How about five (!) convergently evolved proteins?
The carbonic anhydrases are a well-known family of proteins which (to
date) have 5 independently convergently evolved forms: alpha, beta,
gamma, delta, and zeta. (Epsilon got folded into beta after
crystallography revealed it to be a beta-form.)
Regarding question 1, see:
Acta Cryst. (2008). D64, 354-367
Towards a rational approach for heavy-atom derivative screening in
protein crystallography
J.
Agniswamy, M.
G. Joyce, C.
H. Hammer and P.
D. Sun
Cheers.
riya doreen wrote:
I have two questions regarding derivatization:
VMWare is snappier, but VirtualBox is free.
Cheers, Roger Rowlett
Andy Torelli wrote:
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 whil
Title: Computer hardware and OS "survey"
Well, Coot, O, Pymol, CNS, and CCP4i, as well as
Open-EPMR all have Windows versions. The main issues with a Windows
workflow are (1) jobs will run significantly slower than in Linux, and
(2) the DOS command shell is not as powerful as Linux, although it
I usually try the following, in order:
1. Transfer crystals to mother liquor + 30% glycerol or ethylene glycol
(sometimes lower depending on crystallization solution). This did not
work for you.
2. Transfer crystals to mother liquor + 30% glucose (or try sequential
soaks in M.L. + 15% and then
The holy trinity of protein chromatography is ion
exchange (IEX), hydrophobic interaction chromatography (HIC), and gel
exclusion (GEC), assuming no affinity purification is possible.
Depending on the abundance of protein, these three steps, perhaps in
concert with additional chemical steps, in
I would think 30% MPD or more should normally be
enough to cryoprotect. If that is not the case, then you can always
supplement with additional MPD, glycerol, or glucose. If I understand
correctly, your crystals are forming in the presence of some additional
protein precipitate? If so, you migh
Some context would be helpful (essential?). What's
in the crystallization solution? What, if anything, is known about the
protein of interest? What is the ligand interacting with (metal ions,
hydrogen bonding donors/acceptors, charged residues, etc?), and what
are the interaction distances? Do
The CCP4 mtz2various will do this. A sample script
and instructions for using the CCP4i GUI can be found at
http://capsicum.colgate.edu/chwiki/tiki-index.php?page=Phase+Solution#File_preparation.
The example cited is for preparing a file for EPMR, which is a simple
hklF listing.
Cheers,
--
I had a student solve a medium resolution (2.3 A)
data set with (unfortunately) 12 identical protein chains in the
asymmetric unit. To save a little time, and to take advantage of a
large amount of potential averaging we used NCS to do the initial phase
of the refinement. For 10 of the 12 chain
See
http://strucbio.biologie.uni-konstanz.de/ccp4wiki/index.php/Stereo
first. The xorg.conf file must be configured correctly to enable stereo.
Roger Rowlett
Eric Liu wrote:
Hi All,
I would like to explore if anybody had experience using song CRT
multiscan E540 monitor for stereo view? I
I will second noting the loss of arp_waters in
CCP4 6.1. I know there is a "find waters" option in Coot 0.5.2 under
"Calculate...Other modeling tools", and perhaps this is adequate, but
it doesn't seem to have as many options for fine-tuning as arp_waters
did. Coot allows for finding waters abo
I don't know if this is relevant here, but we found that certain CCP4
6.0.2 tasks run under CCP4i suffered poor performance over NFS due to
the frequent writing and reading of temporary files. Starting CCP4i
from a local directory eliminated the problem. (It appeared that the
temporary files we
Does anyone have any experience with an
"incufridge" for storing protein crystallization trays? (e.g.,
http://www.sigmaaldrich.com/catalog/ProductDetail.do?N4=Z708623|SIGMA&N5=Product%20No.|BRAND_KEY&F=SPEC).
