Re: [ccp4bb] ARP/wARP 7.1; ccp 6.1.13 and Mac OSX 10.6 (64bit fink)
On 9 Sep 2010, at 01:57, Leiman Petr wrote: Switch to the 64bit environment (source /sw64/bin/init.sh in a new terminal window). Install ARP/wARP while in this terminal (not sure if this is needed actually, probably not). Then copy all the files from these directories: /sw/share/xtal/ccp4-6.1.13/ccp4i/templates /sw/share/xtal/ccp4-6.1.13/ccp4i/tasks /sw/share/xtal/ccp4-6.1.13/ccp4i/scripts to their 64bit equivalents /sw64/... Then copy /sw/share/xtal/ccp4-6.1.13/ccp4i/etc/UNIX/modules.def to /sw64/... As an alternative (simpler?) method. I installed ARP/wARP with CCP4 environment variables set to find the 64bit versions (i.e having sourced /sw64/bin/init.sh) and then in a separate root shell sourced /sw/bin/init.sh and installed the ARP/wARP task into the users CCP4 task area so that all I had to do was copy ~root/.CCP4 to my home directory (and change permissions). Huw -- Dr Huw Jenkins Astbury Centre for Structural Molecular Biology University of Leeds h.t.jenk...@leeds.ac.uk
Re: [ccp4bb] protein dimensions
pdbset gives it to you too. pdbset xyzin a.pdb end if you want the principal exes I still use Amore table function - that reorientates the model according to ppl axes then gives you the dimensions along each axis.. Tim Gruene wrote: On Wed, Sep 08, 2010 at 08:21:33PM +0200, Nikos Pinotsis wrote: quite straightforward with moleman2 and the command stat ... after the command 'xyz align', but the 'stats'-command tells you about it. Tim On Wed, September 8, 2010 19:37, Brett, Thomas wrote: Hi all: Is there program or utility out there that will give maximum protein dimensions (length and width) from the pdb file? I'm sure there is, just curious what people use. Thanks, -Tom
Re: [ccp4bb] non-identical complexes in the asymmetric unit
I use PISA to analyse this - sometimes the differences depend on the definition of what is a hydrogen bond? and unless you have very high resolution it is risky to say there are significant differences.. But certainly there are examples where the results are very significant indeed - you could look at the extensive heamoglobin literature.. Eleanor Roger Rowlett wrote: I don't think this is uncommon at all. For example, we published a structure where 10 chains did not bind ligand at all, and 2 chains did in the ASU (see PDB 3E3I). We have also recently solved a structure where two active sites in the ASU are in different states. Cheers. On 9/8/2010 12:50 PM, Rongjin Guan wrote: Dear All, I have a structure with two complexes in the asymmetric unit, and the interactions on the interface are not the same in the two complexes. Briefly, there are two additional hydrogen bonds in one complex, but not in the other. This coule be due to crystallization artefact, but may have other explanations. Can anyone direct us to some references where this has been discussed before? Thank you very much Rongjin Guan -- Roger S. Rowlett Professor Department of Chemistry Colgate University 13 Oak Drive Hamilton, NY 13346 tel: (315)-228-7245 ofc: (315)-228-7395 fax: (315)-228-7935 email: rrowl...@colgate.edu
Re: [ccp4bb] fail to install ccp4i task for arp warp
