Dear All,
A 3-year PhD or 4-year MRes/PhD position is available in the recently
established laboratory of Prof. Bert van den Berg in the Institute of Cellular
and Molecular Biosciences (ICaMB) at Newcastle University, UK. The project will
focus on elucidation of the mechanisms of
Lin, consider yourself lucky if you only have one problem...;-)
regarding your question, poor reproducibility is a common problem with membrane
protein crystallography and is most likely caused by different amounts of
lipids copurifying with your protein. If you want to avoid it (may not be
My lab has had a C3 for almost 8 years now. It's very good for coli, not so
good for yeast as 25-30 kpsi is in the extreme range of the instrument and
you'll wear out seals etc pretty quickly. For those pressures you'll also need
high pressure air. You'll also need to be fairly disciplined in
Yes, as Jacob says, alternative conformation of the serine. Quite common.
Bert
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Uma Ratu
[rosiso2...@gmail.com]
Sent: Monday, May 21, 2012 4:57 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Serine
Generally speaking it is quite hard to crystallize DDM since it is so soluble
(20% in water). You most likely have protein crystals (of course containing a
lot of detergent as well) that are just not ordered, presumably because most or
all of the lattice contacts are mediated by detergent and
I have to agree with Ed here. I would take it even further and suggest that the
PDB file(s) and structure factors SHOULD be requested by the reviewer if many
(or even some) of the paper's findings and conclusions depend on map
interpretation. Likewise, I would refuse to review if the authors
I would definitely try gelfiltration (how do you get rid of the cleaved tag
anyway, sample buffer exchange?) but especially ion exchange. A homogeneous
sample on SDS-PAGE and/or gelfiltration is often not (at all) homogeneous in
ion exchange. Beyond that i would make some point mutations on
Is there anything that consistently matters in crystallography? ;-)
From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Frank von Delft
[frank.vonde...@sgc.ox.ac.uk]
Sent: Wednesday, August 24, 2011 5:21 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re:
Just a comment: positive results can also be due to poor experimental skills
and/or lack of attention to detail etc.
Peer review should take care of this, at least to some extent. Negative results
can be very valuable.
Bert
From: CCP4 bulletin board
Negative results are not necessarily criticisms of colleagues, at least I don't
think it should be perceived as such. And if it is, folks should just grow
up...:-)
It may be (too) idealistic, but one could argue that the most important aspect
of scientific experiments is reproducibility. If
I'd say its very likely to be orthorhombic. Refinement should tell you.its
the best way to determine the space group anyway. Why do you doubt its
orthorhombic? Is Vm reasonable?
It could be monoclinic and merohedrally winned with the beta angle very close
to 90 degrees, but my money is on
I think that's impossible to say. Some proteins lyophilize fine, some don't.
Generally your chances are best if the protein is sturdy. Is also depends how
it was lyophilized (any salts, buffer etc). Getting the protein in solution may
be tricky; in my limited experience plain water works best
You may have a fairly long cell edge (or two if you are dealing with P3 or P6),
but you also seem to have high mosaicity (pic spot 1). Try the useful
strategies suggested here. It may also be worthwhile to shoot a few roomtemp
crystals to see if your cryo is at fault for your high mosaicity.
http://hamptonresearch.com/product_detail.aspx?cid=26sid=145pid=439
These work pretty well and are cheap.
Bert
On 3/30/11 10:24 PM, Jiamu Du jiam...@gmail.com wrote:
For side by side stereo figures.
Thanks.
On Wed, Mar 30, 2011 at 9:42 PM, Ingrid Attinost ingrid_attin...@hotmail.com
wrote:
=%22Wallace%20BA%22%5BAuthor%5D ,
Sansom MS
http://www.ncbi.nlm.nih.gov/pubmed?term=%22Sansom%20MS%22%5BAuthor%5D .
-Partha
On Wed, Mar 23, 2011 at 9:02 AM, Van Den Berg, Bert
lambertus.vandenb...@umassmed.edu wrote:
Hello all,
Does anyone know how to get values for pore sizes of membrane channels
Hello all,
Does anyone know how to get values for pore sizes of membrane channels? I'm not
interested in A x B angstrom values measured between atom centers and assuming
a regular pore shape, but a real-life value of either surface area at the
narrowest point or the volume of a block centered
Try different detergents.
