Re: [ccp4bb] : [ccp4bb] SO4 or PO4

[Jim Pflugrath]

Besides the anomalous difference peak which might be similar in height to
the anom diff peak from an intrinsic sulfur found in cysteine or
methionine, also consider hydrogen bonding.

In general, a sulfate will not be donating a hydrogen bond.  A phosphate
probably will.



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

[ccp4bb] CSHL X-ray Methods in Structural Biology Course Oct 16-31, 2017 (CryoEM & SAXS, too!)

[Jim Pflugrath]

The June 15th deadline for applications to the CSHL X-ray Methods in
Structural Biology Course to be held later this year, October 16 through
October 31, 2017 is rapidly approaching.

The official course announcement is here:
https://meetings.cshl.edu/courses.aspx?course=C-CRYS=17
so please pass this on to folks who might be interested and who would
benefit.

This immersive course is an outstanding place to learn both the theoretical
and practical aspects of Macromolecular Crystallography because of the
extensive lectures from world-renowned teachers a continuous nd the
hands-on experiments.

This 2017 course is the 30th year that this course has been taught at CSHL
with an All-Star cast of instructors.  Along with the return of the
long-time instruction team of Alex McPherson, Gary Gilliland, Bill Furey
and myself, we have established additional roles for Tassos Perrakis (NKI),
Paul Adams (LBL), Janet Newman (CSIRO), supplemented by many, many others
(see the course flyer linked above for more name dropping) to help us give
the participants an experience in Macromolecular Crystallography learning
that cannot be found anywhere else.  (The student:teacher ratio ends up to
be about 1:1).

We expect to have the participants crystallize several proteins and
determine their structures all in about two weeks.  Students may also work
on their own projects, but not exclusively.  They will also become
well-versed in the theory of X-diffraction and crystal structure
determination while having lots of fun, but not much sleep.  We may even be
using 2 different synchrotrons this year for the first time along with
in-house diffraction data collection.

The course is limited to 16 participants due to the very hands-on nature of
the experiments and the intimate seminar room and laboratory settings.
Please check the above web link for more details.  In particular, please
note the information about fellowships, scholarships, and stipends that are
available.

This course is supported with funds provided by the National Institute of
General Medical Sciences for which we are grateful.  Also there are
stipends available from the Leona M. and Harry B. Helmsley Charitable Trust
and the Howard Hughes Medical Institute to help offset the cost of tuition.
 (Did I say free money?  You bet I did, but not for everyone.)

If anyone has any questions, please send me e-mail, I will be happy to
answer all queries.

Thanks for spreading the word, Jim

Re: [ccp4bb] CCP4BB Digest - 10 Apr 2017 to 11 Apr 2017 (#2017-100)

[Jim Pflugrath]

Let's step back a little bit first:

Do these 3 to 4 diffraction data sets (when kept separate) process nicely
individually and have good Rmeas and other results when scaled separately
from each other?  If so, then do they also have the same unit cell
dimensions?

Jim


 Date:Tue, 11 Apr 2017 10:46:25 +0800

> From:高艺娜 
> Subject: Some problems in data processing
>
> Dear all,
> For my crystal, I have to merge 3-4 sets of diffraction data using HKL2000
> program to meet requirements for data completeness, but the problem is the
> Rmerge value was too high to go on every time.
> Did anyone have met the same problem and how to solve it? or some tips for
> solve this problem？
>
> All comments will be appreciated!
>
>

[ccp4bb] Unknown blob extended from catalytic serine residue

[Jim Pflugrath]

I didn't see the figure, but I would not be surprised if cacodylate donates
a methyl group to make O-methylserine.  Diethylene glycol is quite a bit
bigger than a methyl though.

Cacodylate is quite labile and undergoes radiolysis.  It should probably
not be used for crystallization.  If you either grow in a different buffer
or harvest into a different buffer before diffraction data collection, what
does the electron density map look like?

Jim

Re: [ccp4bb] Shipping crystals for RT data collection

[Jim Pflugrath]

Ooops, I forgot to mention that oils would be another way to keep the
crystal(s) on the loops.  There are plenty of viscous sticky oils to choose
from.  They are some of the same ones used when flash-cooling crystals.

-j

On Fri, Dec 23, 2016 at 12:17 PM, Jim Pflugrath <jim.pflugr...@gmail.com>
wrote:

> A couple of ways of shipping RT crystals:
>
> We would mount in capillaries, seal the ends with wax, then Duco cement or
> fingernail polish.  Be aware that ambient air pressure will 
>

[ccp4bb] CSHL X-ray Methods in Structural Biology Course Oct 12-27, 2015: Application deadline June 15th

[Jim Pflugrath]

The June 15th deadline for applications to the CSHL X-ray Methods in
Structural Biology Course to be held later this year, October 12 through
October 27, 2015 is rapidly approaching.

The official course announcement is here:
https://meetings.cshl.edu/courses.aspx?course=C-CRYSyear=15
so please pass this on to folks who might be interested and who would
benefit.

I think people will agree that this course is an outstanding place to learn
both the theoretical and practical aspects of Macromolecular
Crystallography because of the extensive lectures from world-renowned
teachers and the hands-on experiments.

This year's course will see the return of the long-time instruction team of
Alex McPherson, Gary Gilliland, Bill Furey and myself along with many
talented experts (see the course flyer linked above for more name dropping)
to help us give the participants an experience in Macromolecular
Crystallography learning that cannot be found anywhere else.  (The
student:teacher ratio ends up to be about 1:1).  We expect to have the
participants crystallize several proteins and determine their structures
all in about two weeks.  They will also become well-versed in the theory of
X-diffraction and crystal structure determination while having lots of fun,
but not much sleep.

The course is limited to 16 participants due to the very hands-on nature of
the experiments and the intimate seminar room and laboratory settings.
Please check the above web link for more details.  In particular, please
note the information about fellowships, scholarships, and stipends that are
available.

This course is supported with funds provided by the National Institute of
General Medical Sciences for which we are grateful.

If anyone has any questions, please send me e-mail, I will be happy to
answer all queries.

Thanks, Jim

Re: [ccp4bb] CCP4BB Digest - 28 Feb 2015 to 1 Mar 2015 (#2015-61)

[Jim Pflugrath]

Instead of Br which has a weak signal at its K edge, why not use Iodide at
some wavelength (or energy) where it gives a stronger signal?  The longer
wavelength used will help with spot spatial resolution as well.

One may be fighting radiation damage, too.
One may be fighting a moving (vibrating?) crystal, too.

Sure, you already have some diffraction images, but a better experiment may
make one's life easier.

My advice would be to not use higher than a 200 mM (4% saturated) KI quick
soak, but you didn't tell us how Br ended up in your crystals.

Jim

Recently, I got some datasets with weak anomalous Br signal. I tried to
 merge them according to  Q. Liu et al Science 336, p1033 (2012). I am using
 the script multiscale@SSRL. The merged dataset has WEAKER anomalous
 signals.

 Liu et al used SCALA for scaling and merging while multiscale@SSRL using
 AIMLESS. Should this cause such a difference? The SCALA@SSRL has a
 limitation on the number of frames it can process. So I cannot directly
 check if this caused the difference.

 Any suggestions?

[ccp4bb] New app deadline for CSHL X-ray Methods workshop: July 15, 2014

[Jim Pflugrath]

We have extended the application deadline for the CSHL X-ray Methods in
Structural Biology Course to be held October 13-28,2014.

Our earlier deadline was based on getting all the paperwork completed in
time for using NSLS, but since NSLS will not be available*, we can extend
the deadline to July 15th.  This may be helpful to folks who got caught up
in World Cup action and missed the earlier deadline.

Below is my previous announcement.

Regards, Jim

*We will collect diffraction data remotely at the APS and in the homelab at
CSHL.  Participants are encouraged to bring their own crystals as well.


Here's a link to the announcement:  http://meetings.cshl.edu/courses/2014
/c-crys14.shtml
The course is supported by a grant from the National Institute of General
Medical Sciences

Some financial assistance is also available for some, see the course
announcement on the details of that.

I think the course is an outstanding place to learn both the theoretical
and practical aspects of Macromolecular Crystallography because of the
extensive lectures from world-renowned teachers and the hands-on
experiments.

This year's course will once again see the return of the long-time
instruction team of Alex McPherson, Gary Gilliland, Bill Furey and myself
along with many talented experts to help us give the participants an
experience in Macromolecular Crystallography learning that cannot be found
anywhere else.  (The student:teacher ratio ends up to be about 1:1).  We
expect to have the participants crystallize several proteins and determine
their structures all in about two weeks.  While an emphasis is placed on
proteins that we provide, participants will be able to work on their own
macromolecules as well in the 2014 course.

The course is limited to 16 participants due to the very hands-on nature of
the experiments and the intimate seminar room.  Please check the above web
site for more details.

If anyone has any questions, please send me e-mail, I will be happy to
answer all queries.

Jim

Re: [ccp4bb] report mosaicity

[Jim Pflugrath]

HKL-2000 has a menu button at the top Report.  If you click on that a report 
is generated with mosaicity range explicitly listed.

It is probably not suitable to describe a crystal as having a single mosaicity 
value because mosaicity may be anisotropic.  It should be obvious that a unit 
cell like 58 x 58 x 150 may have a bigger mosaicity along the 58 directions and 
a smaller mosaicity along the 150 direction and still have the Bragg 
reflections spatially separated.  Thus, the mosaicity reported/used by 
diffraction image processing is probably just the best estimate for the 
mosaicity model used by the algorithm AND the orientation of the crystal in the 
experiment.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry 
[ber...@upstate.edu]
Sent: Friday, May 16, 2014 11:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] report mosaicity

If you refine crystal mosaicity (I assume there is a way to do that in the 
gui)
then scalepack prints out a single value for the crystal (search the logfile 
for mosaicity)
eab

On 05/16/2014 12:07 PM, hongshi WANG wrote:
 Dear all,

   Thanks for all your reply and useful comment on this.
 Harry,
 You are right. I think there are individual mosaicity value corresponding to 
 each image from scalepack.
 Clearly, we can get either average or range for report.

 best,
 Hongshi


 On Fri, May 16, 2014 at 2:26 AM, Harry Powell ha...@mrc-lmb.cam.ac.uk 
 mailto:ha...@mrc-lmb.cam.ac.uk
 wrote:

 Hi

 I'm sure that a real HKL or Denzo/Scalepack expert will correct me, but 
 my recollection is that you
 don't use any of the values from Denzo, but the value from Scalepack (see
 
 http://www.hkl-xray.com/sites/default/files/HKL2000manual/chapter3/step14-1.htm,
  for example).


 On 16 May 2014, at 00:26, hongshi WANG wrote:

 Hello everyone,

 I am gonna report the mosaicity of my data set as required by the 
 journal. I processed the data
 using HKL2000. So I checked the denzo log file. I found many different 
 mosaicity values. The first
 one is default input (0.3), the rest are corresponding to specific 
 images. I think the mosaicity
 value required should be an overall value or averaged value. Could you 
 please let me know how I can
 get it.  Or some other software can determine it.
 I really appreciate your help and response!

 Hongshi

 Harry
 --
 ** note change of address **
 Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick 
 Avenue, Cambridge Biomedical
 Campus, Cambridge CB2 0QH
 Chairman of European Crystallographic Association SIG9 (Crystallographic 
 Computing)

Re: [ccp4bb] Confusion about space group nomenclature

[Jim Pflugrath]

After all this discussion, I think that Bernhard can now lay the claim that 
these 65 space groups should really just be labelled the Rupp space groups.  
At least it is one word.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Bernhard Rupp 
[hofkristall...@gmail.com]
Sent: Friday, May 02, 2014 3:04 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Confusion about space group nomenclature
….

Enough of this thread.

Over and out, BR

Re: [ccp4bb] anomalous signal

[Jim Pflugrath]

d/sig should be above 0.80

There seems to be plenty of signal there with all values above 1.02.  We have 
solved structures with less multiplicity and lower d/sig.

There is a different criteria of signal for when you know the positions of 
the anomalous substructure atoms and when you need to find the positions of the 
anomalous substructure atoms.

As for no signal, I think I am on record that there is always an anomalous 
signal. :)  But can you detect it?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Friday, April 25, 2014 4:06 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] anomalous signal

Dear all

sorry about my previous mail where i forgot to mention that the data was 
collected on home source at Cuk alpha and at 1.54A.

written below is the log file of an anomalous data processed through SHELXC..my 
question is ..what is the strength of anomalous signal ?? as it is said For 
zero signal d'/sig and d/sig should be about 0.80. Then in the present 
case is there really a signal or can be assumed no signal..we are expecting one 
Ca atom bound to the protein at its active site..the redundancy of the data is 
11.6..with this signal strength can we assume Ca to be present there or 
whatever little anomalous if present is due to something elseor there is no 
signal at all ??...

Resl.   Inf - 8.0 - 6.0 - 5.0 - 4.0 - 3.8 - 3.6 - 3.4 - 3.2 - 3.0 - 2.8 - 2.60
 N(data) 375   493   580  1319   450   538   679   866  1081  1414  1709
 I/sig58.8  38.6  32.6  38.3  27.7  27.2  21.9  18.4  12.6   9.5   6.1
 %Complete  94.7  99.0  99.3  99.5 100.0  99.6  99.7  99.8  99.6  99.6  90.9
 d/sig   1.65  1.27  1.18  1.25  1.19  1.12  1.11  1.11  0.97  1.02  1.05

--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] shelxd

[Jim Pflugrath]

I think what one uses will depend on what one expects to be in the structure 
and the resolution and the quality of their diffraction data.

I usually start with 1.8 Å resolution data in case there is chance of having 
disulfides.  Then 1.9, then 2.4, then 2.0, then 2.6, then 2.2, then 1.85, then 
1.75, ….  If there are disulfides, then there is the DSUL option if the 
resolution of the diffraction data is not conducive to separating the 
individual sulfur atoms.

One should also change the number of sites that SHELXD is looking for, perhaps 
in a systematic way.  For example, one might be looking for sulfurs based on 
amino acid sequence, but there may be calcium, zinc, manganese, or other strong 
anomalous scattering atoms in the crystal that would make searching for sulfurs 
moot or at least problematic.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Monica Mittal 
[monica.mitta...@gmail.com]
Sent: Tuesday, April 22, 2014 3:13 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] shelxd

Dear all

I am naive in phasing experiments. CAn anyone please guide how to do the 
following:

In a S-SAD for SHELXD searches, try various high-resolution cutoffs for example 
2.5 to 5 Å in steps of 0.1 Å. Based on these attempts, use a high-resolution 
cutoff at for example 3.8 Å for substructure determination with an E min value 
of for example 1.6 to search for X no. of protein sulfur sites.

Thanx in advance
Monica

Re: [ccp4bb] anomalous signal for Mg and Calcium

[Jim Pflugrath]

Further to what Nat wrote which I completely agree with, you should tell us the 
following:

1. Expecting signal of a Calcium atom and expected signal of a Magnesium atom.

2. Are there any intrinsic anomalous scatterers in the structure that you trust 
such as sulfurs from methionines and cysteines or even selenium at 
selenomethionines?  What are their expected signals and do you see those 
signals?  If not, why not?  Basically, these should give one a positive control 
which they can check their experiment with.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Monday, April 21, 2014 5:36 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] anomalous signal for Mg and Calcium

Dear all

Just in the continuation ...

Re: [ccp4bb] AW: [ccp4bb] relation between redundancy and total reflection

[Jim Pflugrath]

If one is using the HKL GUI, then click on the menu bar button Report and 
read the report that is generated.

One caveat is that HKL will count any systematically absent reflections in the 
input files as part of the total number of observations.  I forget if scalepack 
counts the systematically absent unique reflections that appear at the bottom 
of the log file, but do not show up in the output.sca file, but one can find 
that out really quickly.