Dear Jochen, Huw and others, We have managed to reproduce the problem on a 64bit Mac with fink-installed CCP4. We can confirm that the problem appears to be related to 64bit TCL/bltwish as suggested by Huw. Invoking a 32-bit installation of fink alongside a 64 bit one can be tricky, so we propose the following solution which should work in every case. Firstly, get a different tcl - this could be the version of tcl from a 32 bit installation of fink, or otherwise from a downloaded TCL installation, from, for example, ActiveState: http://www.activestate.com/activetcl The introduction of ARP/wARP into the GUI will have to be done using this version of tcl. I would suggest that the simplest way of doing so is to modify the install_csh.sh script that is in the downloaded ARP/wARP folder. At line 565, replace the line: $CCP4I_TCLTK/tclsh eof | tail -2 to point to whatever tclsh you intend to use. For example, if you have a 32 bit fink installation, based in the folder /sw32, then the line should read: /sw32/bin/tclsh eof | tail -2 A second option would be to modify the setting for CCP4I_TCLTK in the ccp4 setup script appropriate to whatever shell you use. In this instance, both manual installation of ARP/wARP into the GUI and installation using the install.sh script should be possible once the modification has been made and the environment variables reset. If you need any further details on how this could be done for your own shell, let me know. Note that installation using this method will require one to operate in a sudo shell, and of course have all environment variables set up in advance of installation, as laid out in the attachment from Huw. HTH, Ciaran Carolan On 06/09/2010 16:10, Huw Jenkins wrote: On 31 Aug 2010, at 14:56, Jochen Kuper wrote: sorry to bother you with this but I just can't figure out what goes wrong ... I am running the latest version of snow leopard on a MBP unibody with the 64bit kernel. I have an up to date fink installation for native 64bit. I've seen this too - it seems to be due to problems with 64bit bltwish. There is a workaround here: http://proteincrystallography.org/ccp4bb/message13191.html but that unfortunately requires a 32 bit fink installation of CCP4 alongside the 64bit one. I can confirm that this works and once installed ARP/wARP and 64bit CCP4 work together fine. Huw -- Dr Huw Jenkins Astbury Centre for Structural Molecular Biology University of Leeds -- Ciaran Carolan EIPOD Postdoctoral Researcher Lamzin Group EMBL Hamburg Outstation Notkestrasse 85 Hamburg Germany Ph. +49 40 89902340
[ccp4bb] Human Haemoglobin Crystallization Protocol
Hi all, I'm looking for a good (or any) crystallization protocol for human haemoglobin. I've found some resources online but they want me to pay to download the PDFs. Does anyone know of any good protocols or at least where I might find this info? Thanks, ~Brennan~
Re: [ccp4bb] Human Haemoglobin Crystallization Protocol
How aboutAdachi, S., S. Y. Park, J. R. Tame, Y. Shiro, and N. Shibayama. 2003. Direct observation of photolysis-induced tertiary structural changes in hemoglobin. Proc Natl Acad Sci U S A 100:7039-44. Ito, L., T. Kobayashi, K. Shiraki, and H. Yamaguchi. 2008. Effect of amino acids and amino acid derivatives on crystallization of hemoglobin and ribonuclease A. J Synchrotron Radiat 15:316-8. HideakiMoriyamaPh.D.AssociateProfessorSchoolofBiologicalSciencesUniversityofNebraska-Lincoln243ManterHallLincoln,NE68588-0118(402)472-5367telephone(402)472-2083faxSBSofficehmoriya...@unl.edue-mailnpx001.unl.eduhmlabwebpage
[ccp4bb] How to define NCS in REFMAC
