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 replay: glycerol in protein stock solutions

[Brett, Thomas]

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] Post Doc position in Molecular biology

[Preben Morth]

Dear ccp4bb
On behalf of Professor Geir Christensen I would like to draw your attention to 
a post position in molecular biology
http://uio.easycruit.com/vacancy/519990/70331?iso=no

Please contact Geir Christensen for further information
 

J. Preben Morth, Ph.D
Group Leader
Membrane Transport Group
Nordic EMBL Partnership
Centre for Molecular Medicine Norway (NCMM)
University of Oslo
P.O.Box 1137 Blindern
0318 Oslo, Norway

Email: j.p.mo...@ncmm.uio.no
Tel: +47 2284 0794

http://www.ncmm.uio.no/research/ncmm-embl-group-leaders/

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

[Roberto Battistutta]

Dear Phil,
I completely agree with you, your words seem to me the best
"philosophical" outcome of the discussion and indicate the right
perspective to tackle this topic. In particular you write "In the end, the
important question as ever is "does the experimental data support the
conclusions drawn from it?" and that will depend on local information
about particular atoms and groups, not on global indicators". Exactly, in
my case, all the discussion of the structures was absolutely "independent"
from having 1.9, 2.0 or 2.1 A nominal resolution, or to cut at 1.5 or 2.0
or 3.0 I/sigma. This makes the unjustified (as this two-day discussion has
clearly pointed out) "technical" critics of the reviewer even more
upsetting.
Ciao,
Roberto

Re: [ccp4bb] mosflm gain

[James Holton]

I have found that the best way to get the GAIN "right" in MOSFLM is to 
have a look at the optimum "Sdfac" parameter at the end of SCALA (the 
first of the three SDCORRection values).  Specifically, if SDFac is > 1, 
then you need to increase the GAIN.  This is because SDFac>1 means that 
the spots were noisier than MOSFLM thought they should be, and if a 
given number of ADU is noisier than expected, then there must have been 
fewer photons involved in generating the signal.  This means that the 
"true gain" was higher.  Yes, there are other sources of error, like 
shutter jitter, beam flicker, calibration errors, absorption effects, 
scale factor errors, etc.  But these are all directly proportional to 
the intensity, and therefore accounted for by adjusting SDadd (the last 
of the three SDCORR values).  SDfac accounts for noise proportional to 
the square root of intensity, and only shot noise (like photon counting) 
behaves like that.


David Waterman makes an excellent point that the point-spread function 
(PSF) acts like a smoothing filter and makes the background look less 
noisy than photon-counting error permits.  This makes the 
BGRATIO-estimated GAIN lower than the "true" GAIN.  However, one can 
argue that this is not always a bad thing, since the error in measuring 
the intensity of a given area of flat background really is "better than 
photon counting".  This is because you have the smoothing effect of the 
PSF working "for you": bringing in signal from areas outside the region 
you are measuring (prior knowledge of "flatness" if you will).  However, 
this smoothing effect of the PSF does not apply to spots because spot 
photons all arrive in essentially the same place, and no "smoothing" 
will change the intrinsic noise of the total number of photons that 
actually arrived.  The upshot of this is that we really need two 
different values for GAIN, one for the background and one for the 
background-subtracted spot intensity.  The influence on sigma(I) would 
depend on the relative contributions from the spot vs the background 
under it.  I am pretty sure this is not implemented.


It is perhaps interesting that there is also a third type of noise which 
is independent of the spot intensity: "read-out noise".  This used to be 
called "fog" on film detectors.  Despite all the money we spend on 
detectors that minimize it, there is no specific accounting for read-out 
noise in MOSFLM or any other integration package I am aware of.  
However, a "trick" to account for it is to simply lower the ADCOFFSET.  
For example, using 1 A X-rays on an ADSC Q315r detector in hwbin mode, 
the true GAIN is 1.8 ADU/photon, the ADCOFFSET is 40 ADU, and the 
read-out noise is equivalent to the noise deposited by ~2 photon/pixel 
of x-ray background.  This means that a blank image has an average value 
of 40 ADU and rms variation of ~2.5 ADU, but this is equivalent to an 
image from a detector with the same gain, no read-out noise, and 
ADCOFFSET of 36 that was "fogged" by 2 photons/pixel (regardless of 
exposure time).  Yes, this is a small change in ADCOFFSET, and I doubt 
you will notice the difference.  I think this speaks to the fact that, 
on modern detectors at least, read-out noise is essentially negligible.


