Re: [ccp4bb] XDS vs SADABS absorption correction factors
-BEGIN PGP SIGNED MESSAGE- Hash: SHA1 Dear Jens, I recently prepared a cif-file and came across the same question. With empirical absorption correction (_exptl_absorpt_correction_type empirical) the values for T_min and T_max loose a bit of meaning. Yet, I took the maximal and minimal values from ABSORP.cbf and scaled them linearly so that T_max = 1.0 (i.e., I devided the minimal value by the maximal value and used this as T_min). I your user adds the 'SIZE' to the shelxl ins-file, shelxl writes estimated values to the cif-output which in my case were much less spread: _shelx_estimated_absorpt_T_min0.983 _shelx_estimated_absorpt_T_max0.997 _exptl_absorpt_correction_typeempirical _exptl_absorpt_correction_T_min 0.447 _exptl_absorpt_correction_T_max 1.000 _exptl_absorpt_process_details'XDS (Kabsch, 2010)' I commented under _exptl_special_details _exptl_absorpt_correction_T_min and _exptl_absorpt_correction_T_max from ABSORP.cbf, the absorption factors used by XDS. These are largely dominated by scale factors, hence the large variation for this non-cubic crystal shape. Best, Tim On 02/20/2014 11:43 PM, Jens Kaiser wrote: All, Sorry, this is a little bit off topic. I could not find a thorough definition of the Correction Factors for absorption correction in XDS nor of the Transmittance factors in SADABS. We collected small molecule samples on a synchrotron beamline, processed the data with XDS and the user now needs to know the Minimum and Maximum T for their cif. From what I could gather, the XDS correction factors in the ABSORP.CBF are multiplied by 1000; the values are around 1 (i.e a little smaller /and/ a little larger). The cif dictionary for _exptl_absorpt_correction_T_ states The permitted range is 0.0 - 1.0, which clearly has to be a different definition. Any pointers are welcome, Cheers, Jens - -- - -- Dr Tim Gruene Institut fuer anorganische Chemie Tammannstr. 4 D-37077 Goettingen GPG Key ID = A46BEE1A -BEGIN PGP SIGNATURE- Version: GnuPG v1.4.12 (GNU/Linux) Comment: Using GnuPG with Icedove - http://www.enigmail.net/ iD8DBQFTBydxUxlJ7aRr7hoRAsCBAKD961a/da/cXKUkUE/aJkdlDgOIUwCeP4v2 X6JJQtgj0UPxvPpE0Yivu/0= =ce4X -END PGP SIGNATURE-
[ccp4bb]
Hi, Sorry for asking an off-topic question, I have recently purified a protein having a molecular weight of 40kDa and concentration of the protein was 8mg/ml. When I tried to set the protein for crystallisation using micobatch method, the protein started precipitating in most of the buffer conditions of Crystal screen and Peg ion. The precipitation took place very quickly (within 5-10 mins). How should I overcome this problem? Regards Prerana
[ccp4bb]
Hi, You could change the ratio of paraffin and silicon oil (1:1 or 1:2) and/or use lower protein concentration. Best regards, Vicky G. Tsirkone, MSc Laboratory for Biocrystallography Department of Pharmaceutical and Pharmacological Sciences KU Leuven ON II Herestraat 49 - box 822 3000 Leuven | Belgium Tel.: +32 16 3 23419 e-mail: vicky.tsirk...@pharm.kuleuven.bemailto:vicky.tsirk...@pharm.kuleuven.be From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Prerana G. [tracy...@gmail.com] Sent: Friday, February 21, 2014 12:14 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Hi, Sorry for asking an off-topic question, I have recently purified a protein having a molecular weight of 40kDa and concentration of the protein was 8mg/ml. When I tried to set the protein for crystallisation using micobatch method, the protein started precipitating in most of the buffer conditions of Crystal screen and Peg ion. The precipitation took place very quickly (within 5-10 mins). How should I overcome this problem? Regards Prerana
[ccp4bb]
Dear Prerana, before starting the crystallization you could try to check the state of you protein, simply determining the Aggregation Index (AI) from measuring the absorbance at 280 nm and 340 nm. The AI is then computed using this simple formula: AI= 100 x (Abs340/(Abs 280 - Abs 340)). Soluble and non-aggregated proteins solutions typically have an Aggregation Index of 2 and lower, whereas for some aggregation will be 2-5; heavily aggregated proteins show an aggregation index 5. Of course you cannot use Nanodrop for it (because the wavelength for the baseline normalization is 340 nm). I would try to decrease the concentration of your protein to 4-5 mg/ml.Good luck. Best, Mirella Vivoli Mirella Postdoctoral Fellow University of Exeter, Biosciences Biocatalysis Centre, Henry Wellcome Building Stocker Road, Exeter, Ex4 4QD Tel:+ 44 (0)1392 726121 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Prerana G. [tracy...@gmail.com] Sent: Friday, February 21, 2014 11:14 AM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Hi, Sorry for asking an off-topic question, I have recently purified a protein having a molecular weight of 40kDa and concentration of the protein was 8mg/ml. When I tried to set the protein for crystallisation using micobatch method, the protein started precipitating in most of the buffer conditions of Crystal screen and Peg ion. The precipitation took place very quickly (within 5-10 mins). How should I overcome this problem? Regards Prerana
Re: [ccp4bb] Calcium soaking
Have you tried lower concentrations of Calcium soaking untli the crystals do not crack? Or does it crack even at very minute calcium concentration? On Thu, Feb 20, 2014 at 3:29 AM, Masaki UNNO unn...@mx.ibaraki.ac.jpwrote: Dear all Apologies for the off-topic question: We are studying an enzyme that is activated by Ca2+. We obtained the crystals of the substrate and Ca2+-free form and solved the structure at 2.7 A resolution. However, the active site electron density map was not clear, although other regions are clear. We would like to determine the substrate-complex with Ca2+, which will elucidate the active site structure at a higher resolution. Now we have a problem that the crystals of a mutant which can bind the substrate and Ca2+ always have cracks in soaking to the crystallization solution containing CaCl2. Co-crystallization does not work at this time. We estimate the structural change in Ca2+-binding is not so big because the isozyme structure did not change very much when binding Ca2+. An isozyme structure in complex with the substrate was determined by soaking Ca2+ (and the substrate). How should we overcome this problem? Best regards ~~~ Masaki UNNO Graduate School of Science and Engineering, Ibaraki University, Japan
[ccp4bb] FEBS Practical and Lecture Courses - BioCrys2014
