Re: [ccp4bb] Difficult to solve MR - A mysterious case
Chen,
The first of your diffraction patterns looks streaky and I'm
wondering whether your intensities carry a significant error due to
poor peak profiles. I know you used already two alternative data
processing programs, but I'd recommend to give XDS a try. When
dealing with poor peak profiles (for a subset of frames) I got
distinctly better results with the latter. You may need to start the
integration with frames that show good peak profiles. (I had to
renumber frames to get XDS to properly integrate the more streaky ones).
As XDS will integrate in P1, unless you choose otherwise, you can
easily test merging stats in a systematic fashion. If your true
symmetry group may be lower than P622, then it is definitely
worthwhile to try lower symmetry when running phaser.
Klaus
===
Klaus Fütterer, Ph.D.
Reader in Structural Biology
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E: k.futte...@bham.ac.uk
Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/
===
On 30 Jun 2010, at 05:50, Chen Guttman wrote:
Dear Colleagues,
Im on the bring of giving up on a very difficult and puzzling
structure and my last hope lies here with you folks...
Background:
Im working on a point mutated protein with a known w.t. structure.
This specific mutation have been crystallized in the form of plates
(C2221 SG) and hexagons (P622 SG). Whilst the plate form structure
was solved without of a hiccup, the later was a different story. I
am attaching two images of the obtained diffractions of the hexagon
crystal, 90 degrees apart (the 905 images were collected at a
synchrotron beamline with a delta phi of 0.2).
What I've done so far:
Diffracted images were indexed & integrated with iMosflm (used
pointless to asses the correct SG). This was later scaled in SCALA
to 2.4A with the attached statistics.
HKL2000 was also used but it couldn't lock on the P622 symmetry
(gave either C2 or P2 which fit to the first section of the data
set but didn't fit the 90 degree diffraction pattern); Splitting
the data into two data sets at the 90degree gave a P6 option but
once it finished the first 90 degree, it missed-fitting the 2nd
data set.
Scaled data was phased using Phaser with the known w.t. structure
which gave a single solution with the following statistics:
RFZ=7.6 TFZ=33.4 PAK=0 LLG=1316 LLG=1426 and with an initial R-
factor of 48.7
Looking at density map, I could distinguish areas which had
excellent fit while other areas demonstrated poor fit. The ligand,
which was soaked with the crystal and was also solved with the w.t.
protein, fitted almost perfectly with the identified positive
peaks. However, apparently there were also major changes (mainly
positive peaks) that could not be accounted for by the structural
restraints and were hard to figure out. These major changes, which
might as well be ghosts of a problematic data set, are the reason I
want to solve this structure
Rigid body, Restrained refinement and real space refinement didn't
improve Rwork/Rfree beyond 0.4/0.46.
I've also tried the following steps:
Used AMore and Molrep - didn't yield any improvement in density map
nor in the R factor statistics
Used Phenix Refine with different settings including simulated
annealing, didn't work either
Tried rebuilding using Autosol and generation of OMIT map - this
yielded some sort of improvement to 0.39/0.44 but still the map was
not drastically improved and there where areas which were hard to
fit with the current model.
Tried removing stretches of residues and check the map after
refinement - mostly i've noticed spots of positive peaks related to
the backbone.
I've also checked for wrong use of symmetry with several programs,
including Zanuda, and still the best SG is P622.
I've checked for twining with Phenix, CCP4 and web hosted programs
and no twining was detected (not even perfect twining).
So, I've pretty much hit a brick wall. I would be happy to hear any
suggestion to what might be the problem with this Data set.
Thanks for the help and time,
Chen
--
Chen Guttman
The Zarivach laboratory, Building 39, Room 009B
Ben-Gurion University of the Negev
POBox 653
Zip Code 84105
Beer-Sheva
Israel
http://lifeserv.bgu.ac.il/wb/zarivach
Tel. +972-8-6479519
Fax. +972-8-6472970
<45deg.jpg><135deg.jpg>
Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?