Hi there: The REFMAC manual give me a hard time to define the NCS during refinement. Can anybody give a first time user a sample script based on the following PDB header (NCS part only is ok, but please include how todefine tight restrant only for both positional and B ref, for both NCS groups)? Thanks a lot! Best Regards, Hailiang REMARK 3 NCS RESTRAINTS STATISTICS REMARK 3 NUMBER OF DIFFERENT NCS GROUPS : 2 REMARK 3 REMARK 3 NCS GROUP NUMBER : 1 REMARK 3 CHAIN NAMES: A B C D E F G REMARK 3 NUMBER OF COMPONENTS NCS GROUP : 2 REMARK 3 COMPONENT C SSSEQI TO C SSSEQI CODE REMARK 3 1 A 2 A 135 1 REMARK 3 1 B 2 B 135 1 REMARK 3 GROUP CHAINCOUNT RMS WEIGHT REMARK 3 TIGHT POSITIONAL 1A(A): 1806 ; 0.092 ; 0.050 REMARK 3 TIGHT POSITIONAL 1B(A): 1806 ; 0.131 ; 0.050 REMARK 3 TIGHT THERMAL 1A (A**2): 1806 ; 0.192 ; 0.500 REMARK 3 TIGHT THERMAL 1B (A**2): 1806 ; 0.228 ; 0.500 REMARK 3 REMARK 3 NCS GROUP NUMBER : 2 REMARK 3 CHAIN NAMES: A B C D E F G REMARK 3 NUMBER OF COMPONENTS NCS GROUP : 2 REMARK 3 COMPONENT C SSSEQI TO C SSSEQI CODE REMARK 3 1 A136 A 190 1 REMARK 3 1 B136 B 190 1 REMARK 3 GROUP CHAINCOUNT RMS WEIGHT REMARK 3 TIGHT POSITIONAL 1A(A): 1806 ; 0.092 ; 0.050 REMARK 3 TIGHT POSITIONAL 1B(A): 1806 ; 0.131 ; 0.050 REMARK 3 TIGHT THERMAL 1A (A**2): 1806 ; 0.192 ; 0.500 REMARK 3 TIGHT THERMAL 1B (A**2): 1806 ; 0.228 ; 0.500
[ccp4bb] List of commonly used cryoprotectants and buffer molecules
Dear All, I am trying to find out which molecules are frequently used by X-ray crystallographers serving as cryoprotectants or as buffer molecules. The idea behind this is to sort native ligands from molecules that appeared in the electron density just because they were used in the crystallization buffer or as a cryoprotectant. Can anybody point out literature, a web site, or simply provide a (subjective) list extending my collection of GOL, EDO, and different size PEGs? What about sugars? Thanks for your help, Chris
Re: [ccp4bb] List of commonly used cryoprotectants and buffer molecules
ISRDB Cryoprotectant database for protein crystals is a good resource, not only for the cryoprotection methods but the freezing methods used from literature. http://idb.exst.jaxa.jp/db_data/protein/200304E02478000.html It is under maintenance at the moment though... HTH, Matt On Thu, Sep 9, 2010 at 5:33 PM, Chris Weichenberger ch...@came.sbg.ac.atwrote: Dear All, I am trying to find out which molecules are frequently used by X-ray crystallographers serving as cryoprotectants or as buffer molecules. The idea behind this is to sort native ligands from molecules that appeared in the electron density just because they were used in the crystallization buffer or as a cryoprotectant. Can anybody point out literature, a web site, or simply provide a (subjective) list extending my collection of GOL, EDO, and different size PEGs? What about sugars? Thanks for your help, Chris -- Matthew L.H. Chu, PhD Postdoctoral Scholar - Weis Lab Department of Structural Biology Fairchild D143, MC 5126 Stanford School of Medicine Stanford, CA 94305-5432 Lab: 650-724-3306 Alternative Email: matt...@stanford.edu Facebook: http://www.facebook.com/MatthewLingHonChu Skype: matthew.lh.chu
Re: [ccp4bb] List of commonly used cryoprotectants and buffer molecules
This cryocrystallography webinar lists some common cryoprotectants: http://www.rigaku.com/protein/webinar-001.html -Original Message- From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Chris Weichenberger Sent: Thursday, September 09, 2010 7:34 PM To: CCP4BB@JISCMAIL.AC.UK Subject: [ccp4bb] List of commonly used cryoprotectants and buffer molecules Dear All, I am trying to find out which molecules are frequently used by X-ray crystallographers serving as cryoprotectants or as buffer molecules. The idea behind this is to sort native ligands from molecules that appeared in the electron density just because they were used in the crystallization buffer or as a cryoprotectant. Can anybody point out literature, a web site, or simply provide a (subjective) list extending my collection of GOL, EDO, and different size PEGs? What about sugars? Thanks for your help, Chris
