Re: [ccp4bb] Calcium ions in enzymes

[Patrick Loll]

staph nuclease

On 31 May 2013, at 6:25 AM, Wei Liu wrote:

 Dear all,
 
 As we all know, many proteins contain calcium ions. Does anyone know if there 
 are reported cases where calcium ions play a catalytic role rather than a 
 structural role in enzymes?
 
 Best
 Wei Liu



---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Puzzling observation about size exclusion chromatography

[Patrick Loll]

If your protein elutes very late, that means it's binding to the column matrix 
(so all estimates of size go into the trash). Check to see that the ionic 
strength of buffer is reasonable (equivalent to, say, 150 mM NaCl). If so, then 
the only solution is to go to a different matrix type.
Pat

On 20 Jun 2013, at 3:09 PM, Zhang, Zhen wrote:

 Dear all,
 
 I just observed a puzzling phenomenon when purifying a refolded protein with 
 size exclusion chromatography. The protein was solubilized by 8M Urea and 
 refolded by dialysis against 500mM Arginine in PBS. The protein is 40KDal and 
 is expected to be a trimer. The puzzling part is the protein after refolding 
 always eluted at 18ml from the superdex S200 column (10/300), which is 
 calculated to be 5KDal by standard. However, the fractions appear to be at 
 40KDal with SDS PAGE and the protein is functional in term of in vitro 
 binding to the protein-specific monoclonal antibody. I could not explain the 
 observation and I am wondering if anyone has the similar experience or has an 
 opinion on this. Any comments are welcome.
 
 Thanks.
 
 Zhen
 
 
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Re: [ccp4bb] off-topic: bug busting

[Patrick Loll]

I'm joining the pig-pile on the Emulsiflex. I like it because:

a. The folks at Avestin are very helpful (they're Canadian--just 
naturally nice)
b. It'll break yeast as well as E coli
c. It seems pretty gentle to me (we pack it in ice, so there's 
negligible sample heating)
d. It's reproducible--if I put a sample through twice, I KNOW it'll be 
well broken, whereas I'm never really sure with a sonicator (maybe the tip is 
aging/dirty/pitted, maybe it's not tuned properly, etc. etc...of course this 
may just be my OCD talking)
e. It gave me an excuse to go to Sears and buy a big hunkin' air 
compressor

Having said that, it requires regular lubrication, and periodic replacement of 
valves, etc., but the vendor is very helpful about talking you through 
maintenance (and they have at least one person who travels a lot, who will stop 
by while he's in town, should we need hand-holding).

Pat

On 4 Feb 2014, at 11:49 AM, Phoebe A. Rice wrote:

 Some time ago, there was a nice discussion of cost-effective, wimpy 
 protein-friendly ways to break open E. coli.  We're thinking about replacing 
 an aging sonicator.  If people have a favorite gizmo, could they repeat that 
 advice?
 thank you,
   Phoebe Rice
 

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Determining concentration of membrane protein

[Patrick Loll]

No, because Bradford is based on the increase in absorbance when the dye moves 
from a hydrophilic environment to a hydrophobic one (like the protein interior, 
or like the interior of a micelle). When detergents are present in excess of 
their CMC, the change in absorbance from partitioning into the micelles is 
generally large compared to any signal due to protein binding; plus preparing a 
perfectly matched blank solution is challenging when dealing with 
protein-detergent solutions.

I second Michael's recommendation--BCA works well.

On 14 Feb 2014, at 1:45 AM, Niks wrote:

 Dear All,
 May be a stupid question. But if we take buffer with detergent as control 
 (Blank), would not the difference in ODs using any of the methods used e.g. 
 Bradford assay, gives protein concentration? 
 
