[ccp4bb] Prof. Chris Dealwis

Colleagues- We regret to announce the untimely death of Dr. Chris Dealwis, Associate Professor of Pharmacology at Case Western Reserve University (CWRU), in Cleveland, Ohio. Chris worked on several important macromolecular structures, including renin, chemokines, amyloid-recognizing antibodies,

[ccp4bb] Tenure Track Faculty Position in Biochemistry, Samford University

inquiries to Dr. Chris Pursell, chair of the search committee and the department, at chris.purs...@samford.edu. Thank you- Brad Bennett, Ph.D. Search Committee Member Assistant Professor of Biology Samford University bbenn...@samford.edu 205-726-4523

[ccp4bb] Aimless recognition of space group

Hi all- I am trying to merge 3 Scalepack .sca files into 1 MTZ file using Aimless in CCP4i (CCP4 version 6.4.0). However, Aimless fails with the error that it does not recognize my rhombohedral space group (R32). #CCP4I TERMINATION STATUS 0 Spgr_descr: No such HM symbol I'm used to this as in

Re: [ccp4bb] cryo-EM

Yes, but the technique is still in its infancy stage. DOI: 10.1016/j.sbi.2014.03.004 Cheers- Brad On Fri, Aug 29, 2014 at 6:34 AM, rana ibd 0285de88044a-dmarc-requ...@jiscmail.ac.uk wrote: Dear CCP4 I wanted to ask has anyone tested 3D protein structure by cryo-electron microscopy?

Re: [ccp4bb] cryo-EM

, but those are slowly falling. Some reading material http://www.ncbi.nlm.nih.gov/pubmed/23181775 http://www.ncbi.nlm.nih.gov/pubmed/25071206 Cheers Marcus Quoting Brad Bennett bradbennet...@gmail.com: Yes, but the technique is still in its infancy stage. DOI: 10.1016/j.sbi

Re: [ccp4bb] Trouble cleaving SUMO tag off of membrane protein

Hi Raji- To echo what John Lee said, we found that we had to maintain a ratio between 1:2 and 1:10 Ulp1:substrate for several different target proteins, none of which were membrane proteins however. So, I would definitely try maybe a 1:5 ratio and see what happens. This wasn't too cost prohibitive

Re: [ccp4bb] Mac OS 10.8 Mountain Lion and CCP4?

Thanks to Jon, Huw, and Albert for quickly setting my mind at ease about upgrading our Mac OS to Mountain Lion and compatibility with CCP4. Just as an FYI, it seems most of the other commonly used software packages (i.e. Phenix v1.8, PyMol v1.5) are ok running on 10.8 as well. Best- Brad On Fri,

[ccp4bb] Mac OS 10.8 Mountain Lion and CCP4?

Hi all- Sorry if this has been gone over before but I could not find a direct answer after a cursory search in the archives and online. Is the CCP4 suite compatible with Mountain Lion? Specifically, the GUI (ccp4i) and Coot? Our Macs are running Leopard and Snow Leopard and we've been thinking

[ccp4bb] Difference set of contacts

Hi all- I have 2 PDB files of the same protein (in slightly different states) and I want to output a table of interactions (preferably hydrogen bonds, salt bridges) that are unique to a structure i.e. Ser22 OG forms a hydrogen bond to Lys25 NZ in structure 1 but not in structure 2. In other

Re: [ccp4bb] The effect of His-tag location on crystallization

I think it was an N-terminal RGS-type His tag in 3O8Y (human lipoxygenase) that mediated crystal contacts with a symmetry related molecule. As I recall, this tag composed a B-strand that formed a nice interface with a native B-strand of the symmetry related molecule. Pretty cool... -Brad On Wed,

Re: [ccp4bb] Off-topic His-Antibody

I'll just second, third, and fourth the assessment of the Qiagen anti-pentaHis antibody. I pretty much gave up on anti-His antibodies until we tried this out in the lab and now everyone loves it. Reasonable sensitivity and specificity (we use IR dye-conjugated secondaries and a Li-Cor Odyssey