Dear Colleagues, Applications are now open for BIOCRYS 2014 Fundamentals of Modern Methods in Biocrystallography organised by Maria Armenia Carrondo and Thomas R. Schneider *Course sponsored by :* FEBS, BioStruct-X and IUBMB * **Date Location:*20th - 27th September 2014 at the Instituto de Tecnologia Química e Biológica, Oeiras, Portugal. Topics of the course will run from fundamentals such as symmetry, point groups and crystal systems, basic diffraction physics, reciprocal space and the Ewalds sphere, radiation damage, data processing, symmetry in the diffraction pattern, structure factors, Patterson function to modern methodologies including molecular replacement, SAD, MAD, MIR and maximum likelihood phasing, direct methods, density modification, refinement, model building, twinning and structure validation. Main challenges on sample preparation and crystallization of proteins will also be covered. The course will be organized with lectures in the mornings and interactive practicals and tutorials in the afternoons. Evening lectures will address two main important topics within Structural Biology, one covering studies of membrane proteins and the other the beginning of the use of free electron lasers in Macromolecular Crystallography. Participants will be limited to 36, aimed primarily at people at the beginning of their crystallographic research activity as PhD students or others. Selected applicants are invited to present a poster during the course. *Speakers and tutors: * *Margarida Archer*ITQB, Oeiras, PT *Isabel Bento*ITQB, Oeiras, PT *Gabor Bunkoczi *University of Cambridge, UK *Kevin Cowtan*University of York, UK *Zbigniew Dauter*Argonne National Laboratory, USA* Kristina*/*Djinovic-*//*Carugo*//, University of Vienne, AT/ *Carlos Frazão*ITQB, Oeiras, PT *Elspeth Garman*University of Oxford, UK *Gordon Leonard*ESRF, Grenoble, FR *Andrew Leslie*MRC LMB, Cambridge, UK *Bernhard Lohkamp *Karolinska Institutet, SE *Adrian Mancuso*European XFEL GmbH, Hamburg, DE* Rob Meijers,*EMBL-Hamburg, DE *Pedro Matias*ITQB, Oeiras, PT* Poul Nissen*, University of Aarhus, DK *Anastassis Perrakis*NKI, Amsterdam, NL *Célia Romão*ITQB, Oeiras, PT *Thomas Schneider,*EMBL-Hamburg, DE* Philip Willmott*, Paul Scherrer Institut, CH *Clemens Vonrhein*Global Phasing Ltd, Cambridge, UK *How to apply:* A registration fee of 650 Euros for academic and 1200 Euros for non-academic applicants is requested for full board and accommodation. Selected applicants will have to pay the registration fee by bank transfer before arrival. A limited number of grants are available from FEBS. For an application, please fill the form on the web page by the 15th of July. For more information visit the course web page http://biocrys2014.itqb.unl.pt/ We look forward to welcoming you to Oeiras -- *Colin E. McVey, DPhil* Principal Investigator Structural Genomics Lab Macromolecular Crystallography Unit Instituto de Tecnologia Química e Biológica Av. da Republica, EAN | Phone:(351)214469663 Apartado 127 | Fax :(351)214433644 2781-901 Oeiras | email:mc...@itqb.unl.pt PORTUGAL
[ccp4bb] Aggregation assay
I am aware of an assay for aggregation (aggregation rate) that is based on fluorescence measurements. It involves excitation at 280 and emission in the range 260-400. Is there a reference for the absorbance method? See Nominé Y, Ristriani T, Laurent C, Lefèvre JF, Weiss E, Travé G. A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. Protein Eng. 2001 Apr;14(4):297-305. PubMed PMID: 11391022. http://www.ncbi.nlm.nih.gov/pubmed/11391022 Jack Tanner Sent from Jack's iPad On Feb 21, 2014, at 7:23 AM, Vivoli, Mirella m.viv...@exeter.ac.ukmailto:m.viv...@exeter.ac.uk wrote: Dear Prerana, before starting the crystallization you could try to check the state of you protein, simply determining the Aggregation Index (AI) from measuring the absorbance at 280 nm and 340 nm. The AI is then computed using this simple formula: AI= 100 x (Abs340/(Abs 280 - Abs 340)). Soluble and non-aggregated proteins solutions typically have an Aggregation Index of 2 and lower, whereas for some aggregation will be 2-5; heavily aggregated proteins show an aggregation index 5. Of course you cannot use Nanodrop for it (because the wavelength for the baseline normalization is 340 nm). I would try to decrease the concentration of your protein to 4-5 mg/ml.Good luck. Best, Mirella Vivoli Mirella Postdoctoral Fellow University of Exeter, Biosciences Biocatalysis Centre, Henry Wellcome Building Stocker Road, Exeter, Ex4 4QD Tel:+ 44 (0)1392 726121 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Prerana G. [tracy...@gmail.commailto:tracy...@gmail.com] Sent: Friday, February 21, 2014 11:14 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Hi, Sorry for asking an off-topic question, I have recently purified a protein having a molecular weight of 40kDa and concentration of the protein was 8mg/ml. When I tried to set the protein for crystallisation using micobatch method, the protein started precipitating in most of the buffer conditions of Crystal screen and Peg ion. The precipitation took place very quickly (within 5-10 mins). How should I overcome this problem? Regards Prerana
Re: [ccp4bb] I/sigmaI or I/sigmaI
Hi Qixu, Sorry, I don't read this BB very often and missed your message. I assumed the DEC-1-2003 archives would be online somewhere, but I may be mistaken. I printed out the discussion and stored it in a notebook. So if nobody here can point to online archives, I can scan and post the pages on DropBox. These were discussions by Anthony Duff, Phil Evans, Jim Pflugrath, et al. Richard On Feb 19, 2014, at 10:21 AM, Cai Qixu wrote: Dear Folmer Fredslund, Thanks for your help. Actually, I can only find the archives until APR-30-2003 at the archives. Where is the DEC-1-2003 archives? Regards, Qixu Cai 发件人: Folmer Fredslund folm...@gmail.commailto:folm...@gmail.com 日期: 2014年2月19日 星期三 下午10:09 至: Cai Qixu caiq...@gmail.commailto:caiq...@gmail.com 抄送: CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk CCP4BB@jiscmail.ac.ukmailto:CCP4BB@jiscmail.ac.uk 主题: Re: [ccp4bb] I/sigmaI or I/sigmaI Dear Qixu Cai, You can find information about where to find the archives here: http://www.ccp4.ac.uk/ccp4bb.php#archives Best regards, Folmer 2014-02-19 14:44 GMT+01:00 Cai Qixu caiq...@gmail.commailto:caiq...@gmail.com: Dear Richard Gillilan, Where to find the archives of Dec 2003? I can only find the archives until 2007 at jiscmail. Thanks. Regards, Qixu Cai 发件人: Richard Gillilan r...@cornell.edumailto:r...@cornell.edu 答复: Richard Gillilan r...@cornell.edumailto:r...@cornell.edu 日期: 2014年2月16日 星期日 上午12:19 至: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK 主题: Re: [ccp4bb] I/sigmaI or I/sigmaI There was an informative discussion on this very topic back in Dec 1-2, 2003 if you browse the CCP4BB archives. Richard Gillilan MacCHESS On Feb 12, 2014, at 6:43 AM, Cai Qixu wrote: Dear all, Does the I/sigmaI in “Table 1” mean for I/sigmaI or I/sigmaI ? Thanks for your answer. Best wishes, Qixu Cai -- Folmer Fredslund
Re: [ccp4bb] Summary: identifying protein crystals via visible light only
What about this one? http://dx.doi.org/10.1107/S0907444912002946 Although the excitation is x-rays and not visible light, you are still conceivably identifying protein crystals with visible light. Seems to mostly come from aromatics. Not sure how much damage you have to do to get enough XEOL signal. Probably depends on how dark the room is. -James Holton MAD Scientist On 2/15/2014 8:36 AM, Richard Gillilan wrote: My original question was: Some years ago, I remember hearing about a microscope that used *visible* light combined with some proprietary image processing algorithm to distinguish between protein crystals, salt, and background. I can't remember the company name or researchers involved. Has anyone here heard of this? None of the answers I received sound like what I remember. Nonetheless, there are two companies that apparently offer visible-light technology for recognizing crystals: (a) Tritek: proteincrystalimaging.com/index.php (b) Jan Scientific: (VISEX) see smb.slac.stanford.edu/news/Visex.pdf (their listed products seem to be UV-based, but apparently this one is visible.) Both of these are proprietary commercial products, so it is impossible to know exactly what they are doing. Richard Gillilan MacCHESS
