[ccp4bb] CCDs + Re: PILATUS data collection
Hi all, Graeme's message tell the core of the detector usage notes here at diamond. He might have unearthed a can of worms or two, though ;) CCDs still are great detectors! I am not sure about his noise comment as the more recent CCDs (post 2000-ish) are designed to operate with anode sources (and definitely image plates have a very good signal-to-noise ratio in long exposures). The mitigation of the readout deadtime is arguably the main experimental design driver when using CCDs. One other tiny little thing to bring to the surface is that the CCD images presented to the user are not the raw images and my personal opinion is that the end users should be at least aware of what a real image looks like (zingers, taper features, tiling) and their implications in the noise per reflection. I agree that filtering, stitching and smoothing help in judging the quality of the diffraction pattern (and crystal) itself. Regards, Jose' - try other color scales! black and white is good but a rainbow pallete helps identifying weaks and strongs because if discrete jumps in color at different intensities. === Jose Brandao-Neto MPhil CPhys Senior Beamline Scientist - I04-1 Diamond Light Source jose.brandao-n...@diamond.ac.uk +44 (0)1235 778506 www.diamond.ac.uk ===
Re: [ccp4bb] question about CCP4 scripts
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1
Dear Joe,
most ccp4 programs make use of space group and cell information if
present in the input file. The script you list below does not have an
input file (apparently it creates a dummy mtz-file with only a list of
Miller indices), so there is nothing to extract. E.g. a simple test
# scalepack2mtz hklin test.sca hklout test.sca eof
eof
confirms that scalepack2mtz extracts the information from the header
automatically and creates a valid mtz-file (I tend to give programs a
try the way I would like them to work, and if that does not work, I
start reading the manual or check on the web or post to a bb).
Regards,
Tim
On 05/09/2013 05:37 AM, Joe Chen wrote:
Hi All,
I am trying to write a shell script to streamline a few steps, one
of which is Unique, see below. As you can see, this program
requires symmetry and cell parameters. In CCP4 GUI Scalepack2mtz,
these info are automatically extracted from .sca file (first two
lines). But I don't know if there is a way to do this in script,
so I don't need to type these values for each dataset. Thank you
in advance for your help.
#!/bin/sh # unique.exam # # runnnable test script for the program
unique - this will use this # program to generate a reflection
list containing null values. #
set -e
unique hklout ${CCP4_SCR}/unique_out.mtz eof labout F=F
SIGF=SIGF symmetry p43212 resolution 1.6 cell 78.1 78.1 55.2 90.0
90.0 90.0 eof
- --
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 Mozilla - http://enigmail.mozdev.org/
iD8DBQFRi12sUxlJ7aRr7hoRAurSAKDnNjVeJN0GvhDLzKmaYUX0mVUWggCePvTF
s78KpaBbPA9j4XTg4IpOjBE=
=qxVe
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[ccp4bb] Reminder - CCP4 Newsletter submissions
The deadline for articles submitted for the next issue of the CCP4 Newsletter is Friday 14th June 2013. Articles may cover any topic of interest to macromolecular crystallographers, though we are particularly interested in articles on software or methodology. Short items of news are also very welcome - help put the news back into Newsletter! Previous issues of the newsletter, and submission instructions, can be found online: http://www.ccp4.ac.uk/newsletters.php Regards Karen McIntyre Scientific Computing Department - CCP4 R92 1.22 RCaH Science Technology Facilities Council Rutherford Appleton Laboratory Harwell Science Innovation Campus Didcot Oxfordshire OX11 0FA Tel +44 (0) 1235 44 5790 Fax +44 (0) 1235 56 7720 **Please note that I only work mornings until 12.45pm** -- Scanned by iCritical. -- Scanned by iCritical.
Re: [ccp4bb] question about CCP4 scripts
Typo:
To avoid crash/overwrite, should be
scalepack2mtz hklin test.sca hklout test.mtz eof eof
^^^
jbb
Jeffrey Bonanno, Ph.D.
