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CCP4BB  May 2010

CCP4BB May 2010

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Subject:

Re: size of protein in negatively stained TEM?

From:

Alexandra Deaconescu <[log in to unmask]>

Reply-To:

Alexandra Deaconescu <[log in to unmask]>

Date:

Fri, 28 May 2010 23:01:40 -0400

Content-Type:

text/plain

Parts/Attachments:

Parts/Attachments

text/plain (1178 lines)

Hello:

I am not at all surprised by your observations. Negatively stained  
images are sometimes tricky to interpret. Despite a much higher  
contrast, one has to deal with various artifacts of the staining/ 
drying/flattening process, and of course, the stain thickness on any  
grid or mesh is variable which may result in the different "sizes" you  
see (assuming all the data was collected at the same defocus value).  
If you see large differences in not only size, but also shape, that  
may indicate that you have some aggregation in your specimen and that  
the differences in size you see are not due to differential staining  
but heterogeneity in the specimen. In my experience, I have seen  
particles that look slightly smaller when negatively-stained (but then  
of course, all is in the map "thresholding" and therefore  don't think  
there is any rule of thumb) f. If you'd like I can have a look at your  
images/class averages, but I am not sure I will have a definitive  
answer :)

Hope this helps.

Alex


Alexandra Deaconescu, Ph.D.
-----------------------------------
Postdoctoral Fellow of the Damon Runyon Cancer Research Foundation
c/o Grigorieff Laboratory
Brandeis University
http://people.brandeis.edu/~deacona




On May 28, 2010, at 7:00 PM, CCP4BB automatic digest system wrote:

> There are 17 messages totaling 2108 lines in this issue.
>
> Topics of the day:
>
>  1. Catalytic residues in active sites
>  2. size of protein in negatively stained TEM?
>  3. Job Advertisment (PhD position)
>  4. How to calculate real-space CC by section? (2)
>  5. How large should the real space correlation coefficient be?
>  6. mosflm in script mode (3)
>  7. protein monitoring (4)
>  8. The "Total" CC in the output of CCP4 OVERLAPMAP
>  9. to what extent bacterial expression reveals the native  
> oligomerization
>     state of mammalian proteins. (2)
> 10. please recommend a crystallization incubator
>
> ----------------------------------------------------------------------
>
> Date:    Thu, 27 May 2010 18:52:30 -0500
> From:    Donnie Berkholz <[log in to unmask]>
> Subject: Re: Catalytic residues in active sites
>
> On 15:52 Tue 25 May     , Clayton, Gina Martyn wrote:
>> I wonder if anyone can recommend a good review/paper describing
>> crystal structures that show high energy residues in active sites. By
>> that I mean residues that may be in a strained conformation and  
>> rotate
>> between conformations, such that they may even switch into unfavoured
>> ramachandran regions during their activity, but that the strained  
>> high
>> energy state is relevant to the enzyme activity?
>
> Gina,
>
> Here are a few papers discussing strained residues in active sites:
>
> Xu, Q., Buckley, D., Guan, C., Guo, H.C. "Structural insights into the
> mechanism of intramolecular proteolysis." Cell 98, 651-661 (1999).
>
> Lawson, C.L. "An atomic view of the L-tryptophan binding site of trp
> repressor." Nat. Struct. Biol. 3, 986-987 (1996).
>
> Herzberg, O., Moult, J. "Analysis of the steric strain in the
> polypeptide backbone of protein molecules." Proteins: Struct. Func.
> Bioinf. 11, 223-229 (1991)
>
> Merritt, E.A. et al. "The 1.25 Ĺ resolution refinement of the cholera
> toxin B-pentamer: evidence of peptide backbone strain at the
> receptor-binding site." J. Mol. Biol. 282, 1043-1059 (1998).
>
> -- 
> Thanks,
> Donnie
>
> Donald S. Berkholz, Postdoctoral research fellow
> James R. Thompson lab, Physiology & Biomedical Engineering
> Grazia Isaya lab, Pediatric & Adolescent Medicine
> Medical Sciences 2-66
> Mayo Clinic College of Medicine
> 200 First Street SW
> Rochester, MN 55905
> 612-991-1321
>
> ------------------------------
>
> Date:    Thu, 27 May 2010 19:08:58 -0700
> From:    Chad K Park <[log in to unmask]>
> Subject: size of protein in negatively stained TEM?
>
> Apologies for the non-crystallographic question, but I would think  
> the wide
> experience and vitality of this board might have some opinions on this
> topic.
>
> We've sent out some samples for negative staining electron  
> microscopy.  They
> were stained with uranyl acetate on commercial grids and the resulting
> images seem to have a dispersity in the size of the little round  
> blobs seen.
> Some of the blobs look like they might be what we see in our crystal
> structures.  However, some of them are quite large.  What have  
> people's
> experience been with this technique and how close you get to an  
> overall
> dimension for your protein or complex?  How closely has that number  
> been to
> results from other techniques (Xtallography, DLS, other hydrodynamic  
> methods
> ....)?  Is there some rule of thumb that describes if the image is  
> slightly
> smaller or larger than the expected size?
>
> Thanks for your comments.
>
> Regards,
> Chad K. Park,
> Analyt. Biophys. Core
> Chem./Biochem., U. of AZ
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 11:47:37 +0200
> From:    Christine Bentz <[log in to unmask]>
> Subject: Job Advertisment (PhD position)
>
> Dear colleagues,
>
> Please find below a job advertisement for a PhD position at the HZI/ 
> Braunschweig, Germany.
>
> ------------------------------------
>
> Job Advertisment Nr. 26/2010
>
> The Division of Structural Biology at the Helmholtz Centre for  
> Infection Research in Braunschweig/Germany invites applications for a
> PhD position.
>
> Planned project:
> Structural analysis of bacterial infection mechanisms
>
> Goals:
> Structural understanding of the interactions between bacterial  
> virulence factors and host cell proteins during infection
>
> Area of research:
> Structural biology (X-ray crystallography), protein biochemistry
>
> Methods:
> State-of-the-art techniques in recombinant DNA technology (PCR,  
> cloning, protein expression systems, site-directed mutagenesis),  
> protein biochemistry (protein purification, protein analytics,  
> crystallization of proteins), enzyme assays, X-ray structural  
> analysis (data collection, structure solution, model building and  
> refinement), structure-function studies on proteins and protein  