Re: [ccp4bb] List of commonly used cryoprotectants and buffer molecules
Dear All, I am trying to find out which molecules are frequently used by X-ray crystallographers serving as cryoprotectants or as buffer molecules. The idea behind this is to sort native ligands from molecules that appeared in the electron density just because they were used in the crystallization buffer or as a cryoprotectant. Can anybody point out literature, a web site, or simply provide a (subjective) list extending my collection of GOL, EDO, and different size PEGs? What about sugars? Thanks for your help, Chris We have listed and categorized many of the chemicals that you are looking for: http://store.p212121.com/categories/Chemicals/ Disclosure: I am associated with this commercial website. Items that are not listed, but would be worth considering: MES Polyethylene Glycol Monomethylether (350, 550, 750, 2000, 5000) PEG 1,500 PEG 6,000 PEG 10,000 PEG 20,000 Paratone-N 1,4-Dioxane 1,6-Hexanediol (+/-)-2-Methyl-2,4-pentanediol Jeffamine® ED-2001 Ethylene imine polymer Jeffamine® M-600® Reagent Pentaerythritol ethoxylate (15/4 EO/OH) Pentaerythritol propoxylate (5/4 PO/OH) Pentaerythritol ethoxylate (3/4 EO/OH) Pentaerythritol propoxylate (17/8 PO/OH) Perfluoropolyether PFO-X175/08 Poly(acrylic acid sodium salt) 5,100 Polypropylene glycol P 425 Polyvinylpyrrolidone K 15 In addition to the chemicals already mentioned it may be worth including detergents and enzymatic substrates. I hope that helps. Take Care, Sean
Re: [ccp4bb] How to define NCS in REFMAC
Take a look at this part of a script, I added your NCS operators in two options.
So you are working on GroEL ?
Good luck,
Jürgen
#!/bin/csh -f
set prevVer = 01
set prevRun = 00
set currVer = 02
set currRun = 04nolig
set currData = my_latest_structure
#
set xyzin = omitted_ligands.pdb
set xyzot = v{$currVer}r{$currRun}_{$currData}.pdb
#
#XDS processed data
set hklin = v02r02_Pf_Cmp24.mtz
set hklot = v{$currVer}r{$currRun}_{$currData}.mtz
#
set log = v{$currVer}r{$currRun}_{$currData}.log
#
refmac5 \
HKLIN $hklin HKLOUT $hklot \
# LIBIN Cmp24.cif \
# TLSIN tls_def.tlsin TLSOUT tls.out \
XYZIN tmp.pdb XYZOUT $xyzot \
EOF $log
MAKE HYDRogens ALL
MAKE CHECK 0
MAKE CISP N BUILD Y
LABI FP=FP SIGFP=SIGFP FREE=FreeR_flag
REFI TYPE RESTrained RESOlution 25 1.7
#option # 1 over the whole chain
#NCSRestraints NCHAins 7 CHAIns A B C D E F G
#or option #2 two domains per chain
#for definitions of restrain codes
# RTFM
# http://www.ccp4.ac.uk/html/refmac5/keywords/restraints.html#ncsr
NCSRestraints NCHAins 7 CHAIns A B C D E F G NSPANS 2 2 135 1 136 190 1
REFI RESI MLKF
#BFACtor SET_to 90
#REFI TLSC 10
REFI BREF ISOT ! Refine overall B-values
WEIG MATR 0.1
DAMP 0.5 0.5
SCALe TYPE BULK
SCALe LSSCale
SCALe LSSCale ANISotropic
SCAL MLSC
NCYC 10
TEMP 1.0 4.0 6.0 6.0 10.0
MONI MANY DIST 4 TORS 4 ANGL 4 CHIR 4 VDWR 3 NCSR 4 PLAN 4 NCSR 4 BFAC 4
BINS 10
EOF
-
Jürgen Bosch
Johns Hopkins Bloomberg School of Public Health
Department of Biochemistry Molecular Biology
Johns Hopkins Malaria Research Institute
615 North Wolfe Street, W8708
Baltimore, MD 21205
Phone: +1-410-614-4742
Lab: +1-410-614-4894
Fax: +1-410-955-3655
http://web.mac.com/bosch_lab/
On Sep 9, 2010, at 8:29 PM, Hailiang Zhang wrote:
Hi there:
The REFMAC manual give me a hard time to define the NCS during refinement.