JIm


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Thursday, April 17, 2014 9:25 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] AW: [ccp4bb] relation between redundancy and total 
reflection

Dear Herman

Where these values can be located..i.e. total no of reflections and no of 
unique reflections..which processed log file is the optimum one to look into..??

regards

Faisal


On Thu, Apr 17, 2014 at 7:48 PM, 
herman.schreu...@sanofi.commailto:herman.schreu...@sanofi.com wrote:
Dear Faisal,
redundancy is total no. of observed reflections divided by no. of unique 
reflections, i.e. how often each unique reflection has been measured on average.
Herman

Von: CCP4 bulletin board 
[mailto:CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von 
Faisal Tarique
Gesendet: Donnerstag, 17. April 2014 16:12
An: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Betreff: [ccp4bb] relation between redundancy and total reflection

Dear all

Can anybody please tell me how redundancy is related to total no. of 
observations and number of unique observations..what is the best way to 
identify and locate these values in a data processed through HKl2000..I know 
that completeness, redundancy, Rsymm, I/isig etc can easily be located in the 
log file but i am more concerned about locating of total reflections and no of 
unique reflections and its relation to redundancy..
--
Regards

Faisal
School of Life Sciences
JNU



--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] metal ion coordination

[Jim Pflugrath]

And what if the site(s) is(are) a mixture of bound metal ions?  What if Mg++, 
Ca++, Na+, Mn++, et al. are bound at the same site(s)?  Can the diffraction 
data rule that out?



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Faisal Tarique 
[faisaltari...@gmail.com]
Sent: Thursday, April 17, 2014 3:13 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] metal ion coordination

Dear all

Can anybody please explain what is the classical metal ion coordination for 
Mg2+, Ca+ and Na+ with Oxygen atom and the average distance with these metal 
ions..does the distance vary with the type of metal ion and its coordination 
with oxygen atom..what is the best way to identify the correct metal ion in the 
electron density in the vicinity of negatively charged molecule mostly oxygen 
containing molecule..In one of my paper the reviewer has asked me to check 
whether the octahedrally coordinated Mg+ is  Ca+ ion..and similarly raised 
doubt about the identity of the Na+ ion as well..his argument was based on 
metal ion to oxygen distance..I am attaching the figure with this mail..i 
request you to please shed some light on this area and help me in clearing some 
doubts regarding this.

--
Regards

Faisal
School of Life Sciences
JNU

Re: [ccp4bb] Tenure track junior Group Leader Positions in Milan, Italy

[Jim Pflugrath]

Thanks for the clarification.  I thought at first this position might be 
related to Dance Your PhD.
http://news.sciencemag.org/scientific-community/2013/11/dance-your-ph.d.-and-winner-…


Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sebastiano 
Pasqualato [sebastiano.pasqual...@gmail.com]
Sent: Friday, April 04, 2014 11:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Tenure track junior Group Leader Positions in Milan, Italy


Ooopsss!!!
Of course it should read experimental and computational cancer biology but 
with this invasive automatic correctors, one typo can lead to very interesting 
fields of study, I guess…

Ciao,
S

On Apr 4, 2014, at 6:10 PM, Oganesyan, Vaheh 
oganesy...@medimmune.commailto:oganesy...@medimmune.com wrote:

It sounds very interesting: “experimental and computational dance biology”. Any 
type of computational dance or there are style limitations?

Regards,

Vaheh Oganesyan
www.medimmune.comhttp://www.medimmune.com
image001.png

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Sebastiano Pasqualato
Sent: Friday, April 04, 2014 12:01 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Tenure track junior Group Leader Positions in Milan, Italy


Dear all,
there are open positions for junior Group Leaders in the field of experimental 
and computational dance biology at the European Institute of Oncology in milan, 
Italy.
Please, find enclosed the details.

To the extent this electronic communication or any of its attachments contain 
information that is not in the public domain, such information is considered by 
MedImmune to be confidential and proprietary. This communication is expected to 
be read and/or used only by the individual(s) for whom it is intended. If you 
have received this electronic communication in error, please reply to the 
sender advising of the error in transmission and delete the original message 
and any accompanying documents from your system immediately, without copying, 
reviewing or otherwise using them for any purpose. Thank you for your 
cooperation. To the extent this electronic communication or any of its 
attachments contain information that is not in the public domain, such 
information is considered by MedImmune to be confidential and proprietary. This 
communication is expected to be read and/or used only by the individual(s) for 
whom it is intended. If you have received this electronic communication in 
error, please reply to the sender advising of the error in transmission and 
delete the original message and any accompanying documents from your system 
immediately, without copying, reviewing or otherwise using them for any 
purpose. Thank you for your cooperation.

--
Sebastiano Pasqualato, PhD
Crystallography Unit
Department of Experimental Oncology
European Institute of Oncology
IFOM-IEO Campus
via Adamello, 16
20139 - Milano
Italy

tel +39 02 9437 5167
fax +39 02 9437 5990
web http://is.gd/IEO_XtalUnit

Re: [ccp4bb] Rmerge of Images

[Jim Pflugrath]

I think you are talking about the R of the reflections in an image to the set 
of unique reflections resulting from scaling.

Here is a definition of Rmerge from a dtscaleaverage log file:

In the tables below Rmerge is defined as:
Rmerge = Sum Sum |Ihi - Ih| / Sum Sum Ih
  h   i  h   i
where Ihi is the ith used observation for unique hkl h,
and  Ih is the mean intensity for unique hkl h.

Here is a definition of Rmeas from a dtscaleaverage log file:

In the tables below the redundancy-independent merging
R factor Rmeas (or Rrim) is defined as:
Rmeas = Sum [N/(N-1)]^(1/2) Sum |Ihi - Ih| / Sum Sum Ih
 h   i  h   i
where Ihi is the ith used observation for unique hkl h,
and  Ih is the mean intensity for unique hkl h.

Sorry, but that's what text only gives you.

Ih is calculated from the entire scaled data set and is the unique hkl.  If a 
batch or image does not have that unique reflection, then it would not appear 
in the above equations.

Also if only 1 reflection was used to calculate Ih, then it is not included 
in the sums.  Quiz time: Can you tell me why?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, February 12, 2014 10:46 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Rmerge of Images

Dear Crystallographers,

Where can I find the definition of the R vs batch reported in scaling? 
Specifically I am wondering whether it is cumulative (each new frame versus all 
previous ones pooled together) or something else, and also how this metric can 
have any meaning on early frames when one has measured each reflection only 
once (in p1, this would be 180 degrees). I am wondering about its efficacy as a 
radiation-damage metric.

JPK

***
Jacob Pearson Keller, PhD
Looger Lab/HHMI Janelia Farms Research Campus
19700 Helix Dr, Ashburn, VA 20147
email: kell...@janelia.hhmi.org
***

Re: [ccp4bb] Room temperature data collection

[Jim Pflugrath]

Clearly, it is always possible to do non-cryogenic data collection simply by 
not using a cryogenic cooling device and mounting crystals so that they do not 
dehydrate or dry out.

I've been doing quite a lot of room temperature data collection lately because 
in the home lab we can SAD-phase lysozyme with data collection times of about 
30 seconds or so.  It would seem that in 30 seconds one would not have a 
problem with radiation damage from a home source.  It would seem.

There are many benefits with cryogenic diffraction data collection beyond just 
the obvious ones.  I am in agreement with Enrico Stura.  While he may not have 
exactly said this, I think one should take the time to figure out how to 
preserve diffraction of their crystals at cryogenic temperatures.

Re: [ccp4bb] Room temperature data collection

[Jim Pflugrath]

If one is mounting in glass capillaries, we used to put them in a box of blue 
tips (1000 ul pipetteman tips).  Just put a small rolled up wad of kimwipe down 
in the bottom, so that the capillary does not jam itself down in the narrowing 
of the tip point.  Every capillary gets its own tip and one can write with a 
Sharpie on the tip to label the capillary within it.  The tip box top kept them 
all in their separate individual tips when travelling.

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Katherine Sippel 
[katherine.sip...@gmail.com]
Sent: Thursday, February 06, 2014 9:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Room temperature data collection

To answer Mark's question, if your crystals are capillary mounted then planes 
are no problem. My protocol is to mount them, wrap them in cotton batting, put 
them in a 50 ml falcon tube, and pack that in bubble wrap.  

Re: [ccp4bb] Best compounds for heavy atom soaks

[Jim Pflugrath]

And quick iodide soaks may be useful in the 200 mM range.  See the sped-up 
video:
http://www.youtube.com/watch?v=45Qc3jOPaKY

Quiz time:  What wavelength would give iodide a similar signal to that of 
selenium?  Can one get a better signal than selenium by choosing a different 
wavelength for data collection?

and of course the webinar presented by Thomas Edwards of the SSGCID:
http://www.rigaku.com/node/1369



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of RHYS GRINTER 
[r.grinte...@research.gla.ac.uk]
Sent: Wednesday, January 15, 2014 11:18 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Best compounds for heavy atom soaks

Hello message board,

My group has some crystals of an interesting protein to take to the synchrotron 
in a couple of weeks. We won't be able to prepare and crystallise a SelMet 
derivative during that time period, but we have loads of crystals sitting 
around. The diffraction isn't great, we see maybe 3.5 at home but might be 
enough to get over the line.
It will be a very difficult MR target, so we were thinking of soaking so 
crystals with heavy atomic compounds that we have lying around. I was wondering 
if people had any suggestions of compounds that people have used successfully 
for experimental phasing and maybe concentrations to use and soaking time.

Cheers,

Rhys

Re: [ccp4bb] ACA 2014 meeting--Session on engaging students in protein crystallography

[Jim Pflugrath]

Hi Roger,

We have a simple lysozyme project where we collect diffraction images in less 
than 30 seconds total exposure time and solve the structure by SAD phasing.  
I've thought that this might make an ideal lab practical: grow crystals one 
week, everyone collects the data on their own crystal the next week (no cryo 
needed), then process diffraction images and solve the structure.

I don't teach undergraduates, but usually have a few high school interns every 
summer.

Do you think a talk about would fit in with your session?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett 
[rrowl...@colgate.edu]
Sent: Tuesday, January 14, 2014 9:25 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] ACA 2014 meeting--Session on engaging students in protein 
crystallography

I am co-organizing (with Kraig Wheeler) a session at the 2014 American 
Crystallographic Association ….

Re: [ccp4bb] 100% Rmerge in high resolution shell

[Jim Pflugrath]

Graeme wrote:
... Rpim is much more instructive. ... as each of these tells something 
different.

I have to ask:
Why is Rpim much more instructive?  I'm trying to figure this out still.  Can 
one please summarize what are best practices with all these numbers and how 
each of these tells something different?

Another problem that I see is that folks can adjust their sigmas many different 
ways without knowing they have adjusted their sigmas.  And they can be adjusted 
incorrectly when they are adjusted.

BTW, Graeme is correct about lots of multiple low I/sigI observations for each 
Bragg reflection in a resolution shell will lead to 100% (or higher) Rmerge 
with I/sigI of 3.  This assumes no systematic errors and only randomly 
distributed random errors (a rare if not impossible situation, I would think).  
I will defer to others about what the relevance of that is.

Thanks for any insights, Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Graeme Winter 
[graeme.win...@gmail.com]
Sent: Tuesday, November 19, 2013 2:02 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 100% Rmerge in high resolution shell

Usually this means that you have relatively high multiplicity, which 
give-or-take improves the I/sig(I) by sqrt(m) where m is the multiplicity, but 
also increases the Rmerge.

For any given narrow shell of reflections,

Rmerge ~ 0.8 / unmerged(I/sig(I))

merged(I/sig(I)) ~ sqrt(m) * unmerged(I/sig(I))

So it is perfectly possible to have unmerged I/sig(I) of 0.8 which will give 
you an Rmerge of around 1.0, and have I/sig(I) (merged) around 3, by having 
multiplciity 14 or so. I suggest that this is the case: if it is much lower 
than this there is something odd going on.

For the merged I/sig(I) Rpim is much more instructive. I'd love it if people 
reported merged and unmerged I/sig(I), Rmerge, Rmeas, Rpim, CC1/2, ... as each 
of these tells something different.

Best wishes,

Graeme

Possibly useful papers:

http://www.nature.com/nsmb/journal/v4/n4/abs/nsb0497-269.html
http://scripts.iucr.org/cgi-bin/paper?he0191
http://scripts.iucr.org/cgi-bin/paper?he0268




On 19 November 2013 06:43, Shanti Pal Gangwar 
gangwar...@gmail.commailto:gangwar...@gmail.com wrote:
Dear  All


Can anyone explain the meaning and relevance of data when the Rmerge is 100% in 
high resolution shell and I/sig(I) is 3.



Thanks



--

regards
Shanti Pal Gangwar
School of Life Sciences
Jawaharlal Nehru University
New Delhi-110067
India
Email:gangwar...@gmail.commailto:email%3agangwar...@gmail.com

Re: [ccp4bb] how to cut back resolution of a well-refined model

[Jim Pflugrath]

Please tell me why Rpim should be looked at.  Cannot one have meaningless data 
and have lots of multiplicity to drive Rpim lower without any real benefit?  
Under what conditions is Rpim useful?

And suppose one looks at I/sigI (and not I/sigI) and CC1/2.  What of it?

And let me write what Phil wrote in a slightly different way:
Please explain how you think that adding the resolution from 2.6 A to 2.45 A 
will improve your model.

Sorry, but maybe it is too soon after the last CC1/2 discussion to raise these 
points, but I am truly interested in various opinions about all this.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Gerard Bricogne 
[g...@globalphasing.com]
Sent: Thursday, October 10, 2013 5:28 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] how to cut back resolution of a well-refined model

Dear Yafang,

 Is it the case that you collected these data on a Pilatus detector,
using relatively low exposure and high multiplicity? These types of datasets
always give what looks like alarmingly high values of R-merge, and many
people who are set in their ways (like so many reviewers still are) tend to
conclude that the alarm is about the data being bad, whereas it is about
Rmerge being a terrible statistic in these situations. The Rpim statistic,
on the other hand, is the one to look at if you want an R-like quantity, and
it is well behaved in this regime. Of course, look at CC1/2 as well, and
I/sigI as you did.


 With best wishes,

  Gerard.

--
On Thu, Oct 10, 2013 at 04:57:20PM -0400, Yafang Chen wrote:
 Hi All,

 I have a structure at 2.45A which has been well refined. However, since the
 R-merge at the last shell is above 1 (although I/sigmaI at the last shell
 is more than 2), we now decide to cut back the resolution to about 2.6A. Is
 there a way to do this based on the well-refined model instead of doing the
 MR and refinement all over again? Thank you so much for your help!

 Best,
 Yafang

 --
 Yafang Chen

 Graduate Research Assistant
 Mesecar Lab
 Department of Biological Sciences
 Purdue University
 Hockmeyer Hall of Structural Biology
 240 S. Martin Jischke Drive
 West Lafayette, IN 47907

--

 ===
 * *
 * Gerard Bricogne g...@globalphasing.com  *
 * *
 * Global Phasing Ltd. *
 * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
 * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
 * *
 ===

Re: [ccp4bb] from sca to CC1/2

[Jim Pflugrath]

The current version of scalepack already calculates CC1/2 and reports it in the 
log file.



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Francesco 
Angelucci [francesco.angelu...@uniroma1.it]
Sent: Wednesday, September 18, 2013 5:21 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] from sca to CC1/2

Dear All,

I am wondering if is possible to calculate the CC1/2 factor from the denzo .sca 
file.
Thank you in advance
Francesco Angelucci

Re: [ccp4bb] Resolution, R factors and data quality

[Jim Pflugrath]

I have to ask flamingly: So what about CC1/2 and CC*?

Did we not replace an arbitrary resolution cut-off based on a value of Rmerge 
with an arbitrary resolution cut-off based on a value of Rmeas already?  And 
now we are going to replace that with an arbitrary resolution cut-off based on 
a value of CC* or is it CC1/2?