Marcus, May I ask the following: assuming 8 A is obtained from a single crystal on the home source, what diffraction limit would one expect on the PX scanner? Best regards, Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology Undergraduate Admissions School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ === On 30 Sep 2010, at 10:44, Marcus Winter wrote: This recent discussion does tend towards the ideal scenario: of identifying ones best-diffracting crystals... before embarking on the synchrotron trip. The established Oxford Diffraction PX Scanner home laboratory instrument can therefore be most useful. This enables the direct X-ray screening of individual (putative) single crystal objects, in situ, in the (any SBS format) crystallisation plate. Yours sincerely, Marcus Winter (Oxford Diffraction Ltd. – now Agilent Technologies) -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Phil Jeffrey Sent: 28 September 2010 19:20 To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron? Often this reflect crystal size - a small crystal in a big beam (or one with a long path in air) on a home source would see the small diffraction signal drop below the noise level quite quickly - often at the low resolution intensity dip that sits very approximately around 6 Angstrom. On a synchrotron source with a tight low-divergence beam that matches more closely the crystal dimensions that same crystal will appear to do rather better. Also one is more likely to expose the crystal longer (in terms of total photon numbers) at a synchrotron, which itself begets better signal/ noise. Alternatively: everyone tries harder before synchrotron trips Phil Jeffrey Princeton On 9/28/10 1:27 PM, Francis E Reyes wrote: > Hi all > > I'm interested in the scenario where crystals were screened at home and > gave lousy (say < 8-10A) but when illuminated with synchrotron radiation > gave reasonable diffraction ( > 3A) ? Why the discrepancy? > > Thanks > > F
Re: [ccp4bb] Off Topic: Web or e-tools for booking instrument time
We use Google Calendar for booking our X-ray facility. Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology Undergraduate Admissions School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 16 Feb 2011, at 12:25, Darren Hart wrote: Hello, Since we are now part of the "Facebook Generation" (or at least 10% of the planet is), it seems there must be a better way of distributing time on our various Aktas to an institute of scientists than our current system which involves a race for the paper booking sheet every Thursday morning. Inevitably people book in a speculative manner and time slots are not always used due to problems in preparative steps etc. Bits of paper in different buildings make it difficult to see who booked what and when. Has anyone successfully implemented an electronic system, perhaps shared through the "cloud", that allows real time booking in a manner that can be revised easily and has worked well? An electronic calendar (Google..) would partially address this, but it seems some sort of social networking site/tool might be better for negotiating instrument time, swapping timeslots, reallocating unused time if the sample prep fails. Any suggestions would be greatly appreciated. If governments can be toppled through Facebook, I'm sure we can book our Aktas in a better way! Darren -- ** Dr. Darren Hart, Team Leader High Throughput Protein Lab Grenoble Outstation European Molecular Biology Laboratory (EMBL) ** www.embl.fr/research/unit/hart/index.html For funded access to ESPRIT construct screening via EU FP7 PCUBE: http://tinyurl.com/ydnrwg4 Email: h...@embl.fr Tel: +33 4 76 20 77 68 Fax: +33 4 76 20 71 99 Skype: hartdarren Postal address: EMBL, 6 rue Jules Horowitz, BP181, 38042 Grenoble, Cedex 9, France **
Re: [ccp4bb] viewing and scoring diffraction using the PX Scanner
Dear Marcus,
I always feel a bit uneasy about the advertisement-like posts that
Agilent (and others) place on this BB. Of course, there are
interactions between users and suppliers on many fronts, not least the
support you guys provide in the form of sponsorship to meetings and
conferences.
Still, the original purpose of this bulletin board is the exchange of
expertise and advice on a particular software package. No doubt,
company-based crystallographers make valuable contributions to
discussions on the BB. This is, however, different to placing an open
sales pitch. I can remember that some in the community were miffed
when discussions on non-CCP4 software packages became prominent.
I think it is only fair to ask suppliers to minimise marketing of
their products here. After all, the infrastructure for the BB is paid
for by public money.
With the obligatory '2 cents worth',
Klaus
===
Klaus Fütterer, Ph.D.
Reader in Structural Biology
Undergraduate Admissions
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E: k.futte...@bham.ac.uk
Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab
===
On 19 Apr 2011, at 08:22, Marcus Winter wrote:
Dear Artem,
Thanks for your reply. You raise a number of points.
Immediately, I should comment that the price of the PX Scanner is very
considerably less than the $400k that you mention.
Whilst - with the proteins and crystallisation conditions that you
may be working
with, visual inspection may be sufficient to differentiate salt from
protein
crystals (as you suggest), you will accept that generally this may
not be the
case. Thus, ‘direct’ inspection, using X-rays, must surely be the
most appropriate way ?
As you will be aware, the best looking crystals are seldom the best
diffracting.
This is well demonstrated through the PX Scanner ‘Crystal
Challenge’, of course.
Clearly, that’s another prime purpose of the PX Scanner: to identify
the ‘best’ crystals
from amongst a multitude of candidates in a single droplet or across
a plate, etc.
Also, using the PX Scanner, we can check the effect of added cryo-
protect. prior to
freezing.
Therefore, with this range of uses, the PX Scanner is clearly not
intended for full
‘data-collection’ – but rather to most effectively support
crystallisation optimisation and
as a complement to in-house and central facility data-collection
work. From the feedback
that we receive, the PX Scanner is much valued by the number of
groups which are
now using these systems worldwide.