I thought maybe these would be better than an empty lab cabinet or a
cold room that we
We have an Ocean Optics USB-4000 unit in our lab. It does everything
from quantifying protein and nucleic acids, spectrophotometric
titrations, and metalloenzyme spectra at low volume/concentration. It's
not a toy, but a diode array spectrophotometer that has excellent S/N
and resolution. You c
I am getting the following error in imosflm when
attempting to read an image file from an RAxis IV++ detector. I can
read RAxis IV image files just fine from the same facility. Is this a
header formatting issue? And is there a (relatively) simple way to fix
it? In MOSFLM (old GUI) attempting to
Anna S Gardberg wrote:
Dear list,
I haven't seen the "crystallographic computing platform" thread come up
for a while, and I've got a chance to upgrade my desktop to a
workstation, so I thought I'd ask the CCP4BB for advice on:
1. Mac vs. Linux (which flavor?) vs. Windows
2. Graphics cards
3
less than 100% occupancy. From your water
model, however, it looks like you may have enough density for a full
occupancy anion. FYI, the surrounding ligands, Arg + Glu + another
H-bonding donor are certainly compatible with the few known bicarbonate
binding sites in proteins.
Roger Rowlett
Kla
Sebastiano,
The density is possibly consistent with a trigonal planar anion such as
bicarbonate or nitrate. Bicarbonate can enter the solution from CO2 in
the atmosphere.
Cheers,
--
Roger S. Rowlett
Professor
Colgate University Presidential Scholar
Department of Chemistry
Colgate Universit
graphics cards with good cooling. You can
build a very fast dual- or quad-core computer for just over $1000 plus
your graphics card. Assuming you like to do this kind of thing.
Cheers, Roger Rowlett
Christopher Bahl wrote:
A cheaper option than building an entire new computer is to transfer
wrote:
Hi CCP4ers
I know I am churning over a well discussed subject but:
We have been trying to figure out the best way to use our NuVision
graphics glasses on the OPTIPLEX 755 Dell.
Turns out the graphics card (NVIDIA FX 3700) is not compatible with
this type of computer due to size issues.
The low-form foam dewar is really nice to work with. Fills easily
without excessive boiloff, and holds level pretty well while working
with crystal loops and vials. Best of all, not much ice will accumulate
at the top of the container while working. Best of all, my
undergraduates can't break it
Based on my own experience with zinc-metalloenzymes with thiolate
ligands, it's usually more a problem to get the zinc OUT than get it IN.
Zinc is pretty thiophilic, so removing it once ligated in a multiple Cys
environment is often difficult. Have you tried TCEP as a reducing agent
for protect
Oops, yes. Element symbol right-justified in 77-78, charge in 79-80. Out
of curiosity, shouldn't "CL " technically be listed as "CL-1" in the
PDB file for Refmac as well?
Cheers,
Roger Rowlett
Ian Tickle wrote:
Hi, it's not clear why this should be nec
Patrick,
Zinc ions should be identified as ZN+2 at the end of the line for Refmac
(see modified line below):
12345678901234567890123456789012345678901234567890123456789012345678901234567890
ATOM 3048 CLBB DRG X 1 1.461 37.707 15.068 1.00 37.36CL
ATOM 3155 O HOH H 318
My usual first approach to increasing crystal size is to decrease
precipitant and increase protein concentration, keeping the mixture in
the proper nucleation zone, but providing more protein for crystal
growth. Minimizing dust or particulates by filtration and centrifugation
of reagents and p
You are correct. See */Protein Sci/* Thurlkill et al. 15 (5): 1214,
http://www.proteinscience.org/cgi/reprint/15/5/1214, which is a recent
effort to codify "typical" pKa values for protein ionizable groups.
Cheers,
--
Rog
Matthew Alan Bratkowski wrote:
Hello.
I am working with a protein that turns a yellowish-brown color when it is
concentrated to around 2 mg/ml or higher in a small volume (a few hundred
uL). I was wondering if the protein bound a metal or other prosthetic
group that would give it this color? T
I haven't done TA cloning in a very long time. Typically, TA cloning is
done with blunt-ended DNA fragments that have been modified by terminal
transferase. Normally, a blunt-ended vector digest is treated with
terminal transferase and TTP. (If the vector is cut by non-blunt-ended
restriction e
have been set reasonably. I think Open-EPMR will also
examine screw axis combinations now, but I've never used it to do this.