I am asked often:  What value of CC1/2 should I cut my resolution at?  What 
should I tell my students?  I've got a course coming up and I am sure they will 
ask me again.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Arka Chakraborty 
[arko.chakrabort...@gmail.com]
Sent: Tuesday, August 27, 2013 7:45 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Resolution, R factors and data quality

Hi all,
does this not again bring up the still prevailing adherence to R factors and 
not  a shift to correlation coefficients ( CC1/2 and CC*) ? (as Dr. Phil Evans 
has indicated).?
The way we look at data quality ( by we I mean the end users ) needs to be 
altered, I guess.

best,

Arka Chakraborty

On Tue, Aug 27, 2013 at 9:50 AM, Phil Evans 
p...@mrc-lmb.cam.ac.ukmailto:p...@mrc-lmb.cam.ac.uk wrote:
The question you should ask yourself is why would omitting data improve my 
model?

Phil

Re: [ccp4bb] HKL2000 sigma cutoff

[Jim Pflugrath]

I wonder if the rejects file had a large number of reflections in it in one 
case, but not in the other case.  One can be playing with various options and 
create or add to such a large list.  That's why there is the Delete Reject 
file option.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Tim Gruene 
[t...@shelx.uni-ac.gwdg.de]
Sent: Sunday, July 07, 2013 11:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] HKL2000 sigma cutoff

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Ursula,

in your first email you wrote that you were confused by [...] HKL2000
for scaling, and in this email you wrote that your fix is to scale the
data with scalepack. In my understanding scalepack is part of HKL2000
- - would you mind explaining what the difference is to you? Just to
understand what caused a problem and how you fixed it.

Regards,
Tim

On 07/05/2013 11:18 PM, Ursula Schulze-Gahmen wrote:
 I found a fix, but not an explanation. I scaled the same data with
 scalepack and had no problem with loosing reflections. I am not
 sure what happened in HKL2000.

 Ursula

 On Fri, Jul 5, 2013 at 12:38 PM, Phil Jeffrey
 pjeff...@princeton.eduwrote:

 Ursula,

 I/sigI of -3 as I recall.

 Are you sure that the downstream programs you are using aren't
 the ones applying the cutoff ?  Scalepack is, in general,
 perfectly happy to write negative intensities to output.sca and
 certainly is doing so as of HKL3000. Perhaps you need to use the
 TRUNCATE YES option in Truncate ?  Does the output MTZ from
 Scalepack2mtz show the number of reflections you expect ?


 Phil Jeffrey Princeton


 On 7/5/13 3:24 PM, Ursula Schulze-Gahmen wrote:

 Sorry for the non-CCP4 question.

 I am confused about the sigma cutoff used by HKL2000 for
 scaling. I scaled a data set to 3.0 A resolution. I collected a
 complete dataset to 2.8A, but the I/sigma is about 1.0 at 3.0
 A. The scaling logfile in HKL2000 shows 100% completeness in
 the highest resolution shell, but about 50% of the reflections
 are below I/sigma =0 in the highest resolution shell. I am
 guessing that these negative reflections are not being written
 out, because the output file from HKL200 does not have 100%
 completeness anymore. I would like to include these negative
 reflections. Is there a setting in HKL2000 that I can change or
 do I need to switch to a different program.

 Ursula






- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A
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Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFR2ZqvUxlJ7aRr7hoRAvNoAKDhHx+kbYYAzcTFaF+ywPhu8YMgFQCeJ2Bw
NMRDZu6kJdbvKQLJJV4Pe70=
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Re: [ccp4bb] anomalous scattering server down?

[Jim Pflugrath]

How does that compare to something that readily works with Fe, such as horse 
hemoglobin on a home lab copper X-ray system with 2 Fe in 291 residues in the 
asymmetric unit?


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry 
[ber...@upstate.edu]
Sent: Saturday, June 01, 2013 6:46 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] anomalous scattering server down?

That's what I wanted. Results are not promising- Bijvoet differences
would be less than 2% at peak, and this is not lysozyme.
Thanks, all,
eab

Mooers, Blaine H.M. (HSC) wrote:
 Bernhard Rupp has a calculator of Bijvoet ratios:

 http://www.ruppweb.org/new_comp/anomalous_scattering.htm



 Blaine Mooers
 Assistant Professor
 Director Macromolecular Crystallography Lab
 Member Stephenson Cancer Center
 Department of Biochemistry and Molecular Biology
 University of Oklahoma Health Sciences Center
 S.L. Young Biomedical Research Center Rm. 466

 Letter address:
 P.O. Box 26901, BRC 466
 Oklahoma City, OK 73190

   Shipping address:
   975 NE 10th Street, BRC 466
 Oklahoma City, OK 73104-5419

 office: (405) 271-8300   lab: (405) 271-8313  fax:  (405) 271-3910

 e-mail:  blaine-moo...@ouhsc.edu

 Faculty webpage: 
 http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/faculty/blaine-mooers-ph-d-
 X-ray lab webpage: 
 http://www.oumedicine.com/department-of-biochemistry-and-molecular-biology/department-facilities/macromolecular-crystallography-laboratory
 SAXS Links webpage: 
 http://www.oumedicine.com/docs/default-source/ad-biochemistry-workfiles/small-angle-x-ray-scattering-links.html?sfvrsn=0

 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Edward A. 
 Berry [ber...@upstate.edu]
 Sent: Saturday, June 01, 2013 6:09 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] anomalous scattering server down?

 Hmm- looks like crosssec calculates F' and F (and cros-section).
 I guess you're thinking of the x-ray edge page on the same server.
 Ive got local copies and graphs of that in an excel spreadsheet.

 I was thinking about the page where you enter your protein's
 molecular weight, number and type of anomalous scatterers, and
 it tells you how accurate your data has to be to make the
 anomalous signal significant.

 Thanks,
 eab

 Bosch, Juergen wrote:
 seems to be down sorry.
 you should be able to use crossec from ccp4
 Jürgen

 On Jun 1, 2013, at 6:47 PM, Edward A. Berry wrote:

 Is Ethan Merritt's anomalous scattering page at:
 https://urldefense.proofpoint.com/v1/url?u=http://www.bmsc.washington.edu/scatter/k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=vriAf%2FYLAE3OBEruPk8rCrKni5F1ZSfZwaGrrX0lwuk%3D%0As=57fb37f57cec12f6c778c53c6bfbdd04afc795f050605681dca25305998c6af0
 down or moved, or  the firewall I'm behind is blocking it?

 I want to check feasibility of a native-iron MAD experiment,
 and I'm not very good at math.

 thanks,
 eab

 ..
 Jürgen Bosch
 Johns Hopkins University
 Bloomberg School of Public Health
 Department of Biochemistry  Molecular Biology
 Johns Hopkins Malaria Research Institute
 615 North Wolfe Street, W8708
 Baltimore, MD 21205
 Office: +1-410-614-4742
 Lab:  +1-410-614-4894
 Fax:  +1-410-955-2926
 https://urldefense.proofpoint.com/v1/url?u=http://lupo.jhsph.edu/k=7DHVT22D9IhC0F3WohFMBA%3D%3D%0Ar=ftLbjJYpc5s5JQz9Q6qd7uT7FxPLb4V0aIwH4RJhyZU%3D%0Am=vriAf%2FYLAE3OBEruPk8rCrKni5F1ZSfZwaGrrX0lwuk%3D%0As=d80576bcb5bffce1fe3f7ffe04c9ec7a4b3338c452e984d4efcbd8036ce5b83c

Re: [ccp4bb] Crystallisation below 0°C

[Jim Pflugrath]

If one has cryoprotectant in their conditions, one may be surprised that 
crystals can grow at -20 deg C and probably lower.  Practice with lysosyme. :)


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Glenn Masson 
[glennmas...@gmail.com]
Sent: Thursday, May 30, 2013 6:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystallisation below 0°C

Hello CCP4BBers,

I am currently playing with some crystals that seem to enjoy lower 
temperatures, and I was thinking of breaking the 0°C threshold.

...

[ccp4bb] CSHL X-ray Methods in Structural Biology Course Oct 14-29, 2013: Application deadline June 15th

[Jim Pflugrath]

Hey Everybody,

I wanted to draw your attention again to the upcoming application deadline on 
June 15, 2013 for the
CSHL X-ray Methods in Structural Biology Course to be held October 14-29, 2013.

Also please also pass this on to any colleagues, friends, professors, research 
associates, grad students, and I suppose relatives who would benefit from 
attending the course.

Here's a link to the announcement:  
http://meetings.cshl.edu/courses/2013/c-crys13.shtml
The course is supported by a grant from the National Institute of General 
Medical Sciences

Some financial assistance is also available, see the course announcement on the 
details of that.

I think the course is an outstanding place to learn both the theoretical and 
practical aspects of Macromolecular Crystallography because of the extensive 
lectures from world-renowned teachers and the hands-on experiments. An  entire 
always open wet lab is devoted to the Course and we rent 18 iMacs for all the 
computational work, so everyone has simultaneous access for any and all 
computational work. But the real plus of this course is the interactions of the 
participants and the instructors --- just ask any former student.

This year's course will once again see the return of the long-time instruction 
team of Alex McPherson, Gary Gilliland, Bill Furey and myself along with many 
talented experts to help us give the participants an experience in 
Macromolecular Crystallography learning that cannot be found anywhere else.  
(The student:teacher ratio ends up to be about 1:1).  We expect to have the 
participants crystallize several proteins and determine their structures all in 
about two weeks.  In 2012, participants also crystallized a membrane protein, 
collected diffraction data, and solved the structure by molecular replacement.  
We intend to expand on that in 2013.

The course is limited to 16 participants due to the very hands-on nature of the 
experiments and the intimate seminar room.  Please check the above web site for 
more details.

If anyone has any questions, please send me e-mail, I will be happy to answer 
all queries.

Jim

Re: [ccp4bb] reference for true multiplicity?

[Jim Pflugrath]

Precision does not trump accuracy is something Michael Blum told me.  

Also Charles Wheelan wrote in his recently published Naked Statistics: “But 
no amount of precision can make up for inaccuracy.” 

I myself have been pleasantly surprised at how low multiplicity can be nowadays 
and still do S-SAD phasing.  And If one uses iodide, the quality of diffraction 
data does not need to be as high as with sulfur-SAD phasing.

Does one need to collect about different rotation axes?  Not always.  I wonder 
now if it would even hide a signal.

Does one need multiplicity of more than 6 to 8?  Not always.  Be careful about 
radiation damage with increasing multiplicity and exposure.

Does one need to minimize radation damage? I definitely think so.

Does one need ot make sure the cryostream is not moving or vibrating your 
sample? Definitely yes.

Does one need enough counting statistics to tease out the signal?  Yes, but 
this depends on the expected signal.

And so on.

I can provide datasets of images if anyone likes.

Jim

Re: [ccp4bb] A crystallographer on Mars

[Jim Pflugrath]

Most participants in the CSHL X-rays Methods in Structural Biology course have 
seen the powerpoint presentation of Alex McPherson's trip to Mars in 2001.  
Here are a couple of slides from the presentation:
http://i43.tinypic.com/33kx79l.jpg
http://i39.tinypic.com/oic17r.jpg  (Trehalose was pretty bad for the Martian 
polar ice caps)

Also note that the deadline for application to the 2013 Course is coming up: 
June 15th.  The course will be held October 14-29, 2013 at Cold Spring Harbor 
Laboratory.  Here is the announcement: 
http://meetings.cshl.edu/courses/2013/c-crys13.shtml

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ethan Merritt 
[merr...@u.washington.edu]
Sent: Tuesday, May 07, 2013 12:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] A crystallographer on Mars

The _New Yorker_ frequently publishes decently written articles on a
.

  ... the mission includes a nuclear-powered mobile laboratory,
  equipped with lasers, spectrometers, and an X-ray crystallographer.

Wow!  Who's the lucky Mars-going crystallographer?  Anyone we know?

Re: [ccp4bb] Difficult data

[Jim Pflugrath]

Maybe you crystallized something else?  Did you look up that unit cell in the 
PDB?




I am having a few issues with a data set I have been working on recently, and 
was hoping to get some ideas on how to deal with it, if anyone is in the mood.

.

Re: [ccp4bb] CCP4 Update victim of own success

[Jim Pflugrath]

I think James gets to 'fight' like in the old game of rogue by pressing the h, 
j, k, l keys on his keyboard (not a detachable one either).  While Eugene gets 
to use a modern game controller or a Wii.

Ooops, game is already over and James has lost.

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Felix Frolow 
[mbfro...@post.tau.ac.il]
Sent: Thursday, April 11, 2013 11:25 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] CCP4 Update victim of own success

I would serve as a second in this duel, but I respect very much both engaged is 
this duel…
Drop you pistols or swords !
:-)
Dr Felix Frolow
Professor of Structural Biology and Biotechnology, Department of Molecular 
Microbiology and Biotechnology
Tel Aviv University 69978, Israel

Acta Crystallographica F, co-editor

e-mail: mbfro...@post.tau.ac.ilmailto:mbfro...@post.tau.ac.il
Tel:  ++972-3640-8723
Fax: ++972-3640-9407
Cellular: 0547 459 608

On Apr 11, 2013, at 18:46 , 
eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk wrote:

That's really hard. Duel?

Eugene

On 11 Apr 2013, at 16:32, James Holton wrote:


CCP4 has a GUI?

-James Holton
MAD Scientist

On 4/11/2013 5:17 AM, 
eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk wrote:
Sorry that this was unclear. We assume that updater is used primarily from 
ccp4i, where nothing changed (and why it should be used from command line at 
all ?:)). The name was changed because it is reserved in Windows, which caused 
lots of troubles. Now it will stay as is.

Eugene

On 11 Apr 2013, at 05:16, James Stroud wrote:


On Apr 10, 2013, at 9:30 PM, 
eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk
 
eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.ukmailto:eugene.krissi...@stfc.ac.uk
 wrote:

No, it got renamed to ccp4um :) That should have been written in update 
descriptions, was it not?


There was only one mention of ccp4um that I could find in all update 
descriptions that I found (6.3.0-020). I only figured out what information was 
trying to be communicated because of your message (see attachment).

James


um-what.png



On 11 Apr 2013, at 03:54, James Stroud wrote:

Hello All,

I downloaded a crispy new version of CCP4 and ran update until the update 
update script disappeared. Is the reason that CCP4 has reached its final update?

James







--
Scanned by iCritical.

Re: [ccp4bb] High Rmerge and I/sigma values....?

[Jim Pflugrath]

As mentioned lots of reasons for this.

a. Poor crystal
b. Poor mount of the crystal
c. Poor equipment or non-working equipment
d. Poor maintenance of good equipment
e. Improper cryoprotection
f. Vibration or movement of goniometer, goniometer head, mounting pin, mounting 
loop, magnet, etc
g. Temperature fluctuation of the environment during the data collection
h. Not enough exposure time or poor signal to noise (improper experimental 
design)
i. Improper data processing (too many things to mention here)
j. etc.
k. et al.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of hamid khan 
[hamid...@yahoo.com]
Sent: Friday, March 29, 2013 8:19 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] High Rmerge and I/sigma values?

Dear CCP4BB Members,

I am interested in your expert comments/opinions about two values of a protein 
crystal diffraction data. Basically I am new to this field and do not have much 
idea about diffraction data interpretation and crystallography software’s use.


1)What could be the possible reasons for a high Rmerge value, say like 
0.185?


2)Value 6.2 for average I/sigma(I) for higher shell means that the 
resolution of the diffraction data is much higher than actually measured, what 
could be the possible reasons for this?

For your ease I would like to past the table here;

Values in parentheses are for the last resolution shell
Space group P2221
Unit-cell parameters (A°)
  a58.08
  b101.32
  c103.47
Molecules in ASU  1
Resolution range   38.63 - 2.50  (2.59 - 2.50)
Total number of reflections 228902
Number of unique reflections   21600
Completeness (%) 99.1(98.0)
Rmerge0.185 (0.373)
Reduced χ2   0.94(1.01)
Average I/σ(I)9.8  (6.2)

Thanks for the tips..,

Hamid Khan

Re: [ccp4bb] Diffraction data with big rotation angle

[Jim Pflugrath]

5 degrees per image may be problematic for some algorithms since the 
diffraction spots have uncertain positions in three dimensions.  Two dimensions 
will come from the position of the spot on the detector and the position of the 
detector.  The third dimension will come from the rotation angle value of the 
image which will have a large uncertainty.  There are ways around this and 
d*TREK uses some of them.