However, even the proof of Grandma’s apple pie is not until the
eating. Accordingly,
we most cordially invite you to visit one of our application labs –
or perhaps one of our
customer sites (by arrangement), with you, hopefully, being able to
bring one or more
of your crystallisation plates for inspection using the PX Scanner
system. Since you
are based in North America, I believe, one of my responsible
colleagues will take up
this invitation with you, off-BB.
We look forward to our continuing discussions – with yourself, and
all others who
may be interested.
Many Thanks and Best Regards,
Marcus Winter (Agilent Technologies)
From: Artem Evdokimov [mailto:artem.evdoki...@gmail.com]
Sent: 19 April 2011 02:50
To: WINTER,MARCUS (A-UnitedKingdom,ex1)
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] viewing and scoring diffraction using the PX
Scanner
Hi,
So what's your secret - how did you pack an entire synchrotron into
a little box?
OK, so I am being facetious a little. However, I cannot help asking
myself why would I want to spend so much money on a system that is
basically a (vertical) X-ray diffractometer in a box, with fixed
distance, and sans the ability to collect data? I can only guess
that the system costs in the range of $400K (am I right?) and for
that money one could get a pretty nice actual X-ray diffraction set-
up...
Now, if this thing cost say ... $80K I may be interested, although
most of our crystals are so small that this set-up will uniformly
score them as 'no idea' because they don't even diffract at home on
a 'real' X-ray source with a CCD.
Artem
P.S. the day I start routinely confusing protein and salt crystals
is the day I stop working in the lab :)
On Mon, Apr 18, 2011 at 3:00 AM, Marcus Winter > wrote:
Dear Chris,
I’m prompted by your posting just to mention the Agilent Technologies
PX Scanner ‘Crystal Challenge’ at:
www.agilent.com/chem/crystalchallenge
Thus, the only really
Re: [ccp4bb] Oxford Xcalibur Vs Rigaku micromax
Dear Sankar, One thing to consider is that the Oxford Xcalibur system has been on the market and in users hands for quite some time (even though this does not strictly apply to the NOVA source). I'm not sure how many of the MM002 Rigaku has sold, and the Bruker instrument is very recent. Apart from running tests - which in my experience give limited information in terms of comparison (same crystals!) - your best bet is to tap into the user list that every manufacturer will be able to provide. Write (or phone) these people and ask for their opinion - which most will happily provide. A public forum such as this might make some people hesitant to speak up. As an aside - I agree with Gerard and Dirk that ccp4bb users should show good manners. And I also think that companies should not use the bb as a marketing/ promotional tool. Best, Klaus - Klaus Fütterer, Ph.D. School of Biosciences University of Birmingham P: +44-(0)-121-414 5895 Edgbaston, F: +44-(0)-121-414 5925 Birmingham, B15 2TT, UKE: [EMAIL PROTECTED] W: www.biochemistry.bham.ac.uk/ klaus/ - On 1 Mar 2007, at 06:02, Sankar Narayanan Manicka wrote: Dear Dr. Ross, Thanks very much for your suggestion. I was thinking that there must be people who have used both the systems. But, i see that its nearly impossible to find. Oxford system people have agreed to take our crystals by cryo- shipper and try them out in their machine. sincerely sankar Ross Angel wrote: Sankar We had an Oxford Diffraction PX system, with the same goniometer and detector as the Nova, for 3 years until last Fall when we upgraded to the Nova. We also have 3 other Xcalibur instruments with the same goniometer and control systems. All four diffractometers have performed reliably with very little downtime, the oldest now being nearly 6 years old, and with very little maintainance. The Nova itself has worked well, and has stayed in alignment since it was installed 6 months ago. You can see a few more details at www.crystal.vt.edu. I do not have experience of the micromax. Obtaining a valid comparison between any two diffractometers is difficult. I can only suggest that you do what the rest of us do, and that is take some of your typical crystals and some of your poorer crystals around and try them out on each of the instruments that you are considering. Ross Angel At 12:27 AM 2/28/2007, Sankar Narayanan Manicka wrote: Hi, Our lab is planning to buy an X-ray machine for protein crystallography. Which system would be best for home source, Oxford diffraction system Xcalibur Nova or a MSC/Rigaku MicroMax-002. sincerely, sankar -- Sankar narayanan Manicka[EMAIL PROTECTED] C/o Prof. S. Krishnaswamy +91 452 245 9931 - tel School of Biotechnology +91 452 245 9105 - fax Madurai 625 021 +91 94860 88613 - cell TAMIL NADU INDIA http://www.mkuniversity.org/biotech_school.htm l >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<<<<<<<<< <<<<< Ross Angel Research Professor in Crystallography Crystallography Laboratory Tel: 540-231-7974 Dept. GeosciencesFax: 540-231-3386 Virginia Tech Blacksburg VA 24060-0420 USA http:// www.crystal.vt.edu/crystal/ >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>><<<<<<<<<<<<<<<<<<<<<<<<<<<<< <<<<< -- Sankar narayanan Manicka[EMAIL PROTECTED] C/o Prof. S. Krishnaswamy +91 452 245 9931 - tel School of Biotechnology +91 452 245 9105 - fax Madurai 625 021 +91 94860 88613 - cell TAMIL NADU INDIA http://www.mkuniversity.org/biotech_school.htm l