Re: [ccp4bb] off topic detection of oligomerisation

Native PAGE (i.e. BN-PAGE), light scattering (i.e. MALLS) Not quick and easy but could work: AUC (i.e. a sedimentation equilibrium experiment) -Brad On Tue, Jun 26, 2012 at 9:01 AM, Careina Edgooms careinaedgo...@yahoo.comwrote: Dear ccp4 bulletin board I apologise for off topic question. I

Re: [ccp4bb] Neutron data collection

Hello Dr. Palmer- There may be other representatives in the literature by now but the one study I know of that examines the usefulness and limitations of determining cryo-neutron structures is The 15-K neutron structure of saccharide-free concanavalin A, Blakeley et al. (2004), PNAS,

Re: [ccp4bb] TCA or acetone precipitation of proteins

Is it ok for you to heat your sample? If so, you could near-boil your sample in a heating block, say at 95 degrees C, in order to concentrate it. I've done this in regular 1.5 mL eppendorf tubes with either the cap open or where I've punctured the top of the cap with a narrow bore needle (in order

Re: [ccp4bb] How to help crystal grow bigger

Hi SnowDeer- What's your precipitant? Have you tried lowering that as much as possible? This will likely delay nucleation and crystal growth but the crystals will also likely be larger (and perhaps more well ordered). Of course, you may reach a lower limit where you prohibit nucleation. You'll

Re: [ccp4bb] titering baculovirus ?

Though I'm not meaning to turn this into a plaque assay burn session, for many reasons we have also abandoned it and instead titer our baculovirus stocks (P3 and P4) using flow cytometry. We use an antibody against viral gp64 that has been labeled with PE and stain infected cells in 96 well plates

Re: [ccp4bb] X-Ray films

Hi Harry- X-ray crystallography played an integral part in discoveries made in Michael Crichton's Andromeda Strain. Mainly it was used to determine the elemental composition and arrangement of the capsid or shell that the foreign organism was found within. Best- Brad On Thu, Apr 15, 2010 at

Re: [ccp4bb] bacillus protein expression

Hi Amit- We used a well-expressing soluble protein fusion (SUMO) at the N-term of a couple of B. anthracis proteins that we were trying to produce in E. coli, which improved the soluble yield of both targets tremendously. The disadvantage of a fusion is of course you produce a non-native protein-

Re: [ccp4bb] SHELXL refinement

Hi Ajit- Sounds like maybe you haven't defined the refinement to include your waters explicitly. Did you edit the parameter list near the top of the .ins file (like ISOR, CONN, BLOC commands) to include your newly added waters? HTH- Brad On Fri, Feb 12, 2010 at 4:45 PM, Ajit Datta

Re: [ccp4bb] smeared spot in diffraction

Hi Fengxia- How about increasing the PEG% so you don't have to add as much other cryoprotectant? Also, have you tried annealing the crystals? I've had success with this when I've had samples diffract similarly. The simplest way is by blocking the cryostream for 2-3 seconds (repeat 1-2 times) and

[ccp4bb] Summary: Electron density improvement with resolution pictures

Wow, the CCP4BB has proved once again its awesome community spirit. Thanks to all who replied so promptly for my request for graphics showing density improvement (and more accurate modeling) with an increase in data resolution. Below is a summary of the responses: Several people pointed to James

[ccp4bb] Electron density improvement with resolution graphic

Hi all- I recall seeing a long time ago a figure which showed the progressive improvement of electron density as one increases resolution, say from 6 to 3.5 to 2A. I believe I've seen two versions- one a larger scale pic showing the ability to model side chains of alpha helices at higher

Re: [ccp4bb] -RTlnK = delH - TdelS

James- It's: lnKd = deltaH- RT(deltaS) As for Km ~ Kd, here be dragons...Km values cannot be treated as the Kd of a complex because dissociation of a catalytic complex has two potential fates, one that is denoted by a rate constant that is correlated to turnover (so, the forward reaction leading

