Re: [ccp4bb] protein turns brown
Hi Sandy, If your protein is soluble (i.e. we're not dealing with brown precipitate), it is possible that the brown color is due to a bound iron-sulfur cluster. In this case, your protein was always brown, but you couldn't see the color when it was very dilute. As a quick test, you could take a UV-Vis spectra. 4Fe-4S clusters have a peak around 410 nm and are distinctly brown in color. -Andy === Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853 === On 9/24/2010 4:34 AM, sandeep wrote: Dear all, I have purified protein from E.coli. expression system. the protein has been purified with three independant columns. Now during concentration step using amicon, the protein shows brown colour. what could be the reason. best regards and Thanks, sandy http://sigads.rediff.com/RealMedia/ads/click_nx.ads/www.rediffmail.com/signatureline@middle?
Re: [ccp4bb] Cryo Vs crystal size
You've gotten some helpful replies already. I have found the following reference to be helpful in understanding some of the physics behind damage incurred during the crystal cooling process and a general strategy to help avoid it. It expands upon what's already been said - that larger crystals are more prone to distress during cooling. This and other papers from the same group contain useful information and advice. A General Method for Hyperquenching Protein Crystals Matthew Warkentin and Robert E. Thorne Struct Funct Genomics. 2007 December ; 8(4): 141–144. doi:10.1007/s10969-007-9029-0. Best, -Andy On 4/15/2010 4:48 AM, Mark J. van Raaij wrote: and don't forget to check diffraction without freezing. Mark On 15 April 2010 10:37, Anastassis Perrakis a.perra...@nki.nl mailto:a.perra...@nki.nl wrote: Hi - My two cents: First, you say: I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? I think this assumption is confusing. If the crystals were grown in the same drop/condition, they have identical percentage solvent content. Thus, you do not want to look at dehydration, the 'percentage solvent content' is fine. What you want to look at is the mechanics of vitrification. Big crystals, are simply hard to freeze: because of their volume they cannot be vitrified as rapidly and uniformly as smaller crystals. I will not be surprised if there are papers that quantify that, but what I am saying here is only from experience and adding a 'logical' explanation to that experience. Thus, I would simply stay with the smaller crystals (I have a feeling that you 'small' crystals are 'big' for many other people) and be happy they diffract to 2.5 A (is that SR or RA?) A. On Apr 15, 2010, at 3:16, syed ibrahim wrote: Dear Jurgen and Ho Leung To add few more point regarding my question: 1. Crystal was first frozen in LN2 and then transfered to cryo stream (in presence of LN2 in vial) 2. Anealing did not help (both short time and long time) - perhaps the crystal dies. 3. Spots are clear to available resolution (is: 6-7A). In the high resolution region there is no spot but looks like smear in the whole area. 4. The crystal was approximately 1.0mm length and 0.4mm dia. I mounted on 0.5mm loop. So the liquid around the crystal was very less. I deliberately avoided more solvent in the loop to help diffraction. Thanks Syed --- On *Thu, 4/15/10, Jürgen Bosch /jubo...@jhsph.edu mailto:jubo...@jhsph.edu/* wrote: From: Jürgen Bosch jubo...@jhsph.edu mailto:jubo...@jhsph.edu Subject: Re: [ccp4bb] Cryo Vs crystal size To: CCP4BB@JISCMAIL.AC.UK mailto:CCP4BB@JISCMAIL.AC.UK Date: Thursday, April 15, 2010, 3:46 AM There are a couple of additional factors not taken into account here. 1. LN2 versus frozen in strem or propane etc 2. did you try to flash anneal the larger crystal 3. smeary diffraction from the big crystal or not ? 4. how much residual solvent was around your crystal when freezing ? In general smaller crystals are anyhow better in my hands. Jürgen On Apr 14, 2010, at 5:36 PM, syed ibrahim wrote: Hi All I had two crystals grown in same well, one is small and other is 10 times bigger. I treated both crystal in same cryo and same time. The smaller one diffracted to 2.5A and the bigger one to 6-7A. I was expecting the bigger one to diffract high resolution. I assume the bigger crystal might have lot of solvent which prevent for high resolution. If it is true what could be the best way to dehydrate crystal without affecting crystal quality? Thank you Syed PS: Taken care of less solvent to be present in the loop - 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/ http://web.me.com/bosch_lab/ *P** **please don't print this e-mail unless you really need to* Anastassis (Tassos) Perrakis, Principal Investigator / Staff Member Department of Biochemistry (B8) Netherlands Cancer Institute, Dept. B8, 1066 CX Amsterdam, The Netherlands Tel: +31 20 512 1951 Fax: +31 20 512 1954 Mobile / SMS: +31 6 28 597791 -- Mark J van Raaij http://webspersoais.usc.es/mark.vanraaij http://www.ibmb.csic.es