Another way to get the GAIN, of course, is to measure it directly.  I 
did this on an ADSC Q315 detector in swbin mode by comparison to a 
NaI:Tl scintillator (after accounting for the window and sensor 
thickness of the latter device):

http://bl831.als.lbl.gov/~jamesh/pickup/Q315_gain.png
You can see how the GAIN changes appreciably with photon energy, and 
this is largely because lower-energy photons generate less signal.  GAIN 
also changes with the detector read-out mode.  For example, this number 
is 3 times higher for a Q315r in hwbin mode.  I have listed my best 
information on the typical GAIN and read-out noise of common detectors 
on my "minimum crystal size" page here:

http://bl831.als.lbl.gov/xtalsize.html
You can extract the parameters by selecting the "detector type = " you 
want, and then switching it again to "Custom..."


-James Holton
MAD Scientist

On 3/3/2011 12:34 PM, Bryan Lepore wrote:

wondering if mosflm can automatically estimate the gain.

i.e. i gather it is still estimated the usual way.

-Bryan

[ccp4bb] merge datasets

[Florian Schmitzberger]

Dear All,

I have two questions:

1) I have collected multiple, native datasets (5) from the same  
crystal (different parts of the crystals exposed with different  
transmission and oscillation angles). Each dataset on its own is close  
to complete (96-98 %). Naturally, differences in exposure, onset of  
radiation damage (datasets were collected with high transmission),  
local differences in the crystal, will affect the variance of the  
errors in the measurements for the reflections between the different  
datasets; but I would think the redundancy and increased number of  
measurements from all datasets should outweigh this. My tendency is to  
include all datasets.


I am working at 3.8 A resolution (structure is solved; 80 % solvent  
content).  Missing even a few reflection will probably have more of an  
impact at this lower than at higher resolution. Essentially, I am  
trying to obtain better signal in the resolution range 4-3.8 A, where  
there is also diffuse scattering from the solvent between 4 and 3 A  
and ice rings and the signal from the individual datasets is weak.  
Obviously the criterion will be the calculated map quality, but wanted  
to know what some experiences of people have been in such cases.  
Should I merge the datasets or rather use them individually for map  
calculations?


2) What's the quickest/easiest way to ensure equivalent indexing in  
ccp4/imosflm/scala, when merging different datasets together (space  
group P6222) (in XDS there is reference_data_set). Use pointless then  
cad+scala?


Cheers,

Florian

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

[Kay Diederichs]

Am 04.03.2011 11:11, schrieb Kay Diederichs:


There is nothing wrong with R_meas of 147.1% since, as others have said,
R_meas is not limited to 59% (or similar) as a refinement R-factor is.
Rather, R_meas is computed from a formula that has a denominator which
in the asymptotic limit (noise) approaches zero - because there will be
(almost) as many negative observations as positive ones! (The numerator
however does not go to zero)



upon second thought, this explanation is wrong since the absolute value 
is taken in the formula for the denominator.


A better explanation is: in the "noise limit" the numerator is (apart 
from a factor>1 which is why R_meas is > R_sym) a sum over absolute 
values of differences of random numbers. The denominator is a sum over 
absolute values of random numbers. If the random values are drawn from a 
Gaussian distribution then the numerator contributions are bigger by 
square-root-of-two than the denominator contributions. Thus, R_meas can 
be 150-200% .


Kay

[ccp4bb] Fwd: [ccp4bb] XSCALE vs scala

[José Trincão]

Begin forwarded message:

From: Graeme Winter 
Date: March 4, 2011 4/3/11 - 4:51
To: José Trincão 
Subject: Re: [ccp4bb] XSCALE vs scala

Hi Jose,

if you are following the usual XDS procedure, the data will have already been 
scaled by the XDS CORRECT step. XSCALE will perform essentially the same 
scaling, but for more than one sweep.

I usually use scala to merge data from XDS and XSCALE, using "scales constant" 
- however you could also use XDSSTAT.

Best wishes,

Graeme

On 4 March 2011 16:26, José Trincão  wrote:
Hello all,
I've been converting my XDS files (XDS_ASCII.hkl) to mtz using pointless and 
then running scala to get the work mtz. I like this procedure because I get 
useful info, (such as Rpim), that I don't know how to get otherwise. I there 
any advantage/disadvantage of using this procedure instead of scaling and 
converting with XSCALE and XDSCONV?
Thanks!