Re: [ccp4bb] : I on sig I
Phil Evans wrote: *** For details on how to be removed from this list visit the *** *** CCP4 home page http://www.ccp4.ac.uk *** Since this seems to be causing endless confusion, here is the definition used in Scala for I/sigmaI which is what I report to the PDB. This is the table column labelled Mn(I)/sd and printed in the summary at the end (in the latest version only) After scaling, for each unique reflection h we have several observations of the intensity Ih and an estimated corrected error estimate sd(I) From these we calculate a weighted mean Ih and an error estimate of the mean sd(Ih), and a ratio for that unique reflection Ih/sd(Ih) What is printed as Mn(I)/sd is the mean value of that ratio for all reflections (possibly in a resolution bin) ie Ih/sd(Ih) This is an estimate of the average signal to noise. Its value does as has been frequently pointed out depend on the sd estimates being correct, which is always a doubtful proposition, but that's another story . . . Note the the source code of Scala is in the CCP4 distribution and anyone may look in it (look in subroutines ad5sts and prdres) Best wishes Phil Evans Edward A. Berry writes: Edwin Pozharski wrote: I just want to point out that what is requested upon uploading data to the Protein DataBank is NET I OVER AVERAGE SIGMA I To me it sounds pretty much like I/sigmaI. Yes, ADIT asks for: Net I over average sigma(I)19 And for the last resolution shell there is a different definition: Mean I over sigma(I) (observed) 2.71 But when the PDB file is produced, both values are presented as: REMARK 200 I/SIGMA(I) FOR THE DATA SET : 19. ...|. REMARK 200 I/SIGMA(I) FOR SHELL : 2.710 As to whether I/Sigma refers to unique reflections or measurements, there is also the question, before or after adding partials? And the cutoff criterion by which we decide which measurements are classified as observations, which I understand should be -3 for scalepack users: is that a cutoff on the raw (partial) measurement or on the full measurement generated by summing partials? I had the impression that the default -3 sigma cutoff in scalepack was for the raw measurements, although reading the current manual I can't find anything to justify that. As users of proprietary (closed-source) software, we depend on the authors for definition of the output values. As a biologist I thought I could just collect data, run the programs, and deposit my structure. Now I'm getting all confused! Ed Ed. Anthony Duff wrote: When I asked about I on sig I as to whether one should report: (1) I/SIGI; (2) I/SIGI; or (3) I/SIGI, the responses were that it is I/SIGI that should be reported, although it seems that I/SIGI cannot be reasonably interpreted as anything other than I/SIGI. Bart Hazes put it most clearly (noting that Jim Pflugrath is uncertain that reporting I/SIGI has much merit all): - Well because of the first commandment. Thou shalt report I/SigI - Without it a Table 1 wouldn't be a Table 1 would it? As Fred. Vellieux said You can compute a column containing I/SIGI using SFTOOLS, then compute its average value. in detail... sftools read mymtzfile.mtz complete# calculate completeness in 20 bins calc col IoSI = col IMEAN col SIGIMEAN / # polish mathematics. # creates IoSI = IMEAN/SIGIMEAN plot col IoSI versus resol # gives average (IoSI) in 20 bins checkhkl# read average (IoSI) Having said all that, I note that colleagues using scalepack are liable to report: I/SIGI, calculating it themselves using average I and and average error from the last table of the scalepack log file, or I/SIGI, but not for unique reflections, but for all reflections, taken from the last line of the table Summary of reflection intensities and R-factors by batch number. A quick investigation has revealed that I/SIGI for all reflections can be very much greater than I/SIGI for unique reflections. It seems to me that it is impossible to obtain the correct I/SIGI from a scalepack log file. Is this correct? I'm wondering if there is any consistency in the value to be found in the headers of pdb files following REMARK 200 I/SIGMA(I) FOR THE DATA SET Anthony
Re: [ccp4bb] : I on sig I
Eleanor Dodson wrote: Jim's point is that SigI is manipluated in the program, and its value reflects the programmers ideas. If you want to make your hair stand on end process the same data with SCALEPACK and MOSFLM and get the Riso between the 2 SIG estimates! 50% on a good day is the typical value. The reason for this is: SIGI from the imaghes is based on rather uncertain physics and I/SigI for the unmerged data is pretty useless.. But SIGI from the data merging can be estimated much more precisely by comparing the scatter of observations which in an ideal world would be equal. This is done by what scalepack calls the CHI**2 testand SCALA modifying SD to make the SigI reflect a normal distribution . This is quite a good method if you have high enough multiplicity and the symmetry equivalents are not subject to the same systematic errors - not so likely if you have offset your crystal etc.. But of course that is not always the case and then I/SIGI isnt very useful.. Howeve it should be a reasonable measure if there is fair multiplicity.. and you should always quote that! Anthony Duff wrote: When I asked about I on sig I as to whether one should report: (1) I/SIGI; (2) I/SIGI; or (3) I/SIGI, the responses were that it is I/SIGI that should be reported, although it seems that I/SIGI cannot be reasonably interpreted as anything other than I/SIGI. Bart Hazes put it most clearly (noting that Jim Pflugrath is uncertain that reporting I/SIGI has much merit all): - Well because of the first commandment. Thou shalt report I/SigI - Without it a Table 1 wouldn't be a Table 1 would it? As Fred. Vellieux said You can compute a column containing I/SIGI using SFTOOLS, then compute its average value. in detail... sftools read mymtzfile.mtz complete# calculate completeness in 20 bins calc col IoSI = col IMEAN col SIGIMEAN / # polish mathematics. # creates IoSI = IMEAN/SIGIMEAN plot col IoSI versus resol# gives average (IoSI) in 20 bins checkhkl# read average (IoSI) Having said all that, I note that colleagues using scalepack are liable to report: I/SIGI, calculating it themselves using average I and and average error from the last table of the scalepack log file, or I/SIGI, but not for unique reflections, but for all reflections, taken from the last line of the table Summary of reflection intensities and R-factors by batch number. A quick investigation has revealed that I/SIGI for all reflections can be very much greater than I/SIGI for unique reflections. It seems to me that it is impossible to obtain the correct I/SIGI from a scalepack log file. Is this correct? I'm wondering if there is any consistency in the value to be found in the headers of pdb files following REMARK 200 I/SIGMA(I) FOR THE DATA SET Anthony -- Anthony Duff Postdoctoral Fellow School of Molecular and Microbial Biosciences Biochemistry Building, G08 University of Sydney, NSW 2006 Australia Phone. 61-2-9351-7817 Fax. 61-2-9351-4726 --
Re: [ccp4bb] : I on sig I
Mark A. White wrote:
*** For details on how to be removed from this list visit the ***
*** CCP4 home page http://www.ccp4.ac.uk ***
I have a couple of simple awk commands that will calculate both I/sI and
Redundancy, by
parsing the the scalepack output log.
