Re: [ccp4bb] Off-topic: Supplying PDB file to reviewers

2012-04-22 Thread Van Den Berg, Bert
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

Re: [ccp4bb] detergent crystal?

2012-04-26 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Serine

2012-05-21 Thread Van Den Berg, Bert
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

Re: [ccp4bb] OFF TOPIC: recommendations for High Pressure Homogenizers

2012-08-05 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Phase separation and membrane protein crystallization

2012-12-18 Thread Van Den Berg, Bert
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

[ccp4bb] PhD position in outer membrane sugar uptake

2012-12-28 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Protein-detergent micelle sizes

2007-11-20 Thread Van Den Berg, Bert
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

Re: [ccp4bb] detergent concentration used for membrane protein purification

2007-12-21 Thread Van Den Berg, Bert
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

Re: [ccp4bb] crystallization robot

2008-01-09 Thread Van Den Berg, Bert
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

Re: [ccp4bb] crystallization robot

2008-01-09 Thread Van Den Berg, Bert
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

[ccp4bb] low-res cutoff in refinement

2008-01-24 Thread Van Den Berg, Bert
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

Re: [ccp4bb] low-res cutoff in refinement

2008-01-24 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Low resol structure

2008-04-11 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Optimization of needle crystals?

2008-04-16 Thread Van Den Berg, Bert
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

Re: [ccp4bb] SUMARY: Optimization of needle crystals

2008-04-17 Thread Van Den Berg, Bert
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

[ccp4bb] discontinuous data wedges in XDS

2008-04-29 Thread Van Den Berg, Bert
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

Re: [ccp4bb] mutation to cysteines

2008-05-29 Thread Van Den Berg, Bert
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

[ccp4bb] build/refine individual methyl/metylene groups in membrane protein structures

2008-07-04 Thread Van Den Berg, Bert
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

Re: [ccp4bb] spam Re: [ccp4bb] Advice for phasing

2008-07-17 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Cryo with succinate

2008-07-22 Thread Van Den Berg, Bert
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

[ccp4bb] space group stats

2008-07-25 Thread Van Den Berg, Bert
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

Re: [ccp4bb] 27.5% R/29% Rfree for 1.75A structure (details updated)

2008-07-29 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Progresss with Stereo 3D under Mac OS X Leopard

2008-09-17 Thread Van Den Berg, Bert
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!

Re: [ccp4bb] offtopic__which crystals to harvest

2009-01-14 Thread Van Den Berg, Bert
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

Re: [ccp4bb] cryoloops for X-ray data collection from protein crystals at room temperature

2009-01-16 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Se oxidation

2009-02-09 Thread Van Den Berg, Bert
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

[ccp4bb] Questions about (possibly) twinned data

2009-02-16 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Importance of low order reflections?

2009-02-20 Thread Van Den Berg, Bert
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

[ccp4bb] Post-doc position available

2009-02-24 Thread Van Den Berg, Bert
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

Re: [ccp4bb] off-topic detergent hydrolysis?

2009-02-26 Thread Van Den Berg, Bert
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

[ccp4bb] exclude NCS regions in phenix.refine

2009-03-02 Thread Van Den Berg, Bert
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

Re: [ccp4bb] OT: Crystallisation compatible detergents

2009-03-24 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Crystallizing a membrane associated protein

2010-08-01 Thread Van Den Berg, Bert
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

Re: [ccp4bb] protein turns brown

2010-09-24 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Lousy diffraction at home but fantastic at the synchrotron?

2010-09-28 Thread Van Den Berg, Bert
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

Re: [ccp4bb] How to detect the concentration of detergent?

2010-10-04 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Help with Optimizing Crystals

2010-10-26 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Additional band on gel due to his-tag: any references?

2010-10-28 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Strange spots

2010-10-29 Thread Van Den Berg, Bert
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

Re: [ccp4bb] DLS

2010-10-30 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Citations in supplementary material

2010-11-22 Thread Van Den Berg, Bert
?? 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:

Re: [ccp4bb] Citations in supplementary material

2010-11-22 Thread Van Den Berg, Bert
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

Re: [ccp4bb] TEV protease

2011-01-14 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Merging data to increase multiplicity

2011-01-28 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Merging data to increase multiplicity

2011-01-28 Thread Van Den Berg, Bert
://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

Re: [ccp4bb] I/sigmaI of 3.0 rule

2011-03-03 Thread Van Den Berg, Bert
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

Re: [ccp4bb] I/sigmaI of 3.0 rule

2011-03-03 Thread Van Den Berg, Bert
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:

Re: [ccp4bb] I/sigmaI of 3.0 rule

2011-03-03 Thread Van Den Berg, Bert
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

Re: [ccp4bb] off topic: GPCR membane insertion/orientation

2011-03-04 Thread Van Den Berg, Bert
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)

Re: [ccp4bb] off-topic replay: glycerol in protein stock solutions

2011-03-04 Thread Van Den Berg, Bert
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

[ccp4bb] (off-topic) Measurement of channel pore dimensions

2011-03-23 Thread Van Den Berg, Bert
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

Re: [ccp4bb] protein aggregation

2011-03-23 Thread Van Den Berg, Bert
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

[ccp4bb] Structures of crosslinked complexes

2011-03-24 Thread Van Den Berg, Bert
=%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

Re: [ccp4bb] where to buy stereo glasses for making stereo figures?

2011-03-30 Thread Van Den Berg, Bert
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:

Re: [ccp4bb] how to Collecting Data from Long Unit Cell Axes ?

2011-04-05 Thread Van Den Berg, Bert
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.

Re: [ccp4bb] Lyophilized protein sample (purchased)

2011-05-06 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Potential Space Group Issue

2011-07-08 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

2011-07-11 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Fwd: Could Biological Negative Results be published?

2011-07-11 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Aging PEGs

2011-08-24 Thread Van Den Berg, Bert
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:

Re: [ccp4bb] Crystals with Organic solvents

2011-08-26 Thread Van Den Berg, Bert
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

Re: [ccp4bb] phasing with se-met at low resolution

2009-05-10 Thread Van Den Berg, Bert
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).

Re: [ccp4bb] Shredded E coli pellets

2009-07-02 Thread Van Den Berg, Bert
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

Re: [ccp4bb] Lipid content/removal

2009-07-07 Thread Van Den Berg, Bert
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

Re: [ccp4bb] detergent crystals

2009-08-04 Thread Van Den Berg, Bert
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

Re: [ccp4bb] video that explains, very simply, what Structural Molecular Biology is about

2009-11-14 Thread Van Den Berg, Bert
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