Can anybody give a first time user a sample script based on the following
PDB header (NCS part only is ok, but please include how todefine tight
restrant only for both positional and B ref, for both NCS groups)? Thanks
a lot!
Best Regards, Hailiang
REMARK 3 NCS RESTRAINTS STATISTICS
REMARK 3 NUMBER OF DIFFERENT NCS GROUPS : 2
REMARK 3
REMARK 3 NCS GROUP NUMBER : 1
REMARK 3 CHAIN NAMES: A B C D E F G
REMARK 3 NUMBER OF COMPONENTS NCS GROUP : 2
REMARK 3 COMPONENT C SSSEQI TO C SSSEQI CODE
REMARK 3 1 A 2 A 135 1
REMARK 3 1 B 2 B 135 1
REMARK 3 GROUP CHAINCOUNT RMS WEIGHT
REMARK 3 TIGHT POSITIONAL 1A(A): 1806 ; 0.092 ; 0.050
REMARK 3 TIGHT POSITIONAL 1B(A): 1806 ; 0.131 ; 0.050
REMARK 3 TIGHT THERMAL 1A (A**2): 1806 ; 0.192 ; 0.500
REMARK 3 TIGHT THERMAL 1B (A**2): 1806 ; 0.228 ; 0.500
REMARK 3
REMARK 3 NCS GROUP NUMBER : 2
REMARK 3 CHAIN NAMES: A B C D E F G
REMARK 3 NUMBER OF COMPONENTS NCS GROUP : 2
REMARK 3 COMPONENT C SSSEQI TO C SSSEQI CODE
REMARK 3 1 A136 A 190 1
REMARK 3 1 B136 B 190 1
REMARK 3 GROUP CHAINCOUNT RMS WEIGHT
REMARK 3 TIGHT POSITIONAL 1A(A): 1806 ; 0.092 ; 0.050
REMARK 3 TIGHT POSITIONAL 1B(A): 1806 ; 0.131 ; 0.050
REMARK 3 TIGHT THERMAL 1A (A**2): 1806 ; 0.192 ; 0.500
REMARK 3 TIGHT THERMAL 1B (A**2): 1806 ; 0.228 ; 0.500
Re: [ccp4bb] Fab purification and crystallization
I assume you separate your complex over a size exclusion before setting up trays ? If not try that and see if you get better crystals. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 9, 2010, at 10:59 PM, xaravich ivan wrote: Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone experienced such problem? Is there any thing that could be tried to stop this precipitation. thanks in advance. ivan
Re: [ccp4bb] How to define NCS in REFMAC
Dear Jürgen:
Exactly! 1SS8, the tls-refined GroEL. I generated a PDB containing the
total anistropic B values by TLS, based on which I want to reproduce the R
factor by REFMAC as they used. Thanks for your advice, following is my
script. Unfortunately, it failed to reproduce the PDB R factor...
Not sure whether the NCSR part is correct or not, 'cause the PDB file
provides 3 NCS groups where 2 of them containing mutliple domains (see PDB
header after the REFMAC script...).
Thanks lot!