### I/sigma by Shell
awk '{ if ($11 1. ($1$2 || $2==hkl)) printfSig: %7.2f%5.2f %8.1f
\n,$1,$2,(0.5*($4-$3)+($5-$4)+2.5*($6-$5)+4.5*($7-$6)+7.5*($8-$7)+15*($9-$8)+25*$10)/($11+0.01)
}' scalepack.log | tail -16
Redundancy by Shell
awk '{ if ($13 $3 ($1$2 || $2==hkl)) printfRed: %7.2f%5.2f %8.1f
\n,$1,$2,($4+2*$5+3*$6+4*$7+5.3*$8+7.3*$9+10*$10+14*$11+1337*$12)/($13+0.01)
}'
scalepack.log | tail -16
PS. You will probably need to reassemble the above commands. Each one is a
single command
line, which can be placed at the end of your scalepack.com script. Note that
I usually use
15 resolution shells, so that the output of each command is truncated to 16
lines,
otherwise you get a lot of junk, particularly from the latest versions of
scalepack.
Change the tail command to limit your output to the number of resolution
shells plus 1.
david.borh...@abbott.com wrote:
I've written an awk/nawk script that will post-process a Scalepack log file
to produce
pseudo I/SIGI values as a function of resolution, as well as the (pseudo)
overall
value. It does this by massaging the I/Sigma in resolution shells table,
calculating a
pseudo I/SIGI weighted by the number of reflections in each individual
I/SIGI//resolution bin. (Some counting of number of reflections is also
corrected.) This
process is obviously an inaccurate, stopgap measure, but some would argue
that it is
better than nothing. Perhaps the next update of Scalepack will report more
complete
statistics, along the lines of Scala?
David Borhani, Ph.D.
Group Leader, Biochemistry
Vox:508-849-2944
Fax:508-755-8361
Email:david.borh...@abbott.com
Smail: Abbott Bioresearch Center, Inc.
100 Research Drive
Worcester, MA 01605 U.S.A.
http://abbott.com/abbottbioresearch/
# awk/nawk file
# DWB 1Oct2003
#
# Extract I/SigI statistics as a function of resolution from a scalepack
log file.
#
# Usage:
#
# awk -f scalepack_Isig.nawk scalepack.log scalepack.log.I_over_sigma
#
# Typical input data from a Scalepack log file:
#
#ShellI/Sigma in resolution shells:
# Lower Upper No. of reflections with I / Sigma less than
# limit limit 0 1 2 3 51020 20 total
# 50.00 3.21265173 101 163 335 707 8474 9181
# 3.21 2.55 110 214 315 410 609 1131 2521 6690 9211
# 2.55 2.23 206 383 608 815 1253 2197 4277 4879 9156
# 2.23 2.02 195 466 761 1051 1700 3119 5758 3388 9146
# 2.02 1.88 448 941 1464 1959 2863 4859 7434 1757 9191
# 1.88 1.77 664 1511 2344 3100 4420 6558 8520 575 9095
# 1.77 1.68 846 2131 3370 4420 5934 7891 9022 155 9177
# 1.68 1.61 1088 2852 4441 5629 7030 8529 905048 9098
# 1.61 1.54 1281 3455 5197 6204 7313 8206 843511 8446
# 1.54 1.49 685 1986 3076 3847 4671 5115 5162 2 5164
# All hkl 5549 13990 21649 27536 35956 47940 60886 25979 86865
#
#---
#
BEGIN {
# skip to correct start of table...
x = 0
y = 0
nrbins = 0
#
while (x != 1)
{
getline
if ($1 == Shell $2 == I/Sigma $4 == resolution) {x = 1}
}
# Skip to limit limit 0 1 2... line, and load up I/Sigma bin
values...
getline ; getline
nibins = NF-3
for (i = 3; i = NF-1; ++i)
{
# Force 20 to be interpreted as 30.0
if (substr($i,1,1) == ) $i = substr($i,2,length($i)) * 1.5
# Force real value rather than text for all ibin values
ibin[i-2] = $i + 0.