New York Structural Genomics Research Consortium
-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Tim Gruene
Sent: Thursday, May 09, 2013 4:26 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] question about CCP4 scripts
-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1
Dear Joe,
most ccp4 programs make use of space group and cell information if present in
the input file. The script you list below does not have an input file
(apparently it creates a dummy mtz-file with only a list of Miller indices), so
there is nothing to extract. E.g. a simple test
# scalepack2mtz hklin test.sca hklout test.sca eof eof
confirms that scalepack2mtz extracts the information from the header
automatically and creates a valid mtz-file (I tend to give programs a try the
way I would like them to work, and if that does not work, I start reading the
manual or check on the web or post to a bb).
Regards,
Tim
On 05/09/2013 05:37 AM, Joe Chen wrote:
Hi All,
I am trying to write a shell script to streamline a few steps, one of
which is Unique, see below. As you can see, this program requires
symmetry and cell parameters. In CCP4 GUI Scalepack2mtz, these info
are automatically extracted from .sca file (first two lines). But I
don't know if there is a way to do this in script, so I don't need to
type these values for each dataset. Thank you in advance for your
help.
#!/bin/sh # unique.exam # # runnnable test script for the program
unique - this will use this # program to generate a reflection list
containing null values. #
set -e
unique hklout ${CCP4_SCR}/unique_out.mtz eof labout F=F SIGF=SIGF
symmetry p43212 resolution 1.6 cell 78.1 78.1 55.2 90.0
90.0 90.0 eof
- --
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 Mozilla - http://enigmail.mozdev.org/
iD8DBQFRi12sUxlJ7aRr7hoRAurSAKDnNjVeJN0GvhDLzKmaYUX0mVUWggCePvTF
s78KpaBbPA9j4XTg4IpOjBE=
=qxVe
-END PGP SIGNATURE-
Re: [ccp4bb] question about CCP4 scripts
Looks like my signature messed up the script too... scalepack2mtz hklin test.sca hklout test.mtz eof eof ^^^
[ccp4bb] Codon Optimization for Insect Expression
Hi all, Sorry for the off-topic request. I am considering ordering codon-optimized genes for expression in insect cells (High Five). I wonder if anyone could make suggestions regarding: which companies are best to order from (accurate synthesis – cost efficiency) and what optimized codon sequence generators yield the best protein expression (as I understand it, not all algorithms are created equal). Thanks, Randy
Re: [ccp4bb] Codon Optimization for Insect Expression
I hope this isn't too much of a thread hijack, but i also wanted to add a question to Randy's : How big a protein can one express in insect cells? I've read in a few places that it's not size limited but i wonder if that's borne out in practice. On 9 May 2013 16:05, Randy Watson sigepra...@gmail.com wrote: Hi all, Sorry for the off-topic request. I am considering ordering codon-optimized genes for expression in insect cells (High Five). I wonder if anyone could make suggestions regarding: which companies are best to order from (accurate synthesis – cost efficiency) and what optimized codon sequence generators yield the best protein expression (as I understand it, not all algorithms are created equal). Thanks, Randy
Re: [ccp4bb] Codon Optimization for Insect Expression
Hi Randy and Rodger, We have had real success using BlueSky (MA) for insect expression, I believe we have had around 15-20 genes synthesis-optimized with these guys. The expression levels have been rather reasonable too (obviously protein dependent, but on average 1 mg of xtal grad protein per 1g of cell pellet). The largest construct we did I believe was around 90kDa. Cheers, Mat From: Roger Pickman rpick...@googlemail.com To: CCP4BB@JISCMAIL.AC.UK Sent: Thursday, 9 May 2013, 10:31 Subject: Re: [ccp4bb] Codon Optimization for Insect Expression I hope this isn't too much of a thread hijack, but i also wanted to add a question to Randy's : How big a protein can one express in insect cells? I've read in a few places that it's not size limited but i wonder if that's borne out in practice. On 9 May 2013 16:05, Randy Watson sigepra...@gmail.com wrote: Hi all, Sorry for the off-topic request. I am considering ordering codon-optimized genes for expression in insect cells (High Five). I wonder if anyone could make suggestions regarding: which companies are best to order from (accurate synthesis – cost efficiency) and what optimized codon sequence generators yield the best protein expression (as I understand it, not all algorithms are created equal). Thanks, Randy
Re: [ccp4bb] question about CCP4 scripts
At this point you do have the scalepack2mtz output file (BTW,
imosflm/aimless is wholeheartedly recommended by this convert), and you
can easily extract all the info from there. There is mtzdmp, of course,
but it's easier to parse the actual mtz file (hey, the records are
actually text). Like so:
egrep -oa SYMINF.{74} foo.mtz | awk '{print symm $5}'
Gives you the space group *number*, most ccp4 programs I know accept
that in addition to string symbol. But if you really need the latter,
echo symm $(egrep -oa SYMINF.{74} foo.mtz | cut -d' -f 2 | sed
s///g)
will do.
