Rohit,
Yes, you are wise to worry about this coupled reaction for a number of reasons.
Your question allows me to pull my head out of grading lab final exams on this
very concept. I agree with Herman's and Roger's comments. I have a couple of
other items to mention:
Luckily, NAD(H)-linked en
y “selecting for the least soluble” oligomeric state
> we would rarely crystallize proteins as dimers.
>
> Crystallization is such a confusing process J
>
> Thierry
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of R. M.
> Garavito
> Se
I just wanted to disagree with Roger's word choice, but not his argument (this
is a "flame"-free response). Forget about "packing" and "packable" as there is
no outside force doing the work. The molecules are just falling into a local
energy minimum where favorable intra- and intermolecular in
Dear Hay,
And your point is? I am not trying to be snarky (although I am just starting
my morning coffee), but to bring up the fact that CCP4BB readers need more info
to comment on your case, like space group, local interactions, and how packed
is "tightly packed."
I have had two cases of "
Yamei,
This is not unusual, particularly for many proteins that bind nucleotide
derivatives, especially GDP/GTP binding proteins, as Nat said. If it is GDP
that is tightly bound at high occupancy, it should be quite easy to identify
because of the pyrophosphates and the guanine ring. To build
Although three months is a long time, it is no completely unheard of, and does
not require the invocation of proteolysis. The longest time I have heard of is
~1 yr, so count yourself lucky. However to get good advice, as well as to use
it, you need to ask yourself several questions:
1. What
Xiao,
You could be the victim of the dreaded PEG cross-linking effect. One of the
unfortunate by-products of keeping PEG stock solutions in water is that they
will form peroxides and aldehydes. They will slowly cross-link the surface of
some crystals. However, it is dependent on the nature o
Prishant,
Remember that concentrating by almost any method is a non-uniform process. In
your case, right at the membrane the concentration is much higher than in the
surrounding solution. As Chris says, frequent efforts to keep the solution
well mixed can prevent precipitation. As you mix, l
Reza,
If your protein is not too small (>20 kDa), use a spin-column (i.e., desalting
column) with G-25 sephedex. It is CHEAP, fast, and the recovery is good. We
have even used them to adjust buffer concentrations or to remove micellar
detergents; we have used protein concentrations up to 10 m
Avisek,
Besides the advice that Bernhard and Remy have given you, beware of strange
packing arrangements with low symmetry space groups and higher order oligomers.
We have had several cases in P21 and C2 where tetramers pack with 6 or 8
molecules in the asymmetric unit (ASU), but could not be
Shiv,
PEG1500 and PEG3350 are not the same because of the nature of their synthesis
and manufacture. They are a polydisperse and semi-purified products of an
ethylene oxide condensation. So while they are described as
H-(CH2-CH2-O)-OH, the value of and its ranges are quite different. Some
Rhys,
If crystals grow reproducibly and within a reasonable timeframe, I would always
do co-crystallization, particularly if the HA is a good anomalous scatterer.
We have had good success with this method, including a recent membrane protein
structure. Even if you get crystals that are not is
Frank,
It is not just osmotic factors that need balancing, but the fact that polymers
and salts don't always mix well. PEG and high salt can form 2-phase systems
that don't disperse well, particularly in a crystal. Check out Ray et al.
(Biochemistry 1991, 30, 6866-6875) where Bill Ray and and
Jerome,
>> Does anyone know the best way to store crystallization
>> screening blocks that contain PEG 3350?
I would recommend storing them in a fridge or a clean coldroom (mold-free).
Lower temperature and low light does help.