}
}
#
# Process table data...
#
{
while (y != 1)
{
# Process regular lines of the table
if ($1 != All)
{
# Increment resolution bin counter, store resolution limits for this line...
++nrbins
rbin[1,nrbins] = $1
rbin[2,nrbins] = $2
# Store number of reflections in each I/SigI for this line...
for (i = 3; i = nibins + 2; ++i) bin[i - 2,nrbins] = $i
getline
}
else
{
y = 1
}
}
}
#
END {
printf Pseudo I/sigI Statistics from scalepack log file: %s\n\n,
FILENAME
printf bin dmax dmin Nhkl I/sigI\n
printf \n
ntot = 0
isigtot = 0
# Loop over resolution bins...
for (j = 1; j = nrbins; ++j)
{
n[j] = 0
isig[j] = 0
# Loop over I/SigI bins at given resolution...
for (i = 1; i = nibins; ++i)
{
# Total number of reflections, calculate pseudo I/SigI...
n[j] = n[j] + bin[i,j]
isig[j] = isig[j] + bin[i,j] * ibin[i]
isigtot = isigtot
Re: [ccp4bb] High Salt Cryo
I want to start off by thanking everyone. The replies, both on- and off-board, were speedy and numerous. I apologize for the delay in expressing my gratitude; I was implementing your wonderful suggestions. I have put together a summary for the archive. To recap, I was looking for suggestions to cryoprotect crystals from 3 M NaCl crystallization conditions excluding ethylene glycol or PEG completely and avoiding glycerol and sucrose due to apparent crystal instability. -Several people confirmed that 4 M NaCl should be sufficient. -There were several suggestions for cryosalts with malonate and formate being the most frequent. Lithium salts were also put on the table including partial or complete substitution of NaCl with LiCl, both as a soak or a crystallization solution. -As an end run around the apparent glycerol instability, stepwise transfers and quick dips with glycerol were suggested. Glycerol supplemented with xylitol was also thrown into the mix. -There was one tale of sucrose being successfully added into a crystallization condition when direct soaks were unsuccessful -There were several confirmations regarding the success of Paratone and Paraffin oil with pro-tip of dehydrating the paraffin oil in a speed-vac overnight. -Additional suggestions included 50-75% saturated sugars and 6.5 M proline. Since I’m a good scientist I will include the reference section. I found it very useful. -Cryosalts: suppression of ice formation in macromolecular crystallography. K. A. Rubinson et al, Acta Cryst. (2000). D56, 996-1001 -Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. T. Holyoak et. al. *Acta Cryst.*(2003). D *59*, 2356-2358 -Proline: Mother Nature's cryoprotectant applied to protein crystallography. T.A. Pemberton et. al. Acta Cryst. (2012) D68, 1010-8. -Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions. V. Berejnov et.al. J. Appl. Cryst. (2006) 39, 244-251 -A comparison of salts for the crystallization of macromolecules. A. McPherson. Protein Sci. (2001) 10, 418-22 -Strategies for protein cryocrystallography. L. Vera, E. A. Stura. Crystal Growth Design (2013) 14(2), 427-435 Thank you all again. I really appreciate your time and energy. Cheers, Katherine On Wed, Feb 19, 2014 at 9:23 AM, Enrico Stura est...@cea.fr wrote: Dear All, I would like to point out that the conditions 1.8 - 2.0 M NaCl are not considered High Salt as NaCl is soluble to 5M and a 2X solution (i.e. 4M NaCl) is possible. Also NaCl contrary to ammonium sulfate, citrate, phosphate, etc. is compatible with polyethylene glycol without phase separation problems. This means that with 1.8 - 2.0 M NaCl you have an vast repertoire of possible ways to cryo-protect crystals and with the vast repertoire you gain a good possibility of finding conditions that enhance diffraction: see: Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography. Crystal Growth Design, http://pubs.acs.org/doi/full/10.1021/cg301531f For me the definition for High Salt is that 2X for the precipitant component is not possible. Enrico. On Wed, 19 Feb 2014 16:06:27 +0100, Karolina Michalska dzi...@amu.edu.pl wrote: 4M NaCl should work too. It worked for the conditions with 1.8 - 2.0 M NaCl. Karolina W dniu 2014-02-19 06:38, Mooers, Blaine H.M. (HSC) napisał(a): For crystals grown out of a 2 uL drop of 1.2-1.8 M LiSO4 or 1.6-2.4 M AmmSO4, we do in situ cryoprotection with sodium malonate. We add 2-4 uL of 1.9 M Na malonate to the crystallization drop, wait 10 seconds and add 2-4 uL of 2.4 M sodium malonate, repeat with 2.8 M and then 3.4 M. We do not bother withdrawing aliquots to maintain a fixed volume. You may need to tweak the volumes to optimize the resulting diffraction. You can also break the additions at given concentration into smaller aliquots to reduce the osmotic shock. This approach is much gentler than transferring the crystal directly to 3 M sodium malonate. Do not leave the drop exposed to the air for more than 3 minutes or so because salt crystals will start to grow. When there are multiple crystals in a drop, often the unused crystals in the very high salt solution will still diffract well up to a year later if the crystallization chamber is resealed well; their diffraction might even improve with the prolonged exposure to high salt. Blaine Mooers Assistant Professor Department of Biochemistry and Molecular Biology University of Oklahoma Health Sciences Center S.L. Young Biomedical Research Center Rm. 466 Shipping address: 975 NE 10th Street, BRC 466 Oklahoma City, OK 73104-5419 Letter address: P.O. Box 26901, BRC 466 Oklahoma City, OK 73190 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-
Re: [ccp4bb] High Salt Cryo
If you need phases, you might change the salt ion(s) to something with significant anomalous signal, i.e., Rb+, Cs+, Br-, I- instead of Na+ and Cl-. With such high ion concentrations, you should get some really high-occupancy sites. In any case it is sometimes handy to have experimental phases if things don’t go the way you thought with MR. JPK From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Katherine Sippel Sent: Friday, February 21, 2014 11:48 AM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] High Salt Cryo I want to start off by thanking everyone. The replies, both on- and off-board, were speedy and numerous. I apologize for the delay in expressing my gratitude; I was implementing your wonderful suggestions. I have put together a summary for the archive. To recap, I was looking for suggestions to cryoprotect crystals from 3 M NaCl crystallization conditions excluding ethylene glycol or PEG completely and avoiding glycerol and sucrose due to apparent crystal instability. -Several people confirmed that 4 M NaCl should be sufficient. -There were several suggestions for cryosalts with malonate and formate being the most frequent. Lithium salts were also put on the table including partial or complete substitution of NaCl with LiCl, both as a soak or a crystallization solution. -As an end run around the apparent glycerol