Hailiang
*
#!/bin/bash
PDB=1SS8
refmac5 \
HKLIN 1ss8-sf.mtz \
HKLOUT ${PDB}-refmac.mtz \
XYZIN ${PDB}.pdb \
XYZOUT ${PDB}-refmac.pdb eor
#
# Input/output column assignments
#
LABIN FP=FP SIGFP=SIGFP FREE=FREE
LABO FC=FC PHIC=PHIC FWT=2FOFCWT DELFWT=FOFCWT
#
# Type of refinement
#
REFI TYPE REST
REFI RESI MLKF
REFI BREF ISOT METH CGMAT
#
# NCS restraints
#
NCSRestraints NCHAins 7 CHAIns A B C D E F G NSPANS 2 2 135 1 410 525 1
NCSRestraints NCHAins 7 CHAIns A B C D E F G NSPANS 2 136 190 1 375 409 1
NCSRestraints NCHAins 7 CHAIns A B C D E F G NSPANS 1 191 374 1
#
# Scaling, in particular requesting anisotropic scaling
#
SCAL TYPE BULK
SCAL LSSC ANISOT
#
# Other keywords
#
WEIG AUTO
MONI MEDI
NCYC 10
END
eor
***
REMARK 3 NCS RESTRAINTS STATISTICS
REMARK 3 NUMBER OF DIFFERENT NCS GROUPS : 3
REMARK 3
REMARK 3 NCS GROUP NUMBER : 1
REMARK 3 CHAIN NAMES: A B C D E F G
REMARK 3 NUMBER OF COMPONENTS NCS GROUP : 2
REMARK 3 COMPONENT C SSSEQI TO C SSSEQI CODE
REMARK 3 1 A 2 A 135 1
REMARK 3 1 B 2 B 135 1
REMARK 3 1 C 2 C 135 1
REMARK 3 1 D 2 D 135 1
REMARK 3 1 E 2 E 135 1
REMARK 3 1 F 2 F 135 1
REMARK 3 1 G 2 G 135 1
REMARK 3 2 A410 A 525 1
REMARK 3 2 B410 B 525 1
REMARK 3 2 C410 C 525 1
REMARK 3 2 D410 D 525 1
REMARK 3 2 E410 E 525 1
REMARK 3 2 F410 F 525 1
REMARK 3 2 G410 G 525 1
REMARK 3 GROUP CHAINCOUNT RMS WEIGHT
REMARK 3 TIGHT POSITIONAL 1A(A): 1806 ; 0.092 ; 0.050
REMARK 3 TIGHT POSITIONAL 1B(A): 1806 ; 0.131 ; 0.050
REMARK 3 TIGHT POSITIONAL 1C(A): 1806 ; 0.098 ; 0.050
REMARK 3 TIGHT POSITIONAL 1D(A): 1806 ; 0.087 ; 0.050
REMARK 3 TIGHT POSITIONAL 1E(A): 1806 ; 0.108 ; 0.050
REMARK 3 TIGHT POSITIONAL 1F(A): 1806 ; 0.091 ; 0.050
REMARK 3 TIGHT POSITIONAL 1G(A): 1806 ; 0.092 ; 0.050
REMARK 3 TIGHT THERMAL 1A (A**2): 1806 ; 0.192 ; 0.500
REMARK 3 TIGHT THERMAL 1B (A**2): 1806 ; 0.228 ; 0.500
REMARK 3 TIGHT THERMAL 1C (A**2): 1806 ; 0.189 ; 0.500
REMARK 3 TIGHT THERMAL 1D (A**2): 1806 ; 0.190 ; 0.500
REMARK 3 TIGHT THERMAL 1E (A**2): 1806 ; 0.216 ; 0.500
REMARK 3 TIGHT THERMAL 1F (A**2): 1806 ; 0.182 ; 0.500
REMARK 3 TIGHT THERMAL 1G (A**2): 1806 ; 0.193 ; 0.500
REMARK 3
REMARK 3 NCS GROUP NUMBER : 2
REMARK 3 CHAIN NAMES: A B C D E F G
REMARK 3 NUMBER OF COMPONENTS NCS GROUP : 2
REMARK 3 COMPONENT C SSSEQI TO C SSSEQI CODE
REMARK 3 1 A136 A 190 1
REMARK 3 1 B136 B 190 1
REMARK 3 1 C136 C 190 1
REMARK 3 1 D136 D 190 1
REMARK 3 1 E136 E 190 1
REMARK 3 1 F136 F 190 1
REMARK 3 1 G136 G 190 1
REMARK 3 2 A375 A 409 1
REMARK 3 2 B375 B 409 1
REMARK 3 2 C375 C 409 1
REMARK 3 2 D375 D 409 1
REMARK 3 2 E375 E 409 1
REMARK 3 2 F375 F 409 1
REMARK 3 2 G375 G 409 1
REMARK 3 GROUP CHAINCOUNT RMS WEIGHT
REMARK 3 TIGHT POSITIONAL 2A(A):647 ; 0.072 ; 0.050
REMARK 3 TIGHT POSITIONAL 2B(A):647 ; 0.076 ; 0.050
REMARK 3 TIGHT POSITIONAL 2C(A):647 ; 0.073 ; 0.050
REMARK 3 TIGHT POSITIONAL 2D(A):647 ; 0.070 ; 0.050
REMARK 3 TIGHT POSITIONAL 2E(A):647 ; 0.083 ; 0.050
REMARK 3 TIGHT POSITIONAL 2F(A):647 ; 0.071 ;
[ccp4bb] Beamtime at the ALS-BCSB beamlines