> complexes. Close cooperation with cell biologists with background in  
> host-pathogen interaction.
>
> Prerequisite:
> Masters or Diploma in biochemistry, biology or chemistry.  
> Experimental experience in protein biochemisty and recombinant DNA  
> technology. Familiarity with protein crystallography/structural  
> biology advantageous. The successful applicant will become a member  
> of the newly installed graduate school of the centre.
>
> Starting date:
> The position is immediately available but later starting dates are  
> also possible.
>
> Duration:
> The contract will be for 2 years with the possibility to extend for  
> a third year.
>
> Salary:
> TVöD 13/2
>
> Published:          18.05.2010
> Closing date:     30.06.2010
>
> Applications marked for Code 26/2010 should be sent to: Helmholtz  
> Centre for Infection Research, Personalabteilung, Inhoffenstrasse 7,  
> D-38124 Braunschweig/Germany
>
> Applications should contain a CV, copies of university degrees and  
> contact information with two letters of recommendation
>
> Information:
> For further information please contact Prof. Dr. Dirk Heinz ([log in to unmask] 
> , phone: +49(0)531-6181-7000)
>
>
> --------------------------------------------------------
>
> Christine Bentz
> Personal Assistant to Prof. Dr. Dirk Heinz
> Division of Structural Biology
>
> Helmholtz Centre for Infection Research
> Inhoffenstrasse 7
> 38124 Braunschweig, Germany
>
> Fon  0049 (0)531.6181.7002
> Fax  0049 (0)531.6181.7099
> Monday - Friday / 8 am - 4 pm
>
> --------------------------------------------------------
>
> Protect the environment -
> please don't print this e-mail unless you really need to
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 14:11:07 +0100
> From:    Eleanor Dodson <[log in to unmask]>
> Subject: Re: How to calculate real-space CC by section?
>
>   I havent a reference for the "correct" value of the CC - it is just
> based on maps I have seen solved then checked out later.
>
> but if your final model gives a very poor CC with a map calculated  
> from
> experimental phases, either for parts of the structure, or for the
> whole,  it is time to worry. Maybe your phases are bad - poor
> measurements, low solvent content, incorrect heavy atom sites, etc.
> Or maybe your model has some serious errors?
> But of course all crystal structures have some parts better ordered  
> than
> others and for those bits the exptlly phased map may have weak  
> density,
> espec. after solvent flattening.
> eleanor
>
> [log in to unmask] wrote:
>> Hi Eleanor:
>>
>> Do you have some references in mind that discussed the value of CC  
>> (say
>>> 0.5) to be able to build the structure? Didn't find one for right  
>>> now:-(
>>
>> By the way, probably a "weak" question, In the case "a lousy model  
>> will
>> give poor CCs even if the map is brilliant", we still accept this  
>> model
>> dispite the poor CC, right? Sorry that I didn't get practically  
>> involved
>> too much in real model building, but I just heard that model is more
>> frequently built manually by eyes, not CC etc.
>>
>> Best Regards, Hailiang
>>
>>> If you ask for CORR SECTion then overlapmap does just that - the  
>>> CC will
>>> have a certain value for each section regardless of the CHAIN
>>> parameters. If you want correlation residue by residue you must  
>>> ask for
>>> CORR RESI
>>>
>>> As someone said - a lousy model will give poor CCs even if the map  
>>> is
>>> brilliant..
>>> But once your refinement is finished it is intresting to go back and
>>> check the CC of the initial maps.
>>>
>>> There is a belief that you need a CC of >0.5 to be able to build the
>>> structure but different problems and different builders achieve
>>> different results..
>>> Eleanor
>>>
>>> Hailiang Zhang wrote:
>>>> Hi,
>>>>
>>>> I am working on a real space correlation on a specif protein  
>>>> section
>>>> using
>>>> CCP4 OVERLAPMAP. I am using the following scripts, not sure  
>>>> whether it
>>>> is
>>>> good or not (didn't find in OVERLAPMAP documentation).
>>>>
>>>> overlapmap       \
>>>>               mapin1 ${PDB}-1.map    \
>>>>               mapin2 ${PDB}-2.map    \
>>>>               mapin3 ${PDB}-mask.map \
>>>> <<eof
>>>> CORR SECT
>>>> CHAIN A $START $END
>>>> END
>>>>
>>>> There is no error message, but the results make no difference no  
>>>> matter
>>>> how I change $START and $END. I am not sure whether the above  
>>>> script is
>>>> ok.
>>>>
>>>> By the way, more importantly to me, if corr sect works at all,  
>>>> will it
>>>> print out a single CC value by integrating over the WHOLE region  
>>>> define
>>>> by
>>>> the section range?
>>>>
>>>> Thanks!
>>>>
>>>> Best Regards, Hailiang
>>>
>>>
>>
>>