instability, stepwise transfers and quick dips with glycerol were suggested. Glycerol supplemented with xylitol was also thrown into the mix. -There was one tale of sucrose being successfully added into a crystallization condition when direct soaks were unsuccessful -There were several confirmations regarding the success of Paratone and Paraffin oil with pro-tip of dehydrating the paraffin oil in a speed-vac overnight. -Additional suggestions included 50-75% saturated sugars and 6.5 M proline. Since I’m a good scientist I will include the reference section. I found it very useful. -Cryosalts: suppression of ice formation in macromolecular crystallography. K. A. Rubinson et al, Acta Cryst. (2000). D56, 996-1001 -Malonate: a versatile cryoprotectant and stabilizing solution for salt-grown macromolecular crystals. T. Holyoak et. al. Acta Cryst. (2003). D59, 2356-2358 -Proline: Mother Nature's cryoprotectant applied to protein crystallography. T.A. Pemberton et. al. Acta Cryst. (2012) D68, 1010-8. -Effects of cryoprotectant concentration and cooling rate on vitrification of aqueous solutions. V. Berejnov et.alhttp://et.al. J. Appl. Cryst. (2006) 39, 244-251 -A comparison of salts for the crystallization of macromolecules. A. McPherson. Protein Sci. (2001) 10, 418-22 -Strategies for protein cryocrystallography. L. Vera, E. A. Stura. Crystal Growth Design (2013) 14(2), 427-435 Thank you all again. I really appreciate your time and energy. Cheers, Katherine On Wed, Feb 19, 2014 at 9:23 AM, Enrico Stura est...@cea.frmailto:est...@cea.fr wrote: Dear All, I would like to point out that the conditions 1.8 - 2.0 M NaCl are not considered High Salt as NaCl is soluble to 5M and a 2X solution (i.e. 4M NaCl) is possible. Also NaCl contrary to ammonium sulfate, citrate, phosphate, etc. is compatible with polyethylene glycol without phase separation problems. This means that with 1.8 - 2.0 M NaCl you have an vast repertoire of possible ways to cryo-protect crystals and with the vast repertoire you gain a good possibility of finding conditions that enhance diffraction: see: Vera, L., Stura, E. A. (2013) Strategies for protein cryocrystallography. Crystal Growth Design, http://pubs.acs.org/doi/full/10.1021/cg301531f For me the definition for High Salt is that 2X for the precipitant component is not possible. Enrico. On Wed, 19 Feb 2014 16:06:27 +0100, Karolina Michalska dzi...@amu.edu.plmailto:dzi...@amu.edu.pl wrote: 4M NaCl should work too. It worked for the conditions with 1.8 - 2.0 M NaCl. Karolina W dniu 2014-02-19 06:38, Mooers, Blaine H.M. (HSC) napisał(a): For crystals grown out of a 2 uL drop of 1.2-1.8 M LiSO4 or 1.6-2.4 M AmmSO4, we do in situ cryoprotection with sodium malonate. We add 2-4 uL of 1.9 M Na malonate to the crystallization drop, wait 10 seconds and add 2-4 uL of 2.4 M sodium malonate, repeat with 2.8 M and then 3.4 M. We do not bother withdrawing aliquots to maintain a fixed volume. You may need to tweak the volumes to optimize the resulting diffraction. You can also break the additions at given concentration into smaller aliquots to reduce the osmotic shock. This approach is much gentler than transferring the crystal directly to 3 M sodium malonate. Do not leave the drop exposed to the air for more than 3 minutes or so because salt crystals will start to grow. When there are multiple crystals in a drop, often the unused crystals in the very high salt solution will still diffract well up to a year later if the crystallization chamber is resealed well; their diffraction might even
Re: [ccp4bb] Aggregation assay
Analogous to Dr. Mirella, we've used the ratio of A320/A280 (for membrane proteins). In response to a fussy reviewer, I located some relevant references (refs. 10-13, copied below) when we referred to this in A high-throughput differential filtration assay to screen and select detergents for membrane proteins, Vergis et al., Anal. Biochem 407:1 (2010). [10] S.J. Leach, H.A. Scheraga, Effect of light scattering on ultraviolet difference spectra, J. Am. Chem. Soc. 82 (1960) 47904792. [11] Y. Cordeiro, F. Machado, L. Juliano, M.A. Juliano, R.R. Brentani, D. Foguel, J.L. Silva, DNA converts cellular prion protein into the b-sheet conformation and inhibits prion peptide aggregation, J. Biol. Chem. 276 (2001) 4940049409. [12] G.J. Lee, A.M. Roseman, H.R. Saibil, E. Vierling, A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state, EMBO J. 16 (1997) 659671. [13] Y. Panyukov, I. Yudin, V. Drachev, E. Dobrov, B. Kurganov, The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering, Biophys. Chem. 127 (2007) 918. Regards, -MW Michael C. Wiener, Ph.D. Professor Department of Molecular Physiology and Biological Physics University of Virginia PO Box 800886 Charlottesville, VA 22908-0886 434-243-2731 434-982-1616 (FAX) On Fri, 21 Feb 2014 15:05:46 + Tanner, John J. tanne...@missouri.edu wrote: I am aware of an assay for aggregation (aggregation rate) that is based on fluorescence measurements. It involves excitation at 280 and emission in the range 260-400. Is there a reference for the absorbance method? See Nominé Y, Ristriani T, Laurent C, Lefèvre JF, Weiss E, Travé G. A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. Protein Eng. 2001 Apr;14(4):297-305. PubMed PMID: 11391022. http://www.ncbi.nlm.nih.gov/pubmed/11391022 Jack Tanner Sent from Jack's iPad On Feb 21, 2014, at 7:23 AM, Vivoli, Mirella m.viv...@exeter.ac.ukmailto:m.viv...@exeter.ac.uk wrote: Dear Prerana, before starting the crystallization you could try to check the state of you protein, simply determining the Aggregation Index (AI) from measuring the absorbance at 280 nm and 340 nm. The AI is then computed using this simple formula: AI= 100 x (Abs340/(Abs 280 - Abs 340)). Soluble and non-aggregated proteins solutions typically have an Aggregation Index of 2 and lower, whereas for some aggregation will be 2-5; heavily aggregated proteins show an aggregation index 5. Of course you cannot use Nanodrop for it (because the wavelength for the baseline normalization is 340 nm). I would try to decrease the concentration of your protein to 4-5 mg/ml.Good luck. Best, Mirella Vivoli Mirella Postdoctoral Fellow University of Exeter, Biosciences Biocatalysis Centre, Henry Wellcome Building Stocker Road, Exeter, Ex4 4QD Tel:+ 44 (0)1392 726121 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK] on behalf of Prerana G. [tracy...@gmail.commailto:tracy...@gmail.com] Sent: Friday, February 21, 2014 11:14 AM To: CCP4BB@JISCMAIL.AC.UKmailto:CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] Hi, Sorry for asking an off-topic question, I have recently purified a protein having a molecular weight of 40kDa and concentration of the protein was 8mg/ml. When I tried to set the protein for crystallisation using micobatch method, the protein started precipitating in most of the buffer conditions of Crystal screen and Peg ion. The precipitation took place very quickly (within 5-10 mins). How should I overcome this problem? Regards Prerana