>> Is it a good idea to freeze the PEG solutions
>> at -80°C and
You may unleash a deluge of anecdotes and horror stories, but this is quite
common. I have experienced this many times, and you just need to step back and
ask yourself what is being done differently:
1. Are all materials used in the preparation of the protein the same
(suppliers, sources, exp
Bernhard,
One other point, after rereading your email. For step-elution ion exchange
(quick on, quick off), you can use some very cheap and quite robust media the
will resist a lot of stuff. Have them pour their own columns and toss it away
when it gets too dirty. You don't have to use the h
Bernhard,
We have had similar, but not identical issues with some insect cell media, as
well as column interference by lipid. If we see this, we tend to run all the
material with the protein (cell lysate or media) through step-elution ion
exchange (quick on, quick off). While the purification
Matt,
In addition to the suggestions of the others, have you done a simple self
rotation function? It can tell you quite a bit about how things are packed and
give you strict criteria for choosing one solution over another. As Roger
said, choosing an even number of monomers in the ASU is a go
Dear Eugene and other CCP4ers
The recent discussion about superposition has prompted me to ask about a
different kind of superposition problem. We are working on a small dimeric
protein that is entirely made up of helices. Instead of large, concerted
domain movements, which I am quite familia
Rana,
It is hard to answer you question without more details (MW and pI of your
target protein). MBP binds very well to amylose resins and is usually quite
easily bound to anion exchange resins. Did you just run a "standard" ion
exchange protocol or try different pH regimes?
However, you did
Stefano,
Before you address the problem, you need to ask yourself a couple of things.
You say that "on the gel filtration we clearly see two bands corresponding to
holoprotein and free FAD." That is not too odd, but have you ask the question
is all the protein good protein.
Is this an enzy
Roger,
While I agree with your list, the BCA assay does not use molybdate (as we make
it from scratch with bicinchoninic acid, sodium carbonate, sodium bicarbonate,
sodium tartrate, and cupric sulfate pentahydrate). For membrane proteins, I
prefer the BCA assay until the protein is pure enough
Felix,
Although I have passed through George's lab at UCSD several times, I have not
seen that crystal, but it should be pointed out that George's interests did
extend beyond photosynthesis. Some of us older folks remember George's
contributions to protein crystal growth, both theoretical and
There are many caveats to using glutaraldehyde on crystals, either for fixing
crystals or for "staining" them.
First, I would not hang a 1-2uL drop of 25% glutaraldehyde in the vapour
diffusion chamber, but add enough glutaraldehyde into the reservoir to make it
0.5-1.0 % (a 1:25 or 1:50 dilu
Gloria,
Here is how we are now doing it, courtesy of Yu-Jing's and Merlin's fine
development work. We have also tried it to quantitatively remove tags by TEV
cleavage, but we haven't solved that problem yet.
Regards,
Michael
Tan, Yu-Jing, Wei-Han Wang, Yi Zheng, Jinlan Dong, Giovanni Stefano
Dear Andre,
It always impressive to see a near atomic resolution structure with a bound
lipid. Congratulations. However, to identify what lipid you have requires a
bit more analysis than just looking in databases. First, what is the bacterium
you are using as the host? If it is E. coli, the
Pascal,
The reason this phenomenon looks odd to you is that detergent phase separation
is not a micelle size phenomenon, but a micelle surface phenomenon. Like any
colloidal solution, even protein, there can be conditions where the colloidal
particles aggregate (the cloud point), creating a mi
Wei
I heartily second Dale's comment. Since you do know what has been in contact
with your protein, you should be able to make a list of ALL POSSIBLE compounds
your protein has been exposed to. However, don't go off the deep end. You say
that "We guess that the molecular formula should be C8H
; toufic el arnaout
> School of Medicine - 660 S Euclid Ave
> Washington University in St. Louis
> St Louis, MO 63110, USA
>
>
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of R. M. Garavito
> [rmgarav...@gmail.com]
> Sent: Sunday, July 14, 2013 11:17 AM
>
Raji,
One point the most people forget about is that whenever you concentrate any
detergent-solubilized membrane protein is that you will ALWAYS concentrate the
detergent. So regardless of the MWCO, if the protein-detergent complex
concentrates, the overall detergent concentration also increas
Most modern textbooks have sections on the proper protections and measures to
take, although the information may be dated. See chapter 6 in Volume III of
the International Tables for X-ray Crystallography. With the modern equipment
and regulatory measures in most countries, you really have to
Dear Teresa,
In addition to Bert's excellent list, I have found that many membrane proteins
aggregate in regular SDS-PAGE sample buffer, particularly when heated. In the
best case, we always see "dimers" or, in the worst case, a ladder or smear of
aggregates. If you heat your SDS-PAGE samples
Dear Zhen,
I should also point out that the statement Matt made ("Superdex is known to
have some ion-exchange characteristics, so that it can weakly interact with
some proteins.") is not completely correct. Superdex and all chromatographic
media made from carbohydrates (dextran, agarose, etc.)