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 14:19:29 +0100
> From:    Eleanor Dodson <[log in to unmask]>
> Subject: Re: How large should the real space correlation coefficient  
> be?
>
> Maybe it is worth recalling some ancient discussions, involving Real
> Space R factors as defined by Alwyn Jones and Gerard Kleywert.
>
> If I remember properly, they give an "Rfactor" between the density  
> in an
> ATOMMAP generated from a model, but with truncated B factors and the
> density in the map underconsideration - an exptly phased one, a 2mFO
> -DFC or whatever.
> This requires that the electron densities are more or less on the same
> scale, and gave good Real Space R factors for atoms with low B  
> factors,
> and high ones for wrong residues, disordered residues, and those with
> high B factors
>
> The CC is meant to avoid problems of scale - the ATOMMAP is calculated
> taking the b factors into account, so gives a reasonable CC for
> correctly placed atoms with high b factors. However theoretically a
> residue with occupancies =0.00 which lies in a totally empty part of  
> the
> map under consideration could still give a resonable CC .
>
> As Pavel says, it is a very blunt tool which can mislead but also help
> you pinpoint errors.. I have found it most useful when trying to  
> select
> the best phasing procedure..
>   Eleanor
>
>
> Pavel Afonine wrote:
>> Hi Hailiang,
>>
>> On 5/25/10 8:14 PM, Hailiang Zhang wrote:
>>> Have seen the real-space correlation used widely judging the map  
>>> quality.
>>> Generally or empirically, in order to say an map (area) has "good"
>>> quality, how large should the real space correlation coefficient  
>>> be? Say,
>>> is 0.8 good enough on a residue base? Any references about this  
>>> will be
>>> greatly appreciated!
>>
>> why don't you just familiarize yourself with the map CC values  
>> computed
>> per atom or per residue, for a few different structures at different
>> resolutions? It might take you a few hours but from that point on you
>> will have some reference  between the map CC values and actual map
>> appearance. phenix.model_vs_data or phenix.real_space_correlation can
>> compute all these values for you.
>>
>> I did it at some point to educate myself and never regretted about  
>> the
>> time I spent doing this -:)
>>
>> Pavel.
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 07:35:33 -0700
> From:    Jan Abendroth <[log in to unmask]>
> Subject: mosflm in script mode
>
> Hi all,
> I have been trying to use mosflm in script mode, in a quick-n-dirty  
> effort to pipe some information from labelit.index into a simple  
> data collection strategy. While doing that, I run into the following  
> issue. I have not been able to find a way to read in image  
> information without starting the old gui. Is there a command in the  
> current mosflm versions to run it in shell mode only? Below the  
> script that I have been using.
> Any ideas?
>
> Thanks
> Jan
>
>
> ---
> ipmosflm summary integrate02.sum <<eof
> DIRECTORY ../images
> TEMPLATE image_###.img
> IMAGE 1
> HKLOUT integration02.mtz
> GENFILE integration02.gen
> #detector-take defaults
>
> #UIS_PIXEL 0.102400
> #UIS_SIZE 3072
>
> NUSPOT OFF
> BEAM 155.400500 158.807700
> DISTANCE 299.775600
> TWOTHETA 0.0
>
> WAVE 0.977400
> #beam
> SYNCHROTRON POLARIZATION 0.9
> DIVERGENCE 0.100 0.020
> DISPERSION 0.0001
>
>
> MOSAICITY 0.60
> SYMMETRY p2
> RESOLUTION 3.5
> MATRIX integration02.mat
>
> PROFILE OVERLOAD PARTIALS
> RASTER 19 19 9 4 4
> SEPARATION 1.80 1.80 CLOSE
> REFINEMENT RESID 7.5
> REFINEMENT INCLUDE PARTIALS RESID 10.0 #High s/n
>
>
> scanner adsc
> strategy auto
> stats on
> go
> end
> exit
> eof
>
>
> --
> Jan Abendroth
> Emerald BioStructures
> Seattle / Bainbridge Island WA, USA
> home: Jan.Abendroth_at_gmail.com
> work: JAbendroth_at_embios.com
> http://www.emeraldbiostructures.com
>
>
>
>
>
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 07:46:10 -0700
> From:    Bernhard Rupp <[log in to unmask]>
> Subject: Re: How to calculate real-space CC by section?
>
> Ad empirical observations:
>
> I have run and inspected routinely literally hundreds of RSCC plots  
> during
> the progress of MR searches and autobuild/refinement cycles.
>
> Almost always an initial average RSCC less than 0.6 to 0.55  
> indicated a
> non-solution
> or was otherwise unrecoverable. So I am perhaps a tad more  
> pessimistic than
> Eleanor
> as far as MR maps go (the absence of bias in experimental maps may  
> give the
> slightly lower empirical CC threshold of 0.5).
>
> The achievable CC of course is local, and the average depends, amongst
> other factors mentioned, on the type of protein/crystal. Sturdy  
> stuff like
> helix bundles may well give average CCs of 0.95 or higher. Floppy