Try 10% or more glycerol.
Try adding ligands (if present/known).
Try varying ionic strength and/or pH.
Try giving more specifics so people on the board may be able to help you better.
Bert
On 3/23/11 1:51 PM, gauri misra kamga...@gmail.com wrote:
The protein purifies
Hi Justin,
I'm not sure if there are papers regarding this for GPCRs, but the phenomenon
you're referring to is the positive inside rule. This basically means that
the SecY translocon (in a way that is only partially clear) mediates membrane
protein insertion in such a way that the (net)
Hi Tom,
Adding glycerol to (crystallization) buffers is a very common practice when
working with membrane proteins. Many membrane proteins have been crystallized
(perhaps even the majority) with glycerol, even up to 30% v/v. So, at least for
membrane proteins, there is no problem.
Bert
On
There seem to be quite a few rule followers out there regarding resolution
cutoffs. One that I have encountered several times is reviewers objecting to
high Rsym values (say 60-80% in the last shell), which may be even worse than
using some fixed value of I/sigI.
On 3/3/11 9:55 AM, Ed
Does the position of this inflection point depend on the redundancy? Maybe it
does not; for high-redundancy data one would simply get a much higher
corresponding Rsym.
On 3/3/11 11:13 AM, Ed Pozharski epozh...@umaryland.edu wrote:
On Thu, 2011-03-03 at 16:02 +0100, Vellieux Frederic wrote:
We should compile this discussion and send it as compulsive reading to journal
editors...;-)
Bert
On 3/3/11 12:07 PM, Simon Phillips s.e.v.phill...@leeds.ac.uk wrote:
I take the point about a tendency in those days to apply sigma cutoffs to get
lower R values, which were erroneously
I have heard this before. I'm wondering though, does anybody know of a
systematic study where different data processing programs are compared with
real-life, non-lysozyme data?
Bert
On 1/28/11 7:58 AM, Bosch, Juergen jubo...@jhsph.edu wrote:
I was a bit reductive with my statement
://web.me.com/bosch_lab/
On Jan 28, 2011, at 8:37 AM, Van Den Berg, Bert wrote:
I have heard this before. I'm wondering though, does anybody know of a
systematic study where different data processing programs are compared with
real-life, non-lysozyme data?
Bert
On 1/28/11 7:58 AM, Bosch
The way to go is make-your-own, especially since it is a pretty lousy enzyme
(we often use 1:5 to 1:10 molar ratio of TEV:protein). You can get expression
vectors here:
http://mcl1.ncifcrf.gov/waugh_tev.html
Bert
From: CCP4 bulletin board
?? I don't know if I understand the question, but don't most journals have
references that do include the article titles?
Science, Nature, Cell, NSMB, PNAS, JMB, Structure all have references
titlesas they should.
Bert
On 11/22/10 9:35 AM, John R Helliwell jrhelliw...@gmail.com wrote:
OK I get it, its about the supplements...apologies.
Bert
On 11/22/10 9:35 AM, John R Helliwell jrhelliw...@gmail.com wrote:
Dear Jacob,
Additional content, like article titles, whether print or online, need
to be checked properly for accuracy.
Article titles (if supplied by authors) can often
Hi Shukuri,
If you're on a tight budget and you only want to use DLS to verify sample
homogeneity I would save my money or use it for something else (a
crystallization robot, for example). You definitely do NOT need DLS to solve
crystal structures, including those of membrane proteins. Many
Autoindexing in the truest sense of the word? ;-)
On 10/29/10 12:08 PM, David Goldstone david.goldst...@nimr.mrc.ac.uk wrote:
Dear All,
Does anyone have any insight into what the circles around the spots
might be?
cheers
Dave
--
David Goldstone, PhD
National Institute for Medical Research
Hi Seb,
I'm not aware of the notion (and neither are your reviewers apparently) that a
His tag often results in two bands on a lane in SDS page. Why would that be?
Extra SDS binding to the positive patch?
Just wondering if there's any truth to your statement.