Re: [ccp4bb] Crystallization of lysine and arginine rich proteins
Umar, Check out: Czepas et al. The impact of Lys--Arg surface mutations on the crystallization of of the globular domain of RhoGDI, Acta D (2004) 60 275-280. They point out that sulfate ions can help mediate contacts between arginine residues from neighboring molecules in the crystal. You may have already considered the surface entropy server (http://nihserver.mbi.ucla.edu/SER/) to help identify any specific stretches of amino acids that could be mutated to reduce entropy and possibly promote crystallization. Maybe there are a couple regions of your sequence that are flagged as particularly unfavorable in terms of their predicted entropy contribution. There is evidence that lysine is more of a problem in this respect as compared to arginine (Derewenda et al. Acta D (2006) 62 116-124). Good luck, -Andy === Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853 === On 11/2/2009 8:39 AM, Jan Rash wrote: Dear All, I have a question regarding the crystallization of lysine and arginine rich protein around 13%. So far our attempts to crystallize this protein have not been successful although the secondary structure predictions, CD spectroscopy measurements clearly show that this protein is folded. I presume that these lysine and arginine are the sources of the local flexibility in the protein even though the protein is globular overall. Moreover, my attempts to crystallize the limited proteolysis fragments also did not achieve crystals. I have also tried the crystallization with its binding partners and could not succeed. I think any compound that binds to the lysine/arginine side chains might affect the crystallization process thereby reducing the internal flexibility of protein. Can anybody suggest some effective strategy for the crystallization? Thanks Umar
[ccp4bb] [SUMMARY] X-ray diffraction image -- .jpg
To the CCP4 community. As always, I have received a number of very helpful suggestions to my query. I am grateful to all those who replied. My best summary of the suggestions is below. Best Regards, -Andy Torelli A notable consideration (provided by Jim Pflugrath) is that A JPEG has fixed colors for the pixel values. A diffraction image has to use a viewer to convert the pixel values (counts) to a color. One problem with just using a converter to jpeg is how to convert intensities to color (i.e. computer display values). Additionally, Morten Kjeldgaard noted that opening saved images (particularly TIFF images) in Photoshop will require Image - Adjustments - Equalize to see anything other than black. Specific program suggestions: - diff2jpeg - a CCP4 utility within the DiffractionImage library for handling different diffraction file formats. See: http://www.ccp4.ac.uk/html/DiffractionImage.html - idiffdisp - a CCP4 utility. See also CCP4wiki page: http://ccp4wiki.org/~ccp4wiki/wiki/index.php?title=CCP4i_diffraction_image_display_%28idiffdisp%29 - ipdisp - a CCP4 utility. See also: http://www.ccp4.ac.uk/dist/html/ipdisp.html - Mosflm has capability including control over gr[e|a]yscale. See FAQ section for details. - ADXV - reportedly easy to install and can save in TIFF format. Also will save image to match coloring in view window: http://www.scripps.edu/~arvai/adxv.html - Marview - can be installed using Linux_glibc-2.3.3 (RedHat9, WS3, etc). Just download it, unpack (gunzip marView.gz). See also http://www.marresearch.com/download.html#Utilities # chmod a+x marView a) to run the program from terminal # ./marView or # kate .bashrc: alias marview=/home/user/Desktop/marview/./marView b) alternativelly, place the program in the Desktop and just double-click to start it up. - demo version of d*TREK is freely available from Rigaku and has dtdisplay for this purpose. - Labelit - see http://cci.lbl.gov/labelit - labelit.png filename output.png [-large] - ImageMagick - part of most linux distros. See also James Holton's helpful and detailed reply to my original post. - convert -depth 16 -type Grayscale -colorspace GRAY -endian LSB -size 3072x3072+512 GRAY:test_0_001.img test_0_001.jpg