Jose

José Trincão, PhD   CQFB@FCT-UNL
2829-516 Caparica, Portugal

"It's very hard to make predictions... especially about the future" - Niels Bohr


José Trincão, PhD   CQFB@FCT-UNL
2829-516 Caparica, Portugal

"It's very hard to make predictions... especially about the future" - Niels Bohr

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

[Daniel Picot]

In addition to Bert remarks, you can read this paper from the positive 
inside rule instigators.



Seppälä S, Slusky JS, Lloris-Garcerá P, Rapp M, von Heijne G. Control of
membrane protein topology by a single C-terminal residue. Science. 2010 
Jun 25;328(5986):1698-700. Epub 2010 May 27. PubMed PMID: 20508091.


with the cited literature

Daniel



Le 04/03/2011 17:31, Justin Hall a écrit :

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: GPCR membane insertion/orientation

[Pascal Egea]

Hi Justin,

Since GPCRs are polytopic a-helical transmembrane proteins, it is very
likely that (1) insertion into the membrane is primarily performed by the
Sec61 complex AKA translocon and (2) targeting to the membrane would be
controlled by the signal recongition particle and its receptor. the latter
implies that a N-terminal signal sequence (that may very well be the first
TM of a GPCR) would control the insertion process. Does your favorite GPCR
have a predicetd  signal sequence?
Sec61 in theory contributes to signal sequence orientation according to the
positive-inside end rulebut as for any rule they are exceptions.

there is a set of excellent papers dissecting this mechanism by

Skach WR NSMB (2009) 16:6 606-12 (review)
Pitonzo & Skach Mol Biol Cell (2009) 20(2) 685-698 (article)
Sadlish H & Skach NSMB (2005) 12(10) 870-878 (article)
Sadlish and Skach J Membrane Biol 202 115-126 (2004) (review)

You may also want to look in the work of the group of Art Johnson (paper by
Woolhead et al)

describing the insertion process of aquaporin by the sec61 complex. they are
polytopic a-helical membrane proteins and you may want to look into these
articles since they dissect the process of TM insertion, orientation and
protein maturation quite well.

Hope this helps,

Best regards


-- 
Pascal F. Egea, PhD
Assistant Professor
UCLA, David Geffen School of Medicine
Department of Biological Chemistry
314 Biomedical Sciences Research Building
office (310)-983-3515
lab  (310)-983-3516
email pe...@mednet.ucla.edu

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

[ccp4bb] off topic: GPCR membane insertion/orientation

[Justin Hall]

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

[ccp4bb] XSCALE vs scala

[José Trincão]

Hello all,
I've been converting my XDS files (XDS_ASCII.hkl) to mtz using pointless and 
then running scala to get the work mtz. I like this procedure because I get 
useful info, (such as Rpim), that I don't know how to get otherwise. I there 
any advantage/disadvantage of using this procedure instead of scaling and 
converting with XSCALE and XDSCONV?
Thanks!

Jose

José Trincão, PhD   CQFB@FCT-UNL
2829-516 Caparica, Portugal

"It's very hard to make predictions... especially about the future" - Niels Bohr

Re: [ccp4bb] weird crystals, I/sigma of >3.0 rule

[Maia Cherney]

Hi James,

I remember that P1 did not help. That was like 2 years ago. That crystal 
was very important at that time, so I had to use it. There were many 
other crystals since then (native,  mutants and complexes) in the same 
space group without problems. But also I had even a more weird crystal 
that Randy Read was interested to investigate fully. That crystal 
produced a nice diffraction to high resolution, scaled perfectly in any 
program, had no twinning. It had the same space group with slightly 
different cell dimensions . It gave huge LLG in phaser, but could never 
be refined. I sent those data to many people, and Randy figured out the 
type of disorder that crystal had. I guess, some crystals can be very weird.


Maia

[ccp4bb] bit off topic

[Ariel Talavera]

Hi CCP4ers,

Is there any place from where I could download .pdb files of LPS lipid A
from different bactiria?