[ccp4bb]
I'm searching for examples of protein structures with a bound palladium ion. A ligand search on RCSB returns only a single structure (1KS4). Hard to believe that there should not be more examples out there. Does anybody know of any examples off hand? Klaus - Klaus Fütterer, Ph.D. School of Biosciences University of Birmingham P: +44-(0)-121-414 5895 Edgbaston, F: +44-(0)-121-414 5925 Birmingham, B15 2TT, UKE: [EMAIL PROTECTED] W: www.biochemistry.bham.ac.uk/ klaus/ -
[ccp4bb] Research Fellow in Structural Biology of Tuberculosis
Research Fellow School of Biosciences University of Birmingham We seek a highly motivated Research Fellow to join an interdisciplinary research team working on structural and biochemical aspects of cell wall synthesis in the pathogen Mycobacterium tuberculosis, in a thriving research environment with cutting edge facilities in X-ray crystallography, NMR and a range of other spectroscopic techniques. You should have a first degree in chemistry, biochemistry or a related discipline and have earned a PhD in the area of macromolecular X-ray crystallography. Proven experience in all aspects of X-ray crystallographic structural analysis of proteins, and willingness to travel to participate in experiments at synchrotron sources are a must for this post. In addition to this qualification, we are looking for an individual with proven experience in purification and crystallization of membrane-bound proteins. Informal enquiries can be addressed to Professor Gurdyal S. Besra by phone (+44 (0)121 415 8125, email: [EMAIL PROTECTED]) or to Dr. Klaus Fütterer (+44 (0) 121 414 5895, [EMAIL PROTECTED]). Maximum starting salary £24,402 a year, in the range of £24,402 to £31,840 a year (potential progression on performance once in post to £33,799). This post is available for a period of 36 months. Closing date: 6 July 2007 Reference: H39998 Details from 0121 415 9000 or http://www.hroperations.bham.ac.uk/ vacancies/furtherParticulars.htm?refNo=H39998 HR, University of Birmingham, Edgbaston, Birmingham B15 2TT A University of Fairness and Diversity - Klaus Fütterer, Ph.D. School of Biosciences University of Birmingham P: +44-(0)-121-414 5895 Edgbaston, F: +44-(0)-121-414 5925 Birmingham, B15 2TT, UKE: [EMAIL PROTECTED] W: www.biochemistry.bham.ac.uk/ klaus/ -
Re: [ccp4bb] optimizing ncs masks in DM + interpreting output
Francis,
I still like to use Gerard Kleywegt's programme IMP (of the RAVE
suite) to optimise NCS matrices before density modification in DM or
other programs. Initial correlation coefficients (prior to solvent
flattening) should be 20% or better. Below, the matrices are doubtful
and density averaging might not have the desired effect. In my most
recent case, optimisation with IMP increased correlation from <= 10%
to ~ 30%, which made a distinct difference for the subsequent solvent
flattened and density averaged map.
Klaus
===
Klaus Fütterer, Ph.D.
Reader in Structural Biology
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E: k.futte...@bham.ac.uk
Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/
===
On 10 Nov 2009, at 21:36, Francis E Reyes wrote:
Hi all
I have a rotation + translation component for the 2-fold given to me
by GETAX using low resolution low quality experimental maps. I
understand that these are input into DM for NCS averaging. What are
signs of progress as well as how does one usually optimize the mask?
When doing automask is there a certain value of the masked region or
correlation that is considered acceptable?
I'm getting some refined NCS matricies that have a decent correlation
(0.275-0.344). I'd like to see if it's still refining an NCS operator
that is consistent with the self rotation maps. The self rotation maps
for my two fold ncs show a euler coordinate (alpha beta gamma) of 0,
90, 180 which is 48.4% of the crystallographic peaks. The refined NCS
operator from DM is 15.374 73.253 164.235 showing some deviations from
pollarrfn. Is this an indicator of epic failure?
Thanks!
FR
-
Francis Reyes M.Sc.