> structures (without prejudice whether the plasticity is genuine or
> reflects long-range order like packing issues) can be 0.85 without
> real indications from the map what to improve.
>
> In almost all cases, the average RSCC consistently reflected the
> average fo vs fc correlation coefficients that Refmac reports.
>
> In case of very large structures you may run into grid limitations,
> which may also have some effect on the actual mean CC.
>
> BR
>
> -----Original Message-----
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Eleanor Dodson
> Sent: Friday, May 28, 2010 6:11 AM
> To: [log in to unmask]
> Subject: Re: [ccp4bb] How to calculate real-space CC by section?
>
>   I havent a reference for the "correct" value of the CC - it is just
> based on maps I have seen solved then checked out later.
>
> but if your final model gives a very poor CC with a map calculated  
> from
> experimental phases, either for parts of the structure, or for the
> whole,  it is time to worry. Maybe your phases are bad - poor
> measurements, low solvent content, incorrect heavy atom sites, etc.
> Or maybe your model has some serious errors?
> But of course all crystal structures have some parts better ordered  
> than
> others and for those bits the exptlly phased map may have weak  
> density,
> espec. after solvent flattening.
> eleanor
>
> [log in to unmask] wrote:
>> Hi Eleanor:
>>
>> Do you have some references in mind that discussed the value of CC  
>> (say
>>> 0.5) to be able to build the structure? Didn't find one for right  
>>> now:-(
>>
>> By the way, probably a "weak" question, In the case "a lousy model  
>> will
>> give poor CCs even if the map is brilliant", we still accept this  
>> model
>> dispite the poor CC, right? Sorry that I didn't get practically  
>> involved
>> too much in real model building, but I just heard that model is more
>> frequently built manually by eyes, not CC etc.
>>
>> Best Regards, Hailiang
>>
>>> If you ask for CORR SECTion then overlapmap does just that - the  
>>> CC will
>>> have a certain value for each section regardless of the CHAIN
>>> parameters. If you want correlation residue by residue you must  
>>> ask for
>>> CORR RESI
>>>
>>> As someone said - a lousy model will give poor CCs even if the map  
>>> is
>>> brilliant..
>>> But once your refinement is finished it is intresting to go back and
>>> check the CC of the initial maps.
>>>
>>> There is a belief that you need a CC of >0.5 to be able to build the
>>> structure but different problems and different builders achieve
>>> different results..
>>> Eleanor
>>>
>>> Hailiang Zhang wrote:
>>>> Hi,
>>>>
>>>> I am working on a real space correlation on a specif protein  
>>>> section
>>>> using
>>>> CCP4 OVERLAPMAP. I am using the following scripts, not sure  
>>>> whether it
>>>> is
>>>> good or not (didn't find in OVERLAPMAP documentation).
>>>>
>>>> overlapmap       \
>>>>               mapin1 ${PDB}-1.map    \
>>>>               mapin2 ${PDB}-2.map    \
>>>>               mapin3 ${PDB}-mask.map \
>>>> <<eof
>>>> CORR SECT
>>>> CHAIN A $START $END
>>>> END
>>>>
>>>> There is no error message, but the results make no difference no  
>>>> matter
>>>> how I change $START and $END. I am not sure whether the above  
>>>> script is
>>>> ok.
>>>>
>>>> By the way, more importantly to me, if corr sect works at all,  
>>>> will it
>>>> print out a single CC value by integrating over the WHOLE region  
>>>> define
>>>> by
>>>> the section range?
>>>>
>>>> Thanks!
>>>>
>>>> Best Regards, Hailiang
>>>
>>>
>>
>>
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 08:00:54 -0700
> From:    James Holton <[log in to unmask]>
> Subject: Re: mosflm in script mode
>
> Check the log file carefully that MOSFLM has "swallowed" each of your
> script lines.  I don't think "stats on" is a valid command.  The
> strategy command "stats" (with no qualification) is generally run  
> after
> the "go" command:
>
> strategy auto
> go
> stats
> end
>
>
> What is probably happening is MOSFLM is balking on the "stats on" line
> and then exits "strategy".  The next line is a "go", which means load
> the image in graphics.
>
> There is also a program I call "Wedger Elves" which (among other  
> things)
> will take a matrix and an image and give you a strategy using MOSFLM.
> It will also run LABELIT to do indexing if you have it installed.
> http://bl831.als.lbl.gov/~jamesh/elves/
>
> -James Holton
> MAD Scientist
>
> Jan Abendroth wrote:
>> Hi all,
>> I have been trying to use mosflm in script mode, in a quick-n-dirty
>> effort to pipe some information from labelit.index into a simple data
>> collection strategy. While doing that, I run into the following  
>> issue.