Thanks a lot in advanced.

regards,
Ariel 

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

[Maia Cherney]

Kay,

Thank you for your explanation. The radiation damage was not the factor, 
but there was something strange about this crystal (actually two 
crystals had the same strange behavior). I could not process them in 
HKL2000, but it showed the problem (see pictures in the attachment). The 
processing in XDS was done at the CLS (Canadian Light Source). I know 
they always have the latest version of XDS.


Maia

Kay Diederichs wrote:

Maia,

provided radiation damage is not a major detrimental factor, your data 
are just fine, and useful also in the high resolution shell (which 
still has  of 2.84 so you could probably process a bit beyond 
2.25A).


There is nothing wrong with R_meas of 147.1% since, as others have 
said, R_meas is not limited to 59% (or similar) as a refinement 
R-factor is. Rather, R_meas is computed from a formula that has a 
denominator which in the asymptotic limit (noise) approaches zero - 
because there will be (almost) as many negative observations as 
positive ones! (The numerator however does not go to zero)


Concerning radiation damage: First, take a look at your frames - but 
make sure you have the same crystal orientation, as anisotropy may 
mask radiation damage! Then, you can check (using CCP4's loggraph) the 
R_d plot provided by XDSSTAT (for a single dataset; works best for 
high-symmetry spacegroups), and you should also check ISa (printed in 
CORRECT.LP and XSCALE.LP).


HTH,

Kay

P.S. I see one potential problem: "XSCALE (VERSION  December 6, 2007)" 
when the calculation was done 28-Aug-2009. There were quite a number 
of improvements in XDS/XSCALE since that version. The reason may be 
that a licensed, non-expiring version was used - make sure you always 
rather use the latest version available!



>  Original Message 
> Subject: [Fwd: Re: [ccp4bb] I/sigmaI of >3.0 rule]
> Date: Thu, 3 Mar 2011 10:45:03 -0700
> From: Maia Cherney 
>
>
>
>  Original Message 
> Subject: Re: [ccp4bb] I/sigmaI of >3.0 rule
> Date: Thu, 03 Mar 2011 10:43:23 -0700
> From: Maia Cherney 
> To: Oganesyan, Vaheh 
> References: <2ba9ce2f-c299-4ca9-a36a-99065d1b3...@unipd.it>
> <4d6faed8.7040...@ualberta.ca>
> <021001cbd9bc$f0ecc940$d2c65bc0$@gmail.com>
> <4d6fcab6.3090...@ualberta.ca> <4d6fcbff.2010...@ualberta.ca>
> <73e543de77290c409c9bed6fa4ca34bb0173a...@md1ev002.medimmune.com>
>
>
>
> Vaheh,
>
> The problem was with Rmerg. As you can see at I/sigma=2.84, the Rmerge
> (R-factor) was 143%. I am asking this question because B. Rupp wrote
> "However, there is a simple relation between  and R-merge
> (provided no other indecency has been done to the data). It simply is
> (BMC) Rm=0.8/."
> Maybe my data are indecent? This is the whole LP file.
>
> Maia
>
> MMC741_scale-2.25.LP
>
>
>** 


>XSCALE (VERSION  December 6, 2007)  28-Aug-2009
>** 


>
> Author: Wolfgang Kabsch
> Copy licensed until (unlimited) to
>  Canadian Light Source, Saskatoon, Canada.
> No redistribution.
>

<><>

Re: [ccp4bb] cooled incubator for crystallisation plates

[Ed Pozharski]

On Fri, 2011-03-04 at 10:39 +, Hough, Michael A wrote:
> Could anyone recommend a reasonably priced (i.e. less than £3000)
> cooled incubator for crystallisation plates?

Consider wine coolers - many thermoelectric (vibration-free) models are
available. Normally they don't go down to 4 degrees (the one I have has
the lowest setting at 8), but most people use cold rooms for that.
Shelves are normally not flat (they are for bottles after all), but that
is easy to replace. The best thing about them is that they are an order
of magnitude cheaper because they are mass produced. I paid about $80
for mine (it's small, yes), which means you can buy ~45 of them for the
price that you listed. 