Cheers,
Roger Rowlett
Phil Evans wrote:
Perhaps obvious - are you sure the space group is C222 not C2221?
Phil
On 25 Jul 2008, at 14:19, Roger Rowlett
Carl Soja wrote:
Dear all
I tried to solve one structure by ccp4i molrep(resolution at 3.0 A,
space group C222, sequence ID 30%). I can get a good Rfactor 0.528 at
first translation function. However, the second translation function
Rfac is 0.526, the third is 0.525, the fourth is 0.525.
Meg wrote:
Dear All,
I want to prepare 25 mM sodium acetate buffer pH 4.5. can anyone give the
exact composition of how to prepare it. we prepare it using sodium acetate
and acetic acid combination. i am not able to arrive at the calculatation
correctly, so if anyone can explain me with the abo
Tjaard Pijning wrote:
I have crystals grown from 1 M sodium succinate pH 7 and have
been searching for suitable cryo conditions.
Is succinate in itself already a cryoprotectant (maybe at somewhat
higher concentration) or should I
add e.g. glycerol ? Any experience or ideas are welcome.
Thanks
I'll second the recommendation for the Malvern Zetasizer. They are
rock-simple and interface with a computer through USB which makes future
computer upgrades relatively simple. The are however, not cheap--oops,
inexpensive.
Cheers,
--
-
There are actually two very good ways to concentrate dilute protein
while carrying out a purification step. If the solution is low ionic
strength, then IEX is the way to go. If the solution is too high in
ionic strength to do IEX directly, then add ammonium sulfate (usually to
about 1.0 M) and
Guenter Fritz wrote:
A mild and quick method is to use dry Sephadex G-25. The material will
swell and take up all the liquid except molecules larger than ca. 5 kDa.
Dear All,
we have GCSF protein produced in inclusion bodies. we solubilise it refold
it and then concentrate it using proflux
Sampath Natarajan wrote:
Dear All,
I am refining a structure with 2.5A resolution by refmac5.
I could find the solution by MR using molrep. After fitting the model,
I refined the structure again with 0.3 weighting term, but the output
PDB file shows many splits in the residues. So I
On a general note, it is not unusual at all for a random mutation (i.e.,
one not in the active or regulatory site of an enzyme, and not
significantly connected with the catalytic or regulatory mechanism) to
affect the rate constant of an enzyme-catalyzed reaction (kcat or
kcat/Km) by a factor o
Debajyoti Dutta wrote:
Hi all,
I have a dataset giving contradictory results. When I am trying the
molecular replacement with Amore and Molrep autoMR it is automatically
taking trimer but upon running BALBES it is taking tetramer. The cell
content analysis is also supporting the trimer co
xu zhen wrote:
> Hi, everyone,
>
> I am preparing the table of data collection and refinement statics. In this
> table, I need to list the average B factor of protein, ligand and water
> seperately, can you tell me how to calculate thoes numbers?
>
> Zhen
> __
sajid akthar wrote:
Dear All
My protein size is ~30kD and crystallizes with
19%Peg3350, 0.2M Nacl, and 0.1M Na Cacodylate buffer.
Please refer the attached crystal image with this. The
crystal looks like good enough for home source. These
crystals appears in 4-5 days at room temp.
Sometimes I'
Kianoush wrote:
Dear All,
I occasionally get the following error after running refmac:
#CCP4I TERMINATION STATUS 0 Error from script
/home/local/LINUX/ccp4-6.0.2/ccp4i/scripts/refmac5.script: error
writing "stdout": I/O error
The above error is from the last lines of the log file. It seem
Chris Waddling wrote:
Basically, the newest version of HKL2000 won't run on Linux machines that do
not have the libg2c.so.0 library (part of gcc3) in /usr/lib/ . This is a
problem for us, as every new computer we acquire uses a version of Linux
(that is no longer terribly new) that uses gcc4 (w
Narayanan Ramasubbu wrote:
Dear all:
I am noticing that in some of my structures, at 1.5 A resolution, 1)
there is some negative density around the C of the carboxyl groups.