[ccp4bb] X-ray diffraction image -- .jpg
Hi everyone, Is there a free utility that can convert an x-ray diffraction image collected with an ADSC detector to a standard image file format e.g. .jpg, png, etc.? I'm looking for something more elegant than a screen-capture that will yield a higher (graphics) resolution image. I'm sure someone must have done this, but I haven't been able to find one. Thanks, Andy Torelli
Re: [ccp4bb] stacked thin plates
James, I think the standard suggestions apply. Try to tweak your crystallization conditions to get a single crystal (i.e. not a series of stacked plates). PEG 400 can be a good substitute for MPD, but you could also try additive screens as well as co-crystallization with products, cofactors, substrates, analogs, etc. to alter the crystallization. Alternatively, seek out a beamline that offers X-ray beam diameters of 5-20 microns. You can survey your entire crystal(s) for very small diffracting volumes that might only contain a single plate. You may get lucky. -Andy === Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853 === On 9/3/2009 8:40 AM, james09 pruza wrote: Deal all, Sorry for the non-ccp4 query once again. I need suggestions regarding the improvement in crystal quality. I have crystallized a protein in MPD. The crystals grow like a thin plates and the plates are stacked together. So, the mosaicity is very high and also indexing is difficult even at 2 angstrom data set. All suggestions are welcome. Thanks. James.
Re: [ccp4bb] problems of co-crystallization of protein-DNA complex
Ru Heng, It is commonly helpful to combine your protein and DNA under dilute conditions and then concentrate the complex. Combining concentrated DNA and protein together has a very good chance of precipitating in my experience. I completely agree that trying different buffer conditions is a good idea (try to find one that keeps your protein happy as evaluated by DLS). Good luck, -Andy On 8/13/2009 8:23 AM, Raji Edayathumangalam wrote: Couple of things, Ru Heng. 1. What buffer conditions is your protein in? Is it similar to the buffer you describe as using to dissolve your DNA in? In general, you can even get away with dissolving and annealing the oligos in just Tris etc. 2. Play with buffer conditions, particularly NaCl concentrations. 3. Tweak the protein and DNA ratios. For nucleosomes, we always got white precipitate if we did not always titrate the DNA to protein ratios for every individual prep, I believe optimization of the above parameters would help with the white precipitate formation. Hope that helps. Raji --- Raji Edayathumangalam Joint Research Fellow Brigham and Women's Hospital/ Harvard Medical School Brandeis University On Aug 13, 2009, at 12:56 AM, ruheng wrote: Dear CCP4bbers, I am now working on a DNA binding protein and the purity of the protein is quite good, however the results of DLS showed that the protein aggregates terribly in quite a lot of different buffer conditions I tried and still no crystals can be obtained. So I am going to co-crystallize the protein in complex with DNA. I synthesized the oligonucleotides varying different numbers of basepairs to determine the optimal length which can bound to my protein by EMSA. I dissoved the oligos in the buffer containing 50mM Tris-HCl, 100mM NaCl, 10mM MgCl2 and 1mM DTT, pH 7.9 and then annealed the DNA into the double stranded form at a final concentration of 50uM. When I performed the EMSA experiment, I mixed the purified protein with the dsDNA at the molecular ratio approximately 1:1, but white precipitate was generated as I mixed them. Does anyone have this kinds of experience when working on DNA binding proteins and co-crystallizing the protein-DNA complex? Any suggestions from yours will be appreciated. Thank you all. Ru Heng 搜索本应是快乐的，不是么? 快乐搜索，有问必应！微软隆重推出! 立即试用！ http://bing.com.cn?FORM=M00HCNPubl=WLHMTAGCrea=TEXT_Search_Where_You_Are_1X1
Re: [ccp4bb] {Spam?} quick purification?