-- 
"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

[Marjolein Thunnissen]

hi

Recently on a paper I submitted, it was the editor of the journal who wanted 
exactly the same thing. I never argued with the editor about this (should have 
maybe), but it could be one cause of the epidemic that Bart Hazes saw


best regards

Marjolein

On Mar 3, 2011, at 12:29 PM, Roberto Battistutta wrote:

> Dear all,
> I got a reviewer comment that indicate the "need to refine the structures at 
> an appropriate resolution (I/sigmaI of >3.0), and re-submit the revised 
> coordinate files to the PDB for validation.". In the manuscript I present 
> some crystal structures determined by molecular replacement using the same 
> protein in a different space group as search model. Does anyone know the 
> origin or the theoretical basis of this "I/sigmaI >3.0" rule for an 
> appropriate resolution?
> Thanks,
> Bye,
> Roberto.
> 
> 
> Roberto Battistutta
> Associate Professor
> Department of Chemistry
> University of Padua
> via Marzolo 1, 35131 Padova - ITALY
> tel. +39.049.8275265/67
> fax. +39.049.8275239
> roberto.battistu...@unipd.it
> www.chimica.unipd.it/roberto.battistutta/
> VIMM (Venetian Institute of Molecular Medicine)
> via Orus 2, 35129 Padova - ITALY
> tel. +39.049.7923236
> fax +39.049.7923250
> www.vimm.it

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

[Phil Evans]

This is very closely related to the way in which I would like to think about 
this: if you consider adding another thin shell of data, are you adding any 
significant information? Unfortunately as Garib Murshudov has pointed out, we 
don't have any reliable way of estimating the information content of data.  
(Also it should be considered anisotropically.)

Another way of thinking about this is to consider that if we had perfect error 
models and weighted the data perfectly, then adding a shell of data with 
essentially no useful information should at least do no harm (ie weights are 
close to zero).  But we do not have perfect error models, so adding too much 
data may in the end degrade our structural model.

Much of the problem arises from our addiction to R-factors as a measure of 
quality, when they are unweighted and therefore very misleading. We are are 
also too quick to judge the quality of a structure by its nominal "resolution", 
whatever that means. In the end, the important question as ever is "does the 
experimental data support the conclusions drawn from it?" and that will depend 
on local information about particular atoms and groups, not on global indicators

Phil


On 4 Mar 2011, at 10:35, John R Helliwell wrote:

> Dear Roberto,
> Overnight I recall an additional point:-
> In chemical crystallography, where standard uncertainties are
> routinely avaliable for the molecular model from the full matrix
> inversion in the model refinement, it is of course possible to keep
> extending your resolution until your bond distance and angles su
> values go up. Thus if you distrust or do not wish to slavishly follow
> a Journal's Notes for Authors, such as for Acta Crystallographica
> Section C to which I referred yesterday, you can, in this way, check
> yourself the good sense of the data quality criteria required. [This
> is a similar test to the one that Phil mentioned yesterday ie with
> respect to scrutinising electron density maps for your protein ie do
> they show more detail by adding more diffraction data.]
> Best wishes,
> John
> 
> On Thu, Mar 3, 2011 at 11:29 AM, Roberto Battistutta
>  wrote:
>> Dear all,
>> I got a reviewer comment that indicate the "need to refine the structures at 
>> an appropriate resolution (I/sigmaI of >3.0), and re-submit the revised 
>> coordinate files to the PDB for validation.". In the manuscript I present 
>> some crystal structures determined by molecular replacement using the same 
>> protein in a different space group as search model. Does anyone know the 
>> origin or the theoretical basis of this "I/sigmaI >3.0" rule for an 
>> appropriate resolution?
>> Thanks,
>> Bye,
>> Roberto.
>> 
>> 
>> Roberto Battistutta
>> Associate Professor
>> Department of Chemistry
>> University of Padua
>> via Marzolo 1, 35131 Padova - ITALY
>> tel. +39.049.8275265/67
>> fax. +39.049.8275239
>> roberto.battistu...@unipd.it
>> www.chimica.unipd.it/roberto.battistutta/
>> VIMM (Venetian Institute of Molecular Medicine)
>> via Orus 2, 35129 Padova - ITALY
>> tel. +39.049.7923236
>> fax +39.049.7923250
>> www.vimm.it
>> 
> 
> 
> 
> -- 
> Professor John R Helliwell DSc

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

[Roberto Battistutta]

Dear all,
just to say that I really appreciate and thank the many people who spent time 
responding to my issue. I have read with much interest (and sometimes with fun) 
all comments and suggestions, very interesting and useful.
Thanks a lot,
Bye,
Roberto.