215 UCB
University of Colorado at Boulder
gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D
8AE2 F2F4 90F7 9640 28BC 686F 78FD 6669 67BA 8D5D
Re: [ccp4bb] Refining against images instead of only reflections
A long time ago I did a bit of Rietveld refinement and I see some similarities between this approach and what people have been proposing in this thread. Refining against the profile of the 1-d powder diffraction pattern rather than extracting integrated intensities helped to improve the quality of the refined structures significantly. Finding the correct (or best) profile function, however, took a while, at least for the X-ray case. Klaus === Klaus Fütterer, Ph.D. Reader in Structural Biology School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: www.biochemistry.bham.ac.uk/klaus/ === On 20 Jan 2010, at 20:29, Edward Snell wrote: Hi Paul, I'll probably open myself up to criticism (welcomed) but I think I'd disagree with this somewhat. While crystallography from the Bragg reflections provides a nice static picture of the structure, looking at the diffuse scatter in more detail may give more knowledge about mechanism - i.e. if there are any characteristic modes associated with significant motion etc. Higher resolution is not always good, one of my enlightening experiences came from paying attention to collecting very complete, very low resolution data. Similarly, after collecting 0.8A data from a large protein I leant a lot about data processing but even more about how to not tell anyone, move the detector back, and then attenuate the beam :) The high-res provided a lot more work and didn't provide any more useful structural knowledge than a 1.2A data set collected in a fraction of the time. However, it did provide a window into how X- rays can perturb the structure - being greedy is not always good. Diffuse scattering has been neglected in the field (for good reason) but I think we have the processing power to take advantage of it now. To misquote Richard Feynman, "there is plenty of room at the bottom", make sure you get the low resolution information as well as the high. I do agree that we may have to rethink image storage somewhat. Looking over a paper not too long ago that had over 30,000 images involved in the analysis made me remember the days when the tape drives were slower writing data than the detectors producing it. That mad scramble to start backup before starting collection ;) Realtime readout, continuous rotation etc., may need to redefine our thoughts of images. Cheers, Eddie Edward Snell Ph.D. Assistant Prof. Department of Structural Biology, SUNY Buffalo, Hauptman-Woodward Medical Research Institute 700 Ellicott Street, Buffalo, NY 14203-1102 Phone: (716) 898 8631 Fax: (716) 898 8660 Skype: eddie.snell Email: esn...@hwi.buffalo.edu Telepathy: 42.2 GHz Heisenberg was probably here! -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Paul Smith Sent: Wednesday, January 20, 2010 3:00 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Refining against images instead of only reflections Hi Jacob, I see you're still in the crystallography business. While you have an interesting idea, I doubt refining structures against entire images would be of any use in obtaining higher quality macromolecular structures. Much of what you see on the screen is a function of parameters completely unrelated or irrelevant to the structure being studied. Diffuse scattering can come from the cryo liquid surrounding the crystal as well as the fibers of the mounting loop itself. Background scattering is related to beam collimation. Spot size/shape is a function of crystal morphology among other things. In addition, every detector has its own peculiarities that make the intensities observed apart from diffraction spots particular to that detector. Also, you would have to take into account other physical properties such as ambient temperature, detector dark current fluctuations, variations in air absorption, etc. So, you could conceivably fit all of these various parameters to the images on hand, but none of them give you any actual information about your structure. As always, if you want more information about your structure, get higher resolution data. Nonetheless, I do think some thought could be put in to exactly how data are reduced. Perhaps the impending era of real time detector readout will help us rethink about spot profiles and intensity integration in a more sophisticated way. We may see a return to thinking about ccd readouts like an area detector which makes the process of analyzing images moot. --Paul --- On Wed, 1/20/10, Jacob K
Re: [ccp4bb] Why nobody comments about the Nobel committee decision?
Perhaps because the (macromolecular) crystallography community is becoming a bit blase about Nobel prizes being awarded to one or several of its distinguished colleagues almost every year? (Ok, not quite). Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 9 Oct 2013, at 22:07, Alexander Aleshin wrote: > Sorry for a provocative question, but I am surprised why nobody > comments/congratulations laureates with regard to recently awarded Nobel > prizes? However, one of laureates in chemistry contributed to a popular > method in computational crystallography. > CHARMM -> XPLOR -> CNS -> PHENIX->… > > Alex Aleshin
[ccp4bb] Marie Curie Early Stage Research
Dear Colleagues, I would like to make you aware of the following opportunity for final year undergraduate and Masters-level students. The University of Birmingham and GSK have initiated Coopera-TB, an EU-funded consortium focussing on hit to lead optimisation of novel anti-TB scaffolds. Details can be found here: http://www.jobs.ac.uk/job/AHQ297/marie-curie-early-stage-researchers/ I would be grateful, if you could forward this information to those potentially interested in this opportunity. Kind regards, Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab ===
Re: [ccp4bb] How it could be possible?