Re: [ccp4bb] Contact Surface Area

Hi Dan- There's an online server and program called DPX which will calculate the depth of all the atoms in your PDB file. An atom's depth is defined as the minimum distance it is to the nearest solvent accessible atom, with depth = 0 meaning the atom is not buried at all (most likely at the

Re: [ccp4bb] bond lengthening in atomic resolution structure

Hi Ivan- Since you most likely have the data to back it up and I assume you have the computational firepower to do it, why don't you run a full matrix refinement in SHELX to get estimates of bond lengths and their deviations? It's worked for me in calculating carboxylate C=O bond lengths to

Re: [ccp4bb] problem with CCP4i

Hi Atlanta- Yep, this one bit me (on Mac OS X), too. To fix it, I had to go to /usr/local/ccp4-6.1.2/share/ccp4i/etc/UNIX and edit my configure.def file. Almost at the end of the file, there is a line that says USE_DBCCP4I_ON_STARTUP. Change the value from 1 to 0. This should refresh your project

Re: [ccp4bb] xplot 84 driver and Bad plot84 file format error

install (as does xloggraph and xccpjiffy2idraw). On Wed, Aug 12, 2009 at 10:49 AM, Brad Bennett bradbennet...@gmail.comwrote: Hi all- CCP4 distribution: 6.1.1 OS: Mac OS X, version 10.5.7 (Intel) I wanted to look at a plot output (.plt file) from a POLARRFN run invoked within ccp4i. When

Re: [ccp4bb] selenomethionine labeled protein expression in insect cells

Hi Riya- Expression Systems has a protocol for SeMet incorporation (Click on the Selenomethionine Incorporation pdf file at http://www.expressionsystems.com/?mvcTask=documents) and they advertise that they do just about anything you would want to do with insect cells (see

[ccp4bb] xplot 84 driver and Bad plot84 file format error

Hi all- CCP4 distribution: 6.1.1 OS: Mac OS X, version 10.5.7 (Intel) I wanted to look at a plot output (.plt file) from a POLARRFN run invoked within ccp4i. When attempting to open the plot file, I get a Bad plot84 file format but the archaic xplot84 driver window does open just with no plot

[ccp4bb] Disulfide bond survival in the presence of DTT?

Hi all- Got a perplexing thiol chemistry/phasing issue. To prevent non-native disulfide bonds from forming between free Cys but to preserve native disulfides, I have heard of using a very low (say 0.5 mM) concentration of DTT. I've recently come across a paper where the assignment of 3 disulfides

Re: [ccp4bb] purification

Hi Kien- Are you basing extensive proteolysis (degradation) on an SDS-PAGE result alone? Are you monitoring the elution profile from Ni/NTA? Do you see numerous A280 peaks for your elution? Sample prep of membrane proteins for SDS-PAGE is very trial-and-error. Heating the samples may cause weird

Re: [ccp4bb] Eukaryotic Membrane Proteins

Hi Raji- A number of GPCRs have been overexpressed in E. coli. Examples are Reinhard Grishammer's work with neurotensin receptor and Christopher Tate's work with B2-adrenergic and adenosine 2a receptors. The problem was not necessarily expression but proper outer membrane targeting of *active*

Re: [ccp4bb] Off topic: Mammalian gene expression in E. coli

Like most things (and scientists in general I suppose), SUMO tags have both a good and a naughty side. Like GST, in my hands, they enhanced expression and solubility of a couple of different target proteins. Their primary advantage is that when you clip off SUMO with the SUMO protease (aka Ulp1)

Re: [ccp4bb] Off topic: Crystal degredation with age

FWIW, many of the crystals used in classical neutron diffraction experiments were pretty elderly samples by the time data collection was initiated, partly to allow large crystals to grow ever larger, partly because of the mandatory deuterium exchange process and partly because the experiments

Re: [ccp4bb] Removal of bacterial Hsp70 contaminant from recombinant protein

Hey Yong... add 10 mM ATP + 2.5 mM MgCl2 in your purification buffer and wash it off during your affinity step...this has worked for me (of course make sure that you're not unlucky and your protein doesn't elute during this step; don't worry it shouldn't) if that doesn't work, maybe try