Roberto Battistutta
Associate Professor
Department of Chemistry
University of Padua
via Marzolo 1, 35131 Padova - ITALY
tel. +39.049.8275265/67
fax. +39.049.8275239
roberto.battistu...@unipd.it
www.chimica.unipd.it/roberto.battistutta/
VIMM (Venetian Institute of Molecular Medicine)
via Orus 2, 35129 Padova - ITALY
tel. +39.049.7923236
fax +39.049.7923250
www.vimm.it

Il giorno 03/mar/2011, alle ore 12.29, Roberto Battistutta ha scritto:

> Dear all,
> I got a reviewer comment that indicate the "need to refine the structures at 
> an appropriate resolution (I/sigmaI of >3.0), and re-submit the revised 
> coordinate files to the PDB for validation.". In the manuscript I present 
> some crystal structures determined by molecular replacement using the same 
> protein in a different space group as search model. Does anyone know the 
> origin or the theoretical basis of this "I/sigmaI >3.0" rule for an 
> appropriate resolution?
> Thanks,
> Bye,
> Roberto.
> 
> 
> Roberto Battistutta
> Associate Professor
> Department of Chemistry
> University of Padua
> via Marzolo 1, 35131 Padova - ITALY
> tel. +39.049.8275265/67
> fax. +39.049.8275239
> roberto.battistu...@unipd.it
> www.chimica.unipd.it/roberto.battistutta/
> VIMM (Venetian Institute of Molecular Medicine)
> via Orus 2, 35129 Padova - ITALY
> tel. +39.049.7923236
> fax +39.049.7923250
> www.vimm.it
> 

[ccp4bb] PhD studentships for October 2011: University of Essex

[Hough, Michael A]

Applications are invited for 3 year PhD studentships in my lab, available for 
October 2011 in the Department of Biological Sciences at the University of 
Essex, Colchester.

The studentship titles are:

"Defining the structural factors that control ligand discrimination in 
haemoproteins"
"Using X-ray radiolysis of haem and copper proteins with single crystal 
spectroscopy to generate and characterise reaction intermediates"
Both projects will involve the opportunity to perform experiments at leading 
international X-ray facilities including the Swiss Light Source, near Zürich 
and Diamond Light Source, UK.
Informal enquiries are welcome, please contact me at maho...@essex.ac.uk or 
visit

Full details may be found at: 
http://www.essex.ac.uk/bs/prospective_students/pg_research/Current_Opportunities.aspx
Applications are invited from candidates holding, or expecting to be awarded 
1st class, upper second class or a masters degree in a relevant subject.  Full 
details on how to apply online are available at 
https://www.essex.ac.uk/pgapply/.
The closing date for applications is 18th March 2011.
For information on eligibility and the application procedure contact the 
graduate school:
E-mail: bsgradresea...@essex.ac.uk, Tel: 01206 873321.



Dr Michael Hough
Lecturer in Molecular Biophysics
Department of Biological Sciences
University of Essex
Wivenhoe Park, Colchester
CO4 3SQ
United Kingdom

http://www.essex.ac.uk/bs/staff/profile.aspx?ID=2216

PhD studentships available for October 2011
http://www.essex.ac.uk/bs/prospective_students/pg_research/Current_Opportunities.aspx

[ccp4bb] cooled incubator for crystallisation plates

[Hough, Michael A]

Hi,

Could anyone recommend a reasonably priced (i.e. less than £3000) cooled 
incubator for crystallisation plates? We plan to initially use this at 4C but 
flexibility would be useful.

One that we have considered is the LEEC LT2J

http://www.appletonwoods.co.uk/acatalog/LEEC_Cooled_Incubator.html

Does anyone have experience of this unit?

Thanks!

Mike






Dr Michael Hough
Lecturer in Molecular Biophysics
Department of Biological Sciences
University of Essex
Wivenhoe Park, Colchester
CO4 3SQ
United Kingdom

http://www.essex.ac.uk/bs/staff/profile.aspx?ID=2216

PhD studentships available for October 2011
http://www.essex.ac.uk/bs/prospective_students/pg_research/Current_Opportunities.aspx

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

[John R Helliwell]

