmm...I probably have a bit of experience in this business of 'negative 
results'.
Let me just say that it definitely has negative impact on your career when you 
criticize colleagues.
It is extremely difficult to cut through the 'defendant's' and editorial 
denial. In my experience
a lot of lip service is paid to the modern viewpoint of the scientific method 
which Tim optimistically voices,
but alas, we live in a real world of postmodern relativism and subjective 
interest, and reality looks different.

For those attending the IUCr meeting (shameless advert to follow), I have 
changed the title of my
talk in the validation MS to
"Transgressing Conventional Boundaries: Postmodern Structural Biology".

The absence of the word 'crystallography' in the title is not a coincidence.

Cheers, BR


On Mon, Jul 11, 2011 at 4:31 AM, Tim Gruene 
mailto:t...@shelx.uni-ac.gwdg.de>> wrote:
On Mon, Jul 11, 2011 at 04:35:31PM +0530, Partha Chakrabarti wrote:
> What if the negative results contradict some recent papers in big journals?

In that case it is certainly not a "negative result" and I am actually surprised
someone even comes up with the idea of not publishing a result just for lip
service. A good scientist is happy to deal with criticism of his or her
results - if they would not get criticised, they would not have been worth
publishing.

Cheers, Tim

> Would the PI risk his / her contacts & connections? Of course for the PhD
> student or postdoc, it matters a lot to get it 'published'..
>
>
>
> On Mon, Jul 11, 2011 at 4:27 PM, Bosch, Juergen 
> mailto:jubo...@jhsph.edu>> wrote:
>
> > http://www.jnrbm.com/
> >
> > Might this be 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 Jul 11, 2011, at 6:03 AM, F.Xavier Gomis-Rüth wrote:
> >
> >  Dear CCP4ers,
> > I think this is a very interesting initiative and it could potentially lead
> > to a discussion within the board.
> > Best,
> > Xavier
> >
> >
> >
> >  Mensaje original   Asunto: Could Biological Negative
> > Results be published?  Fecha: Sun, 10 Jul 2011 21:39:52 -0500  De: David
> > Alcantara 
> > mailto:listmas...@mail.arjournals.com>>mailto:listmas...@mail.arjournals.com>>
> >   Para:
> > f...@ibmb.csic.es
> >
> >  Dear colleague,
> >
> > As you are well aware it is common in our field that many of our
> > endeavors do not lead to the results we want or expect. Numerous tests
> > and experiments have outcomes that we share with our immediate
> > colleagues during informal meetings, but that we are not considering
> > material for publication. As a result a wealth of information is never
> > brought to the attention of the greater public, which is not only
> > unfortunate, but also has others repeating similar studies to produce
> > the same negative results. Not only are a lot of resources like time
> > and money wasted in this way, but it also leads to frustration that
> > could have been prevented if only the scientists would have been aware
> > of the negative results of earlier studies.
> >
> > My name is David Alcantara and, on behalf of our editorial board,
> > I’d like to invite you to submit your articles to The All Results
> > Journals: Biology, a new journal that focuses on publishing the grey
> > literature that has never been published. It is our goal to compile
> > and publish those experiments that led to negative results or to
> > outcomes that were not expected and were not before considered for
> > publication. We of The All Results Journals feel that it is equally
> > important to publish these results together with interpretations of
> > the scientists involved and in this way offer a solutio

Re: [ccp4bb] Aging PEGs

[Van Den Berg, Bert]

Is there anything that consistently matters in crystallography? ;-)

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft 
[frank.vonde...@sgc.ox.ac.uk]
Sent: Wednesday, August 24, 2011 5:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Aging PEGs

And now,   does anybody know of systematic data indicating how consistently 
all this matters?
phx

On 24/08/2011 21:45, Prince, D Bryan wrote:

For those of us truly controlling types :), I used to make the PEG
solutions and filter them over a Bio-Rad resin that filtered out all the
junk added to stabilize the PEG solution. Then, of course I had to
freeze all my PEG solutions in aliquots, or wrap them in foil and store
at 4C in the dark. This would take several days, depending on the FW of
the PEG. If you are really sensitive about what is in your PEG
solutions, try GC-grade PEG's. The FW profile is much more restricted
around the reported value (i.e. PEG 3350 molecular biology grade has a
broad peak centered on Mr=3350. PEG 3350 GC-grade has a much tighter
peak profile.) Back before you could buy Crystal Screen I, II or HT, you
had to make the stock solutions, then make the screen. But at least when
you did that, you had all the stocks. Now, I just buy pre-made
solutions, and keep them in a drawer with a date opened written on the
bottle. Isn't progress grand? :)

Bryan


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-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Jacob Keller
Sent: Wednesday, August 24, 2011 3:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Aging PEGs

A while ago I measured the pH's of old and new PEGs and found them
very different, and internally attributed all "old vs new PEG issues"
to pH. Upon reflection, this seems too simplistic. Are there other
known mechanisms of crystallization capacities of PEGs of various
ages?