[ccp4bb] High Rwork/Rfree vs. Resolution
Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
Chris, what you get is not unheard of but clearly you are not in majority: at around 1.95A resolution distribution of R-factors in PDB is: Histogram of Rwork for models in PDB at resolution 1.85-2.05 A: 0.093 - 0.118 : 3 0.118 - 0.143 : 75 0.143 - 0.168 : 821 0.168 - 0.193 : 2617 0.193 - 0.218 : 2950 0.218 - 0.242 : 1147 0.242 - 0.267 : 201 your case 0.267 - 0.292 : 21 0.292 - 0.317 : 2 0.317 - 0.342 : 1 Histogram of Rfree for models in PDB at resolution 1.85-2.05 A: 0.138 - 0.160 : 12 0.160 - 0.183 : 106 0.183 - 0.205 : 742 0.205 - 0.227 : 1971 0.227 - 0.249 : 2566 0.249 - 0.272 : 1676 0.272 - 0.294 : 616 0.294 - 0.316 : 119your case 0.316 - 0.339 : 24 0.339 - 0.361 : 6 Histogram of Rfree-Rwork for all model in PDB at resolution 1.85-2.05 A: 0.001 - 0.011 : 67 0.011 - 0.021 : 428 0.021 - 0.031 : 1324 0.031 - 0.041 : 2220 0.041 - 0.050 : 1975 0.050 - 0.060 : 1059 your case 0.060 - 0.070 : 459 0.070 - 0.080 : 201 0.080 - 0.090 : 75 0.090 - 0.100 : 30 Pavel P.S.: Command to the statistics as above is: phenix.r_factor_statistics 1.95 On Fri, Feb 21, 2014 at 4:41 PM, Chris Fage cdf...@gmail.com wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
Thanks for the assistance, everyone. For those who suggested XDS: I forgot to mention that I have tried Mosfim, which is also better than spot fitting than HKL2000. How does XDS compare to Mosflm in this regard? I am not refining the high R-factor structure with NCS options. Also, my unit cell dimensions are 41.74 A, 69.27 A, and 83.56 A, so there isn't one particularly long axis. I'm guessing the low completeness of the 1.65 angstrom dataset has to do with obstacles the processing software encountered on a sizable wedge of frames (there were swaths of in red in HKL2000). I'm not sure why this dataset in particular was less complete than the others. Thanks, Chris On Fri, Feb 21, 2014 at 6:41 PM, Chris Fage cdf...@gmail.com wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
Hi Chris, I personally would go with your thick dataset. 90% completeness is not stellar, but in my opinion not detrimental, either. I had one project that persistently yielded crystals that diffracted to rather high resolution (2.3), but in one direction no lunes were discernible and - consistent with that - the other direction's diffraction consisted of lines that had little beads on them - i.e. extremely smeary spots. XDS was the only program to integrate this data at an Rmerge better than 25% (it actually got below 10%). I was able to phase this data experimentally (Fe-MAD), use NCS and end up with amazing maps. Nevertheless, refinement was a bitch: It never went significantly below 30 for Rfree and messed up the geometry of the model, even though the electron density was clearly showing where the model should be. My explanation for this was that this was a rare case were the phases were actually determined better than the Fs. If you look back, in the days before refinement, reflection intensities were not measured, they were classified as weak, medium and strong - and that was enough to generate meaningful electron densities. In a cases like that, were the accurate determination of integrated intensities is a a problem, there should be a mechanism to submit experimental electron density instead of refined models, as the latter will make way less sense. So again - you got lucky with your thick dataset -- use it and don't sweat the 90% completeness! HTH, Jens On Fri, 2014-02-21 at 18:41 -0600, Chris Fage wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
Re: [ccp4bb] Aggregation assay
Hi, Two things i would like to add: 1) Due to the dependence of A280 on amino acid composition, a simple two-wavelength 280 and 340 or 320 comparison is not very ideal for determining the scatter component of the UV absorption of protein/protein aggregate particles (the usefulness of this simple method for determining the degree of aggregation is not questioned). Instead, we use data points in the 320-350nm range from a UV absorption/scattering scan to plot a curve for determining the scattering component, which serves two purposes: a) to be used for subtracting the A280 to get real UV absorption of the protein at 280nm, b) to have an idea of how aggregated the sample is. To do this, we plot a double log curve with absorption values A and wavelengths lambda at 320 330 340 350 nm, and fit it to the equation log(A)=C - k*log(lambda) Then the resulting equation can be used for extrapolating the scatter component at 280 nm. Of course the larger this component is, the more aggregated the protein is. The degree of aggregation (particle size and population) is also reflected by the k value. The larger the k is, the less aggregated the solution is. An explanation of the theory behind this can be found here: http://www.chem.agilent.com/Library/applications/59633927.pdf Our favorite method is the one in the scattering model part, note that in figure 7 their molecule has UV absorption peak at 300, while in most of our cases, the proteins have peak at 280. There should be an academic article on this method. However I can not locate it right now. Apologies for that. In fact, a quick look at the UV curve in the 300-350 nm range can directly tell us how badly aggregated a protein solution is. A perfectly not aggregating protein solution should have a UV absorption curve that quickly drops to nearly zero at 300 nm. One can test this by measuring an aggregation sample before and after filtration or centrifugation and see the dramatic change on the UV curve. (0.22um filter's pore sizes are quite comparable to the 200-400nm UV wavelengths, while most globular proteins are roughly 2-10 nm in diameter.) 