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Niu Tou 
[niutou2...@gmail.com]
Sent: Thursday, March 14, 2013 6:01 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Diffraction data with big rotation angle

Hi Tim,

I only tried HKL2000, and index with different resolution and different number 
of images. I am not quite familiar with XDS or d*trek.

One thing I am not sure is if this large oscillation angle will cause problem 
in indexing?
If this is true, any method to overcome it?

The situation I met was when I chose different settings to do index, HKL2000 
would give different cell dimensions while most of them were not small enough 
for a peptide crystal. The unit cell should be pretty small since even 
collecting data with 5 degree oscillation angle, the spots in one image were 
still fewer than a normal protein crystal case, so there is no spots overlap.

Best,
Yang

On Thu, Mar 14, 2013 at 5:20 AM, Tim Gruene 
t...@shelx.uni-ac.gwdg.demailto:t...@shelx.uni-ac.gwdg.de wrote:
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear Niu,

could you let us know more about what you have already tried?

- - use more images, maybe all images for indexing
- - try a different program: xds instead of mosflm instead of hkl200
instead of d*trek instead of xds  depending what you have tried.
- - try to find the unit cell dimensions manually from adxv
- - try cell_now with the SPOTS.XDS

in XDS I would check the output of IDXREF.LP to figure out if indexing
seems possible, i.e. if the input parameters seem stable or if they
are just floating around

and many more - it depends on the data set, really!

Best,
Tim

On 03/13/2013 09:12 PM, Niu Tou wrote:
 Dear colleagues,

 We have some diffraction data from small peptide crystals, the
 shape of diffraction spots looks normal, and resolution is beyond
 2A. The data were collected with 5 degree rotation per image. Later
 on we found it is hard to do index. Does anybody know some skills
 to figure this problem?

 Best wishes, Niu


- --
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

GPG Key ID = A46BEE1A

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Version: GnuPG v1.4.12 (GNU/Linux)
Comment: Using GnuPG with Mozilla - http://enigmail.mozdev.org/

iD8DBQFRQZZFUxlJ7aRr7hoRAmr3AKD28Mml3XY2LIWkDknKrkJFToLDvwCgu1DI
LDaPuAMfGlEIEObuWcckM7Y=
=yuwC
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Re: [ccp4bb] protein crystals or salt crystals

[Jim Pflugrath]

If one tries to use a dye to determine if crystals are protein or salt, then I 
recommend that they use both a positive and a negative control.  So have some 
handy salt or sugar crystals ready along with some known protein crystals to 
use as controls.

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ganesh Natrajan 
[ganesh.natra...@ibs.fr]
Sent: Monday, February 11, 2013 3:37 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] protein crystals or salt crystals

Dear Amro,

What you could try is this. Make a solution of 0.5 % (w/v) commassie brilliant 
blue in 10% (v/v) ethanol in water. Pipet 1 ul of this into your drop and close 
the cover slip. If the crystals are protein, they should turn blue after some 
time (typically 30 mins). Salt crystals will not turn blue as they are not 
stained by commassie.

You could also try using Hampton's Izit crystal dye for this, but the problem I 
have faced with it is that the izit itself crystallizes (gives lovely blue 
crystals) under certain buffer conditions.

cheers

Ganesh






Hallo my colleagues.
 i hope every one doing ok . i did screening since two weeks . i noticed today 
this crystals. i don`t know either it salt or protein crystal . my protein has 
zero tryptophan so i could distinguish by UV camera.
the condition was conditions:
0.1M SPG buffer pH 8 and 25%PEG 1500. in addition to Nickle chlorid 1mM.


best regards
Amr

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

[Jim Pflugrath]

Suggestion: What does your plunge of a loopful of the buffer (no crystal) into 
liquid nitrogen tell you?


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yuri Pompeu 
[yuri.pom...@ufl.edu]
Sent: Friday, November 30, 2012 7:22 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] To cryo or not to cryo...

Dear community,
I have what seems to be a pretty decent single crystal that grew from a screen 
set up 2 weeks ago.
I am trying to reproduce it but so far I have not succeeded. I am however 
afraid the crystal that did form will start to deteriorate. So this brings me 
to dilemma, I feel like I should try and mount this crystal and shoot it. But 
since I only have 1 sample, I do not want to mess this up...  I am inclined to 
try cryo conditions, but I am afraid the addition of a cryo such as glycerol 
could destroy the little guy.
The crystal formed in 30% PEG 4000, 0.1M NaCitrate pH5.6 and 0.2M NH4AcO, I 
wonder if this is a cryo condition already?
Any suggestions would be appreciated.

best,

Re: [ccp4bb] model protein for ligand soaking

[Jim Pflugrath]

Sucrose co-crystallized in lysozyme is wonderful. I think a soak will crack the 
crystals, but sometimes not.

Here's a pic of electron density from an orthorhombic crystal of lysozyme 
solved in the recent 2012 CSHL X-ray Methods in Structural Biology course:

http://i46.tinypic.com/2e4xk7k.jpg


Jim

  
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ed Pozharski 
[epozh...@umaryland.edu]
Sent: Friday, November 09, 2012 8:44 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] model protein for ligand soaking

On Fri, 2012-11-09 at 15:12 +0100, Ulrich Zander wrote:
 Does anybody have a suggestion for a protein/ligand combination that
 could be used for that and that is commercially available?

Perhaps lysozyme complexed with some sugar?

--
Bullseye!  Excellent shot, Maurice.
  Julian, King of Lemurs.

[ccp4bb] What to put on Custom Declaration for shipped samples?

[Jim Pflugrath]

I was asked by our shipping folks what we should put on the Customs Declaration 
so that samples that we ship or that are shipped to us (in dewars, styrofoam 
boxes, and/or padded envelopes) would not be held up in Customs.

I had them put:  

Scientific samples of less than 1 mg of non-infectious, non-hazardous protein. 
 No health hazard.

but it has been so long that I have had to do so.  I suppose I could name the 
exact protein, (e.g. hen egg white lysozyme), but maybe that is not a good idea.

What wording do folks put on these forms nowadays?  What works?  Do I need to 
put the buffer components?  

Thanks for responses.

Jim

Re: [ccp4bb] low-resolution data and SG

[Jim Pflugrath]

This looks like an output from SCALEPACK.  Unfortunately, one has no way to 
know from the output if 21 and 90 are strong intensities or not.  One cannot go 
by the I/sigmaI alone.  For example, suppose there is thermal diffuse scatter 
at these positions or perhaps there is a cosmic ray or radioactive decay 
(zinger) or a spot from a split crystal or the tail of a nearby streaky spot or 
other error during integrating of these Bragg reflections.

I recommend you look at
(a) neighboring reflections to get a sense of what a strong reflection value 
is.  Maybe it is 20,000 or more so that 90 would be a weak reflection, and
(b) the raw image itself at these reflection positions to see what the actual 
appearance of the pixel values in these positions are.

Jim


...
 0   0  17   2.4   2.0   1.2

  0   0  18  21.1   4.5   4.7

  0   0  19  90.2   6.0  15.0

...

Re: [ccp4bb] Rpim and how its related to anomalous signal

[Jim Pflugrath]

In my opinion Rpim is not related directly to anomalous signal, so perhaps that 
is why there is some confusion.

Also I think some folks confused Rpim with Rrim.  The latter is also called 
Rmeas.  But once again, these are not related directly to anomalous signal.  I 
do not find Rpim very useful for anything since when the data has high 
multiplicity Rpim gets very low, but as Michael Blum told me, Precision does 
not trump accuracy.  I would personally rather have accurate data than highly 
redundant but inaccurate data.

I like to look at the deltaF / sigma(deltaF) value reported by SHELXC and other 
programs, where deltaF is ||F+| - |F-||.
This might also be called d/sig.  There are other things to look at as well.

Jim

From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Vijayakumar.B 
[vijaybioscie...@gmail.com]
Sent: Tuesday, September 18, 2012 4:36 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Rpim and how its related to anomalous signal

Dear CCP4BB users,

I am very much interested to work in Anomalous scattering technique for 
macromolecular structure determination. I have already gone through some 
literature in which they have explained about a parameter called “Rpim”.  I am 
little bit confused about Rpim values.

Can anyone tell me about the use of this parameter in structure determination 
by anomalous techniques and how Rpim is related to anomalous signal?  Thanks in 
advance

With kind regards

B. Vijayakumar

Re: [ccp4bb] Ca or Zn

[Jim Pflugrath]

How would you distinguish between a mixture of Ca and Zn in the same locations?


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Kumar, Veerendra 
[veerendra.ku...@uconn.edu]
Sent: Tuesday, October 30, 2012 1:55 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Ca or Zn

Dear CCP4bb users,

I am working on a Ca2+ binding protein. it has 4-5 ca2+ binding sites.  I 
purified the protein  in presence of Ca2+ and crystallized the Ca2+ bound 
protein. I got crystal and solved the structure by SAD phasing at 2.1A 
resolution. I can see the clear density in the difference map for metals at the 
expected binding sites. However I had ZnCl2 in the crystallization conditions. 
Now i am not sure whether the observed density is for Ca or Zn or how many of 
them are ca or  zn? Since Ca (20 elctron) and Zn (30 electron), is this value 
difference can be used to make a guess about different ions?
is there any way we can find the electron density value at different peaks?

Thank you

Veerendra

Re: [ccp4bb] d*trek

[Jim Pflugrath]

d*TREK can process single crystal diffraction images from all the common 
detectors including not only Rigaku detectors, but many different detectors 
found at synchrotron beamlines around the world such as the APS, ALS, NSLS, 
CHESS, SLS, ESRF, Diamond, Soleil, PhotonFactory, SPring8, CAMD, Trieste, DESY, 
AS, LNLS, and you name it.



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Harry Powell 
[ha...@mrc-lmb.cam.ac.uk]
Sent: Friday, August 24, 2012 4:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] d*trek

Hi

It should do. Jim will know for sure.

On 23 Aug 2012, at 23:08, jlliu liu wrote:

My real questions is:

Does d*trek recognize the img file format collected at ALS or APS and process 
the data?

Thanks!



On Thu, Aug 23, 2012 at 6:00 PM, jlliu liu 
jlliu20022...@gmail.commailto:jlliu20022...@gmail.com wrote:
Do anybody know if d*trek can process data collected from APS?  Thanks a lot in 
advance.


Harry
--
Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre, Hills Road, 
Cambridge, CB2 0QH

Re: [ccp4bb] Large unit cell, overlaps

[Jim Pflugrath]

For large unit cells, one must take particular care with the X-ray beam and the 
orientation of the crystal.  The latter has already been mentioned in previous 
response.  For the beam, some things to do are:
1. Make crystal smaller.
2. Make beam smaller (use a smaller collimator size).
3. Reduce beam divergence (change the divergence setting of your optic).
4. Focus beam on the detector and not on the crystal.

Also be aware that by definition large unit cells have small mosaicity.  If 
they didn't, one would not even be collecting data because the spots would be 
all smeared together due to overlap.  No amount of fine-slicing will help 
resolve spots that are overlapped in the rotation direction.

Jim
  
...
I've collected a Pt soak data set on our home source with a 0.5˚ oscillation 
angle, but the anomalous signal drops off after about 8Å.  I am wondering if 
this is a problem due to overlaps at higher resolution.

Should I be concerned with this?  The crystal mosaicity from XDS is 0.25, so 
fairly low.  What can I do about this, should I try smaller oscillation angles?

Thanks,

Jason.

Re: [ccp4bb] cryo for high salt crystal

[Jim Pflugrath]

Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that w/v or 
v/w?), try using 100% saturated in reservoir, 75% saturated in reservoir, or 
50% saturated in reservoir.  You will have to TEST these.  See also this 
webinar on cryocrystallography which shows how to make these solutions: 
http://www.rigaku.com/node/1388

You could also try high salt solutions with similar technique.

Good luck!

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang 
[mzhang...@hotmail.com]
Sent: Tuesday, July 10, 2012 11:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo for high salt crystal

regaentDear All,

I am sure this question was discussed before. But I am wondering if anyone got 
the same experience as I do.
I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at pH7. I 
tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil, or ammonium 
sulfate itself: The problem is that all the cryo plus original reagents in the 
reservoir precipitate the salts out. And more serious problem is because of 
high salt in the condition, while I am trying to loop the crystal, both the 
drop and cryoprotectant drop form salt crystals (not sure it is KCl or ammonia 
sulfate) significantly and very quickly, that cause my crystal dissolved. My 
crystal doesn't seem to survive paraton-N oil. Does anyone here have similiar 
case? any suggestion will be appreciated.

Thanks,
Min

[ccp4bb] CSHL X-ray Methods in Structural Biology Course Oct 15-30, 2012: Application deadline June 15th

[Jim Pflugrath]

Once again I wanted to draw everyone's attention to the 
Cold Spring Harbor Laboratory 2012 X-ray Methods in Structural Biology 
course which will take place
October 15 through October 30, 2012.

The official course announcement is here:
http://meetings.cshl.edu/courses/c-crys12.shtml 

I think the course is an outstanding place to learn both the theoretical and 
practical aspects of Macromolecular Crystallography because of the extensive 
lectures from world-renowned teachers and the hands-on experiments.

Just recently, David Piston of Vanderbilt noted (Understanding how it works 
(2012) Nature 484, pp 440-441) that
We need to do a better job of teaching students how techniques work before 
they start using them.”  The CSHL course has tried to do that since it was 
first held in 1988 25 years ago when Alex McPherson, Hans Deisenhofer, Alwyn 
Jones, and Jim Remington joined me for 2 weeks of fun and hard work.  

This year's course will see the return of the long-time instruction team of 
Alex McPherson, Gary Gilliland, Bill Furey and myself along with many talented 
experts to help us give the participants an experience in Macromolecular 
Crystallography learning that cannot be found anywhere else.  (The 
student:teacher ratio ends up to be about 1:1).  We expect to have the 
participants crystallize several proteins and determine their structures all in 
about two weeks.

The course is limited to 16 participants due to the very hands-on nature of the 
experiments and the intimate seminar room.  Please check the above web site for 
more details.

If anyone has any questions, please send me e-mail, I will be happy to answer 
all queries.

Jim

Re: [ccp4bb] Fwd: [ccp4bb] Fun Question - Is multiple isomorphous replacement an obsolete technique?

[Jim Pflugrath]

And for more Personal Reflections, one may wish to take a gander at the Rigaku 
Webinar series with presentations by Brian Matthews and Michael G. Rossmann.

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Carter, Charlie 
[car...@med.unc.edu]
Sent: Wednesday, June 06, 2012 2:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Fwd: [ccp4bb] Fun Question - Is multiple isomorphous 
replacement an obsolete technique?



Begin forwarded message:

Date: June 6, 2012 3:05:16 PM EDT
To: aaleshin aales...@burnham.orgmailto:aales...@burnham.org
Subject: Re: [ccp4bb] Fun Question - Is multiple isomorphous replacement an 
obsolete technique?

There are four such papers in Methods in Enzymology, Vols 368 and 374:

David Blow:  How Bijvoet Made the Difference:  The Growing Power of Anomalous 
Scattering V. 374, pp. 3-22

Brian Matthews:  Transformations in Structural Biology:   A Personal View  V. 
368 pp. 3-10

Michael Rossmann:  Origins V. 368, pp. 11-21

Ulrich W. Arndt:  Personal X-ray Reflections  V. 368, pp. 21-45

These reminiscences are there entirely because my co-Editor Bob Sweet felt 
exactly the same way Alex does.

Charlie

On Jun 6, 2012, at 2:12 PM, aaleshin wrote:

I wonder if anyone attempted to write a historic book on development of 
crystallography. That generation of crystallographers is leaving this world and 
soon nobody will be able to say how the protein and non-protein structures were 
solved in those days.

Alex
...

Re: [ccp4bb] Propane still?