As for unit cell parameters, this should work
egrep -oa CELL.{76} foo.mtz | sed -n 1p
Keep in mind that this extracts the global cell and will be
problematic if you have multi-dataset file (which I presume you don't).
If you need Nth dataset grab DCELL record, e.g. for the dataset #1
egrep -oa DCELL.{75} foo.mtz | sed -n 2p
Cheers and
https://xkcd.com/208/
Ed
On Wed, 2013-05-08 at 23:37 -0400, Joe Chen wrote:
Hi All,
I am trying to write a shell script to streamline a few steps, one of
which is Unique, see below. As you can see, this program requires
symmetry and cell parameters. In CCP4 GUI Scalepack2mtz, these info
are automatically extracted from .sca file (first two lines). But I
don't know if there is a way to do this in script, so I don't need to
type these values for each dataset. Thank you in advance for your
help.
#!/bin/sh
# unique.exam
#
# runnnable test script for the program unique - this will use this
# program to generate a reflection list containing null values.
#
set -e
unique hklout ${CCP4_SCR}/unique_out.mtz eof
labout F=F SIGF=SIGF
symmetry p43212
resolution 1.6
cell 78.1 78.1 55.2 90.0 90.0 90.0
eof
--
Best regards,
Joe
--
Edwin Pozharski, PhD, Assistant Professor
University of Maryland, Baltimore
--
When the Way is forgotten duty and justice appear;
Then knowledge and wisdom are born along with hypocrisy.
When harmonious relationships dissolve then respect and devotion arise;
When a nation falls to chaos then loyalty and patriotism are born.
-- / Lao Tse /
Re: [ccp4bb] Codon Optimization for Insect Expression
You can always synthesize your own too! Artem Or idtdna. Dna2.0, blue sky, and many others On May 9, 2013 10:01 AM, Mathew Martin mathew.mar...@rocketmail.com wrote: Hi Randy and Rodger, We have had real success using BlueSky (MA) for insect expression, I believe we have had around 15-20 genes synthesis-optimized with these guys. The expression levels have been rather reasonable too (obviously protein dependent, but on average 1 mg of xtal grad protein per 1g of cell pellet). The largest construct we did I believe was around 90kDa. Cheers, Mat -- *From:* Roger Pickman rpick...@googlemail.com *To:* CCP4BB@JISCMAIL.AC.UK *Sent:* Thursday, 9 May 2013, 10:31 *Subject:* Re: [ccp4bb] Codon Optimization for Insect Expression I hope this isn't too much of a thread hijack, but i also wanted to add a question to Randy's : How big a protein can one express in insect cells? I've read in a few places that it's not size limited but i wonder if that's borne out in practice. On 9 May 2013 16:05, Randy Watson sigepra...@gmail.com wrote: Hi all, Sorry for the off-topic request. I am considering ordering codon-optimized genes for expression in insect cells (High Five). I wonder if anyone could make suggestions regarding: which companies are best to order from (accurate synthesis – cost efficiency) and what optimized codon sequence generators yield the best protein expression (as I understand it, not all algorithms are created equal). Thanks, Randy
[ccp4bb] Membrane Protein Optimisation
Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys
[ccp4bb] 2 positions available for software developers in cryoEM
Dear All, I have 2 positions available for software developers in cryoEM: 1) A developer to work alongside Chris Wood on the CCP-EM project. The post will support a number of software development projects (see e.g. http://www.ccpem.ac.uk/ccpem_projects.php), as well as providing general community support. 2) A developer to work on Work Package 9 of the BioMedBridges grant (http://www.biomedbridges.eu/), concerned with developing a database of volumes with associated tools, in collaboration with developers at the EBI. We are looking for talented developers of scientific software. While knowledge of cryoEM and structural biology would be an advantage, applicants from other backgrounds are welcomed if you can demonstrate an ability to learn and a flexibility in approach. The projects are collaborative in nature, so you must be a good communicator and be able to work as part of a team. The posts are located at the Research Complex at Harwell (http://www.rc-harwell.ac.uk/), within the group of Martyn Winn in the Scientific Computing Department of STFC (http://www.stfc.ac.uk/scd/), and alongside the core team of CCP4. The Research Complex is located on the Harwell Science and Innovation Campus, adjacent to the Diamond synchrotron and provides an exciting multi-disciplinary work environment, including many relevant structural biology groups. Applications need to be made via the TopCareer site (see links on http://www.ccpem.ac.uk/positions.php) and informal enquiries can be made to me. Cheers Martyn * * Dr. Martyn Winn * STFC Daresbury Laboratory, Daresbury, Warrington, WA4 4AD, U.K. * Tel: +44 1925 603455 (DL) or +44 1235 567865 (RcaH) * E-mail: martyn.w...@stfc.ac.uk Skype: martyn.winn * -- Scanned by iCritical.