Dear Prem, If your protein has 2 domains, it is possible that they their relative orientation is different in your target compared to the search model. Therefore searching with the two domains separately can improve your Z-score in Phaser and give you a better map. However, you did not tell us what your Z-score was after phaser (>= 8.0 after translation search?) and how your map looks like after phaser, but before buccaneer. For instance, if you simply refine your initial MR solution in refmac, do you get density for side chains that are not part of your search model? Or do you see extra bits of backbone density that are not in the search model.?What about searching with just one domain, do you see (recognisable) density for the second? Finally, an R-value (free or cryst?) of 38% after an initial MR solution is not unreasonable. You may have to manually rebuild your model in Coot with subsequent refinement in Refmac to lower your R-values. Of course, rebuilding in this case requires that you see some extra density you can build into or some parts of the map where your current model does not fit the density. Have you tried to do a rigid body refinement of your initial MR solution, where you allow each domain to move independently? All the best with this structure. Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 2 Dec 2013, at 04:11, Prem Prakash wrote: > Dear All, > > The density obtained after molecular replacement using phaser at 2.5 Angstrom > and then used buccneer for autobuilding of the model. I am not getting > reasonable R value (it is 38.5 %) but the figure of merit is 0.629. > > As My protein has two domains. So is it possible to fragment the individual > domain and then again perform the molecular replacement. How it will improve > my phase more and how R-factor will be reduced ? > > Please help and direct me in proceeding in a right way, and if possible > provide a protocol for doing that. Thank you > > With kind regards > >
Re: [ccp4bb] Best compounds for heavy atom soaks
Did anybody mention native gel electrophoresis to select suitable HA ions? Worked for us really nicely in a situation were speed was essential. Here's a reference: PMID 14646083 Klaus === Dr. Klaus Fütterer Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 15 Jan 2014, at 20:49, Matthew Franklin wrote: > Hi Rhys - > > Don't forget to try sulfur-SAD, especially the multi-crystal version > published recently: > > http://journals.iucr.org/d/issues/2013/07/00/ba5189/index.html > > This seems well suited to your situation. > > - Matt > > > On 1/15/14 12:18 PM, RHYS GRINTER wrote: >> 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 >> > > > -- > Matthew Franklin, Ph. D. > Senior Scientist > New York Structural Biology Center > 89 Convent Avenue, New York, NY 10027 > (212) 939-0660 ext. 9374
[ccp4bb] Intergrown crystals
Dear ccp4bb contributors, We are dealing with the problem of a protein (~ 50 kDa) that crystallises readily, but has an annoying habit of forming highly intergrown rods or needles. Even when the crystals look optically homogenous under the microcsope, diffraction is so so (3.5 Å or so on the synchrotron), but patterns reflect several crystal lattices that the processing software cannot resolve properly. We have tried this: - additive screens - switching the His-tag from N- to C-terminus - cutting the tag - thermal stability screens in a variety of buffers - growth in the presence of potential ligands/substrates Any suggestions for tricks that we haven't thought of so far? Thank you. Klaus === Dr. Klaus Fütterer School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham F: +44-(0)-121-414 5925 Edgbaston E: k.futte...@bham.ac.uk Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab ===
[ccp4bb] Observations-to-parameter ratio in Refmac
Hi CCP4BB readers, Does someone know whether Refmac outputs the observations-to-parameters ratio or, failing that, the number of refined parameters in the log file? Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham E: k.futte...@bham.ac.uk Edgbaston T: @KFbrumbio Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab ===
Re: [ccp4bb] SAXS facility and Cryo EM facility
Dear Ruby, Most synchrotrons will have a beamline designed for small angle solutions scattering. It would depend on your geographical location, which facility you may prefer to look at. Their websites have detailed information about what beamlines are available to external users and what type of experiments you could do. Evidently, cryo-EM is a completely different technique, and not normally attached to a synchrotron source. You may want to check out academic institutions around your geographical base to find a lab that does cryo-EM. Kind regards, Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham E: k.futte...@bham.ac.uk Edgbaston T: @KFbrumbio Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 12 Jun 2014, at 17:42, Ruby Sharma wrote: > > > Hello folks can u please help me by telling that where can i avail SAXS > facility?? > and Cryo EM too?? > > > > Regards > > Ruby > >
Re: [ccp4bb] comparisons - views