Dear Roberto,
Overnight I recall an additional point:-
In chemical crystallography, where standard uncertainties are
routinely avaliable for the molecular model from the full matrix
inversion in the model refinement, it is of course possible to keep
extending your resolution until your bond distance and angles su
values go up. Thus if you distrust or do not wish to slavishly follow
a Journal's Notes for Authors, such as for Acta Crystallographica
Section C to which I referred yesterday, you can, in this way, check
yourself the good sense of the data quality criteria required. [This
is a similar test to the one that Phil mentioned yesterday ie with
respect to scrutinising electron density maps for your protein ie do
they show more detail by adding more diffraction data.]
Best wishes,
John

On Thu, Mar 3, 2011 at 11:29 AM, Roberto Battistutta
 wrote:
> Dear all,
> I got a reviewer comment that indicate the "need to refine the structures at 
> an appropriate resolution (I/sigmaI of >3.0), and re-submit the revised 
> coordinate files to the PDB for validation.". In the manuscript I present 
> some crystal structures determined by molecular replacement using the same 
> protein in a different space group as search model. Does anyone know the 
> origin or the theoretical basis of this "I/sigmaI >3.0" rule for an 
> appropriate resolution?
> Thanks,
> Bye,
> Roberto.
>
>
> Roberto Battistutta
> Associate Professor
> Department of Chemistry
> University of Padua
> via Marzolo 1, 35131 Padova - ITALY
> tel. +39.049.8275265/67
> fax. +39.049.8275239
> roberto.battistu...@unipd.it
> www.chimica.unipd.it/roberto.battistutta/
> VIMM (Venetian Institute of Molecular Medicine)
> via Orus 2, 35129 Padova - ITALY
> tel. +39.049.7923236
> fax +39.049.7923250
> www.vimm.it
>



-- 
Professor John R Helliwell DSc

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

[Kay Diederichs]

Maia,

provided radiation damage is not a major detrimental factor, your data 
are just fine, and useful also in the high resolution shell (which still 
has  of 2.84 so you could probably process a bit beyond 2.25A).


There is nothing wrong with R_meas of 147.1% since, as others have said, 
R_meas is not limited to 59% (or similar) as a refinement R-factor is. 
Rather, R_meas is computed from a formula that has a denominator which 
in the asymptotic limit (noise) approaches zero - because there will be 
(almost) as many negative observations as positive ones! (The numerator 
however does not go to zero)


Concerning radiation damage: First, take a look at your frames - but 
make sure you have the same crystal orientation, as anisotropy may mask 
radiation damage! Then, you can check (using CCP4's loggraph) the R_d 
plot provided by XDSSTAT (for a single dataset; works best for 
high-symmetry spacegroups), and you should also check ISa (printed in 
CORRECT.LP and XSCALE.LP).


HTH,

Kay

P.S. I see one potential problem: "XSCALE (VERSION  December 6, 2007)" 
when the calculation was done 28-Aug-2009. There were quite a number of 
improvements in XDS/XSCALE since that version. The reason may be that a 
licensed, non-expiring version was used - make sure you always rather 
use the latest version available!



>  Original Message 
> Subject: [Fwd: Re: [ccp4bb] I/sigmaI of >3.0 rule]
> Date: Thu, 3 Mar 2011 10:45:03 -0700
> From: Maia Cherney 
>
>
>
>  Original Message 
> Subject: Re: [ccp4bb] I/sigmaI of >3.0 rule
> Date: Thu, 03 Mar 2011 10:43:23 -0700
> From: Maia Cherney 
> To: Oganesyan, Vaheh 
> References: <2ba9ce2f-c299-4ca9-a36a-99065d1b3...@unipd.it>
> <4d6faed8.7040...@ualberta.ca>
> <021001cbd9bc$f0ecc940$d2c65bc0$@gmail.com>
> <4d6fcab6.3090...@ualberta.ca> <4d6fcbff.2010...@ualberta.ca>
> <73e543de77290c409c9bed6fa4ca34bb0173a...@md1ev002.medimmune.com>
>
>
>
> Vaheh,
>
> The problem was with Rmerg. As you can see at I/sigma=2.84, the Rmerge
> (R-factor) was 143%. I am asking this question because B. Rupp wrote
> "However, there is a simple relation between  and R-merge
> (provided no other indecency has been done to the data). It simply is
> (BMC) Rm=0.8/."
> Maybe my data are indecent? This is the whole LP file.
>
> Maia
>
> MMC741_scale-2.25.LP
>
>
>**
>XSCALE (VERSION  December 6, 2007)  28-Aug-2009
>**
>
> Author: Wolfgang Kabsch
> Copy licensed until (unlimited) to
>  Canadian Light Source, Saskatoon, Canada.
> No redistribution.
>

--
Kay Diederichshttp://strucbio.biologie.uni-konstanz.de
email: kay.diederi...@uni-konstanz.deTel +49 7531 88 4049 Fax 3183
Fachbereich Biologie, Universität Konstanz, Box M647, D-78457 Konstanz

This e-mail is digitally signed. If your e-mail client does not have the
necessary capabilities, just ignore the attached signature "smime.p7s".



