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 <[email protected]>
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 <[email protected]>
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 <[email protected]>
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 ([email protected]
, 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 <[email protected]>
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
[email protected] 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 <[email protected]>
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 <[email protected]>
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 <[email protected]>
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:[email protected]] On Behalf Of
Eleanor Dodson
Sent: Friday, May 28, 2010 6:11 AM
To: [email protected]
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
[email protected] 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 <[email protected]>
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 <[email protected]>
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 <[email protected]>
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 <[email protected]>
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 <[email protected]>
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." <[email protected]>
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:[email protected]] On Behalf Of
Sollepura Yogesha
Sent: Friday, May 28, 2010 12:04 PM
To: [email protected]
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 <[email protected]>
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
_________________________________________________________________
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------------------------------
Date: Fri, 28 May 2010 11:56:02 -0700
From: Ursula Schulze-Gahmen <[email protected]>
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
[email protected]
------------------------------
Date: Fri, 28 May 2010 14:02:46 -0700
From: Jerry McCully <[email protected]>
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: [email protected]
Subject: [ccp4bb] to what extent bacterial expression reveals the
native oligomerization state of mammalian proteins.
To: [email protected]
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
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Date: Fri, 28 May 2010 18:09:29 -0400
From: lei feng <[email protected]>
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.
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End of CCP4BB Digest - 27 May 2010 to 28 May 2010 (#2010-144)
*************************************************************