Dear All, September 15, 2010 is the deadline for the November/December 2010 Rapid Access Proposal cycle. All Berkeley Center for Structural Biology(BCSB) beamlines are equipped with ADSC Q315/Q315R detectors, automated sample changers and data collection software enabling high-throughput crystal screening and data collection. Remote data collection is available on all BCSB beamlines, providing the user with the full complement of sample visualization, sample manipulation, beamline control, data acquisition and data analysis tools exactly as they would see them if they were stationed at the beamline. The main difference between local operation and remote operation, is the length of the network cable! This enhanced remote operation capability is coupled with *22hr onsite support* by BCSB staff who are able to assist immediately with loading additional samples for remote users or troubleshoot any issues that might arise. Remote users can furthermore be kept up-to-date on changes in ring status via an SMS service (http://bcsb.als.lbl.gov/wiki/index.php/Ring_Status_Notifications_to_Cell_Phone) or via twitter (@AlsRingStatus). Specific features are summarized below. Beamlines 5.0.1, 5.0.2, 5.0.3: -- Beamline 5.0.2 is equipped with a novel variable collimator allowing users to adjust the beamsize continuously and on the fly between 25 and 100 micron, both horizontally and vertically. With a collimator setting of 30x30 micron, typical exposures are around 1 to 2 second. The Berkeley Automounter sample handling system has a routine capacity of 96 samples (6 pucks). In a typical high-throughput screening mode, the mount-to-mount time is around 2.5 minutes per sample, allowing users to screen a full puck within 45 minutes. The sector 5 beamline user stations are equipped with fully high-adjustable, ergonomically friendly work stations. Beamlines 8.2.1 and 8.2.2: --- To facilitate studies on small crystals, a microdiffractometer was installed in the beamline 8.2.1 endstation. The new equipment allows precise sample positioning to within 2 microns, excellent sample viewing of very small crystals, and an off-axis crystal positioningstage. Both beamlines 8.2.1 and 8.2.2 feature a Rigaku sample changer (Actor), allowing remote operations to now be a routine mode of access for these beamlines. Data analyses in the BCSB is facilitated by software maintained by sbgrid (http://www.sbgrid.org). A 16 core linux machine is available for our users to process their data and solve/refine their structure. An additional mode of access to the BCSB beamlines is through the Collaborative Crystallography (CC) Program. Users apply for beamtime via the general user program, and collaborate with an expert crystallographer who will conduct the experiments and data reduction on behalf of the researchers. Depending on the users, structure solution, model building and refinement can be carried out as well. Please contact bcsbbeamt...@lbl.gov for more information. Please visit http://bcsb.lbl.gov/ for more details about the Center and its beamlines. To find out more, click on: http://www.als.lbl.gov/als/quickguide/independinvest.html We invite you to submit a proposal at: http://alsusweb.lbl.gov/ Scroll down to Structural Biology beamines (includes protein SAXS). Click on New Proposal. If you have any questions or would like to request open beamtime, please e-mail bcsbbeamt...@lbl.gov. Please note that executed user agreements must be received by LBNL prior to beamtime. Proprietary fees, if applicable, must be received by LBNL at least five working days prior to scheduled beamtime. -- - P.H. Zwart Research Scientist Berkeley Center for Structural Biology Lawrence Berkeley National Laboratories 1 Cyclotron Road, Berkeley, CA-94703, USA Cell: 510 289 9246 BCSB: http://bcsb.als.lbl.gov PHENIX: http://www.phenix-online.org SASTBX: http://sastbx.als.lbl.gov -
[ccp4bb] why I can't reproduce R based on the same program?