2) I also notice the negative density around S in a disulfide region. It
is as though the disulfide is broken.
Could some
Chu-Young Kim wrote:
Hello everyone,
I have not done much crystallography in the past five years but I'm
trying to get back into it now because we stumbled upon a very
interesting enzyme. It seems a lot has changed in the computer
hardware world. I was trained on an SGI back in graduate scho
杨柳青 wrote:
> Hello,everyone!
> I have a question,is the expression vector effect crystallization much?
> For example,"PQE2-protein" structure has 2 molecules in one
> unit-cell,"PQE70-protein single mutant" has 5molecules in one
> unit-cell.Is the vector effect the results or the mutant itself?
> C
EPMR (now Open-EPMR, http://www.epmr.info) is an excellent alternative
for finding difficult, high-dimensional MR solutions. Experiment with
various resolution limits. We solved an asymmetric unit with 3
difficult-to-place dimers of low sequence homology by gradually
increasing the high-resolut
Ngo Duc Tri wrote:
Dear CCP4 experts,
I'm working on a kind of cystein peptidase. It shows a little
degradation after 2 days storing at 4 degree even I tried many kinds
of protease inhibitors, or glycerol and DTT.
So I decided to work fast (1 day) then set xtal. After one week I pick
up some c
James Stroud wrote:
Hello All,
I have a tough ~3.5 Å (pushing it) MR problem where I have a solution
of sorts, but because I'm working with a heterodimer of two closely
related subunits (with two such heterodimers in the ASU) I have a 2**2
possibilities for the arrangement of these subunits in t
Andrew Dore wrote:
Dear ccp4 comrades,
I have a problem I hope someone can help with. I am attempting to
index and integrate
a number of datasets which will all index (with mosflm) and scale
(with scala) in P21 with
low penalty scores and low Rmerge respectively.
However, when ipmosflm integrat
Zheng Zhou wrote:
Hi, All
Could any tell me how CCP4 handle free R flag? I know It is important
to select the same** FreeR reflections if I move to next step of
refinement. But everytime I start from fresh, the freeR Flag remains
unchanged. The Rwork and Rfree of my models are fine ( 20.7% a
sajid akthar wrote:
Hi All
Sorry for non CCP4 question. I want to make Pottasium Nitrate solution
of pH 6.9 (ofcourse for crystallisation trails only). I'm adjusting pH
by using NaOH. But it seems the pH is not stable after sometime. Is
there any good way to do it.
Thank you
Sa
Huiying Li wrote:
I tried to scale, using SCALA through CCP4i GUI, three blocks of data
collected with one crystal (3 mtz files output from MOSFLM). The GUI
has only one MTZ input slot. Which program can be used to combine the
3 unmerged mtz files together? CAD refused to handle these raw mtz f
at purpose. My total cost was jut for the HDDs, about $300.
Works great.
Cheers,
Roger Rowlett
Professor
Colgate University
Dept of Chemistry
-Original Message-
From: "Green, Todd" <[EMAIL PROTECTED]>
Subj: [ccp4bb] off topic - back-up servers
Date: Mon Dec 10,
If suppression of adventitious protein binding to Ni-NTA with elevated
imidazole concentration is not practical (depending on the protein you
can use up to 50 mM or higher), then you might try separating the
proteins using hydrophobic interaction chromatography, which is rapid
and very powerful
Go to the Pymol community wiki (www.pymolwiki.org) and install SLERPY, a
python plugin that allows you to create multiple scenes and transitions
in Pymol. It is possible to create some pretty sophisticated animations
using slerpy, including fading transitions of surfaces. If you ray-trace
the m
Is it possible that your crystal is not really a single crystal, or has
multiple domains? I had a similar problem with an otherwise beautifully
diffracting crystal (beyond 2.0 A) whose integration rapidly broke down
after a few frames. On close inspection, it was possible to visualize
multiple
One approach is to try modifying your current condition with additives.