 Regards
 Nishant
 
 
 On Thu, Feb 13, 2014 at 9:09 PM, Roger Rowlett rrowl...@colgate.edu wrote:
 Your basic choices for protein assays are:
 Alkaline copper methods (e.g., Biuret and micro-biuret)
 alkaline copper + molybdate methods (e.g., Lowry, BCA assays)
 Hydrophobic dye methods (e.g. Bradford)
 UV methods (e.g., A280, A230, A210, etc.)
 Method 1 is least sensitive to amino acid composition, but is also has 
 highest detection limits. Thiols interfere. Method 2 is very idiosyncratic 
 with amino acid composition, and also subject to interference by thiols. 
 Method 3 is not usable in detergent solutions. Method 4 has many inteferences 
 as most everything absorbs in the far UV region.
 If you have some special protein cofactors, metals, chromophores, etc. these 
 can be exploited for better measurements. For ecample metalloproteins are 
 easy to quantify by ICP-OES or TXRF if they are reasonably pure.
 Cheers,
 ___
 Roger S. Rowlett
 Gordon  Dorothy Kline Professor
 Department of Chemistry
 Colgate University
 13 Oak Drive
 Hamilton, NY 13346
 
 tel: (315)-228-7245
 ofc: (315)-228-7395
 fax: (315)-228-7935
 email: rrowl...@colgate.edu
 On 2/13/2014 10:06 AM, Raji Edayathumangalam wrote:
 Dear CC4BBers,
 
 I am trying to figure out what is the best way to determine the protein 
 concentration of my membrane protein. My purified membrane protein is in 
 20mM Tris pH 7, 150mM NaCl and 0.02% DDM (CMC of DDM=0.0076%).
 
 After reading the friendly manuals and searching online, I've learned that 
 detergents interferes with assays like Bradford but can't find good 
 descriptions of what works best. For now, I am trying to estimate 
 concentration from absorbance at 280nm and using molar extinction 
 coefficients based on aromatic amino acids, but again suspect detergent 
 interference. I would like to know what other folks working on membrane 
 proteins are doing.
 
 Thanks very much.
 Raji
 
 -- 
 Raji Edayathumangalam
 Instructor in Neurology, Harvard Medical School
 Research Associate, Brigham and Women's Hospital
 Visiting Research Scholar, Brandeis University
 
 
 
 
 
 -- 
 The most difficult phase of  life is not when No one understands you;It is 
 when you don't understand yourself

Re: [ccp4bb] question on charge charge interactions

[Patrick Loll]

Two Arg side chains stack next to each other in ferritin and in GST (see, for 
example, Arg-59 and its symmetry mate in 3F33). I expect there are other 
examples, but these two come readily to mind.

Cheers,

Pat


On 27 Mar 2014, at 7:11 PM, Tom Peat wrote:

 Hello All, 
 
 I am appealing to the community as I don't seem to be able to find through 
 Google what I am looking for, and I just don't have the ability to look 
 through every structure in the PDB to find this. 
 I have what I think is an interesting case: a two domain protein structure 
 with a mostly hydrophobic interface between the two domains- the kicker is 
 that I have two charged residues buried in this domain and they are 
 identical. 
 That is, I'm looking for an Arg-Arg or Asp-Asp type interaction (not Arg-Asp) 
 where these residues are less than 4 Angstroms apart. So I was wondering if 
 anyone had seen this in any other structure(s)?  
 The really interesting bit is that this interaction actually regulates the 
 activity of the enzyme domain although the domain interface isn't that close 
 to the catalytic site.  
 
 Thanks in advance to all those who can find a reference to this kind of 
 interaction. 
 Cheers, tom




---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pl...@drexelmed.edu

[ccp4bb] Fwd: [ccp4bb] : MTZ for a protein fragment and R factors, Masking and Maps

[Patrick Loll]

George,

Remember that scattering from every point in the cell contributes to every 
reflection; the R-value is a global metric of agreement between the model and 
the data. Hence, calculating the R-value for a few selected residues is not a 
sensible thing to do, unless you want to ask how well your particular fragment 
explains the scattering from the entire crystal (which I'm guessing is not the 
case for a 23 amino-acid fragment).

If you're interested in assessing the agreement between model and map for a 
particular fragment, you might want to consider instead the real-space R value 
and the related real-space correlation coefficient. 