>> I have not been able to find a way to read in image information
>> without starting the old gui. Is there a command in the current  
>> mosflm
>> versions to run it in shell mode only? Below the script that I have
>> been using.
>> Any ideas?
>>
>> Thanks
>> Jan
>>
>>
>> ---
>> ipmosflm summary integrate02.sum <<eof
>> DIRECTORY ../images
>> TEMPLATE image_###.img
>> IMAGE 1
>> HKLOUT integration02.mtz
>> GENFILE integration02.gen
>> #detector-take defaults
>>
>> #UIS_PIXEL 0.102400
>> #UIS_SIZE 3072
>>
>> NUSPOT OFF
>> BEAM 155.400500 158.807700
>> DISTANCE 299.775600
>> TWOTHETA 0.0
>>
>> WAVE 0.977400
>> #beam
>> SYNCHROTRON POLARIZATION 0.9
>> DIVERGENCE 0.100 0.020
>> DISPERSION 0.0001
>>
>>
>> MOSAICITY 0.60
>> SYMMETRY p2
>> RESOLUTION 3.5
>> MATRIX integration02.mat
>>
>> PROFILE OVERLOAD PARTIALS
>> RASTER 19 19 9 4 4
>> SEPARATION 1.80 1.80 CLOSE
>> REFINEMENT RESID 7.5
>> REFINEMENT INCLUDE PARTIALS RESID 10.0 #High s/n
>>
>>
>> scanner adsc
>> strategy auto
>> stats on
>> go
>> end
>> exit
>> eof
>>
>>
>> --
>> Jan Abendroth
>> Emerald BioStructures
>> Seattle / Bainbridge Island WA, USA
>> home: Jan.Abendroth_at_gmail.com
>> work: JAbendroth_at_embios.com
>> http://www.emeraldbiostructures.com
>>
>>
>>
>>
>>
>>
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 16:41:50 +0000
> From:    Harry Powell <[log in to unmask]>
> Subject: Re: mosflm in script mode
>
> Hi Jan
>
> The "IMAGE " command turns the old gui on - always has done, as far  
> as I know.
>
> With "Strategy auto" you shouldn't need to give an image number.
>
> HTH
>
> On 28 May 2010, at 14:35, Jan Abendroth wrote:
>
>> Hi all,
>> I have been trying to use mosflm in script mode, in a quick-n-dirty  
>> effort to pipe some information from labelit.index into a simple  
>> data collection strategy. While doing that, I run into the  
>> following issue. I have not been able to find a way to read in  
>> image information without starting the old gui. Is there a command  
>> in the current mosflm versions to run it in shell mode only? Below  
>> the script that I have been using.
>> Any ideas?
>>
>> Thanks
>> Jan
>>
>>
>> ---
>> ipmosflm summary integrate02.sum <<eof
>> DIRECTORY ../images
>> TEMPLATE image_###.img
>> IMAGE 1
>> HKLOUT integration02.mtz
>> GENFILE integration02.gen
>> #detector-take defaults
>>
>> #UIS_PIXEL 0.102400
>> #UIS_SIZE 3072
>>
>> NUSPOT OFF
>> BEAM 155.400500 158.807700
>> DISTANCE 299.775600
>> TWOTHETA 0.0
>>
>> WAVE 0.977400
>> #beam
>> SYNCHROTRON POLARIZATION 0.9
>> DIVERGENCE 0.100 0.020
>> DISPERSION 0.0001
>>
>>
>> MOSAICITY 0.60
>> SYMMETRY p2
>> RESOLUTION 3.5
>> MATRIX integration02.mat
>>
>> PROFILE OVERLOAD PARTIALS
>> RASTER 19 19 9 4 4
>> SEPARATION 1.80 1.80 CLOSE
>> REFINEMENT RESID 7.5
>> REFINEMENT INCLUDE PARTIALS RESID 10.0 #High s/n
>>
>>
>> scanner adsc
>> strategy auto
>> stats on
>> go
>> end
>> exit
>> eof
>>
>>
>> --
>> Jan Abendroth
>> Emerald BioStructures
>> Seattle / Bainbridge Island WA, USA
>> home: Jan.Abendroth_at_gmail.com
>> work: JAbendroth_at_embios.com
>> http://www.emeraldbiostructures.com
>>
>>
>>
>>
>>
>>
>
> Harry
> --
> Dr Harry Powell, MRC Laboratory of Molecular Biology, MRC Centre,  
> Hills Road, Cambridge, CB2 0QH
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 12:03:57 -0400
> From:    Sollepura Yogesha <[log in to unmask]>
> Subject: protein monitoring
>
> Dear All,
> I have expressed 30-40 aa region my protein fused to GST.
> I subjected it to precision protease cleavage. On the gel I can see  
> the band.
>
> When I  looked for ProtParam in expasy it shows that  my peptide   
> doesn't have Extinction coefficients as " there are no Trp, Tyr or  
> Cys in the region considered, your protein should not be visible by  
> UV spectrophotometry."
>
> I need to separate GST from the cleavage mixture.
>
> How can I monitor my peptide during FPLC and after that.
>
> AA composition is Ala (A)   8,    Arg (R)   2,       Asn (N)   3,    
> Asp (D)   2,   Gln (Q)   2,       Glu (E)   2,  Gly (G)   4,   Ile  
> (I)   1,   Leu (L)   4,   Lys (K)   4,    Phe (F)   2,      Pro  
> (P)   2,   Ser (S)   5,   Thr (T)   5,   Val (V)   3.
>
> I am looking for some suggestions
>
> Thanks in advance
>
> Yogi
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 11:08:20 -0700
> From:    Eric Larson <[log in to unmask]>
> Subject: Re: protein monitoring
>
> Hi Yogi,
>
> You can see your peptide on a gel so why can't you "monitor" it by  
> SDS-PAGE?  A little time consuming, yes, but then you have the extra  
> benefit of also seeing if there are contaminating proteins in your  
> sample.
>
> good luck,
> Eric
> __________________________
> Eric Larson, PhD
> MSGPP Consortium
> Department of Biochemistry
> Box 357742
> University of Washington