Emma,
You need to provide more information. Luckily, I222 is orthorhombic, so a* is
along a, b* is along b, and c* is along c. This makes things so much easier to
interpret. I hope the plotting conventions you used are typical: b along +y
(North pole), a along +x (to the right), and c perpen
Ed,
> Protein crystals are fragile but not soft.
> If your crystals are like gelatine it's unusual.
I hate to disagree with the disagreement, but there are many exceptions to this
rule. I have seen many protein crystals that are quite malleable and bendable.
One protein produced rod-shape
Actually, to echo Ron, many low-temperature/freezing/vitrification crystal
experiments were done in the 1970's, some by Tsernoglou and Petsko, when they
were both at Wayne State, I believe. However, the direction Jacques Dubochet
was looking at was an extension of work from the early 1960's. E
Theresa,
In addition to the comments provided, you do need to consider the vapor
diffusion experiment process as a whole. The primary reasons why we put the
precipitant mixture in the reservoir, aside from being lazy, is (1) it provides
a straight forward and partially accurate starting point
Dear Rana,
I think you need to clear up some confusion about this experiment. MBP fusions
suffer from a number of drawbacks depending on what you are doing. First, did
you use the MPB domain to purify the fusion protein (with an amylose column)?
If so, you also purified native MBP from the E
Vitali,
Echoing what Dan said, I am not sure why you have chosen detergents first, as
there are many other agents which stabilize proteins. Is the instability due
to hydrophobic surfaces (e.g., made worse at higher salt) or not. Some
non-detergent suggestions are:
1) diols like MPD (see work
Rajan,
This is old news. Ron Kaplan published a lot about sarcosyl extraction of
native mitochondrial carrier proteins from "inclusion bodies" in the late
1990's, but there is an equally robust literature stating that sarcosyl is a
denaturant. It is very much dependent on a protein's characte
Rashmi,
What you ask is impossible to answer because you have not given us any details.
Sodium sarcosyl (or sarcosine) is a very problematic, but useful detergent.
For some proteins, it is an effective denaturant (hence the
denaturing/refolding protocols in the literature); for others, it is
Ho,
A second the vote for OmpF, but many porins could do. Although it is a little
harder to purify from native membranes, OmpF has the advantage that it can be
crystallized in about 1-2 hours from a simple detergent solution with different
PEGs AND (!!!) it is as stable as a rock (you can drop
Uma,
Before this discussion goes much further, you need to provide more details:
1) putative space group?
2) observed resolution and diffraction anisotropy?
3) how big was the crystal and what was its shape? Was the crystal split?
4) were the data sets taken at different points on the crystal?
Evette,
I think the primary issue is what kind of analysis was being reported on. That
is what I look for when I review a manuscript. If the authors are doing a
broad structural analysis (homology of TIM barrels, X-ray refinement protocols,
etc.), I wouldn't expect citations beyond stating th
Roger's note reminded me of some older literature (old in the sense that this
problem extends back into the mid-1970's). Dealing with cryopreservation of
crystals grown in "high" salt can be a real problem, but as many people have
pointed out, the normal cryoprotectants can work, although many
Most of the comments you will get will be anecdotal in that people will report
the successful results and do not take the time or effort to characterize the
less successful results. This often occurs because the tagged portion of the
protein is most often disordered, even in the best crystals.