[Jim Pflugrath]

Here is a trick which I will attribute to Cambridge:

Fill balloon with gas.  Put end of balloon over 15 ml Falcon tube.  Put Falcon 
tube in LN2.  No wasted gas.

I would recommend CF4 or carbon tetrafluoride instead of propane though.  CF4 
is cheap and non-dangerous.

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Prince, D Bryan 
[dbryan.pri...@astrazeneca.com]
Sent: Monday, May 21, 2012 4:15 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Propane still?

Good afternoon fellow ccp4bb’rs,

I was wondering if anyone knows if a still to condense gaseous propane to 
liquid propane using dry ice is commercially available. I want to make sure 
that it is not something I can purchase before I build one fit to purpose. I 
appreciate any advice and knowledge you can share.

Regards,
Bryan Prince



Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.

Re: [ccp4bb] Propane still?

[Jim Pflugrath]

As with all reagents used in the lab it is best to understand the health 
hazards.  Thanks for note.

Jim


From: Jacob Keller [j-kell...@fsm.northwestern.edu]
Sent: Monday, May 21, 2012 5:22 PM
To: Jim Pflugrath
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] Propane still?

Well, not to be a downer, but wikipedia has some comments on the hazards...

Environmental effects

Tetrafluoromethane is a potent greenhouse 
gashttp://en.wikipedia.org/wiki/Greenhouse_gas that contributes to the 
greenhouse effecthttp://en.wikipedia.org/wiki/Greenhouse_effect. It is very 
stable, has an atmospheric lifespan of 50,000 years, and a high greenhouse 
warming potentialhttp://en.wikipedia.org/wiki/Greenhouse_warming_potential of 
6500 (CO2http://en.wikipedia.org/wiki/Carbon_dioxide has a factor of 1); 
however, the low amount in the atmosphere restricts the overall radiative 
forcinghttp://en.wikipedia.org/wiki/Radiative_forcing effect.

Although structurally similar to 
chlorofluorocarbonshttp://en.wikipedia.org/wiki/Chlorofluorocarbons (CFCs), 
tetrafluoromethane does not deplete the ozone 
layerhttp://en.wikipedia.org/wiki/Ozone_depletion. This is because the 
depletion is caused by the chlorine atoms in CFCs, which dissociate when struck 
by UV radiation. Carbon-fluorine bonds are stronger and less likely to 
dissociate. According to Guinness World 
Recordshttp://en.wikipedia.org/wiki/Guinness_World_Records Tetrafluoromethane 
is the most persistent greenhouse gas.

[edithttp://en.wikipedia.org/w/index.php?title=Tetrafluoromethaneaction=editsection=7]Health
 risks

Depending on the concentration, inhalation of tetrafluoromethane can cause 
headaches, nausea, dizziness and damage to the cardiovascular 
systemhttp://en.wikipedia.org/wiki/Cardiovascular_system (mainly the heart). 
Long-term exposure can cause severe heart damage.

Due to its density, tetrafluoromethane can displace air, creating an 
asphyxiationhttp://en.wikipedia.org/wiki/Asphyxiation hazard in inadequately 
ventilated areas.

On Mon, May 21, 2012 at 5:08 PM, Jim Pflugrath 
jim.pflugr...@rigaku.commailto:jim.pflugr...@rigaku.com wrote:
Here is a trick which I will attribute to Cambridge:

Fill balloon with gas.  Put end of balloon over 15 ml Falcon tube.  Put Falcon 
tube in LN2.  No wasted gas.

I would recommend CF4 or carbon tetrafluoride instead of propane though.  CF4 
is cheap and non-dangerous.

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] 
on behalf of Prince, D Bryan 
[dbryan.pri...@astrazeneca.commailto:dbryan.pri...@astrazeneca.com]
Sent: Monday, May 21, 2012 4:15 PM
To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Propane still?

Good afternoon fellow ccp4bb’rs,

I was wondering if anyone knows if a still to condense gaseous propane to 
liquid propane using dry ice is commercially available. I want to make sure 
that it is not something I can purchase before I build one fit to purpose. I 
appreciate any advice and knowledge you can share.

Regards,
Bryan Prince



Confidentiality Notice: This message is private and may contain confidential 
and proprietary information. If you have received this message in error, please 
notify us and remove it from your system and note that you must not copy, 
distribute or take any action in reliance on it. Any unauthorized use or 
disclosure of the contents of this message is not permitted and may be unlawful.





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

Re: [ccp4bb] effective iodide conc. for SAD data

[Jim Pflugrath]

Iodide is a fantastic derivative.  One does not need a lot with modern X-ray 
equipment, careful data collection, and great software.


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Rajesh Kumar 
[ccp4...@hotmail.com]
Sent: Thursday, May 03, 2012 8:51 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] effective iodide conc. for SAD data

Dear All,

I have very thin crystals but diffracting. I was not able to handle them easily 
for iodide soak. I always lost the crystals during manipulation and other big 
crystals obtained after seeding doesn't even give any diffraction. I tried for 
co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul 
drop of 1 ul protein and 2ul reservoir).
Is this concentration of iodide is enough for SAD data  ( if it had good 
incorporation) ?
I appreciate your help.

Thanks
Rajesh

Re: [ccp4bb] effective iodide conc. for SAD data

[Jim Pflugrath]

Please see my poster at the ACA 2012 meeting.  See also:
(1) Dauter, Z., Dauter, M.  Rajashankar, K.R. (2000) Acta Cryst. D56, 232-237.
(2) Nagem, R.A.P, Dauter, Z.  Polikarpov, I. (2001) Acta Cryst. D57, 996-1002.

:)


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett 
[rrowl...@colgate.edu]
Sent: Thursday, May 03, 2012 11:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] effective iodide conc. for SAD data

Interesting idea. The only caveat that springs to mind is that the more useful 
anions (e.g., iodide and bromide) are on the chaotropic end of the Hofmeister 
series and may potentially destabilize protein structure or protein-protein 
interactions, which might complicate co-crystallization starting from known 
conditions, especially at higher concentrations of anion. Alternate cations may 
be less problematic.

Cheers,

___
Roger S. Rowlett
Gordon  Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edumailto:rrowl...@colgate.edu

On 5/3/2012 11:29 AM, Jacob Keller wrote:
I have wondered for a long time now why it is not standard practice for all 
crystallization protein stocks to contain either Br- or I- ions instead of Cl-, 
even for cationic buffers like TRIS, which could be titrated with HBr or HI to 
get in the 10+ mM range. Also, one could use Cs or Rb for the cations (and 
titrate anionic buffers with the respective hydroxides). What's there to lose? 
The gain is obviously the possible anomalous signal (always helpful), and one 
might pick up additional interesting and possibly physiologically-relevant 
halide or alkali metal sites. Seems that structural genomics people might 
standardize this into the pipeline as well, and thereby potentially cut out 
SeMet protein production in many if not most cases.

JPK

On Thu, May 3, 2012 at 9:05 AM, Jan Abendroth 
jan.abendr...@gmail.commailto:jan.abendr...@gmail.com wrote:
Hi Rajesh,
it can be a bit all over the place:
For quick soaks, we typically use 500mM-1000mM. A good starting point might be 
to simply replace the NaCl concentration in your protein buffer. By some 
serendipity we also managed to solve a structure by I/S SAD after a 1mM NaI 
soak. One iodide had found its way into a nice binding pocket.
For co-crystallization, mostly 200mM should be fine.
Another approach could be to supplement your cryo buffer with iodide, replacing 
NaCl. NaI is highly soluble in ethylene glycol.

Also see here: http://www.ncbi.nlm.nih.gov/pubmed/21359836

Good luck!
Cheers,
Jan
--
Jan Abendroth
Emerald Bio
Seattle / Bainbridge Island WA, USA
home: Jan.Abendroth_at_gmail.comhttp://Jan.Abendroth_at_gmail.com
work: JAbendroth_at_embios.com
http://www.emeraldbiostructures.com

On May 3, 2012, at 6:51 AM, Rajesh Kumar wrote:

Dear All,

I have very thin crystals but diffracting. I was not able to handle them easily 
for iodide soak. I always lost the crystals during manipulation and other big 
crystals obtained after seeding doesn't even give any diffraction. I tried for 
co-crystallizing with NaI. The crystals appear only up to 20 mM in 1:2 (3ul 
drop of 1 ul protein and 2ul reservoir).
Is this concentration of iodide is enough for SAD data  ( if it had good 
incorporation) ?
I appreciate your help.

Thanks
Rajesh




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

Re: [ccp4bb] Off-pick: suggesiton of puck

[Jim Pflugrath]

Rigaku ACTOR robots can accept many different styles of pucks depending on the 
LN2 dewar baseplate that the particular ACTOR is equipped with.  Rigaku in 
China will sell ACTOR pucks, but if you are looking for ALS-style pucks or 
ESRF-style pucks or Unipucks or ???, then I am not sure where to get them.

Anyways, I have forwarded your query to my colleagues in China.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Frank 
[fanshil...@hotmail.com]
Sent: Monday, April 16, 2012 8:35 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Off-pick: suggesiton of puck

Hi guys:

Recently we need purchase some puck for Actor, unfortunately we can't find any 
agency in China.

I really appreciate that if anyone can give us some suggestion about that? such 
like brand, price, and necessary accessory?

Thanks for help

Best regards

Frank

Re: [ccp4bb] unique reflections vs unique observations

[Jim Pflugrath]

I am aware that some programs report interesting numbers.

In addition to what Andrew wrote about resolution cutoffs and truncated 
reflections, I'd like to mention another issue with systematically absent 
reflections.

The number of observations or reflections that come from an integration program 
are fed into a scaling program.  However, the integration program may use a 
different space group than the scaling program.  I am aware that in HKL  
systematically absent reflections are counted as observations when given to the 
scaling program SCALEPACK, and are reported in the log file as a unique 
reflection, but do not appear in the output *.sca file.

For example, when the integration program uses spacegroup P4 to integrate, then 
the user scales in spacegroup P4(1)2(1)2.  What happens to the (0 0 5), (0 0 
6), (0 0 7), (0 0 9), (0 11 0), and other systematically absent reflections in 
P4(1)2(1)2 that are passed from the integration program to the scaliing 
program?  I can tell you that they disappear from the output file, but appear 
in the total number of observations in the log file.  So if the input file to 
scaling has 169 systematically absence observations that when reduced to unique 
reflections yield 69 unique reflections, then SCALEPACK will report 169 extra 
input observations and 69 extra output unique reflections than are present.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Naveed A Nadvi 
[nnad2...@uni.sydney.edu.au]
Sent: Sunday, April 15, 2012 3:06 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] unique reflections vs unique observations

Dear CCP4 users,

I am a bit confused about the use of these terms in regards to structure 
refinement statistics. When I process my data with SCALA, the program outputs 
statistics in terms of total and unique numbers of observations. However, 
when I use the MTZ files with REFMAC, the final PDB file has number of 
reflections. These values are of similar magnitude, but not identical. The 
issue is even more complicated when I look at tables of statistics between 
different journals. Authors often report unique reflections or unique 
observations.

My questions are:

1) Are these two terms interchangable?
2) Are they relevant to the different stages of processing (e.g. data 
collection vs structure refinement)?
3) How do I rationalize the difference between the two values?

I have read some of the widely used textbooks, but I am still confused when 
looking at publications. Any comments would be highly appreciated!

Kind regards,

Naveed Nadvi

Faculty of Pharmacy,
University of Sydney.

Re: [ccp4bb] Rigaku R-axis IIC X-ray diffraction system

[Jim Pflugrath]

The first Rigaku R-AXIS IIC in North America were installed in late 1990 at 
Yale University and McMaster University.  That's the same year the Hubble 
Telescope went into space and the first search engine Archie was released.

The last R-AXIS IIC shipped in 1994.  So these are really vintage systems if 
anyone is still using them.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Ng Chyan Leong 
[c...@mrc-lmb.cam.ac.uk]
Sent: Friday, April 13, 2012 11:48 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Rigaku R-axis IIC X-ray diffraction system

Dear all,
Do you know is anyone still using Rigaku R-axis IIC X-ray diffraction
system with support services? It seems Rigaku no longer support this
systems.

Thank you in advance.

Best Regards,
Leong

Re: [ccp4bb] How to reduce no. of overlaps

[Jim Pflugrath]

In addition to reducing the beam divergence, you may wish to use a smaller beam 
size by using a smaller collimator or making the slits smaller.  A smaller 
crystal can also help to spatially separate the Bragg spots as can moving the 
detector closer to the crystal. Yes, closer to the crystal.  This is not 
intuitive, but arises since modern homelab beams are not parallel but are 
diverging from a focal point near the sample position.  It is just something 
else you may wish to try.

How you flashcool your sample will also have a large effect on the spot 
sizes/shapes.

Jim

.
Another thing:  most in-house sources allow you to reduce divergence of the 
beam.  You lose intensity, but no matter, just expose longer.  That also 
improves overlap.

Cheers
phx

Re: [ccp4bb] HKL2000 indexing problem

[Jim Pflugrath]

There could be many causes for this.  Perhaps you do not have the best def.site 
file for your detector / beamline / hardware.  What do your local HKL2000 
experts tell you?

You could e-mail me an image and I can help you.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Peter Hsu 
[hsuu...@u.washington.edu]
Sent: Monday, February 20, 2012 6:28 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] HKL2000 indexing problem

Hi all,

I recently collected a data set off a single crystal and have had problems with 
indexing it. Every time I go pick peaks for indexing it constantly picks peaks 
that are just slightly off the actual peak. After indexing, it would always be 
that 2 of the 3 cell dimensions would be fairly normal, while the 3rd would 
have some impossible value such as 1.

On some other occasions if it manages to pick peaks properly, and every time I 
go to index it, it gives back an error that I don't have enough peaks picked to 
index (picked nearly 500).

I've tried using a number of different images to index from and have run into 
the same problem.

Has anyone else run into these problems? Does anyone have any idea what might 
be wrong w/my dataset and/or crystal?

Thanks in advance for any insight,

Peter

Re: [ccp4bb] Freezing crystal

[Jim Pflugrath]

Just a thought for those that mentioned propane and ethane, I would like to 
suggest that they try carbon tetrafluoride (CF4) instead.  It certainly should 
be much safer.  It melts at 90 K and boils at 145 K, so you know you are below 
145 K if you see it as a liquid.

Re: [ccp4bb] Lysozyme/Xenon derivatives Phenix.

[Jim Pflugrath]

When you crystallized your lysozyme, did you have any other anomalous 
scattering atoms in the conditions?  I am thinking of chloride ions in 
particular, but there could be many others.  How did you distinguish between 
sites of xenon and sites of other kinds of atoms in your analysis?

Did you do a control experiment without xenon?  Maybe with air?  Maybe with and 
without pressurization?

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Brennan Bonnet 
[brennan.bon...@lightsource.ca]
Sent: Thursday, February 02, 2012 10:43 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Lysozyme/Xenon derivatives  Phenix.

Hi all,

I have a strange result using Phenix's AutoSol to look for xenon sites in 
lysozyme.

Re: [ccp4bb] scalepackvirus rejections and Rrin

[Jim Pflugrath]

Folks may wish to watch a webinar I gave on using HKL3000.  In the webinar I 
describe how to calculate an Rmeas from scalepack output and also discuss a few 
other how-to operations.

See www.rigaku.com/protein/webinars-past.html for more viewing pleasure.

Jim




From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Yarrow Madrona 
[amadr...@uci.edu]
Sent: Tuesday, December 06, 2011 10:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] scalepackvirus rejections and Rrin

Hello,

I am using scalepackvirus and I noticed that the rejection list grows but
does not disappear in later rounds of scaling from the log file as in
scalepack. I am assuming that the rejections are treated the same way as
in scalepack but for some reason are not removed from the log file.  Does
anyone know if this is correct?

Also I wondered if there is any way to get a redundancy dependent R value
from scalepack.  Thank you.