Still very happy with our Mosquito. We have had it for 8 years. Minimal maintenance. Significant consumables cost (pipette reel), but very worth the money. Just make sure you train people properly on the instrument and don't let anybody use it, who does not know what they are doing. Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham E: k.futte...@bham.ac.uk Edgbaston T: @KFbrumbio Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === On 17 Oct 2014, at 19:41, Roger Rowlett wrote: > I've had a Gryphon for 2+ years and use it in an undergraduate environment. > It's been trouble-free, and there are no instrument consumables, just blocks > and trays. OK, I do have to feed it deionized water and a PCR tube of diluted > protein for each set. It can set a 96-well tray in under 2 minutes. The basic > protocol I use is 200+200 nL drops. The software is easy enough to use that > my undergrads know how to program it to do 1 or 2 drop screens or partial > plates. I don't have the LCP module but you can get that installed or > retro-fitted. > > I'm pretty sure the acquisition cost of the Gryphon is much less than the > Mosquito and NT8. I squeezed mine on a NSF-RUI grant as project research > equipment. > > Cheers, > > ___ > Roger S. Rowlett > Gordon & Dorothy Kline Professor > Department of Chemistry > Colgate University > 13 Oak Drive > Hamilton, NY 13346 > On 10/17/2014 1:56 AM, Dean Derbyshire wrote: >> Apologies for the slightly off topic… thought this was the best way to get >> views from a wide (relevant) audience. >> Any views on differences – pros and cons – and experiences with: >> Mosquito; Gryphon and NT8? >> And similarly with Minstrel and Rock imager. Related to that last >> ‘comparison’ what’s the prevailing thoughts on SONICC vs standard UV laser >> technology… any experiences with coping phase separation or condensation ? >> Thanks >> Dean >> >> >>Dean Derbyshire >> >>Box 1086 >>SE-141 22 Huddinge >>SWEDEN >>Visit: Lunastigen 7 >>Direct: +46 8 54683219 >>www.medivir.com >> >> -- >> This transmission is intended for the person to whom or the entity to which >> it is addressed and may contain information that is privileged, confidential >> and exempt from disclosure under applicable law. If you are not the intended >> recipient, please be notified that any dissemination, distribution or >> copying is strictly prohibited. If you have received this transmission in >> error, please notify us immediately. >> Thank you for your cooperation. >
Re: [ccp4bb] FW: [ccp4bb] Fwd: [ccp4bb] just out of totally idle curiosity ...
I would concur with Ed. I can understand the emotions, but political shouting should be done elsewhere. Let’s remain civil. Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham E: k.futte...@bham.ac.uk Edgbaston T: @KFbrumbio Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === > On 9 Nov 2016, at 17:02, Edward Snell wrote: > > As a Brexit and Trumpet affected person having a foot in both countries ,this > topic is too far off the normal discussion on CCP4 and probably better taken > up privately. CCP4 is not a political discussion site. With CCP4 the signal > is unusually high and the noise low when compared to any discussion board. I > for one would like to keep it there. Political views aside, we’re all trying > to achieve the same scientific goals. Let’s remember that and keep that the > focus. > > Edward Snell Ph.D. > President and CEO Hauptman-Woodward Medical Research Institute > Assistant Prof. Department of Structural Biology, University at Buffalo > 700 Ellicott Street, Buffalo, NY 14203-1102 > Phone: (716) 898 8631 Fax: (716) 898 8660 > Skype: eddie.snell Email: esn...@hwi.buffalo.edu > > Heisenberg was probably here! >
Re: [ccp4bb] delete subject
All, I personally would feel awkward at depositing my hard fought data before the associated paper is accepted. However, a recent paper from our institution (I was not part of that study) originated from precisely the model that has been suggested in this thread: http://www.nejm.org/doi/full/10.1056/NEJMoa1107643 Given the success of various 'open source' endeavours in recent years, I'm slowly coming around to a view that with smart procedures in place, an 'open data' approach could indeed help to solve structures that would otherwise remain unsolved, unpublished and unused. Klaus On 28 Mar 2013, at 08:46, Anastassis Perrakis wrote: > Dear all, > > Let me start by apologizing for finally making this email longer than I > intended - I did not have the time to make it shorter. > > I must say I am humbled by the amount of positive energy and constructive > thinking that Bill has. That must explain also how he manages to keep up a > fantastic resource for the community, what is a largely thankless task with > little academic reward, but still so helpful. His response did get me > thinking, as his opinions often do. In principle, in an era of information > sharing, why don't we indeed solve structures with the collective brain of > world crystallographers? We could share data, and educate people, and also > get better structures at the end of the day. As indeed many people are in a > small institution somewhat off the beaten path - I work in a cancer research > institute after all and I am the weirdo here - and as indeed I see my > knowledge getting obsoleted in a steady pace, this idea sounds great! > > However, its a bit like 'true' socialism - a grant idea, that we may find it > will not work too well in practice, at least not under the constraints of > human nature. > > I will keep advocating the greatness of ccp4bb. Its a fantastic resource, > which is made truly amazing by the many questions that are being asked, and > keep educating everybody. > But, at the same time, I will keep advocating the usefulness of the typical > constructions we have in science, whereupon people work in teams. These teams > are there > to share experience and help each other with overlapping expertise. Why such > a basic question (and others in the past) need to come out of the 'team'? > Is there no competence within the team to address it, or is there no correct > communication? > In either case, should we as scientists