Also, since in this case there
Hi Matt,
You'll probably get many different answers to a question like this, but what I
would do is go back to your protein and make different constructs; chop off
termini, surface mutations etc, maybe cleave off the tag. Of course more
screening and optimization might work, but my sense is
Hi YB,
For membrane protein crystallization it is common practice (although not always
necessary) to dialyze the protein after the final concentration step (against
GF buffer). The problem with DDM is that dialysis is slow due to the low cmc,
and in general it is advisable to finish the prep
I find this interesting as well, mainly because I have never seen this myself
and I have looked at plenty of badly diffracting crystals. In my hands,
synchrotron data at most end up being ~1.5 angstrom better in terms of
resolution than the same crystals on our home source. I'm wondering if
Maybe you should give us a hint about the identity of your protein (if you
dare;-)). I'm sure there are folks around who may be able to say whether or
not your protein is supposed to be brown. You can't expect too much help if you
don't provide (m)any details.
Cheers, Bert
On 9/24/10
Hi Daniel,
Whether or not I would introduce detergents would depend on the behavior of the
protein during purification and crystallization. Does it behave well during
purification in the absence of detergents? In your crystallization screens, do
most of the drops have heavy precipitation? If
I wonder, just as a side note, whether there will still be a (big) need for
X-ray crystallography in a couple of decades?
What will be the state of the art then in structure prediction?
How much of structure space will have been covered by then, so that homology
modeling can do most of the
This has been elaborated before, but you can safely assume they are NOT
detergent crystals. DDM may be harder to crystallize than your average membrane
proteinI hope those crystals diffract!
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech
Hi Jacob,
there are ways, but simple and fast, I don't know. You can do extraction of
some purified protein with organic solvents and doing TLC. Quantitation may be
harder. In the case of phospholipids you can do a phosphorous determination
(with molybdenum, the protocol should be easy to
Hi Jacob,
take some (few ul) of the lysed culture and spread that together with regular
cells onto an agar plate so that you (would) get a lawn of bacteria after O/N
incubation. If it is phage you'll get clear plaques in your lawn, very
distinctive. If it is regular cell lysis induced by the
Hi Engin,
first off, i would not consider an overall Rmerge of 6-10% lousy data, but
quite acceptable for most real-life, interesting problems (so no lysozyme,
thaumatin etc). Our structure of the protein translocation channel SecY is an
example of de novo low-res Se phasing (PDB code 1RHZ).
Hi Darren,
I'm not aware of any (membrane) protein crystal structures solved with tween20.
It's heterogeneous, and its color suggests it contains impurities and/or
oxidation products, making it even more heterogenous. It would be better to
test the behavior of your complex in the presence of
Hello all,
I'm refining a structure with 4 molecules in the AU. The molecules have
substantial differences in certain regions, so I want to exclude those regions
from the NCS restraints calculation and usage. How do i do this? As far as I
can see, by selecting residues via for example chain A
I wouldn't use any stock solution that has been sitting around for a year at
4C. Why take the risk that something has happened with it/grown in it? Use a
freshly made solution and do not store it at 4C.
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
A postdoctoral position is available in the laboratory of Bert van den Berg at
UMass Medical School, Worcester (MA), to work on a NIH-funded project
investigating the mechanism by which hydrophobic molecules, such as toxic
xenobiotics, cross the bacterial outer membrane. The focus of the
Regarding the merging of regular datasets and low-res ones, what is the
proper thing to do? Merging both complete datasets no matter what or cutting
out the low-res data of the regular dataset? I.e. if the low-res data is not
correctly measured in one dataset, would merging all data not be
Hello all,
we have a dataset collected from multiple (2 or 3) parts of the same crystal
with a microbeam (20 micron). The merged data scales OK (not great) in
monoclinic (1-3% rejections). The resolution is 3.2-3.3 A, so the data is not
fantastic. This is the cell (similar for other
Hi,
I wouldn't worry about Se oxidation. In principle having a mix of
oxidized/reduced seleniums is unfavorable, as you'll have less signal at the
edge (broadening). However, all-oxidized Se apparently makes things better
(sharper and more intense peak; I forgot the reference, i think it may
Hi Cedric,
I haven't read this paper, but there's already a system available for roomtemp
data collection that works quite well. Check out
http://www.mitegen.com/products/micrort/micrort.shtml
Instead of a capillary they use thin polyester tubing that you slide over
(special) bases so that
True. It also appears that most people will be looking for less expensive ways
to distinguish between protein and salt crystals
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
From: CCP4 bulletin board on behalf
On Sep 16, 2008, at 5:26 PM, Warren DeLano wrote:
Also, do crystallographers still consider stereo 3D to be a
high-priority or must-have feature in a graphics workstation?