-Yarrow

--
Yarrow Madrona

Graduate Student
Molecular Biology and Biochemistry Dept.
University of California, Irvine
Natural Sciences I, Rm 2403
Irvine, CA 92697

Re: [ccp4bb] how to add Cadmium ion to structure and do the refinement

[Jim Pflugrath]

If you edit the PDB file by hand, then you should have no problems.  Some 
folks might cringe at this, but this does work:

1. Like Roger Rowlett said, place atom at pointer.  You can place a chloride 
there.  Be sure to add to your molecule and not a new molecule.
2. Save the coordinates to a PDB file.
3. Edit the newly saved PDB file with the Chloride designation with an editor 
(emacs is great) and change the Chloride to a Cadmium.  What does a Cadmium 
look like in a PDB file? I don't have a clue, but I would search for cadmium 
at rcsb.org and save a PDB file with a cadmium in it.  Then you can see what a 
cadmium looks like in the example PDB file and mimic that in your file.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Roger Rowlett 
[rrowl...@colgate.edu]
Sent: Monday, November 28, 2011 3:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] how to add Cadmium ion to structure and do the refinement

Lu,

To add a metal ion to a structure, you can use place atom at pointer
or Get Monomer I usually use the latter. For instructions see 

Re: [ccp4bb] dark progression of radiation damage

[Jim Pflugrath]

Any cacodylate buffer will cause gas to be produced.  One only needs a minute 
exposure on a modern home lab source to see this happening.  I suggest that 
everyone avoid cacodylate in their crystallization drops that end up being 
exposed to X-rays.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Sanishvili, 
Ruslan [rsanishv...@anl.gov]
Sent: Wednesday, November 23, 2011 11:49 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] dark progression of radiation damage

I think I need to clarify couple of things in my recent post about
exploding crystals during re-mounting by a robot. First, it was a bit

Re: [ccp4bb] 1.95A, different phases, maps look the same, R/Rfree 22/24, wrong space group? - No alternative origin

[Jim Pflugrath]

A source of confusion is that we are using two different definitions of 
origin.   In the a graphics program sense (coot, pymol), the origin is always 
at xyz coordinate (0, 0, 0).  I think that's what Napo means by present the 
same cell and origin.

However, imagine a crystal upon which we put a unit cell box with one corner 
labelled (0, 0, 0) and the opposite corner labelled (1, 1, 1) in fractional 
coordinates.  We can use the unit cell box as a ruler to read out positions 
in the crystal.  Within the crystal are our molecules in a repeating lattice.  
Now suppose we move our unit cell box around the crystal while keeping the 
molecules fixed in the lattice.  Do you see how the relative coordinates of the 
molecules with respect to that unit cell box will change even though the 
crystal does not change nor do the molecules in the crystal?

I know this doesn't explain everything about origins as not every point in 
most crystal lattices can be an origin, but I will save that concept for 
another day.  I just wanted to note the apparently different definitions of 
origin in this thread.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Napoleão 
Valadares [n...@if.sc.usp.br]
Sent: Sunday, November 20, 2011 9:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] 1.95A, different phases, maps look the same, R/Rfree 
22/24, wrong space group? - No alternative origin


About the same origin:
The pdbs of both Solution-1 and Solution-2 present the same space group and 
cell, as observed opening the pdbs as text files or in pymol. When I open both 
maps on coot they are not superposed but present the same cell and origin.

Re: [ccp4bb] crystal orientation during data collection

[Jim Pflugrath]

All the currently used diffraction image processing programs can tell the 
crystal orientation from a single diffraction image.

Some programs like d*TREK can calculate new orientations for the crystal if the 
crystal is mounted on a reasonable goniometer such as the Rigaku Kappa 
goniostat or the Rigaku quarter-chi goniostat. :)  And the strategy program in 
d*TREK can use a list of previously collected and/or processed Bragg 
reflections to make sure that the new strategy fills in the missing parts of 
reciprocal space.

Jim


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of yanwu huo 
[applehu...@gmail.com]
Sent: Thursday, November 17, 2011 4:00 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] crystal orientation during data collection

Hi,
I worked on a crystal sensitive to radiation damage, So I need to merge many 
crystal to obtain complete dataset, Does anyone know such program that can tell 
crystal orientation after first frame exposure.
Thank you in advance.


--
Thank you very much and all the best,

Yanwu Huo
Postdoctoral Associate
Department of Molecular Biology and Genetics
Cornell University
Ithaca, NY, 14853
Email:yh...@cornell.edumailto:email%3ayh...@cornell.edu

Re: [ccp4bb] cryo protection

[Jim Pflugrath]

For some ideas on cryocrystallography, one can watch an online webinar on the 
subject:
http://www.rigaku.com/protein/webinar-001.html

Maybe some unbiased folks can comment? ;)

Jim



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Leonard Thomas 
[lmtho...@ou.edu]
Sent: Wednesday, October 26, 2011 11:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo protection

Hi All,

I have run into a very sensitive crystals system when it comes to cryo
protecting them.  I have run through the usual suspects and trays are
...

Re: [ccp4bb] Aging PEGs

[Jim Pflugrath]

PEGs have small amounts of anti-oxidants added to them by the manufacturer.
I think different manufacturers may use different compounds.

And you might imagine that PEGs themselves with their -OH groups go from
alcohol to adelhyde to carboxylate.


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jacob
Keller
Sent: Wednesday, August 24, 2011 2:18 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] data collection

[Jim Pflugrath]

Many programs such as d*TREK (free download) can predict the actual reflns
that would appear on detector given a particular experiment (crystal,
spacegroup, unit cell, detector, detector position, wavelength, etc).

A quick prediction shows that one will get about 3.9-fold redundancy with
the rotation method for spacegroup P1 and about 95% completeness (circle
inscribed in square) for the one orientation I tested.

Jim

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of john
peter
Sent: Tuesday, August 16, 2011 1:20 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] data collection

Hi All,
 I wish to confirm that when we collect native dataset 0-360 for a protein
crystal (P1 space group) , the redundancy could very well be
2 since many reciprocal points cross Ewald sphere twice, right ?
Thanks a lot.
John

Re: [ccp4bb] anomalous scatterer

[Jim Pflugrath]

There is always an anomalous signal to be seen if one measures things well
enough.  One does not need to be at the edge in order to detect anomalous
scatterers.  For example, most (if not all) of the sulfur SAD phasing is
done well away from the sulfur edge.
 
I will note that you stated that the f' and f of Fe at this wavelength was
not zero, thus there should be a signal and a peak.  

When you phase your data, you will get better phases if you describe ALL the
anomalous scatterers and signals that are detectable and not just the Tb.
 
Jim

  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Huiming Li
Sent: Tuesday, August 09, 2011 11:38 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] anomalous scatterer


Hi All,

  I am working on a Tb binding protein on which I collected anomalous data
at Tb edge of 1.648 A.  Each protein is designed to bind one Tb. There are
two copies of the protein in an ASU. I have two questions. First, I am only
able to see one copy of the protein with Tb bound, and no density on the
other copy.  Isn't this a bit surprising? Second, there is one additional
peak on each monomer at the site where Fe is known to bind, and Fe has an
edge of 1.739A. At 1.648A, f' and f'' of Fe is only about 1/5 of Tb. Is it
possible Fe also shows some anomalous signal at Tb edge?

Thank you,

Huiming Li, Ph.D.
Immune Disease Institute
Children's Hospital Boston
Harvard Medical School
Boston, MA 02115

Re: [ccp4bb] output individual redundancies

[Jim Pflugrath]

For each observation or measurement, HKL, d*TREK, and all other common
programs to my knowledge add up the bits of each relfection from its parts.
They do not add parts of of other reflections (even if symmetry-related)
into a reflection.  Reflections for which only part of the Bragg peak is
measured are tossed out.  For example, at the beginning and end of a scan
there are partial reflections which cannot be made full.  
 
(I am aware that one can take a partial measurement and scale it up by the
inverse of its so-called partiality to make it into a fake full reflection.)
 
One can use the NO MERGE ORIGINAL INDEX macro of scalepack and output the
individual measurements of denzo or HK that have had scale factors applied.
This file can be a converted to a d*TREK reflectiion file with SCA2DTREK and
then the individual measurements averaged with dtscaleaverage to get
statistics such as Rmeas, completeness, reduced ChiSq, multiplicity, and a
Table 1 suitable for framing in your Nature paper.  The unique reflection
list output could have the multiplicity of each unique reflection added
pretty trivially if there is a demand for this.
 
Jim


  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Shya
Biswas
Sent: Friday, July 15, 2011 6:20 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] output individual redundancies



Hi,

I was wondering if anyone knows what HKL 2000 does? Does it merge all
partials and treat it as one, because often times I noticed with increase in
partials the redundancy increases.

Shya



On Fri, Jul 15, 2011 at 1:24 PM, James Holton jmhol...@lbl.gov wrote:


At the risk of asking a question to which I should already know the answer:

do partials count as redundancy?

That is, in SCALA, is the number of observations the number of recorded
spots?  Or is it the number of recorded spots after adding partials?   If it
is the latter, what happens if you collect more than 360 degrees of data?
Does the second pass through a given unmerged hkl index count as more
partials or is it now somehow upgraded to an independent observation?

Then again, in Eastern English the word redundancy has a negative
connotation, and the output of SCALA actually uses the word multiplicity.
I wonder if that makes unmerged partials redundant?

-James Holton
MAD Scientist 


On 7/15/2011 8:09 AM, Ed Pozharski wrote:


On Fri, 2011-07-15 at 09:26 +0100, Phil Evans wrote:


Ed. You could count them from the unmerged output as you say, or I
could make you a special version of SCALA or Aimless maybe next week



Phil,

that would be fantastic!  Hope there is broader interest in such option
(beyond Robbie and myself). I'll try unmerged output in the meantime.

Ed.

Re: [ccp4bb] abnormal I/Sigma over phi rotation range

[Jim Pflugrath]

Please clarify:

Did you process the sets separately from the images?

Or did you process 360 degrees of images all the way to scaled, but
unaveraged results, then average reflections from the 5 different rotation
ranges?

Or something else?

With most processing programs that I am aware of, one can adjust their
sigmas to anything they want to.  What happens when you use the same
adjustments for the 180 degrees that you did for the others?


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Vennila Natesan
Sent: Sunday, July 10, 2011 7:54 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] abnormal I/Sigma over phi rotation range

Dear CCP4BB users,

In order to determine the redundancy at which the structure can be solved, I
divided the master data set of my protein into five sets with phi rotations
of  45,65,90, 180 and 360 degrees. I got the mean I/Sigma value as
22.4(11.4), 21.7(11.0), 22.7(11.2), 60.9(45.2) and 15.6(8.1) respectively.
I will be very helpful if i get any idea/possible reasons for the abnormal
value for 180 degree dataset.

For the information, the completeness values are
75.7(78.5),92.6(92.7),99.5(97.7),99.6(97.7)and 99.5(96.4) respectively and
values inside brackets are for highest resolution shell. There was no
noticable change in mosaicity
value. The Rmerge values are 2.2,   2.4,2.7,3.2, and 5.7 (8.7)
respectively.

Thanks in advance

Re: [ccp4bb] Same protein, different molecule numbers per ASU

[Jim Pflugrath]

OK, same space group, but you didn't indicate what the unit cells were.
They are different, right?

  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ferrol
shariff
Sent: Sunday, July 10, 2011 3:10 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Same protein, different molecule numbers per ASU


Hello and good day to everyone! :)


I have some general questions on crystallography work. I hope you don't mind
giving me some ideas. 

I have solved my lipase protein both ground-grown crystals and space-grown
crystals with good resolutions (1.4A and 2.2A). They are the same protein
from the same source, same purification methods, and produced crystals from
the same crystallization conditions (except the gravity part). 

From the data, it shows that both of them belong to the same space group
P212121. But they have different number of molecule per asymmetric unit.
Ground crystal= 1 molecule/ASU, Space crystal= 2 molecules/ASU. At the
moment i have problem explaining this issue. Is it normal to have such
results? Same protein with different number of molecule/ASU? 

I've been trying to get some references on this matter but so far i don't
really get anything that can directly explain it. Furthermore, do i need to
relate this with the gravity effect?

I hope you don't mind sharing some experiences on crystallography especially
regarding this matter.

Thank you very much

-- 
FAIROLNIZA

The advantage of the emotions is that they lead us astray, and the
advantage of science is that it is not emotional
-Oscar Wilde, The Picture of Dorian Gray, 1891

Re: [ccp4bb] How to evaluate Fourier transform ripples

[Jim Pflugrath]

I would be very careful about any bond to As.  I would imagine such 
bonds would be very susceptible to radiolysis with typical radiation used 
in the diffraction data collection.  Have you performed some 
diffraction experiments of various time lengths (or flux) and seen what 
happens around this region of the electron density map?


Jim

On Wed, 6 Jul 2011, conan wrote:


Dear All,
         Hi. I was asked in a manuscript revision to discuss about the possible 
effects of
Fourier transformation ripples on the crystallographic results. Specifically, 
the reviewers
question whether ripples may affect on the electron density around heavy metal 
center which has
a Mo-S-As connection. From which angle or in which way this problem should be 
addressed most
convincingly ?

         Thank you for any suggestion.

Best,
Conan

Re: [ccp4bb] Y-Chi2 running out of chart

[Jim Pflugrath]

Instead of an imperfect crystal, this can also occur if one chooses the
wrong Bravais lattice type (or spacegroup) to integrate.  For example, if
you choose tetragonal when it is really orthorhombic with a ~ b, or if you
choose orthorhombic and beta is 90.2, then you can see that trying to force
that unit cell will lead to higher residuals during positional refinement.
If one reciprocal lattice is oriented mostly along Y, then I think what Bing
observes can happen in such cases.  This is easily tested by integrating in
triclinic.

  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Shiva
Bhowmik
Sent: Friday, June 24, 2011 11:42 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Y-Chi2 running out of chart


Hi Bie,
 
Curious to know what are the cell parameters obtained after scaling? You
mention observing perfect Chi2 statistics with lysozyme crystals. But are
you observing the same Chi2 statisctis with the crystal that yielded unusual
Y-Chi2 if you collect another dataset. If there is a consistency of
observing the unusual Y-Chi2 with that crystal again then it is likey that
the crystal maybe highly imperfect. If not, then there could have been a one
time un-nailed problem occurred during that collection.
 
Cheers,
 
Shiva 
 
On Thu, Jun 23, 2011 at 8:59 AM, bie gao gao...@gmail.com wrote:


Dear colleagues, thank you all for your help! I really appreciate it.
I did perform a lysozyme test after the repair. I collected ~50 degree
(99%). Everything seems to be fine.  Maybe I should have done it for an
entire round.
As for the current collection, as I suspended, it did go out of the chart
but then came down to normal. Overall Rmerge is 7.9% (4% square). As Zbyszek
and others mention, it is probably due to imperfect crystal and also uneven
cooling.
If our field engineer discovers anything else, I'll post it here. Thanks
again for your help. 



On Wed, Jun 22, 2011 at 9:00 PM, Artem Evdokimov artem.evdoki...@gmail.com
wrote:


As a follow up to the excellent suggestions made by others I would suggest
that a close examination of x-file headers may reveal abnormalities in e.g.
crystal orientation -- suspecting an unlocked or drifting goniostat. It may
also indicate a precession around the phi, which should also manifest itself
in a systematic deviation of average intensities (i.e. scale factors) in a
similar pattern (assuming uneven illumination of the crystal). Sometimes the
precession is caused by a bubble or a tiny chunk of ice trapped under the
pin, it can melt unevenly and re-align the pin a few minutes into the run
(something similar used to happen a lot at one or two beam lines and it
drove me nuts until I figured out the need to re-align the crystal after the
initial screening).
 

Artem


On Wed, Jun 22, 2011 at 11:22 AM, bie gao gao...@gmail.com wrote:


Dear Colleagues,

I'm collecting a dataset on our recently repaired Rigaku home source. 
Crystal diffracts to 2.2A. Indexing seems to be all fine. However, during
integration, I realize Y-Chi2 is increasing constantly (from 2 to 4.5,
almost linear) within 60 degree collection, whereas X-Chi2 stays the same.
An image is attached. There are still another 60 degree to go. Although the
prediction fits the images well so far, I'm afraid the Y-Chi2 will
eventually run out of the chart. 
My question is could it be related to any hardware malfunctioning, i.e.,
goniometer, image plates, etc, which may be a side effect of the recent
major repair? Or what else it can be?