Dear Sonali,
I think that first item on your possible to-do list is to verify that you have
indeed crystallized the protein you purified. We, too, got great crystals once
with protein X (100 kD) and noticed that 1) the lattice constants, space group
symmetry, and Matthew's coefficient were wit
Lisa,
As others have said, using careful data collection and the modern program
suites available (SHARP, Phenix, etc.), a 300 KD complex with 111 Se-Met
residues should be quite solvable. But you didn't state is what is in the
asymmetric unit (the important figure): one complex with 111 Se-Me
Dear Anna,
I know that you already have gotten replies from some top experts, but your
intriguing problem brought up some issues I have run across in the past.
First, from you experience with single crystal diffraction, your results are
not that much different from those seen in virus structu
Anita,
To answer a lot of your questions, you need to provide some more information
about your protein, at least some of its biophysical characteristics (size,
oligomeric state, an integral membrane protein, etc.). Just because you are
using detergents does not mean all your problems are deter
rmgarav...@gmail.com
On Apr 27, 2012, at 10:54 AM, Jacob Keller wrote:
> Wouldn't the lack of solubility of the alpha form of DDM suggest an easy
> purification protocol for the beta form?
>
> JPK
>
> On Fr
Hongjun,
I am in agreement with Bert as DDM is exceedingly difficult to crystallize,
even in organic solvents. This is one of the reasons it is so expensive.
However, you can produce a lot of "quasi"-crystals that do show low resolution
diffraction. As Bert said, you may have protein/deterge
Rex,
Can give us a bit more detail (OS version, source of CCP4i, and type of
errors)? I am in the midst of reorganizing our little Mac computer cluster, and
CCP4 with CCP4i installs nicely on OS 10.6.8 and 10.7.1. However, there were
some permissions that need resetting to allow you to fire up
Jacob,
One of the problems with glutaraldehyde is the its chemistry is so
bizarre. It actually forms quite long transient polymers in
solution. You also have to ask yourself why formaldehyde also "fixes"
tissues. This is why glutaraldehyde works so well for tissue fixation
for EM as op
Nian,
Before you dump the column, clean it and run some protein standards on
it. If everything looks OK, run a small sample of your protein
again. If it behaves the same way, then you may have a protein with
hydrophobic patches. Anomalous binding to and elution from polymeric
SEC media
Time to start digging in the archives. Try looking at work by Fran
Jurnak in 1986 (J. Cryst. Growth 76, 577) and Bill Ray's work in 1985
(Analytical Biochem 146, 307), and then the works that cite them. I
thought this was common knowledge, but I guess it goes in phases.
Aging of poly(olig
Chris,
As others have said, using sugars as cryoprotectants is a good first
choice. However, we have run into problems freezing crystals with
sugars when the primarily crystallization reagent is a salt at high
concentrations (0.8-2M). Although 0.4M ammonium phosphate, is not
particularl
Weikai,
What you might be experiencing is a detergent effect, i.e., you are
near a detergent-dependent crystallization boundary. We have been hit
with this many times. Under vapor diffusion conditions, sitting or
hanging drop, the protein-detergent complex crystallizes and the free/
bound
Dear Hari,
You might look at Lucy Waskell's experiences with full length
mammalian Cytochrome P450 2B4. While she got >50 mg of purified
protein per liter of culture, many other heme proteins are much harder
to express in E. coli with heme assembly proteins, chaperones, etc.
You just ha
Vinson,
As Dale and Randy pointed out, you cannot change the ΔG of a reaction
by mutation: enzyme, which is a catalyst, affects only the activation
barrier (ΔE "double-dagger"). You can just make it a better (or
worse) catalyst which would allow the reaction to flow faster (or
slower) to
Tony,
As Ed said, DDM is not, nor will ever be cheap. However, the Anatrace
sol-grade is primarily for the early stages of a protein's isolation
and purification, but is not recommended (or intended) for
crystallization or biophysical experiments. Use their purer grade for
the latter.
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