You might also consider some reading to help bone up on the basics:

http://www.amazon.com/Protein-Crystallography-Eaton-E-Lattman/dp/0801888085  (a 
primer, and a shameless plug)

http://www.amazon.com/Biomolecular-Crystallography-Principles-Application-Structural/dp/0815340818
  (the detailed stuff; essential)


Cheers,

Pat Loll


On 5 Jun 2014, at 11:10 AM, George Devaniranjan wrote:

 Hi,
 
 First off I am pretty new to CCP4/X-ray crystallography so please bear with 
 me as I try to explain my question.
 
 I was looking at a protein structure from the PDB (let's say 1aho.pdb).
 
 I have the corresponding MTZ file. I wanted to calculate the R-factor for 
 some selected residues (lets say 17-40).
 
 I can calculate the R factor for the whole protein using the (MTZ + pdb file) 
 SFCheck tool.
 
 Is there a way to calculate the R-factor for a segment of the protein?
 
 I can generate a masked map using COOT as follows:
 Extensions-- Maps-- Mask Map by Atom Selection (inverse)
 But the result is a map and not a MTZ file 
 (the format I need for the next step SFCheck)
 
 I tried using NCSMask but it did not work out.
 Could someone suggest where I am going wrong in trying to calculate R for a 
 small part of the protein?
 
 Thank you,
 George



---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] How to transfer non-frozen crystals with less disturbance?

[Patrick Loll]

You can cut a small piece of sponge and put that into the reservoir; this 
prevents the reservoir buffer from splashing up into the drop.

The sitting drops should be reasonably safe, but the 10 uL hanging drops are 
big; they'll be vulnerable to falling off if the tray is jarred.

Good luck!

Pat

On 2 Jul 2014, at 4:17 PM, Meisam Nosrati wrote:

 Dear CCP4ers
 
 I need to transfer some crystals mainly in sitting drops to the site of data 
 collection without freezing them.
 
 I do not know what is the best solution to secure the boxes in their place to 
 minimize the disturbance. 
 
 I am using the 24 well VDX plates with 10-80 microliter sitting drops. I have 
 one or two hanging drop boxes as well with 10 microliter size drops.
 
 If you have any experience about this matter, I greatly appreciate, if you 
 share it with me.
 
 Thanks
 
 Meisam Nosrati



---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] very informative - Trends in Data Fabrication

[Patrick Loll]

Ron makes an excellent point. Many institutions devote far more energy to 
limiting risk than to doing the right thing. This leads administrators to a 
frightening, but logical conclusion: The less science we do, the less chance of 
our doing something that could invite a penalty on the university. This 
translates into rules intended to head off bad behavior, but which in fact make 
it more difficult to do honest science, and increase the administrative burden 
(our IT group has already made great strides in this direction--if you can't 
connect to the network, then you can't use it to violate HIPAA!).
So I agree that we should be cautious about improvements.
Pat


On 6 Apr 2012, at 12:23 PM, Ronald E Stenkamp wrote:

 Dear John,
 
 Your points are well taken and they're consistent with policies and practices 
 in the US as well.  
 
 I wonder about the nature of the employer's responsibility though.  I sit on 
 some university committees, and the impression I get is that much of the 
 time, the employers are interested in reducing their legal liabilities, not 
 protecting the integrity of science.  The end result is the same though in 
 that the employers get involved and oversee the handling of scientific 
 misconduct.  
 
 What is unclear to me is whether the system for dealing with misconduct is 
 broken.  It seems to work pretty well from my viewpoint.  No system is 
 perfect for identifying fraud, errors, etc, and I understand the idea that 
 improvements might be possible.  However, too many improvements might break 
 the system as well.
 