Jacob Keller

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
cel: 773.608.9185
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] Crystals with Organic solvents

[Van Den Berg, Bert]

I would definitely try gelfiltration (how do you get rid of the cleaved tag 
anyway, sample buffer exchange?) but especially ion exchange. A homogeneous 
sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in 
ion exchange. Beyond that i would make some point mutations on surface residues 
and focus on those (ie try the surface entropy method).
Good luck, Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of eswar reddy 
[eswar.uo...@gmail.com]
Sent: Friday, August 26, 2011 6:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystals with Organic solvents

Dear All

I was working on a Human protein and expression and 
solubility is good in E.coli  and purification is One step (His-Tag), and i 
need to cleave the Histag before screens, if not the protein will precipitated 
and Aggregated, but after trying for 1.2 years i have crystals and they are 
with Organic solvents, (10 conditions), these crystals are inter grown like 
broccoli  shaped  and i tried seeding, but it is not successful, and even i 
tried  with additive screen but the result is the same  is there is any 
idea to increase the size and shape of my protein crystals.

Any suggestions will be helpful for me

Thanks in Advance

Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers

[Van Den Berg, Bert]

I have to agree with Ed here. I would take it even further and suggest that the 
PDB file(s) and structure factors SHOULD be requested by the reviewer if many 
(or even some) of the paper's findings and conclusions depend on map 
interpretation. Likewise, I would refuse to review if the authors would not 
comply (which has not happened to me thus far).

Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ed Pozharski 
[epozh...@umaryland.edu]
Sent: Friday, April 20, 2012 1:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers

It seems that this discussion has somehow reached the conclusion that if
a reviewer asks for model/data, there absolutely must be an ulterior
motive to cheat you out of your high profile publication.

On the other hand, it seems like the intent of such reviewer is also
misunderstood as if the only reason would be to catch you fabricating
data.

I dare to suggest that neither is correct and while this discussion
seems to have developed along these lines, both only represent a small
fraction of real life situations.

I routinely request unreleased data/models.  I do it to stem the tide of
subprime models in the PDB (outright fabrication is very very rare) and
it helps me to form judgment on presented model interpretation (which is
more difficult/often impossible to do from 2D figures).

If an author refuses to provide data, I would refuse to review.  Don't
mind my name disclosed in exchange for data, secrecy is for totalitarian
governments.

Cheers,

Ed.


On Thu, 2012-04-19 at 20:18 -0400, Edward A. Berry wrote:
> Bosch, Juergen wrote:
> > To pick a bit on George's point with MR & citation.
> >
> > Here's how you can read it in the paper from your favourite competitor:
> >
> > A homology model was generated using [fill in any program for ab initio
> > prediction] and subsequently used for molecular replacement with Molrep.
> > The structure was refined to an Rwork of 21% and Rfree of 24 %.
> >
> Or maybe the structure was solved by MIR, using a lot of heavy atom data that
> they had been unable to solve until a fortuitous MR result gave phases which
> located the heavy atoms -- No, No, it was that new improved version of 
> autosol!
> Anyway, who cares how the heavy atoms were located- the structure was solved
> entirely using their data and they have the raw data (image files even) to
> prove it. It was just bad luck with the derivatives that kept them from
> solving it 6 months earlier. They really deserve to have the first 
> publication!
> >
> >

--
After much deep and profound brain things inside my head,
I have decided to thank you for bringing peace to our home.
Julian, King of Lemurs

Re: [ccp4bb] detergent crystal?

[Van Den Berg, Bert]

Generally speaking it is quite hard to crystallize DDM since it is so soluble 
(>20% in water). You most likely have protein crystals (of course containing a 
lot of detergent as well) that are just not ordered, presumably because most or 
all of the lattice contacts are mediated by detergent and not by protein. 
Unfortunately this is the norm for membrane protein crystallization.

Good luck, Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of 于洪军 
[hongju...@moon.ibp.ac.cn]
Sent: Friday, April 27, 2012 6:07 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] detergent crystal?


Hi,

I am trying to screen crystals of membrane protein in DDM solutions. I got 
crystals and its diffraction pattern as I enclosed. Membrane protein 
crystallization seems quite different from soluble protein. The condition 
contains PEG400. I learn from other topic here that PEG400 can easily produce 
DDM detergent crystals. Is it detergent crystal ?  Can I tell this from the 
diffraction pattern?  Advices would be greatly appreciated.  Thank you.


Hongjun

Re: [ccp4bb] Serine

[Van Den Berg, Bert]

Yes, as Jacob says, alternative conformation of the serine. Quite common.

Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Uma Ratu 
[rosiso2...@gmail.com]
Sent: Monday, May 21, 2012 4:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Serine

Dear All:

Some of serine residues in my model have extra positive Fo-Fc density at the 
edge of side chain. Some don't have. It is not like from phosphates.

I am wondered what is the cause for these extra density. Could these serines be 
post-translational modified?

I have the images attached. P289ser-0512-1 does not have the extra green, where 
P140ser-0512-1 has.