Mark,
I agree with Artem in that you may find significant differences in the
purity of eluants from the 'high fidelity' IMAC resins (I use pre-packed
HisTrap columns).
You may also want to have a look at the following reference for a
comparison of a variety of different affinity tags in terms of purity,
yield, cost, etc.
Comparison of affinity tags for protein purification. Protein Expr
Purif. 2005 May;41(1):98-105.
-Andy
On 8/10/2009 11:56 PM, Artem Evdokimov wrote:
Hi,
Since you're considering a serious undertaking, it would be good to know
whether your protein is decently expressed and reasonably characterized in
its native state - before advising you on the process :)
If your protein is normally expressed well, is soluble, and not aggregated -
there's going to be little to no difference between the results of major
methods used for primary affinity capture. If your protein is not very
abundant in the lysate, then higher fidelity methods may give you better
purity - in this case StrepII is somewhat better than GST. You can always
try the good old Biotin tag - this will however require that your E. coli
carry excess of BirA (typically supplied from a separate plasmid) as normal
biotinylation levels in most E. coli strains are fairly low.
With 40+ mutants you can expect a certain amount of attrition - some of the
mutants will not express well and some will not purify well.
In general there's nothing wrong with single-step IMAC provided that you can
work out reasonable purification conditions in advance and can assume that
most of your 40+ mutants behave reasonably well (and close to the test
case). For outlier mutants there will undoubtedly be issues with
purification however primary capture method is not likely to fix them since
it did not cause them in the first place.
For abundant proteins - if you use a 'high fidelity' IMAC resin such as
His-SELECT or Talon and if your protein of interest is present (in the
lysate) in reasonable quantity then you can expect the purity of your
single-step product to be compatible between GST and IMAC purification.
StrepII tag is of course a good choice as well.
Regards,
Artem
Nothing is built on stone; all is built on sand, but we must build as if
the sand were stone
Jorge Luis Borges
-Original Message-
From: CCP4 bulletin board [mailto:ccp...@jiscmail.ac.uk] On Behalf Of Mark
Collins
Sent: Monday, August 10, 2009 10:37 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] {Spam?} quick purification?
Hi All
A little off topic, but I am thinking about expressing and purifying 40+
mutants, for an assay, at maybe 1mg total protein each. I'd like the
purification to be quick and easy (ie. one step) but cleaner than just a
6his-tag purification.
Currently, my options are either GST, a longer his tag or a strep-tag.
Any thoughts on the comparison between GST and strep and Ni purifications
would be much appreciated. OR are there any other (better) high affinity
purifications, I've missed, excluding expense Ab resins.
Thanks,
Mark Collins
Re: [ccp4bb] off topic
Hi Kien, You may also want to try to wash your column with elution buffer (without reducing agents), equilibrate it with binding buffer (plus reducing agents) and then bind your protein. In the case of HisTrap columns (a pre-packed, Ni(II) resin column), this procedure is recommended if you wish to use reducing agents. I was told by tech. support that washing with high imidazole elution buffer first (without reducing agents) helps wash out weakly-bound nickel ions that are left over from the charging process. These ions, which are not fully chelated to the resin, are more susceptible to the reducing agents and may undergo the process described by Guenter to result in your brown color. Good luck, -Andy On 6/19/2009 9:19 AM, Guenter Fritz wrote: Hi Kien, DTT coordinates very nicely the Ni(II) on the column. However the DTT-Ni(II) is prone to oxidation which gives almost immediately Ni(III) which causes the brownish colour. As Jürgen pointed out TCEP chelates only weakly Ni(II) and is a better choice. Also beta-mercapto-ethanol does not bind Ni(II) so tightly, i.e causes less trouble. Regarding your 4 Cys residues, there might be some redox going on, but not necessarily. The Cys SGs are often inside in an hydrophobic environment. You can do easily the Ni(II) column WITHOUT any reductant in the buffer. Simply add DTT after the Ni(II) column. You can also check the oxidation state of your Cys by good old