Thanks,
Bing

Re: [ccp4bb] Y-Chi2 running out of chart

[Jim Pflugrath]

If you make your images available via ftp, I can take a closer look and come
up with plausible explanations and fixes.

Did you lock down the phi-axis?

  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of bie
gao
Sent: Wednesday, June 22, 2011 11:23 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Y-Chi2 running out of chart


Dear Colleagues,

I'm collecting a dataset on our recently repaired Rigaku home source. 
Crystal diffracts to 2.2A. Indexing seems to be all fine. However, during
integration, I realize Y-Chi2 is increasing constantly (from 2 to 4.5,
almost linear) within 60 degree collection, whereas X-Chi2 stays the same.
An image is attached. There are still another 60 degree to go. Although the
prediction fits the images well so far, I'm afraid the Y-Chi2 will
eventually run out of the chart. 
My question is could it be related to any hardware malfunctioning, i.e.,
goniometer, image plates, etc, which may be a side effect of the recent
major repair? Or what else it can be?

Thanks,
Bing

[ccp4bb] CSHL X-ray Methods in Structural Biology Course late Oct 2011: Application deadline June 15th

[Jim Pflugrath]

I wanted to draw everyone's attention to the Cold Spring Harbor Laboratory
2011 X-ray Methods in Structural Biology course which will take place
October 17 through November 1, 2011.

The official course announcement is here:
http://meetings.cshl.edu/courses/c-crys11.shtml Astute viewers of that link
will also note that A Special Symposium Celebrating the 40th Anniversary of
the Protein Data Bank will be included this year's course.

I think the course is an outstanding place to learn both the theoretical and
practical aspects of Macromolecular Crystallography because of the extensive
lectures from world-renowned teachers and the hands-on experiments.

A good description of the course is found at the web link above as well as
in the 3rd edition of Gales Rhodes Crystallography Made Crystal Clear
where he writes on page xvii:

... the Cold Spring Harbor course who, for sixteen years, have offered what
many crystallographers tout as the best classroom and hands-on diffraction
training session on the planet -- 2.5 weeks, 9 AM to 9 PM, packed with labs,
lectures, and computer tutorials, with homework for your spare time.

If any former participants in the course want to add to that description,
please feel free to respond to this thread.

Thanks! Jim

Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo

[Jim Pflugrath]

I always try sugars for everything.  There is a cryocrystallography webinar
at rigaku.com with embedded videos on how to do this.  50% to 100% saturated
sugar (sucrose, glucose, trehalose, sorbitol, et al.) in reservoir buffer is
usually what I try. :)

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Chris
Ulens
Sent: Thursday, May 26, 2011 5:27 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] recommendation for ammonium dihydrogen phosphate cryo

Hi,
I would like to get recommendations for a proper cryo solution for a
crystallization hit from the Hampton crystal screen Ammonium di- hydrogen
phosphate 0.4M. I tried increasing glycerol up to 30% with the same ammonium
phosphate concentration or increasing glycerol up to 30% in the presence of
1.3M ammonium phosphate. Both gave iced up drops (I only tried quick and
dirty tests by dipping a cover glass in liquid nitrogen).

Thank you.
-Chris

Re: [ccp4bb] Difficulty indexing diffraction, cell too small

[Jim Pflugrath]

Yes, reciprocal lattice coordinates are available for reflections with
d*TREK.
My colleague has also written a reciprocal lattice viewer which takes
image pixels and transforms them to a reciprocal lattice.  I'm sure others
have similar programs.

But in this modern internet age, I would say enlist the help of experts with
strange images.  I think that usually saves one from going down an
unproductive path.

Jim 

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Doug
Ohlendorf
Sent: Wednesday, May 18, 2011 11:00 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Difficulty indexing diffraction, cell too small

Related to manual indexing, 20 years ago I wrote a program to transform a
list of peaks from the original Xentronics detector to points in reciprocal
space. The peaks were written as a PDB file so the peaks (now waters) could
be displayed on any graphics program. It was very useful for seeing and
separating multiple lattices. Is there an option in MOSFLM or D*Trek to
output peaks in reciprocal x*, y*, z*?

Doug

Douglas H. Ohlendorf   Phone:
612-624-8436
Professor  FAX:
612-624-5121
Dept. of Biochemistry, Molecular Biology  Biophysics Twin Cities Campus,
University of Minnesota Lab web site:
http://biosci.cbs.umn.edu/bmbb/ohlen_lab/index.html


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Pete
Meyer
Sent: Wednesday, May 18, 2011 9:46 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Difficulty indexing diffraction, cell too small

There are a couple of tricks I know for dealing with datasets that are
problematic to index (ignore if you've tried them):

1. double-check the machine parameters (detector to crystal distance and
beam center in particular).

2. Index in mosflm using widely separated images (usually n,n+45,n+90)
starting from a strong zone.

3. In really problematic cases, I've sometimes had to resort to manual spot
picking (thankfully not for many years).

Good luck,

Pete

Jason Busby wrote:
 Hi,
 
 I have a diffraction data-set from a hexagonal rod shaped crystal, to
about 2.0 Å.  The problem comes when I try to process the data - Mosflm
won't index it, and XDS indexes it as P622, but the unit cell is too small
to contain even a single molecule of my protein.  I have tried integrating
it in some different space groups that XDS suggested (P2, C2, P1) but in all
cases the Rmerge and Rmeas are worse than for P622.  
 
 If I scale in P622 (or any of the other space groups) I get odd 
 results
from the twinning tests.  For example, the 4th moment of E (expected values
of 2 for untwinned, 1.5 for perfect twin) is around 1.1, and the L-test and
cumulative intensity distribution are unusual as well (uploaded images here:
http://tinypic.com/r/65rfr7/7 and here:  http://tinypic.com/r/30adgyr/7 )
 
 Has anyone else had similar issues?  Any ideas would be appreciated.
 
 Thanks,
 
 Jason.
 
 --
 Jason Busby
 PhD Student
 Laboratory of Structural Biology
 School of Biological Sciences
 University of Auckland
 Thomas Building 110
 3a Symonds St
 Private Bag 92019
 Auckland  1142
 New Zealand
 
 ph:  +64 9 3737599 ext 83888
 fx:  +64 9 3737414

Re: [ccp4bb] Difficulty indexing diffraction, cell too small

[Jim Pflugrath]

In general, the Rmerge and Rmeas should get better with a lower symmetry
spacegroup, so that's weird.

Maybe you didn't crystallize what you thought you crystallized.  Do people
runs gels anymore on crystals to get an idea of what's in the crystal or do
they do MassSpec?

I think another way to go at this is to make images available and have folks
try to process your data for you.  That's starting to become more common
nowadays.

Jim

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jason
Busby
Sent: Tuesday, May 17, 2011 4:44 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Difficulty indexing diffraction, cell too small

Hi,

I have a diffraction data-set from a hexagonal rod shaped crystal, to about
2.0 Å.  The problem comes when I try to process the data - Mosflm won't
index it, and XDS indexes it as P622, but the unit cell is too small to
contain even a single molecule of my protein.  I have tried integrating it
in some different space groups that XDS suggested (P2, C2, P1) but in all
cases the Rmerge and Rmeas are worse than for P622.  

If I scale in P622 (or any of the other space groups) I get odd results from
the twinning tests.  For example, the 4th moment of E (expected values of 2
for untwinned, 1.5 for perfect twin) is around 1.1, and the L-test and
cumulative intensity distribution are unusual as well (uploaded images here:
http://tinypic.com/r/65rfr7/7 and here:  http://tinypic.com/r/30adgyr/7 )

Has anyone else had similar issues?  Any ideas would be appreciated.

Thanks,

Jason.

--
Jason Busby
PhD Student
Laboratory of Structural Biology
School of Biological Sciences
University of Auckland
Thomas Building 110
3a Symonds St
Private Bag 92019
Auckland  1142
New Zealand

ph:  +64 9 3737599 ext 83888
fx:  +64 9 3737414

[ccp4bb] 250 years of Bayes' Theorem: Link

[Jim Pflugrath]

This link should be helpful to many folks here:

http://blog.revolutionanalytics.com/2011/04/250-years-of-bayes-theorem.html

Re: [ccp4bb] Reproducing crystals.

[Jim Pflugrath]

Frances Jurnak published a paper in 1986 on PEG impurities and purification.


As I recall, it turns out that different manufacturers put different
additives in PEGs as preservatives.  These are generally anti-oxidants.
PEGs do get oxidized.

I suggest you heat up your new PEG solutions to say 80 deg C and cool them
down, then use them.  Let us know what happens.

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Jun
Yong Ha
Sent: Tuesday, April 12, 2011 6:57 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Reproducing crystals.

Hi all,

Recently, I produced crystals with MBClass1-64 which contains PEG4000,
HEPES-Na and NaCl. But, I struggled to reproduce crystals. I tried to set up
tray with different batch of solution. I got the crystals only from 2008
solution, but not from fresh ones. I asked technical service of Qiagen, but
they did not have any stock.

pH between fresh and old solution is the same. I could reproduce crystals
with this old solution 100% when setting up.

Do you have any experience like this? Is PEG4000 degraded or oxidized?

Please help me.

Thanks in advance.

Re: [ccp4bb] metal binds?

[Jim Pflugrath]

Collect all your diffraction data in your home lab, solve the phase problem,
fit the map, refine, deposit coordinates in the PDB.

  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Careina Edgooms
Sent: Friday, March 25, 2011 2:40 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] metal binds?


Dear ccp4 users

I would like to know, is there a way to check that heavy metal has bound to
crystals before I take it to synchrotron which is far away?

thanks
Careina

Re: [ccp4bb] glycerol

[Jim Pflugrath]

Glycerol is just another additive to crystallizations and a reasonably good
cryoprotectant.  Sometimes it helps to grow crystals, sometimes it has no
effect, and sometimes it interferes with crystal growth.  Have I covered all
the possiblities?
 
One thing is the glycerol often makes a protein more soluble, so one will
often need a higher protein concentration or higher precipitant
concentration in order to get crystals.  If someone tells me that glycerol
prevented crystal growth, I always ask them if they increased the protein
concentration or precipitant concentration or if they just used their old
recipe.  That is a revealing question.
 
Also note that ethylene glycol has a similar effect, yet is sometimes very
different.
 
Furthermore, these compounds can bind in active sites and elsewhere on
proteins and interfere with assays and other stuff.  Nevertheless, they are
always one of the first additives to try.

  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Ray
Brown
Sent: Thursday, March 10, 2011 4:05 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] glycerol


Hi all,
 
I was intrigued by the recent question of whether glycerol had any adverse
effects on the final purity of protein isolated by chromatography. Glycerol
certainly helps to solubilize some proteins. Does anyone know of any
negative effects of glycerol in protein purification, on protein crystal
quality or use in cryocrystallography and on X-ray diffraction results?
 
Cheers.
 
Ray Brown 

Re: [ccp4bb] I/sigmaI of 3.0 rule

[Jim Pflugrath]

As mentioned there is no I/sigmaI rule.  Also you need to specify (and
correctly calculate) I/sigmaI and not I/sigmaI.

A review of similar articles in the same journal will show what is typical
for the journal.  I think you will find that the I/sigmaI cutoff varies.
This information can be used in your response to the reviewer as in, A
review of actual published articles in the Journal shows that 75% (60 out of
80) used an I/sigmaI cutoff of 2 for the resolution of the diffraction
data used in refinement.  We respectfully believe that our cutoff of 2
should be acceptable. 

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Roberto Battistutta
Sent: Thursday, March 03, 2011 5:30 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] I/sigmaI of 3.0 rule

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] while on the subject of stereo

[Jim Pflugrath]

I will offer my view.

I hate stereo glasses and hate stereo in general.  

One should be able to see 3D from the depth-cueing and by keeping the view
in motion.  For fitting, I like to flip the view by 90 degrees.  I know I am
going to move in displayX and displayY, but never in displayZ.  I then
rotate the view around the vertical axis so thatn the old displayZ becomes
displayX.

Furthermore, I don't waste too much time fitting.  I know the software can
fit the map better than me, so I let it do its job.  I only need to get the
coordinates within the radius of convergence of the refinement program.  I
also know that 9 times out of 10, the displayed electron density is probably
suspect, so I believe in stereochemistry more than I believe in the map.

The main trick is to realize that as a human being, you really are not that
good at fitting the map or that it is unnecessary to waste your time since
the software is really so much better than you.  Refinement is quick enough
that you can try various hypotheses as in:  If I move this here, then
refinement will do the trick and Well, that didn't work, so I will move
that over there and see if refinement will do the trick.

As for stereo figures, you should be able to convey what you want to say
from a good figure with depth-cueing, shadows, etc.  Don't ever use stereo
glasses in a public seminar.  Maybe my opinion will change with better
stereo technology.

OK, I know quite a lot of people will disagree with me. :)

Jim

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of David
Roberts
Sent: Tuesday, March 01, 2011 10:29 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] while on the subject of stereo

Hi again,

I'd like to ask a question about the pedagogy of stereo.  That is, using
stereo with students in the classroom.

Do you all find that, after setting up these elaborate stereo devices,
students really use the stereo or do they tend not to?

I am a huge fan of stereo - and frankly here we have quite a few options for
doing stereo - from the active Nvidia systems that people have recently been
discussing to passive zalmans. ...

As I mentioned, I like stereo a lot, but really projecting on a nice bright
lcd monitor also has it's advantages, and with the ease of moving things
using the mouse (or whatever device you use), the overall need for stereo
seems to be decreasing.  I don't know - I just wonder what peoples views are
out there for the actual need for stereo.  It's incredibly cool - and I
think is a very powerful way to show things - but I'm wondering if we focus
too much on it because it's cool and not because it's pedagogically
necessary.

Just wondering, no worries.  Thanks

Dave

Re: [ccp4bb] Detaching crystals from glass cover slides

[Jim Pflugrath]

Cool the coverslip on the opposite side of the crystal with a chip of dry 
ice.  Do not freeze the drop.  I learned this from Gary Gilliland.  Also I 
wonder if you can simply move the whole tray into a cooler temperature?


You can imagine that the thermal expansion coefficient of the glass 
coverslip and the crystal are different.  I have not tried heating, but 
maybe that works, too?


Or bend the coverslip without breaking is by applying pressure from the 
opposite side.


Jim


...
Any suggestions for detaching crystals from cover slides will be 

greatly

appreciated.

Wataru

Re: [ccp4bb] Merging data to increase multiplicity

[Jim Pflugrath]

You should know that your crystal mosaicity is a physical property of your
crystals and the diffraction experiment.  Generally, it is anisotropic
though most programs output a single value.  How can that single value
describe what is really happening in your experimental data?
 
You can do anything you want to with the processing programs.  
You can fix mosaicity to any value you want.   
You can restrict it to a small value to lie to the program that your spots
are not overlapped.  This should help completeness and redundancy while
perhaps degrading accuracy.
Will that help you solve the structure?  Will that help to find the
anomalous substructure?  WIll that help to get an initial map for chain
tracing?
Will you get a better Rfree if you use data that is merged from several
crystals?
Will you get a better Rfree if you mix and match different mosaicities when
processing the diffraction images from different crystals?
 
These are all hypotheses that you can test.  I am not sure how to test these
hypotheses by querying the internet.
 
  _  

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Anastassis Perrakis
Sent: Friday, January 28, 2011 8:11 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Merging data to increase multiplicity
 

... but, back to the main point, my advice would be to only limit the
mosaicity, to get better completeness by avoiding overlaps. 
Its not ideal, in the sense that you would be over-estimating the partial
fraction of most partial reflections, and thus systematically
underestimating intensities.
(I hope I got my overs and unders right here ...)

But these errors would not matter much for refinement purposes, where you
would rather have a slightly systematically wrong estimate
for all data, rather than not have the 15% of the data at all. 