> Seattle, WA 98195
>
> On Fri, 28 May 2010, Sollepura Yogesha wrote:
>
>>
>> Dear All,
>>
>> I have expressed 30-40 aa region my protein fused to GST.
>>
>> I subjected it to precision protease cleavage. On the gel I can see  
>> the band.
>>
>> When I  looked for ProtParam in expasy it shows that  my peptide   
>> doesn’t have Extinction coefficients as “ there are no Trp, Tyr or
>> Cys in the region considered, your protein should not be visible by  
>> UV spectrophotometry.”
>>
>> I need to separate GST from the cleavage mixture.
>>
>> How can I monitor my peptide during FPLC and after that.
>>
>> AA composition is Ala (A)   8,    Arg (R)   2,       Asn (N)   3,    
>> Asp (D)   2,   Gln (Q)   2,       Glu (E)   2,  Gly (G)   4,   I
>> le (I)   1,   Leu (L)   4,   Lys (K)   4,    Phe (F)   2,      Pro  
>> (P)   2,   Ser (S)   5,   Thr (T)   5,   Val (V)   3.
>>
>> I am looking for some suggestions
>>
>> Thanks in advance
>>
>> Yogi
>>
>>
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 14:30:50 -0400
> From:    Hailiang Zhang <[log in to unmask]>
> Subject: The "Total" CC in the output of CCP4 OVERLAPMAP
>
> Hi all:
>
> Thanks for all kindly helps with real space CC. Now I have a new  
> question
> again. In the output of OVERLAPMAP in CCP4, there is a almost last  
> line
> saying "Total...":
>
> #######################################
> .......
>    1243             0.9528   0.9249
>    1244             0.9741   0.8591
>    1360             0.9483   0.9145
>  $$
> Total:              0.8853   0.8676
> <B><FONT COLOR="#FF0000"><!--SUMMARY_BEGIN-->
> OVERLAPMAP:   Normal termination
> #########################################
>
> Now what does the "Total" CC mean? Is it a single CC generated by
> integrating over the whole system? Or just an average of each  
> individual
> CC values? I do need to first one, and hope that's it.
>
> Best Regards, Hailiang
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 13:40:28 -0500
> From:    "Radisky, Evette S., Ph.D." <[log in to unmask]>
> Subject: Re: protein monitoring
>
> Try a Bradford-type assay, scaled down to microplate format.  You can
> buy reagents pre-made; Pierce makes a good one .  It is fast-- you  
> pipet
> 10 microliters or so from each chromatography fraction into a well  
> with
> the detection reagent, and wait 5 minutes.  If protein concentrations
> are moderate to high in your peaks, you won't even need to use a plate
> reader; the fractions with protein will be quite visibly blue  
> against a
> white background.
>
> You would probably want to check the MW of your protein peaks on a gel
> anyway, but at least you won't need to run lanes with a lot of  
> fractions
> containing no protein.
>
> Evette S. Radisky, Ph.D.
> Assistant Professor
> Mayo Clinic Cancer Center
> Griffin Cancer Research Building, Rm 310
> 4500 San Pablo Road
> Jacksonville, FL 32224
> (904) 953-6372
>
>
>
> ________________________________
>
> From: CCP4 bulletin board [mailto:[log in to unmask]] On Behalf Of
> Sollepura Yogesha
> Sent: Friday, May 28, 2010 12:04 PM
> To: [log in to unmask]
> Subject: [ccp4bb] protein monitoring
>
>
>
> Dear All,
>
> I have expressed 30-40 aa region my protein fused to GST.
>
> I subjected it to precision protease cleavage. On the gel I can see  
> the
> band.
>
> When I  looked for ProtParam in expasy it shows that  my peptide
> doesn't have Extinction coefficients as " there are no Trp, Tyr or Cys
> in the region considered, your protein should not be visible by UV
> spectrophotometry."
> I need to separate GST from the cleavage mixture.
> How can I monitor my peptide during FPLC and after that.
> AA composition is Ala (A)   8,    Arg (R)   2,       Asn (N)   3,    
> Asp
> (D)   2,   Gln (Q)   2,       Glu (E)   2,  Gly (G)   4,   Ile (I)    
> 1,
> Leu (L)   4,   Lys (K)   4,    Phe (F)   2,      Pro (P)   2,   Ser  
> (S)
> 5,   Thr (T)   5,   Val (V)   3.
> I am looking for some suggestions
> Thanks in advance
> Yogi
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 11:40:31 -0700
> From:    Jerry McCully <[log in to unmask]>
> Subject: to what extent bacterial expression reveals the native  
> oligomerization state of mammalian proteins.
>
>
> Dear ALL:
>
>      I am sorry for this stupid question.
>
>    I guess bacterial expression system is still  most popular in  
> structural biology.
>
>    If you get a very good soluble E.coli expression of a human  
> protein without disulfide bonds,
> to what extent do you believe that the oligomerization state of this  
> bacterial expression will reflect the real physiological state of  
> this protein in humans?
>
>     Can someone give comments or refer some literature?
>
>      Thanks a lot,
>
> Jerry McCully
> 		 	   		