Re: [ccp4bb] Membrane Protein Optimisation
Hi Rhys, I suspect that what you call a gel might be phase separation (correct me if this is wrong) like an oily phase enriched in protein and detergent. you may have too much detergent in your drop. may I ask what detergent you are using (low or high CMC) and at what concentration of detergent do you think you are when you setup your drops. you might have concentrated more detergent than you think along your protein. the MW cutoff of the membrane you are using to concentrate is important relative to the size of the free detergent micelle and of course the protein-detergent micelle you are trying to concentrate. too much detergent staying around is a major cause of trouble (i.e. poor diffraction and phase separation competing with productive crystal growth) besides many other parameters Hope this helps -- Pascal F. Egea, PhD Assistant Professor UCLA, David Geffen School of Medicine Department of Biological Chemistry Boyer Hall room 356 611 Charles E Young Drive East Los Angeles CA 90095 office (310)-983-3515 lab (310)-983-3516 email pe...@mednet.ucla.edu On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys
Re: [ccp4bb] Membrane Protein Optimisation
With information you've provided I have multiple suggestions for you, some of which you may have already tried: 1. OMPs can be fairly particular about which detergents they will crystallize in. Try exchanging the protein into a different detergent/detergent mixture and then set up new trays in your favorite broad matrix screens. DDM, DM, LDAO, OG, and C8E4 are a few of my favorites for OMPs, but there are many others. This can be done using a size exclusion column as the final purification step for your protein where the column is equilibrated with the appropriate detergent. This step will also let you know how well behaved the protein is in that detergent via the shape/height of the peak. This won't help you optimize your current condition, but it may lead to different/new conditions with even better crystals. As Pascal suggested, try to carefully choose which MW cut-off concentrator you end up using. Minimizing the amount of detergent concentration that occurs during your concentration step(s) is optimal. 2. Attempt crystallization using DHCP/CHAPSO bicelles. You can buy them pre-made from MemX Biosciences or make them yourself using a published protocol from David Bowie's lab. These have been used to crystallize the mitochondrial beta barrel protein VDAC and I had success crystallizing intimin in them. David Bowie also has a JoVE article on the bicelle crystallization method. 3. Attempt crystallization using Lipidic Cubic Phases. This was the saving grace for my post-doc project. Neither detergent nor bicelle crystallization produced crystals that were of sufficient quality, but the crystals from LCP all diffracted to the 2.0 angstrom resolution range. If you're unfamiliar with the technique, there are several nice videos on JoVE describing it by Vadim Cherezov and Martin Caffrey. 4. Alter your construct. Small changes in the construct (ie: deletion or addition of 1-2 residues on the N- or C-terminus) often led to drastically different crystallization behavior of several OMPs in my hands. Cheers, Jim On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER r.grinte...@research.gla.ac.uk wrote: Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructures http://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.com jfair...@embios.com
Re: [ccp4bb] Membrane Protein Optimisation