2) Nanodrop's baseline function in the scan mode (Protein 280 for example) is actually an all-point zeroing. At the blanking step, the instrument records a blank absorption curve and later uses this curve to subtract the sample curve. So when using a proper solution as blank, the sample curve is quite faithfully reflecting the UV absorption + scattering. I believe most UV spectrometers allow the users to do the same in the scan mode. So they are all good for this task. Zhijie -Original Message- From: Michael C. Wiener Sent: Friday, February 21, 2014 12:16 PM To: CCP4BB@JISCMAIL.AC.UK Subject: Re: [ccp4bb] Aggregation assay Analogous to Dr. Mirella, we've used the ratio of A320/A280 (for membrane proteins). In response to a fussy reviewer, I located some relevant references (refs. 10-13, copied below) when we referred to this in A high-throughput differential filtration assay to screen and select detergents for membrane proteins, Vergis et al., Anal. Biochem 407:1 (2010). [10] S.J. Leach, H.A. Scheraga, Effect of light scattering on ultraviolet difference spectra, J. Am. Chem. Soc. 82 (1960) 4790–4792. [11] Y. Cordeiro, F. Machado, L. Juliano, M.A. Juliano, R.R. Brentani, D. Foguel, J.L. Silva, DNA converts cellular prion protein into the b-sheet conformation and inhibits prion peptide aggregation, J. Biol. Chem. 276 (2001) 49400–49409. [12] G.J. Lee, A.M. Roseman, H.R. Saibil, E. Vierling, A small heat shock protein stably binds heat-denatured model substrates and can maintain a substrate in a folding-competent state, EMBO J. 16 (1997) 659–671. [13] Y. Panyukov, I. Yudin, V. Drachev, E. Dobrov, B. Kurganov, The study of amorphous aggregation of tobacco mosaic virus coat protein by dynamic light scattering, Biophys. Chem. 127 (2007) 9–18. Regards, -MW Michael C. Wiener, Ph.D. Professor Department of Molecular Physiology and Biological Physics University of Virginia PO Box 800886 Charlottesville, VA 22908-0886 434-243-2731 434-982-1616 (FAX) On Fri, 21 Feb 2014 15:05:46 + Tanner, John J. tanne...@missouri.edu wrote: I am aware of an assay for aggregation (aggregation rate) that is based on fluorescence measurements. It involves excitation at 280 and emission in the range 260-400. Is there a reference for the absorbance method? See Nominé Y, Ristriani T, Laurent C, Lefèvre JF, Weiss E, Travé G. A strategy for optimizing the monodispersity of fusion proteins: application to purification of recombinant HPV E6 oncoprotein. Protein Eng. 2001 Apr;14(4):297-305. PubMed PMID: 11391022. http://www.ncbi.nlm.nih.gov/pubmed/11391022 Jack Tanner Sent from Jack's iPad On Feb 21, 2014, at 7:23 AM, Vivoli, Mirella m.viv...@exeter.ac.ukmailto:m.viv...@exeter.ac.uk wrote: Dear Prerana, before starting the crystallization you could try to check the
[ccp4bb]
Dear CCp4b users, I have a protein which has been crystallized in two different conditions. In one of those conditions, the structure shows the domain shifting. Is there any programme or online server which calculates the angular shift in domain when campared to the other condition crystallized structure which shows no such domain shift. Thanks in advance Avinash Singh
[ccp4bb]
Dear Avinash, Try DnyDom server for domain motion analysis.. DynDom http://fizz.cmp.uea.ac.uk/dyndom/ -Best, Babu -- www.bm14.eu On Sat, Feb 22, 2014 at 7:19 AM, avinash singh avns.si...@gmail.com wrote: Dear CCp4b users, I have a protein which has been crystallized in two different conditions. In one of those conditions, the structure shows the domain shifting. Is there any programme or online server which calculates the angular shift in domain when campared to the other condition crystallized structure which shows no such domain shift. Thanks in advance Avinash Singh
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
Chris, First, I would try NCS restraints even at ~ 2 A. Second, any outliers in your diffraction data set that might skew the R values? Third, have you checked that your refined bulk solvent model is reasonable? Axel On Feb 21, 2014, at 6:13 PM, Chris Fage cdf...@gmail.com wrote: Thanks for the assistance, everyone. For those who suggested XDS: I forgot to mention that I have tried Mosfim, which is also better than spot fitting than HKL2000. How does XDS compare to Mosflm in this regard? I am not refining the high R-factor structure with NCS options. Also, my unit cell dimensions are 41.74 A, 69.27 A, and 83.56 A, so there isn't one particularly long axis. I'm guessing the low completeness of the 1.65 angstrom dataset has to do with obstacles the processing software encountered on a sizable wedge of frames (there were swaths of in red in HKL2000). I'm not sure why this dataset in particular was less complete than the others. Thanks, Chris On Fri, Feb 21, 2014 at 6:41 PM, Chris Fage cdf...@gmail.com wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
Re: [ccp4bb] High Rwork/Rfree vs. Resolution
I can't help but suggest to also try PDB_REDO for tuning refinement. http://xtal.nki.nl/PDB_REDO/index.jsp One of the things you get, is exactly what Pavel explains below, how your structure looks in comparison with others in similar resolution, but also with the PDB_REDO data bank structures. I will also agree that the 90% complete data set might be best - just refine both models, compare, and choose the best one according to validation criteria (you get a few for free in PDB_REDO and of course in eg Molprobity) Tassos Sent from my iPad On 22 Feb 2014, at 02:20, Pavel Afonine pafon...@gmail.com wrote: Chris, what you get is not unheard of but clearly you are not in majority: at around 1.95A resolution distribution of R-factors in PDB is: Histogram of Rwork for models in PDB at resolution 1.85-2.05 A: 0.093 - 0.118 : 3 0.118 - 0.143 : 75 0.143 - 0.168 : 821 0.168 - 0.193 : 2617 0.193 - 0.218 : 2950 0.218 - 0.242 : 1147 0.242 - 0.267 : 201 your case 0.267 - 0.292 : 21 0.292 - 0.317 : 2 0.317 - 0.342 : 1 Histogram of Rfree for models in PDB at resolution 1.85-2.05 A: 0.138 - 0.160 : 12 0.160 - 0.183 : 106 0.183 - 0.205 : 742 0.205 - 0.227 : 1971 0.227 - 0.249 : 2566 0.249 - 0.272 : 1676 0.272 - 0.294 : 616 0.294 - 0.316 : 119your case 0.316 - 0.339 : 24 0.339 - 0.361 : 6 Histogram of Rfree-Rwork for all model in PDB at resolution 1.85-2.05 A: 0.001 - 0.011 : 67 0.011 - 0.021 : 428 0.021 - 0.031 : 1324 0.031 - 0.041 : 2220 0.041 - 0.050 : 1975 0.050 - 0.060 : 1059 your case 0.060 - 0.070 : 459 0.070 - 0.080 : 201 0.080 - 0.090 : 75 0.090 - 0.100 : 30 Pavel P.S.: Command to the statistics as above is: phenix.r_factor_statistics 1.95 On Fri, Feb 21, 2014 at 4:41 PM, Chris Fage cdf...@gmail.com wrote: Dear CCP4BB Users, I recently collected a number of datasets from plate-shaped crystals that diffracted to 1.9-2.0 angstroms and yielded very nice electron density maps. There is no major density unaccounted for by the model; however, I am unable to decrease Rwork and Rfree beyond ~0.25 and ~0.30, respectively. Probably due to the more 2-dimensional nature of my crystals, there is a range of phi angles in which the reflections are smeared, and I am wondering if the problem lies therein. I would be grateful if anyone could provide advice for improving my refinement statistics, as I was under the impression that the R-factors should be ~5% lower for the given resolution. A few more pieces of information: -Space group = P21, with 2 monomers per asymmetric unit; -Chi square = 1.0-1.5; -Rmerge = 0.10-0.15; -Data were processed in HKL2000 and refined in Refmac5 and/or phenix.refine; -PHENIX Xtriage does not detect twinning, but hints at possible weak translational pseudosymmetry; -I was previously able to grow one atypically thick crystal which diffracted to 1.65 angstroms with Rwork/Rfree at 0.18/0.22. Unfortunately, the completeness of the dataset was only ~90%. Regards, Chris
