[ccp4bb] 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 (dirk.he...@helmholtz-hzi.de, 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
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
zhan...@umbc.edu 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
Re: [ccp4bb] 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.
[ccp4bb] 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
Re: [ccp4bb] 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:ccp...@jiscmail.ac.uk] On Behalf Of
Eleanor Dodson
Sent: Friday, May 28, 2010 6:11 AM
To: CCP4BB@JISCMAIL.AC.UK
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
zhan...@umbc.edu 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
Re: [ccp4bb] 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
Re: [ccp4bb] 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
[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
Re: [ccp4bb] 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
[ccp4bb] 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 BFONT COLOR=#FF!--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
Re: [ccp4bb] 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:ccp...@jiscmail.ac.uk] On Behalf Of Sollepura Yogesha Sent: Friday, May 28, 2010 12:04 PM To: CCP4BB@JISCMAIL.AC.UK 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
[ccp4bb] 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
Re: [ccp4bb] 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: for-crystallizai...@hotmail.com Subject: [ccp4bb] to what extent bacterial expression reveals the native oligomerization state of mammalian proteins. To: CCP4BB@JISCMAIL.AC.UK 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
[ccp4bb] 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=multiaccountocid=PID28326::T:WLMTAGL:ON:WL:en-US:WM_HMP:042010_4
Re: [ccp4bb] size of protein in negatively stained TEM?
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 dberkh...@gentoo.org 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 Å 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 ckp...@email.arizona.edu 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 christine.be...@helmholtz-hzi.de Subject: Job Advertisment (PhD position) Dear colleagues, Please find below a job advertisement for a PhD position at the HZI/ Braunschweig, Germany.