Thanks for the suggestions so far, I should have given a little more information in my initial post. The protein I'm working on is an Gram-Neg Beta-Barrel about 100kDa, likely with 22 strands. I'm currently crystallising in bOG, although my next optimisation step is to try a range of detergents. I'm using a refolding protocol which relies heavily in LDAO (one of the only cost effective detergents for the volumes I'm using), I refold, nickel purify (N-terminal tag, signal peptide removed) , do a size exclusion step (S200), then detergent exchange into bOG using a nickel column. The BOG concentration is obviously high 0.8-1%, which might be causing the gels? The gels don't look like classic phase separation to me (from soluble proteins that is, this is my first membrane protein), they are not spherical, the tend to float or sit on the bottom and are semi-solid, around 30-100uM diameter. These screens I've set up are classic screens for membrane proteins (mem-plus, mem-start, PGA etc.) and the drops aren't dried up. The gels are strongly birefringent, but the drop is not. Additionally the conditions in my initial screen which yielded crystals seemed biased for the formation of the gels. Thanks again for the help, I feel I might have a long raod to optimisation ahead of me. Rhys From: Jim Fairman [fairman@gmail.com] Sent: 09 May 2013 20:58 To: RHYS GRINTER Cc: ccp4bb Subject: Re: [ccp4bb] Membrane Protein Optimisation With information you've provided I have multiple suggestions for you, some of which you may have already tried: 1. OMPs can be fairly particular about which detergents they will crystallize in. Try exchanging the protein into a different detergent/detergent mixture and then set up new trays in your favorite broad matrix screens. DDM, DM, LDAO, OG, and C8E4 are a few of my favorites for OMPs, but there are many others. This can be done using a size exclusion column as the final purification step for your protein where the column is equilibrated with the appropriate detergent. This step will also let you know how well behaved the protein is in that detergent via the shape/height of the peak. This won't help you optimize your current condition, but it may lead to different/new conditions with even better crystals. As Pascal suggested, try to carefully choose which MW cut-off concentrator you end up using. Minimizing the amount of detergent concentration that occurs during your concentration step(s) is optimal. 2. Attempt crystallization using DHCP/CHAPSO bicelles. You can buy them pre-made from MemX Biosciences or make them yourself using a published protocol from David Bowie's lab. These have been used to crystallize the mitochondrial beta barrel protein VDAC and I had success crystallizing intimin in them. David Bowie also has a JoVE article on the bicelle crystallization method. 3. Attempt crystallization using Lipidic Cubic Phases. This was the saving grace for my post-doc project. Neither detergent nor bicelle crystallization produced crystals that were of sufficient quality, but the crystals from LCP all diffracted to the 2.0 angstrom resolution range. If you're unfamiliar with the technique, there are several nice videos on JoVE describing it by Vadim Cherezov and Martin Caffrey. 4. Alter your construct. Small changes in the construct (ie: deletion or addition of 1-2 residues on the N- or C-terminus) often led to drastically different crystallization behavior of several OMPs in my hands. Cheers, Jim On Thu, May 9, 2013 at 8:34 AM, RHYS GRINTER r.grinte...@research.gla.ac.ukmailto:r.grinte...@research.gla.ac.uk wrote: Hi All, A quick question if you've ever worked on membrane proteins, I'm trying to optimize crystals for bacterial integral outer membrane protein I'm working on. I'm getting some fairly modest rod like crystals in a 0.1M Tris pH 7.5, 30% PEG 600, 0.03M MgCl2 condition. However I get lots and lots of birefringent gel forming in the same condition, I get the feeling that this is Detergent/Protein complex and is robbing my crystals of material to grow bigger. These crystals diffract to 5 A so I'd quite like to make them bigger and better, Cheers, Rhys -- Jim Fairman, Ph D. Crystal Core Leader I Emerald BioStructureshttp://www.emeraldbiostructures.com/ Tel: 206-780-8914 Cell: 240-479-6575 E-mail: fairman@gmail.commailto:fairman@gmail.com jfair...@embios.commailto:jfair...@embios.com