Hi there: I want to reproduce the R factor provided by PDB file. The structure was refined by REFMAC, and so I think if I try a REFMAC refinement based on the pdb file and reflection data, the initial R factor given by REFMAC should be it. The pdb file provides residual B factors with TLS given by the header. I therefore generated the PDB with the total anisotropic B, based on which I tried REFMAC. However, the initial R_free was higher than provide by PDB (0.226 vs 0.216). Not sure why I can't reproduce R based on the same program. Thanks for any advice. Best Regards, Hailiang
Re: [ccp4bb] why I can't reproduce R based on the same program?
I see in your script you used Weight Auto - play a bit with that value and you will find a better fit. Have you used the same TLS groups ? I don't see that you are using TLS refinement in your script. Jürgen - Jürgen Bosch Johns Hopkins Bloomberg School of Public Health Department of Biochemistry Molecular Biology Johns Hopkins Malaria Research Institute 615 North Wolfe Street, W8708 Baltimore, MD 21205 Phone: +1-410-614-4742 Lab: +1-410-614-4894 Fax: +1-410-955-3655 http://web.mac.com/bosch_lab/ On Sep 10, 2010, at 1:33 AM, Hailiang Zhang wrote: Hi there: I want to reproduce the R factor provided by PDB file. The structure was refined by REFMAC, and so I think if I try a REFMAC refinement based on the pdb file and reflection data, the initial R factor given by REFMAC should be it. The pdb file provides residual B factors with TLS given by the header. I therefore generated the PDB with the total anisotropic B, based on which I tried REFMAC. However, the initial R_free was higher than provide by PDB (0.226 vs 0.216). Not sure why I can't reproduce R based on the same program. Thanks for any advice. Best Regards, Hailiang
Re: [ccp4bb] Fab purification and crystallization
Dear Ivan, We faced the same problem for our Hya/Fab complex (clustered needles). By change the pH and doing microseeding, we were able to improve the crystals quality. For more information see our paper Padavattan, S., Schirmer, T., Schmidt, M., Akdis, C., Valenta, R., Mittermann, I., Soldatova, L., Slater, J., Mueller, U., and Markovic-Housley, Z. (2007). Identification of a B-cell epitope of hyaluronidase, a major bee venom allergen, from its crystal structure in complex with a specific Fab. J Mol Biol 368, 742-752. for detailed protocol see the supplementary information in my thesis : http://edoc.unibas.ch/651/ We also faced the problem of antibody precipitation during dialysis and concentration with bacterial expressed IgG. This could be due to absence of glycosylation. So I avoided extensive dialysis and did papain digestion with diluted IgG. Digested sample load on to Mono-S column to separate the Fab isoforms from (Fab)2 and undigested IgG. The purified Fab mixed with antigen 1:1.2 molar ratio and used the gel filtration to separate the complex from unbound antigen. Hope this will help you Cheers Sivaraman Padavattan On Fri, Sep 10, 2010 at 8:29 AM, xaravich ivan xaravich.i...@gmail.comwrote: Hi CCP4bb, I have two questions regarding Fab purification and Fab-antigen complex crystallization and would really appreciate any input from the experienced board. 1) I have got some hits for Fab-antigen complex (150 kD) but they are all needle clusters. Whatever fine screen I formulate, it always gives me these needle clusters. Are there some better common ways to change needles to single crystals? 2) I have certain IgGs from which I purify the Fab by papain digestion (resin from ThermoSci). One of the first steps is to dialyze the IgG with the digestion buffer,( 20mM Na-phosphate and 10mM EDTA pH 7.0) Over night. I always get 30-60% of the IgG precipitated during this overnight dialysis. I tried to increase the salt by adding 200mM NaCl but of no effect. Have anyone experienced such problem? Is there any thing that could be tried to stop this precipitation. thanks in advance. ivan