biochemistry (http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Thiols_and_disulfides). In order to avoid oxidation of Cys or Met on the Ni(II) column, strip your Ni(II) and charge your IMAC column with Zn(II) instead of Ni(II). Zn(II) is redox inert. HTH Guenter Try TCEP as it does not interfere with the NiNTA resin whereas DTT does. But why do you care if your column is brown or not - can you elute your protein ? Are there other metal ions e.g. iron which bind to your resin perhaps ? Jürgen On 19 Jun 2009, at 02:25, Kn Ly wrote: Hello everyone, I am trying to purify a 13 KDa membrane protein using Ni NTA. The protein is solubilised in Triton X 100, 20 mM phosphate buffer, 150 mM NaCl and binds very well to the column. However, it also turns the column brownish. The protein contains 4 cysteine residues so I suspect that this causes cross-linking with other proteins and thus brownish precipitation on the column. So I included 5 mM beta-ME in my buffer to prevent disulfide bond formation but this doesn't help. I tried 1 mM DTT and this ruined the column. Help!! Is there anyway to prevent this brownish problem? Thanks a lot in advance Kien - 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.me.com/bosch_lab/
[ccp4bb] Offtopic: Alscript question
To the CCP4bb, I have an off-topic request regarding Alscript, a program used to make postscript images from sequence alignments. Does anyone have an Alscript script file (.als) that successfully implements the RELATIVE_TO and mask ILLEGAL commands that I could look at? I haven't been able to get these commands to work and I haven't found a fix from the documentation, CCP4wiki or Google searches. I would be grateful to anyone with an example script using these commands they could share. If so, please spare the bulletin board and respond to me directly. Best Regards, -Andy Torelli === Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry Chemical Biology Baker Laboratory, Cornell University Ithaca, NY 14853 ===
Re: [ccp4bb] SUMMARY:vSystem virtual machine recommendation for crystallography?
Hello Everyone, Thank you for the replies to my questions regarding system virtual machine software. I have organized the replies into subheadings and summarized the comments below. The original question was: I would like to install a system virtual machine to run Ubuntu Linux as a guest OS on a 32-bit Vista laptop. The idea is to allow occasional use of crystallographic refinement programs while I'm away from lab. The laptop has an Intel Core 2 duo processor (2.0 GHz) and 3 GB RAM. There are popular software programs available (VMWare, Parallels, VirtualBox, etc.), but is anyone aware of any considerations that would make one better for the above purposes? For example, will one offer easy control over distributing hardware resources to prevent crippling Vista while running refinement within the guest Linux? The organized/summarized responses: Individual user experiences: - VirtualBox 2.2.2 to run Ubuntu 9.04 (guest) on WinXP 32-bit (native). Runs well with 512 Mbyte assigned memory with 64 Mbyte assigned graphics memory (dual core 2.4 GHz machine with 2 Gbyte total RAM). - VMWare used for various guest OSs. Found to work well. - Parallels useful for Linux as a guest; VMWare for Windows as a guest. - Qemu/Kvm and VirtualBox work well with Linux as a guest OS. Kvm found to be troublesome for Windows as a guest OS. - VMWare server 2 running CentOS (guest) on Linux (native) Software considerations: - VirtualBox is free - VirtualBox supports hardware virtualization, but it is off by default. Some others do as well. - Some distributions of VMWare are free - VMWare reported to be stable, has wide user-base, good documentation and can run dual-CPU General considerations: - No accelerated graphics performance (i.e. Coot/Pymol run slowly at ~15 frames/sec. with non-native graphics. - Previous threads recommend running graphics in native OS to take full advantage of hardware acceleration - Recommended to set up shared folders to transfer files between OSs to facilitate using native graphics software/hardware. - You have to reboot the machine to change the memory configuration, but people agreed the software is generally easy to configure for resource allocation. Thanks again to all who replied, -Andy Torelli -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry and Chemical Biology Cornell University =
[ccp4bb] System virtual machine recommendation for crystallography?