Or at least thats what I thought back in '99 refining MutS ... where I did
refine a lot with both datasets and liked the 'fixed mosaicity' one better.

A.


On Jan 28, 2011, at 13:26, José Trincão wrote:


Ah, yes, I was missing that. The statistics will be wrong. But in principle
I will get an mtz with better data, because I am integrating more
observations which would have been rejected by being missed at low
resolution if the mosaicity was set too low or being rejected by overlaps at
high resolution if the mosaicity is increased.
So the question is - can I use this data for refinement? Or should I stick
with the best of the datasets (the one with the highest completeness and
multiplicity)?

Thanks!

Jose

On Jan 28, 2011, at 28/1/11 - 11:59, Ian Tickle wrote:

Jose - you're missing the fact that the same dataset processed in
different ways are not statistically independent datasets!  Increasing
the multiplicity for independent data reduces the uncertainty because
the calculation of the SU assumes statistical independence.

Cheers

-- Ian

On Fri, Jan 28, 2011 at 11:46 AM, José Trincão trin...@dq.fct.unl.pt
wrote:


Hello all,


I have been trying to squeeze the most out of a bad data set (P1,
anisotropic, crystals not reproducible). I had very incomplete data due to
high mosaicity and lots of overlaps. The completeness was about 80% overall
to ~3A. Yesterday I noticed that I could process the data much better fixing
the mosaicity to 0.5 in imosflm. I got about 95% complete up to 2.5A but
with a multiplicity of 1.7. I tried to integrate the same data fixing the
mosaicity at different values ranging from 0.2 to 0.6 and saw the trend in
completeness, Rmerge and multiplicity.


Now, is there any reason why I should not just merge all these together and
feed them to scala in order to increase multiplicity?


Am I missing something?



Thanks for any comments!



Jose




José Trincão, 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






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

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



P please don't print this e-mail unless you really need to
Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member
Department of Biochemistry (B8)
Netherlands Cancer Institute, 
Dept. B8, 1066 CX Amsterdam, The Netherlands
Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791

Re: [ccp4bb] Heavy atom salt at low pH

[Jim Pflugrath]

Crystals of tendamistat were grown from hydrochloric acid and solved by MIR.

I do not recall anything special about the heavy atom soaks, so try
everything in your heavy atom closet.
What have you tried that has not worked?
 
  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Debajyoti Dutta
Sent: Wednesday, November 17, 2010 3:32 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Heavy atom salt at low pH



Hi All,

Sorry for a non CCP4 question. I have been trying to phase a protein
structure using different heavy atom derivatives. The problem is the
crystallization pH is very low (from 2.8 to 3.5). I will be highly benefited
if anybody kindly suggests me the possible heavy atom salts to try

sincerely
Debajyoti

Re: [ccp4bb] how to optimize small rod-shaped crystals

[Jim Pflugrath]

With Izit or other dyes, you might wish to do a positive control with bona
fide protein crystals and a negative control with bona fide salt crystals.
 
  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Matthew Bratkowski
Sent: Tuesday, November 16, 2010 7:58 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] how to optimize small rod-shaped crystals


I like using Izit dye from Hampton
(http://hamptonresearch.com/product_detail.aspx?cid=4
http://hamptonresearch.com/product_detail.aspx?cid=4sid=41pid=33
sid=41pid=33) to check if crystals are protein or salt.  If the crystals
are protein, the dye should absorb rather readily into the crystals and turn
them blue, while the rest of the drop will eventually turn clear.  Quite
likely, excess dye will also crystallize out as well.  Salt crystals will
not soak in the dye, and the rest of the drop may remain blue for several
days. 

Using Izit is easy and saves a lot of time.  In my experience, I have gotten
a lot of false positives from phosphate crystallization conditions, so you
want to be sure that the crystals are not salt before you waste any time on
optimizing them.

Matt

 

Re: [ccp4bb] High Rmerge with thin frames

[Jim Pflugrath]

In general, if the Rmeas or Rmerge is high in the low resolution shells,
then something is not optimal with the data collection.

Bill Shepard has already mentioned the loop vibrating or moving in the
cryogenic gas flow.  Other problems could be the goniometer head was loose,
the magnet was loose, the pin was loose, etc.  There could be excessive
shutter shutter.  The shutter and the crystal rotation could be poorly
synchronized.  There could be some other vibration in the system which could
cause the X-ray flux to vary quite a bit.  It could be as simple as the
cycling of the cooling for the monochromator, the room or hutch, or the
X-ray source itself.  All of these would affect non-thin-images as wells.

As already mentioned, combining thin-images into wider images will not
overcome these problems.

If Rmerge or Rmeas was 40% or more in the low resolution shells, then the
diffraction pattern is probably mis-indexed, but that kind of high value was
not reported here.

OTOH, if The diffraction is quite weak, one may be limited by counting
statistics.  This also cannot be overcome by processing.

Jim

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Sergei
Strelkov
Sent: Friday, November 05, 2010 3:41 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] High Rmerge with thin frames

Dear All,

I am processing a dataset collected (not by me) with 0.1 degree
oscillations.
The diffraction is quite weak even though there is a clean diffraction
pattern to about 3A.
...

Re: [ccp4bb] Crystal gel band

[Jim Pflugrath]

It reads like you need to run a lane or two with a positive control of some
kind.  Can you grow lysozyme, glucose isomerase, hemoglobin or other
crystals of a protein around the same expected molecular weight and try run
on the gel lanes with about the same amount of crystalline volume as your
putative protein crystals?

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
xaravich ivan
Sent: Monday, November 01, 2010 9:51 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystal gel band


Hi everyone,
I have grown some crystals after micro-seeding starting from thin-small
needles from needle-clusters. These crystals are larger in size than the
needles but are comparable to the shape and don't look like salt crystals.
But I cannot see the bands( its a complex) in the SDS-PAGE.I do not have a
home source,handy and would like to send these to the synchrotron.

Is it possible to NOT see a band of protein crystals in SDS-PAGE, if, say,
the amount of protein is  1uG? 
Has anyone experienced such a thing (no band in gel, but crystal diffracts)?

It would be nice if I get observations/suggestions.

ivan

Re: [ccp4bb] Strange spots

[Jim Pflugrath]

Are these very strong reflections?  Do they appear on more than one image?

Are they an artefact of the detector or the image display program?

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

[Jim Pflugrath]

Zbyszek, 

Since you mention I/sigmaI in your PDF, do you mean I/sigmaI or
I/sigmaI?  
Do you mean I/sigmaI (in whatever rendition you choose) for the averaged
unique reflections or the I/sigmaI for the observations?
Also since one can adjust sigmaI in your scalepack program through the use
of the Error Scale Factor or the Error Model, how can a reviewer believe any
of the I/sigmaI that are reported by authors?

Thanks for any insights into these questions, 

Jim

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Zbyszek Otwinowski
Sent: Thursday, October 28, 2010 7:41 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)

Feel free to use it as you wish.

--

DUFF, Anthony wrote:
 I reckon you could share hypothetical review comments for educational 
 purposes.
 
 
 -Original Message-
 From: CCP4 bulletin board on behalf of Bernhard Rupp (Hofkristallrat 
 a.D.)
 Sent: Thu 10/28/2010 12:22 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: Re: [ccp4bb] Rules of thumb (was diverging Rcryst and Rfree)
 
 Why not double open review? If I have something reasonable to say, I 
 should be able to sign it. Particularly if the publicly purported 
 point of review is to make the manuscript better.  And imagine what 
 wonderful open hostility we would enjoy instead of all these hidden 
 grudges! You would never have to preemptively condemn a paper on 
 grounds of suspicion that it is from someone who might have reviewed 
 you equally loathful earlier. You actually know that you are creaming the
right bastard!
 
 A more serious question for the editors amongst us: Can I publish 
 review comments or are they covered under some confidentiality rule? 
 Some of these gems are quite worthy public entertainment.
 
 Best, BR
 


--
Zbyszek Otwinowski
UT Southwestern Medical Center  
5323 Harry Hines Blvd., Dallas, TX 75390-8816
(214) 645 6385 (phone) (214) 645 6353 (fax) zbys...@work.swmed.edu

Re: [ccp4bb] Is the Rmerge invalidate by twinned data?

[Jim Pflugrath]

It may be time for our annual I/sigmaI discussion.

Please note that I/sigmaI is what the RCSB expects from you and it is 
generally lower than I/sigmaI.  Some packages do not output I/sigmaI 
in an obvious place for you to put in your Table 1.  :)



On Wed, 6 Oct 2010, Ed Pozharski wrote:


You don't need twinning to invalidate the Rmerge as a criterion for the
resolution cutoff, there are other reasons why you should use I/sigma
instead.  If you process data all  the way to 3A, what's the I/sigma in
the highest resolution shell?

On Wed, 2010-10-06 at 11:28 +0200, fulvio saccoccia wrote:

Dear all,
I have a data set collected at 3A resolution. I processed the data but I
had to cut the resolution at 3.6A for the high value of Rmerge (at 3.6A
it is 0.18). After scaling and MR I realized that my data were twinned.
This is my question: can I  reprocess all the data set using all the
reflections up to 3A resolution even if the Rmerge is very high, knowing
that data are twinned? and also, is the Rmerge invalidate by the twinning?

Re: [ccp4bb] List of commonly used cryoprotectants and buffer molecules

[Jim Pflugrath]

This cryocrystallography webinar lists some common cryoprotectants:
http://www.rigaku.com/protein/webinar-001.html  

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Chris
Weichenberger
Sent: Thursday, September 09, 2010 7:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] List of commonly used cryoprotectants and buffer molecules

Dear All,

I am trying to find out which molecules are frequently used by X-ray
crystallographers serving as cryoprotectants or as buffer molecules. The
idea behind this is to sort native ligands from molecules that appeared in
the electron density just because they were used in the crystallization
buffer or as a cryoprotectant. Can anybody point out literature, a web site,
or simply provide a (subjective) list extending my collection of GOL, EDO,
and different size PEGs? What about sugars?

Thanks for your help,

Chris

Re: [ccp4bb] Crystallization of low solubility proteins from glycerol-containing solutions

[Jim Pflugrath]

Have you tried to use glycerol or ethylene glycol as the precipitant?  What
happens when you go to 50% or higher concentrations?

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Roger
Rowlett
Sent: Wednesday, August 25, 2010 9:19 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Crystallization of low solubility proteins from
glycerol-containing solutions


Does anyone have practical experience crystallizing low solubility proteins
from solutions containing significant (10-20%) glycerol? 

Re: [ccp4bb] Another scaling question

[Jim Pflugrath]

It might be instructive to draw a precession-like diagram of your
reflections in reciprocal space.  Remember that reciprocal space dimensions
are generally in reciprocal Angstrom and volumes raise those dimensions to
the third power.  Thus (1/12)^3 to (1/15)^3 is not a big volume.

How many reflections should you have between 15 Angstrom and 12 Angstrom?
Between 12 Angstrom and 4 Angstrom?

Suppose only 4 unique reflections exist between 12 and 15 Angstrom. What
happens to your completeness if you are missing just one of them?


-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Simon
Kolstoe
Sent: Thursday, June 24, 2010 10:05 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Another scaling question

Dear CCP4bb,

I am still playing around scaling two datasets together and have noticed
another interesting behavior in scala. If I scale all my data (from 1.5A to
51A) I get 100% completeness in my outer shell, 98% in my inner shell and
99.9% overall, stats that I am normally quite happy with. I tend to also
look at the table in the log file which in this case reports above 98%
completeness in all shells between 1.5 and 4.7A. Rmerges are 0.054 overall
with 0.29 in the outer shell, which again I think is OK.

However I then ran scala again in an attempt to scale with the strongest
overlapping reflections in my two datasets, so limited the resolution to
between 15A and 4A. Now when I look at my completeness I get 97% overall,
but only 32% in the highest 15-12A shell! Is something funny going on in the
program or am I really missing 70% of my data in this resolution, and if so
how come the scala run with all the data doesn't report this? I am now
worrying that all the data I previously thought was complete might be
lacking many lower resolution reflection!

Thanks,

Simon

Re: [ccp4bb] Processing compressed diffraction images?

[Jim Pflugrath]

d*TREK will process compressed images with the following extensions: .gz
.bz2 .Z .pck and .cbf
 

-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Ian
Tickle
Sent: Thursday, May 06, 2010 6:25 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Processing compressed diffraction images?

All -

No doubt this topic has come up before on the BB: I'd like to ask about the
current capabilities of the various integration programs (in practice we use
only MOSFLM  XDS) for reading compressed diffraction images from
synchrotrons.  A...

cluster: the disk I/O is the bottleneck.  Now you could argue that we should
spread the load over more disks or maybe spend more on faster disk
controllers, but the whole point about disks is they're cheap, we don't need
the extra I/O bandwidth for anything else, and you shouldn't need to spend a
fortune, particularly if there are ways of making the software more
efficient, which after all will benefit everyone.

Cheers

-- Ian

[ccp4bb] Help with Bayes's theorem

[Jim Pflugrath]

I thought some of you would enjoy a little conditional probability
discussion found in the NY Times today, since this is a big part of
crystallography nowadays.  I'm always on the lookout for good ways to teach
Bayes's theorem.

http://opinionator.blogs.nytimes.com/2010/04/25/chances-are/

Jim 

Re: [ccp4bb] Substitute for Lithium Sulfate

[Jim Pflugrath]

I would test several hypotheses.  You have a multitude of salts to choose
from.  Test them all.
Let me ask you this:  What anions can you test?   What's already on your
shelf of chemicals?  What did you test already?
 
Jim

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
Yi-Liang Liu
Sent: Friday, April 23, 2010 12:53 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Substitute for Lithium Sulfate


Hi Everyone,


I have a question about the crystallization condition. Currently the
condition I use contains 0.2M lithium sulfate. However, the ligand there
seems to compete the site where sulfate binds in the active site. Are there
any good substitute to replace the Lithium sulfate in my crystallization
buffer?

Best,

Lucas

Re: [ccp4bb] Rsym problems...maybe???

[Jim Pflugrath]

Yeah, but how was I/sdI determined?  Most programs allow you to multiply 
your sdI by any number you want which in turns means that you can create 
any I/sdI that you want.


A multiplicity of 11 does not explain a high Rsym to me.

Jim

On Thu, 22 Apr 2010, Frank von Delft wrote:


Yeah, stop worrying!  Your I/sdI is all that matters.
phx


On 22/04/2010 18:06, Daniel Bonsor wrote:

Hello again.

At first I was not worry but maybe now I am. I have completed a structure 
and submitted to the PDB. They queried my Rsym value in the highest 
resolution bin, 2.5-2.37A (may I dare say it 100%). I was not worried at 
the time as I had:


99.4% completeness
Mean(I/sdI) of 2.5
and a redundancy of 11 (which would explain the high Rsym)
Space group I422

My Rpim in this shell is 30%.

Should I reduce the resolution and start from scratch again or is 
everything fine and dandy and I should stop worrying?

Re: [ccp4bb] phosphate v sulfate

[Jim Pflugrath]

One might also check the pH of your crystal and the number of hydrogen bond
donors from the putative sulfate (zero?) or the putative phosphate
(non-zero).
I'm alluding to the structures of the sulfate-binding protein and the
phosphate-binding protein from Quiocho's group.
 
Jim

  _  

From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of
wtempel
Sent: Saturday, April 10, 2010 9:49 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] phosphate v sulfate


Dear colleagues, 
I would like to poll the community on the prevailing practice of
distinguishing between phosphate and sulfate in their structures.
Suppose all of the following apply:
1) a well contoured tetrahedral density in your model-phased 2Fo-Fc map in
the active site of your kinase or GTPase protein.
2) a significant, say 5*sigma, coincident peak in your model-phased
anomalous difference fourier map from data collected at a Cu rotating anode
source.
3) The crystal grew in 2M ammonium sulfate.
Please post your answers to the list if you feel this question is of general
interest.*
Thank you for your input.
Wolfram Tempel

 *If it is not, I apologize to everyone for wasting their valuable time.