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://i39.tinypic.com/oic17r.jpg (Trehalose was
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
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
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
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
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Edward A. Berry
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
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
The current version of scalepack already calculates CC1/2 and reports it in the
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Francesco
Sent: Wednesday, September 18, 2013 5:21 AM
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
... Rpim is much more instructive. ... as each of these tells something
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
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
And quick iodide soaks may be useful in the 200 mM range. See the sped-up
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
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
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
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
Thanks for the clarification. I thought at first this position might be
related to Dance Your PhD.
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of
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
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
Further to what Nat wrote which I completely agree with, you should tell us the
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
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
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
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.
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Bernhard Rupp
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
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
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
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
The number of observations or reflections that come from an integration
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
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
Sent: Thursday, May 03, 2012
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
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.
As with all reagents used in the lab it is best to understand the health
hazards. Thanks for note.
From: Jacob Keller [j-kell...@fsm.northwestern.edu]
Sent: Monday, May 21, 2012 5:22 PM
To: Jim Pflugrath
Subject: Re: [ccp4bb
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:
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.
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Carter, Charlie
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
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).
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,
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
Sent: Tuesday, October 30, 2012 1:55 PM
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
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
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
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:
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
Sent: Friday, November 30, 2012 7:22 PM
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.
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
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
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.
Maybe you crystallized something else? Did you look up that unit cell in the
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.
Although the peak height of S atoms can be used as an internal yardstick,
one has to worry about differences in occupancy and possibly hetergeneous
sites (i.e. Ca, Mn and Mg) which can confuse the interpretation of the
On Mon, 16 Apr 2007, Eleanor Dodson wrote:
In cases like this I
d*TREK from Rigaku can process these images.
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Sent: Wednesday, May 16, 2007 2:21 AM
Subject: [ccp4bb] .sfrm image files
I have image files with the format
X-ray Methods in Structural Biology
Cold Spring Harbor Laboratory
October 15th-30th, 2007
Deadline for applications: June 15th
This year marks the 20th time this course has been taught at Cold Spring
Harbor Laboratory. It is supported with funds
I just wanted to re-post this in case you know someone who wants some
travel money. Jim
Rigaku Americas Corporation is pleased to announce the availability of five
(5) awards of $500 each to post-doctoral research fellows to help with the
costs of travel to upcoming summer meetings such as
What is the mosaicity of the unfrozen crystal?
From: Mary Fitzgerald [EMAIL PROTECTED]
Date: Mon, 9 Jul 2007 18:05:10
Subject: [ccp4bb] Help with reducing crystal mosaicity
I'm looking for some new ideas. I have
Is there any advantage to bzip2-ing the individual images rather than
making one bzip2-ed tarball with tar cvfj?
Yes. If you have folks sending you images by sftp or e-mail, then you
don't have ensure a tarball of Giga or Tera bytes works. You can send the
smaller multiple files and restart
It probably goes back to the days of using a single-counter diffractometer
where one didn't have multiple Bragg reflections on an image or film pack.
That is, each reflection was collected by itself. Even in a small molecule
crystal data collection nowadays, it would not hurt to have the crystal
Folks, I wanted to call your attention to the following position(s)
available at Rigaku.
Rigaku, the world's largest analytical X-ray company,
is seeking experienced scientific programmers to join
well-recognized Rigaku scientists such as Jim Pflugrath
Bong angels are probably ideal already!
Please explain, so that I can teach this to my students. :) Jim
Did you grow Malic Acid crystals?
I see no reason why glycerol cannot be the precipitant. I am aware that
at least one protein is crystallized with ethylene glycol as the
On Wed, 9 Jan 2008, JINJIN ZHANG wrote:
I have got large and nice crystals in a condition
Is there any way to do a search of crystal orientation matrices with a known
cell to find the best fit to the diffraction pattern? The data were collected
on the Pilatus6M detector so I am limited to mosflm and XDS for processing.
d*TREK will easily process Pilatus6M images.
There are many excellent review articles about cryocrystallography and
cryoprotectants. Do labs generally have these articles handy in a methods
folder? Do lab heads help their colleagues by making them read them?
Mineral oil is generally not a good oil to use because it changes volume
I received a lot of positive e-mails about my post on cryo
methods. One theme was, Well, what are the papers I should read?
I know of one such paper published in Methods. I have made the PDF of it
downloadable from our web site at
I would use all the data myself and report that the model was built from a
a dataset with 74% completeness in the 2.25 to 2.15 Anngstrom shell. I
would not put the number 2.15 A in the manuscript title nor in the poster
For me the acceptable completeness is 90% in the highest
That's an easy hypothesis to test. Simply set up your drops with the same
conditions except without protein and see if you get crystals. Please let
us know the results. Thanks!
On Tue, 29 Apr 2008, Sam Stephenson wrote:
Are calcium citrate crystals a common false positive in trays
From: Jim Pflugrath [EMAIL PROTECTED]
Subject: 2008 Rigaku Post-doc Summer Travel Bursary Awards
Rigaku Americas Corporation is pleased to announce the availability of five (5)
awards of $500 each to post-doctoral research fellows to help with the costs of
travel to upcoming summer meetings
Have you thought about phasing off the sulfurs? This is quite a common
On Tue, 27 May 2008, Joe Smith wrote:
Sorry for an off-topic query.