encourage such teams with low-level > competence? May I remind you that Tom is in Lueven/Belgium, > a large and outstanding University, with at least two very competent (and > friendly) crystallographers in campus. Does the fact he has to post the > ccp4bb with a basic question > testify for a complete failure of his supervisor to either help him or get > other people on site to help him? Should such supervisors be left to guide > students? > > I have nothing against sharing data. I am the fool that submits data at the > same time as I submit my paper, a practice that is followed by surprisingly a > few people, > as most people wait a few weeks until the paper is accepted to submit their > structure (some data mining shows that 1/3 of the PDB entries associated with > papers > even in journals like Acta D are only deposited to the PDB at least a week > after the paper submission date!.. no think what this % is in other > journals). > And the mild consequence of this is that somebody picks the structure up, > panics to be scooped, submits his/her story, and scoops you while your paper > is being rejected > for reasons that are not connected to the structure (I am not implying foul > play here, but suggesting a consequence of basic openness). > > Are we finally, at the end, with this open and sharing spirit, encouraging > people to think that crystallography is too trivial? It has once been said in > this bb, that > 'solving a structure is trivial in the same way that climbing mountain > Everest is trivial: it has been down before, its being done now, and it will > be done again, > by many well-trained and determined people'. Many people have read and > trained for this task. If you do not read a couple of books and train before > attempting the climb, > and you send an email asking the everestbb 'does anybody know how to open > this oxygen valve?' you are asking for trouble though > … and the people that let you attempt the climb without that knowledge, are > also in the wrong. > > The end result of this open and sharing spirit, which downgrades the > importance of competence in major methodologies like X-ray crystallography, > was summarized recently is some text I recently got by email from Brussels: > "... advanced methods for X-ray crystallography and Electron Microscopy is a > very narrow field that will limit the employability of the graduates" > There are the wise words of the referees of a joined grant (with 10 other > people fro
[ccp4bb] Organic solvents for ligand solubilisation
Dear CCP4BB followers,
We are currently trying to obtain ligand-bound complexes for one of our
proteins by soaking and/or co-crystallisation. We have had prior success for
this protein, but using a different class of ligands. The new ligand (in DMSO)
remains in solution (more or less) when mixed with the
reservoir/cryoprotectant, and the diffraction pattern survives the soaking
nicely. Annoyingly though, all we see are density peaks that match the size of
DMSO and become more pronounced when increasing [DMSO] (to help solubilisation
of the ligand). Soaking times varied between minutes to 16 hours. Kd was
measured ( ~ 10 uM) in the solution state.
We have tried pyridine (which keeps the ligand in solution, but kills
diffraction in an instant), and DMF (which doesn't keep the ligand in solution
when mixing with cryoprotectant).
I am wondering whether the community has suggestions for alternative organic
solvents that have been used to solubilise hydrophobic ligands, and are
reasonably gentle to the protein crystal.
Thank you.
Klaus
===
Klaus Fütterer, Ph.D.
Reader in Structural Biology
Undergraduate Admissions
School of Biosciences P: +44-(0)-121-414 5895
University of Birmingham F: +44-(0)-121-414 5925
Edgbaston E: k.futte...@bham.ac.uk
Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab
===
Re: [ccp4bb] Co-purified ligand present in protein crystal
Wenhe Potentially, mass spectrometry in combination with electron capture dissociation and/or collision-induced dissociation might be helpful. See doi: 10.1007/s13361-013-0662-5. Best wishes, Klaus === Dr. Klaus Fütterer Deputy Head of School Undergraduate Admissions Room 717, Biosciences Tower School of Biosciences P: +44-(0)-121-414 5895 University of Birmingham E: k.futte...@bham.ac.uk Edgbaston T: @KFbrumbio Birmingham, B15 2TT, UK W: http://tinyurl.com/futterer-lab === > On 21 Aug 2017, at 04:41, > wrote: > > Dear CCP4BB members, > > We would like to identify a ligand that is present in crystal structure > (according to strong positive densities at active site) but absent in > crystallization condition. We already have some candidates in mind based on > our knowledges on this protein but we need to validate further. The general > method we are using now is to use methanal to precipitate protein and extract > ligand from the precipitated protein. Then we analyse the methanol extraction > sample on LC-MS. One problem of this method is that the methanol extraction > will not be 100% efficient which means there is only a small portion of > bound-ligand can be extracted from the protein— particularly if the ligand > binds very tightly to the protein. So I would like to know whether anyone has > experience to efficiently extract tighly-bound ligands from protein for > downstream analysis. One method is to digest protein with protease such as > trypsin. Or use urea to denature the protein. However, these methods require > relatively long processing time which is not optimal when the ligand that we > want to analyse is unstable (degrade overtime). Anyone has more suggestions? > > Thank you! > > Kind regards, > Wenhe