Hello everyone, I would like to install a system virtual machine to run Ubuntu Linux as a guest OS on a 32-bit Vista laptop. The idea is to allow occasional use of crystallographic refinement programs while I'm away from lab. The laptop has an Intel Core 2 duo processor (2.0 GHz) and 3 GB RAM. There are popular software programs available (VMWare, Parallels, VirtualBox, etc.), but is anyone aware of any considerations that would make one better for the above purposes? For example, will one offer easy control over distributing hardware resources to prevent crippling Vista while running refinement within the guest Linux? My Google and CCP4bb searches have not turned up anything so far. Thanks in advance for any advice or reference material. I will post a summary e-mail as well. Best Regards, -Andy Torelli -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry and Chemical Biology Cornell University =
Re: [ccp4bb] Cryo-protectant
Hi Liew, There have already been some very good suggestions. I agree with Tim Gruene that a great starting point is to test the diffraction properties of your crystal at room temperature. This can serve as a baseline for comparing/evaluating cryoprotecting agents and methods. You can also test your potential cryoprotection solutions to see if they freeze clear or even take a few X-ray snapshots of them to confirm there are no ice rings. There are lots of publications that can be helpful. One very helpful reference is: Garman, E.F. and Doublie, S. Cryocooling of Macromolecular Crystals: Optimisation Methods. Methods in Enzymology (2003) 368, 188-216. Be aware that, as mentioned previously, the method for freezing can make a difference (e.g. freezing in cold-stream vs. plunging in nitrogen). An obvious difference is different cooling rates (you can find references for this) or less obvious reasons, for example differences in dehydration that occur during longer/shorter transfer through air from drop to cold-source for either method. Finally, you can check out the database for cryoprotecting solutions: http://idb.exst.jaxa.jp/db_data/protein/search-e.php Good luck, -Andy -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Laboratory of Steven E. Ealick Department of Chemistry and Chemical Biology Cornell University = On 4/24/2009 10:44 AM, Liew Chong Wai wrote: Hi all Thanks for your precious suggestions and ideas. The crystallization buffer condition is 0.1M BIS-TRIS pH 5.5, 0.2M MgCl.6H2O, 35% PEG3350 Now, my crystal seem ok in 20% ethylene glycol, but only after a couple minutes of dehydration at room temperature. For sure, i will try other cryoprotectant that was suggested here. I just wondering why MPD kills the crystal. Many thanks *LIEW* -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Laboratory of Steven E. Ealick Department of Chemistry and Chemical Biology Cornell University =
[ccp4bb] Scaling of intensities
To the CCP4 community, I have a question about ImportScaled. When I select both the Keep the input intensities in the output file and the Run Truncate... options, the output MTZ file contains IMEAN and SIGIMEAN values that are different from the input intensity file. Specifically, the values are multiplied by 1/100th of the SCALE term reported in the log file that is calculated from the Wilson Plot during the Truncate procedure. However, when the Run Truncate... option is not selected, the output MTZ file contains unaltered IMEAN and SIGIMEAN values that match the input file. After reading the recent CCP4 threads regarding Truncate as well as the program documentation, I still have a few questions: 1. My understanding is that this scaling of the intensities is done to bring them to an (approximate) absolute scale and therefore can only be performed when Truncate is run simultaneously (because the Wilson Plot is necessary to calculate the appropriate scale factor). However, why is the scaling equal to 1/100 of the SCALE term from the Wilson Plot (i.e. why not exactly the SCALE term)? 2. For programs that use intensities as a target for refinement, is it necessary to have the intensities scaled in this way or is it also valid to use the unaltered (scaled only) intensities? 3. On a related note, when is it best to refine against intensities vs. amplitudes? I have not been able to find recent literature that pertains to macromolecular crystallography and the documentation I've looked at for popular refinement programs that offer both targets do not provide guidelines as far as I can tell. If anyone could recommend some literature, I would really appreciate it. Thank you very much for your time, Best Regards, -Andy Torelli -- = Andrew T. Torelli Ph.D. Postdoctoral Associate Department of Chemistry and Chemical Biology Cornell University =