Thank you for you advice and comment

Uma

Re: [ccp4bb] OFF TOPIC: recommendations for High Pressure Homogenizers

[Van Den Berg, Bert]

My lab has had a C3 for almost 8 years now. It's very good for coli, not so 
good for yeast as 25-30 kpsi is in the extreme range of the instrument and 
you'll wear out seals etc pretty quickly. For those pressures you'll also need 
high pressure air. You'll also need to be fairly disciplined in cleaning, so 
its not great for sharing between different labs (we all knows how that 
goes...). Cleaning yourself is possible, but to open it you'll need a big 
wrench to get sufficient torque. But again, for Ecoli it's very good. We've 
also had good experiences with the avestin people coming in to do maintenance 
in terms of waiting time etc.

Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Marcelo Carlos 
Sousa [marcelo.so...@colorado.edu]
Sent: Saturday, August 04, 2012 2:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] OFF TOPIC: recommendations for High Pressure Homogenizers

Sorry for the off topic question, but it is relevant to those that produce 
proteins for crystallographic purposes:

We are looking to replace an old French Press with a high pressure homogenizer 
for cell disruption (E. coli and yeast). We are currently looking at the 
Avestin C3 and the Niro Panda 2000. I would appreciate any feedback (positive 
or negative) from users of these instruments (or any other competing 
homogenizer).

Thanks in advance

Marcelo

Re: [ccp4bb] Phase separation and membrane protein crystallization

[Van Den Berg, Bert]

Lin, consider yourself lucky if you only have one problem...;-)
regarding your question, poor reproducibility is a common problem with membrane 
protein crystallography and is most likely caused by different amounts of 
lipids copurifying with your protein. If you want to avoid it (may not be 
possible altogether) do your preps as reproducible as possible, especially 
using the same amount of detergent per cell weight, stirring at the same speed, 
for the same time etc, etc. Even then it may be hard to control. I tend to use 
this "feature" as a screening variable and always do several preps to hopefully 
get one that works well and will allow you to solve a structure.

good luck, Bert

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yibin Lin 
[yyb...@gmail.com]
Sent: Monday, December 17, 2012 11:29 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Phase separation and membrane protein crystallization

Dear all,

I am working with one membrane protein. I met one problem. From
different purification protein (following same methods, never changing
anything except "date and time"), I setup crystal plate using same
conditions, why sometime I can get crystals and sometime only phase
separation?

Thanks,
Lin

[ccp4bb] PhD position in outer membrane sugar uptake

[Van Den Berg, Bert]

Dear All,

A 3-year PhD or 4-year MRes/PhD position is available in the recently 
established laboratory of Prof. Bert van den Berg in the Institute of Cellular 
and Molecular Biosciences (ICaMB) at Newcastle University, UK. The project will 
focus on elucidation of the mechanisms of energy-dependent uptake of complex 
oligo- and polysaccharides by members of the human gut microbiota. Applicants 
should have a strong interest in Biochemistry.

For more details and application instructions, please see: 
http://www.findaphd.com/search/ProjectDetails.aspx?PJID=41882&LID=1125
For recent publication from the van den Berg lab please see: 
http://www.ncbi.nlm.nih.gov/pubmed?term=van%20den%20Berg%20AND%20outer%20AND%20membrane

Interested candidates can contact me for informal information, but applications 
should be made using the University’s online postgraduate application form.

Best wishes and a happy 2013!

Bert


Bert van den Berg
Professor of Membrane Protein Structural Biology
ICaMB, Newcastle University, UK
email (until 02/2013): bert.vandenb...@umassmed.edu


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Jochen Zimmer 
[jochen_zim...@virginia.edu]
Sent: Thursday, December 27, 2012 9:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Postdoctoral Position on Biopolymer Translocation

Dear All,

An NIH-funded postdoctoral position on
membrane-integrated
glycosyltransferases is available in
the Zimmer laboratory at the
University of Virginia
(https://jobs.virginia.edu, posting #
0611274). Projects focus on the
biosynthesis and membrane
translocation of nature’s most
abundant polymers, including
cellulose, chitin, and hyaluronan.
Please have a look at our most recent
publications for further details:
http://www.ncbi.nlm.nih.gov/pubmed/23222542
http://www.ncbi.nlm.nih.gov/pubmed/22343360

The Zimmer laboratory is part of a
newly created Center for Membrane
Biology
(http://pages.shanti.virginia.edu/Ctr_for_Membrane_Biology/),
which offers excellent resources for
crystallography, EM, NMR, EPR, SAXS,
as well as single molecule
fluorescence microscopy.
Crystallization robotics for
detergent- and LCP-based
crystallization are also available.

The successful candidate should be
highly motivated, have extensive
experience in molecular biology and
protein expression and purification,
be able to work independently while
integrating well in a diverse research
group, and should have at least one
first-author publication in a peer
reviewed scientific journal.

Interested candidates can contact me
directly (jochen_zim...@virginia.edu)
and should respond to the
aforementioned posting.

Sincere regards & Happy Holidays,
Jochen

___
Jochen Zimmer, D. Phil.
Molecular Physiology & Biological
Physics
University of Virginia
480 Ray C. Hunt Drive
Snyder Building #360/362
Charlottesville, VA 22908
Phone: 434 243 6506