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Re: [ccp4bb] mosflm gain

[A Leslie]

Dear All,

spotted a mistake in my response, please see the correction below (in  
bold):


There are a host of caveats in this procedure. For example, if the  
images contains significant diffuse scatter around the Bragg spots,  
the BGRATIO may be above 1.0 ... this is probably the commonest  
effect, but does not mean the gain is wrong. If for any reason the  
mask definition (defining the boundary between background and spot)  
has not worked correctly so the spot extends into the background, this  
will also give a BGRATIO of GREATER THAN one (in this case, the  
BGRATIO will tend to be close to 1.0 for weak spots but greater than  
1.0 for strong ones). The boundary is controlled by the "Profile  
tolerance" parameters, which are sometimes set artificially high to  
help process images where the spots are not fully resolved.


Andrew

On 3 Mar 2011, at 20:34, Bryan Lepore wrote:


wondering if mosflm can automatically estimate the gain.

i.e. i gather it is still estimated the usual way.

-Bryan

Re: [ccp4bb] mosflm gain

[A Leslie]

Dear Bryan,

The quick answer is no. As David Waterman  
mentioned, it has a default value for the gain for each type of  
detector that it can deal with.


A more detailed answer. An incorrect value for the gain can be  
indicated by values of the BGRATIO which differ significantly from  
unity (1.0). BGRATIO  is the ratio of the rms variation in the  
background to the variation expected on the basis of Poisson  
statistics, using the gain to convert from digitised values in the  
image to X-ray photons. This is calculated for all measured spots, and  
binned as a function of intensity for each image measured (and printed  
in the full logfile). Mosflm prints a warning message if this differs  
from 1.0 by more than 10% and will suggest an "improved" value for the  
gain that should be used.


There are a host of caveats in this procedure. For example, if the  
images contains significant diffuse scatter around the Bragg spots,  
the BGRATIO may be above 1.0 ... this is probably the commonest  
effect, but does not mean the gain is wrong. If for any reason the  
mask definition (defining the boundary between background and spot)  
has not worked correctly so the spot extends into the background, this  
will also give a BGRATIO of one (in this case, the BGRATIO will tend  
to be close to 1.0 for weak spots but greater than 1.0 for strong  
ones). The boundary is controlled by the "Profile tolerance"  
parameters, which are sometimes set artificially high to help process  
images where the spots are not fully resolved.


This is why Mosflm does not automatically update its default value for  
the gain based on the BGRATIO.


As David has mentioned, this procedure also assumes that adjacent  
pixels are independent, which they most certainly are not (except  
possibly for some pixel detectors), due to the point spread function  
of the detectors and corrections that are applied to the raw images.


Does it matter ? The gain is used to identify outliers in the  
background plane determination (eg due to zingers, shadows, ice spots,  
hot pixels etc) which are rejected from the calculation, so if it is  
significantly in error this will introduce systematic errors in the  
integrated intensities. This can show up in the cumulative intensity  
distribution in Truncate if the gain is a very long way off. I have  
not done a proper study of this, but I think it would need to be out  
by more than 20% to have a significant effect. The gain is also used  
to calculate sig(I), however, the sig(I) values from Mosflm are  
adjusted in SCALA to reflect the true variation between symmetry  
related reflections so that providing the multiplicity is high enough  
for this to work correctly this will not have any real effect on the  
final merged data.


The bottom line is that the estimates for sig(I) that emerge for this  
procedure seem to be quite good, in that the correction factors that  
are subsequently applied in SCALA for cases where other systematic  
errors are small (ie no radiation damage, absorption etc etc) are very  
close to 1.0.


Best wishes,

Andrew




On 3 Mar 2011, at 20:34, Bryan Lepore wrote:


wondering if mosflm can automatically estimate the gain.

i.e. i gather it is still estimated the usual way.

-Bryan