We have had some occasional successes with polyol, salt, or solvent
additives modifying crystal morphology from needles to something more
tractable. Hampton research has a good list of 10X additives in their
catalog. In our
Our AKTA FPLC has been trouble-free for 9 years, and we bought one of
the early models. It has lived in a cold room, withstood periods of
heavy use, periods of inactivity, and ham-handed undergraduates without
incident. I think we have replaced only the injector channels and the
solvent filters
Instructions for reading CCP4-format maps into pymol can be found in the
pymol wiki. Point your browser to
http://www.pymolwiki.org/index.php/Display_CCP4_Maps.
--
Roger S. Rowlett
Professor
Colgate University Presidential
ssage-
From: CCP4 bulletin board [mailto:[EMAIL PROTECTED] On Behalf Of
Roger Rowlett
Sent: Monday, June 25, 2007 11:16 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Fedora core 6 install problem
Both of these issues are related to the proper definition of the local
host in your /etc/
Both of these issues are related to the proper definition of the local
host in your /etc/hosts file. Check this file to make sure local host is
defined. You should see a line like either of the following:
::1 localhost.localdomain locahost
or
127.0.0.1 localhost.localdomain localhost
I just bui
I've found that many of my undergraduates like the stereo capability, although
I personally rarely use it. So I guess it's worth the pain of getting the
stereo hardware to play nice with the OS and specialized applications. We put
the cheapest stereo-ready cards available at the time(Quadro 980X
CCP4-ers,
I am building CCP4 in FC6 on some new workstations. When I compile from
source, using either the automated install.sh or manually using
configure-make-make install, Phaser does not get installed (configure
doesn't detect it) even though the Phaser and CCBTX packages are present
or unpack
Frank,
You will get a variety of opinions here, but I really like my Olympus
SZX-12. (I used many different models when I was training, and I by far
preferred the Olympus scopes for their ergonomics. YMMV) For
xtallography you will want a separate (cold) light source with fiber
optic cable. I hav
It appears that CCP4i is accessing an older version of Phaser, which
will generate this error. Phaser 1.3.3 is current for CCP4 6.0.2. I had
a similar problem when I had a stand-alone install of Phaser and later
upgraded to a CCP4 package that contained its own version of Phaser. I
think you have t
What have you tried so far for obtaining an MR solution? You have many
options of software, search models, and parameters to use in the MR
search.
Software: Phaser and EPMR should be on your list to try. These two
programs can crack most problems efficiently,
Models: The highest homology models a
Here is one approach:
1. Export the movie frame png files from Pymol or other movie-making
program.
2. Use ImageMagick to mogrify the files to gifs
3. Combine the gifs into an animated gif using the ImageMagick convert
-adjoin command (you can put together several different sequences using
the adj
James,
The "stars" are atoms in a residue that are no longer within
recognizable bonding distances of other atoms. Somewhere along the way
these residues were mangled to the point that some atoms are no longer
within bonding distance of each other. (In Refmac, for example, this can
happen if the X
One major factor that increases the difficulty of finding a satisfactory
MR solution (assuming a good search model) is the number of molecules
that need to be placed in the ASU. Most programs can typically easily
and quickly find a solution for one molecule per ASU. Placing three
molecules per ASU
I think a "tweak script" button or option (before running) would be an
excellent idea.
Cheers,
___
Roger S. Rowlett
Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346
tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228
IMO, the GUI is a lifesaver for getting undergraduates involved in
crystallography in an efficient way. While it is possible to teach my
undergraduates to write scripts for CNS, CCP4, and O, it is much, much
easier for them to learn the CCP4 GUI. Sooner or later, real problems
require you to look u
Ed,
I would highly recommend Phaser to do what you want. Phaser can be
configured to search all possible related space groups, or the ones you
designate. Open-EPMR can also do automated related space-group searching
as well.
Cheers,
___
Roger S. Rowlett
Pr
Manish,
In practice, we have found that it is very helpful to grow up an
overnight starter culture in minimal media to acclimatize the cells for
growth under minimal conditions. (Cells transferred from LB to M9 do not
perform well for overexpression). We inoculate 1 L of modified M9 (see
below) w
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