I have been unable to crystallize a Se-met containing protein (8 Met
in 206 amino acids) in the native crystallization
I would like to point out that flash-cooling in liquid propane has the
added complication that the liquid propane can have a range of temperature
and still be liquid. If you use propane you may not know which
temperature you are actually using. The temperature in the exposed layer
Folks, sorry for the belated announcement, but I noticed the application
deadline is creeping up on us. Jim
X-ray Methods in Structural Biology
Cold Spring Harbor Laboratory
October 13th-28th, 2008
Deadline for applications: June 15th
Whatever dye(s) you use, be sure to run some positive and negative
controls to see how the dye really works.
On Sat, 14 Jun 2008, Mark Del Campo wrote:
Before I place an order for some Izit, are there some other dyes I can
use to check if I've got a protein crystal?
I've been in that cold room / hutch. I never heard of it being flushed
with LN2. I think that is just to make the room sound cooler.
On Thu, 19 Jun 2008, Mischa Machius wrote:
Sadly, I have never seen the room being used. Perhaps one of the 'older'
Martinsrieder on the forum has seen
How would you tell the difference between a unit cell shift and a wavelength
shift when collecting diffraction data at a synchrotron beamline? Well, all
the cell length would scale by the wavelength, so that would be one hint
that the wavelength changed. If a got longer and c got shorter, then
There is ALWAYS anomalous scattering. You do not have be at the
absorption edge to get it. The question is just whether your experiment
is good enough to detect it.
So your question of overlapping always has the answer Yes, but I would
remove the words absorption edge from your question.
A good cover -- judiciously used -- goes a very long way in reducing ice
in dewars whether the dewar is made of foam, glass or stainless steel.
If you keep your dewar covered with a lid that extends on the outside of
the dewar down a few centimeters below the dewar edge, you will have
Ian, are you saying that one might need to know some crystallographic
theory in order to determine space groups? Or can we simply click a few
buttons in a graphical user interface and be done with it?
On Mon, 15 Dec 2008, Ian Tickle wrote:
But that only means that the SF contribution
I wonder if the early use of the shortened structure amplitude is
because it was a pain to do any typing, word processing, typesetting, etc
But soon crystallographers will be solving all their structures on their
cell phones and also just text messaging manuscripts to
Put a small piece of dry ice on the opposite side of the plastic from the
crystal. Perhaps the difference in thermal expansive coefficient will let
the crystal(s) break away. Don't overdo it though. This is a trick that
Gary Gilliland taught me.
On Wed, 28 Jan 2009, Savvas Savvides
Well, what do you expect the anomalous signal contributed from your 45
seleniums in a perfect world to be when the asymmetric unit contains 1300
aa? Do you think a dataset with Rmerge of ~15% is good enough to detect a
signal of even 2%? (Note: I did not do the calculation, so I just made up
On Mon, 23 Mar 2009, Phil Evans wrote:
I'm happy to change the column titles if it makes it clearer. Actually the
I/sigma column in the Scala output is not very useful:
it is I / RMSscatter, ie the mean intensity/mean error, for individual
observations, not taking into account multiple
It's a bit early in the year to have our annual I/sigI discussion on the
CCP4BB, isn't it?
Usually, we wait until at least April 1. :-)
d*TREK could confuse people further as to which one should be used for
reporting and decision making.
The Big Question Again:
If I understand you correctly, you are writing about sucking a crystal up
into a small capillary. This is an ancient technique used with the very
first protein crystal diffraction data collection in the 1950s and 1960s
that is still used today. You can use a small 1 ml plastic syringe for the
That leaves the question of how to label these statistics in a consistent,
clear and concise way: suggestions?
Here is a suggestion. :)
FWIW, dtscaleaverage labels the mean I/sigmaI of the individual input
reflections as I/sig unavg. That is, before any averaging is
The cryoprotectant is 1,6 hexanediol.
On Thu, 2 Apr 2009, HanJie_Heng Chiat Tai wrote:
I have a crystal grown in 2.1M 1,6 hexanediol/0.1 M tro-sodium citrate (pH 6.5).
What's the cryoprotectant can be used to flash cool this crystal?
Any online protein crystal
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.
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
Sent: Wednesday, August 25, 2010 9:19 AM
This cryocrystallography webinar lists some common cryoprotectants:
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Chris
Sent: Thursday, September 09, 2010 7:34 PM
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:
Since you mention I/sigmaI in your PDF, do you mean I/sigmaI or
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
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?
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
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,
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
Sent: Tuesday, November 16, 2010 7:58 PM
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
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
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
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
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
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
One thing is the glycerol often makes a protein
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
Sent: Friday, March 25, 2011 2:40 AM
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
This link should be helpful to many folks here:
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
Yes, reciprocal lattice coordinates are available for reflections with
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
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