Yes, I do. Really, how can you do without?
As for LCD stereo: yes, please!
By R-fac you mean Rsym? If it is, a value of 0.3 seems way too high. How many
rejections do you have with scaling?
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech II, 373 Plantation Street, Suite 115
Worcester MA 01605
Phone: 508 856 1201
Regarding Phil's comment about the space group, check the PBD stats and you'll
see that c222 is pretty rare. It occurs only in 0.24% of all cases, versus 5.1%
of C2221. So i guess you could say that without doing any analysis, there's a
95% chance that a centered orthorhombic cell is c2221
It should be pretty straightforward to figure that out by freezing and shooting
loops with mother liquor only. You can easily fine-tune by including any
additional buffer components you may have.
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
Biotech
For the SeMet dataset, which wavelength did you collect first? If it is the
peak, you could try doing SAD with just the peak wavelength. Maybe combine the
Se peak data with the Hg dataset (provided they are isomorphous) and do MIRAS
as Jacob suggested.
Bert van den Berg
University of
Hi all,
a common issue with membrane protein strustures is that at the end of
refinemnt, there are (almost) always blobs of unassigned density covering the
hydrophobic region that can't be built (because they are too small for a
complete detergent molecule for example). Would it be
Try the program disulfide by design: http://www.ehscenter.org/dbd/
Its easy to install (at least on windows) and to run. You put in a PDB file and
the program will look in the structure where disulphide bonds are possible. It
also ranks the candidates in energies. Finally, it can generate a PDB
Hi all,
is it possible to input discontinuous data wedges into XDS (obtained from for
example inverse beam sweeps)? (So wedge se1 goes from 0-90 deg (image 1-90),
se2 from 180-270 (image 1-90), etc). Or do I have to rename everything so that
I get one data file in which the rotation ranges are
Another possible, and easy, way to optimize initial hits is to take your hit
condition in the reservoir and add small volumes (say 10% or so) of other
screen solutions. In this way you get small deviations from the initial hit
condition that may lead to better crystals or at least gives you
Hi Ngo,
your needles actually look like very thin plates. They seem promising to me.
From appearance its impossible to tell whether the crystals are detergent or
not. DDM is apparently known to form crystals, but I've never really gotten any
(DDM is very soluble). What temperature have you
We had a similar issue recently where we tried to draw conclusions about a
domain having the same conformation in wildtype and mutant proteins. One
reviewer got back saying the resolution of the structures was too low (3-3.5 A)
to say anything meaningful. Of course everything will depend not
Hi all,
during refinement of our (membrane protein) structures, basically in all cases
the R/Rfree values depend a lot on the low resolution cutoff. Putting the
cutoff at lower res (20-50 A) results in substantially higher R/Rfree values
(sometimes few percent). For this reason we mostly
No, we have been using version 1.1 so far. Thanks for the suggestion, we'll use
version 1.2 from now on and try Phenix as well.
Bert
-Original Message-
From: Axel Brunger [mailto:[EMAIL PROTECTED]
Sent: Thu 1/24/2008 7:35 PM
To: Van Den Berg, Bert
Cc: CCP4BB@JISCMAIL.AC.UK
Subject: Re
Hi Lisa,
is the Phoenix capable of setting up hanging drops? For me that is one of the
big plusses of the Mosquito. Are there any other robots out there capable of
doing hanging drops?
Cheers, Bert
Bert van den Berg
University of Massachusetts Medical School
Program in Molecular Medicine
The mosquito has special (albeit fairly pricey at $13 each) plastic sheets that
allow setup of hanging drops in a 96-well format. It can also do multiple drops
per well. As far as I know this is a capability unique to the Mosquito but I
may be wrong.
Bert van den Berg
University of
Hi Rongjin,
the concentration (and total amount) of detergent during purification depends a
lot on what stage of purification you're at. For membrane extraction (1st
step), people typically use anything from 1-5%. This depends on your budget, on
the cmc of the detergent, and of course on the
Jacob,
Whether the calbiochem/anatrace catalogues will work for you depends on what
you want to know. They do have useful info on micelle sizes (aggregation #s,
cmc's etc), but these are values for micelles alone, either in water or 0.1 M
salt or something like that. If your question is how
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