> _________________________________________________________________
> Hotmail has tools for the New Busy. Search, chat and e-mail from  
> your inbox.
> http://www.windowslive.com/campaign/thenewbusy?ocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_1
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 11:56:02 -0700
> From:    Ursula Schulze-Gahmen <[log in to unmask]>
> Subject: Re: protein monitoring
>
> You have to be careful with the Bradford detection assays because some
> of them have a lower detection limit of 3-5 kDa and may not work for
> your large peptide.
>
> For FPLC you may be able to detect your protein using absorption at  
> 215
> nm which detects the peptide bond. Just choose a buffer that does not
> absorb in this range.
>
>
> Ursula
>
> On 5/28/10 9:03 AM, Sollepura Yogesha wrote:
>>
>> Dear All,
>>
>> I have expressed 30-40 aa region my protein fused to GST.
>>
>> I subjected it to precision protease cleavage. On the gel I can see
>> the band.
>>
>> When I  looked for ProtParam in expasy it shows that  my peptide   
>> doesn't have *Extinction coefficients as* " there are no Trp, Tyr  
>> or Cys in the region considered, your protein should not be visible  
>> by UV spectrophotometry."
>> I need to separate GST from the cleavage mixture.
>> How can I monitor my peptide during FPLC and after that.
>> AA composition is Ala (A)   8,    Arg (R)   2,       Asn (N)   3,    
>> Asp (D)   2,   Gln (Q)   2,       Glu (E)   2,  Gly (G)   4,   Ile  
>> (I)   1,   Leu (L)   4,   Lys (K)   4,    Phe (F)   2,      Pro  
>> (P)   2,   Ser (S)   5,   Thr (T)   5,   Val (V)   3.
>> I am looking for some suggestions
>> Thanks in advance
>> Yogi
>
> -- 
> Ursula Schulze-Gahmen, PhD.
> QB3, Tjian Lab
> MCB, 16 Barker Hall #3204
> University of California Berkeley
> Berkeley, CA 94720-3204
> Phone: (510) 642 8258
> [log in to unmask]
>
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 14:02:46 -0700
> From:    Jerry McCully <[log in to unmask]>
> Subject: Re: to what extent bacterial expression reveals the native  
> oligomerization state of mammalian proteins.
>
>
>
> A little update on my own project:
>
> It is a secreted protein without any modification reported. Size  
> exclusion shows that it is an oligomer. CD spectrum shows well- 
> defined secondary structure.
>
> It is stable and soluble.
>
> It may have different states in diseases' condition.
>
> Now I was trying to figure out the native state of this protein. As  
> told by folks on CCP4bb, Most likely, the bacterial expression  
> reveals its original state.
>
> Am I right?
>
> Thanks a lot,
>
> Jerry
> Date: Fri, 28 May 2010 11:40:31 -0700
> From: [log in to unmask]
> Subject: [ccp4bb] to what extent bacterial expression reveals the  
> native oligomerization state of mammalian proteins.
> To: [log in to unmask]
>
>
>
>
>
>
>
>
> Dear ALL:
>
>      I am sorry for this stupid question.
>
>    I guess bacterial expression system is still  most popular in  
> structural biology.
>
>    If you get a very good soluble E.coli expression of a human  
> protein without disulfide bonds,
> to what extent do you believe that the oligomerization state of this  
> bacterial expression will reflect the real physiological state of  
> this protein in humans?
>
>     Can someone give comments or refer some literature?
>
>      Thanks a lot,
>
> Jerry McCully
> 		 	   		
> Hotmail has tools for the New Busy. Search, chat and e-mail from  
> your inbox. Learn more. 		 	   		
> _________________________________________________________________
> The New Busy is not the old busy. Search, chat and e-mail from your  
> inbox.
> http://www.windowslive.com/campaign/thenewbusy?ocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_3
>
> ------------------------------
>
> Date:    Fri, 28 May 2010 18:09:29 -0400
> From:    lei feng <[log in to unmask]>
> Subject: please recommend a crystallization incubator
>
>
> hello everyone
>
>
>
> can anyone recommend an affordable low temp. incubator that could be  
> used for crystallography?
>
>
>
> we do not have cold room, so hope the incubator can go to 4 degree  
> withouth too much vibration
>
>
>
> any size from 10 cubic ft -->20 cubic ft will be fine
>
>
>
> any help is highly appreciated.
> 		 	   		
> _________________________________________________________________
> The New Busy is not the too busy. Combine all your e-mail accounts  
> with Hotmail.
> http://www.windowslive.com/campaign/thenewbusy?tile=multiaccount&ocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_4
>
> ------------------------------
>
> End of CCP4BB Digest - 27 May 2010 to 28 May 2010 (#2010-144)
> *************************************************************

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