 Ron 
 

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

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

[Patrick Loll]

 
 Well, it is clear from this comment that in different fields there are 
 different rules... . In macromolecular Xtallolgraphy, where some people deal 
 with biologists from biomedical sciences, the impact of journals is an 
 important aspect during evaluation and, unfortunately, pre-publication review 
 of structures has no actual value in their field. For a structural BIO-logist 
 in biomedical sciences, a paper it is not just a paper, it is an effort of 
 years reduced to a (or few) paper(s).  The non-structural BIO-people 
 understand what is a Cell paper, but not at all about what it is a 
 pre-publication of a structure. My thougts go in the direction of grant 
 applications, fellowships, promotion, all filtered by the impact factor but 
 not by pre-publication of structures which, btw, it is neither considered in 
 the h-index of a researcher.
 

Oh what the hell, someone else poured the gasoline, I may as well supply a lit 
match:

What Maria says is absolutely true--I dwell among biologists, so I fully 
understand the rules of the field. But it's not so clear that these rules are 
good ones. 

Biology is obsessed with high impact, and I argue science is ill served by this 
preoccupation. The highest impact-factor journals seem to have the highest 
number of retractions (see this past Tuesday's New York Times Science section 
for a discussion). And in this forum it's certainly germane to note that the 
technical quality of published structures is, on average, poorer in the highest 
impact journals (at least by some criteria; see the paper from Brown  
Ramaswamy in Acta Crystallogr D63: 941-50 (2007)).

Pat
---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Anaerobic glovebox crystal cryo-cooling

[Patrick Loll]

Think about hyperquenching (a high-falluting name meaning to use a stream of 
gas to blow off the cold layer that accumulates above the surface of the liquid 
nitrogen). We just blow some nitrogen at the surface of the dewar, very low 
tech...but it works. Importantly, you can use a more leisurely pace when 
plunging, so that you place the loop straight into the vial without fear of 
catastrophe.

Warkentin M, Berejnov V, Husseini NS, Thorne RE (2006) Hyperquenching for 
protein cryocrystallography. Journal of Applied Crystallography 39: 805-811

Warkentin M, Thorne RE (2007) A general method for hyperquenching protein 
crystals. Journal of Structural and Functional Genomics 8: 141-144


On 24 Apr 2012, at 10:20 AM, David Gallagher wrote:

 Hi all, 
 
 I've been using a Belle technologies anaerobic glovebox with in built 
 microscope and liquid nitrogen dewar port in an attempt to harvest and 
 cryo-cool crystals in an oxygen free environment.  
 
 I've been having some problems with icing and think this is most likely due 
 to slow speed that it is possible to plunge loops into the liquid nitrogen.  
 I've been plunging loops straight into vials preloaded into an ESRF puck 
 (with pusher).   This requires more care than what I would ordinarily do 
 (i.e. plunging into bulk liquid nitrogen then and manouvering into a vial 
 afterwards and then clipping said vial into a cane) and so takes longer - 
 perhaps leading to icing.  The latter technique is not available as the 
 glovebox set up means that the dewar can only be reached by my right hand - 
 the microscope is in the way!
 
 Does anyone have any experience of a similar set up and if so have some 
 advice as to how I can overcome this problem?
 
 Many thanks, 
 
 Dave Gallagher
 
 
 -- 
 David Gallagher 
 PhD Student 
 
 MRC Mitochondrial Biology Unit 
 Wellcome Trust / MRC Building 
 Hills Road 
 Cambridge 
 CB2 0XY 
 
 Email: dt...@mrc-mbu.cam.ac.uk 
 Phone: 01223 252913

Re: [ccp4bb] selective protection of C-terminal carboxylate

[Patrick Loll]

If you express the protein as an intein fusion you can make a C-terminal 
thioester, which you can then modify quite specifically with various reagents .
Pat

On 25 May 2012, at 10:00 AM, Sebastiano Pasqualato wrote:

 
 Dear all,
 is anyone aware of a way to selectively protect the carboxylate at a protein 
 C-terminus, while leaving unaffected those of Asp and Glu side chains?
 Thanks a lot in advance,
 have a nice weekend,
 ciao,
 s
 
 
 -- 
 Sebastiano Pasqualato, PhD
 Crystallography Unit
 Department of Experimental Oncology
 European Institute of Oncology
 IFOM-IEO Campus
 via Adamello, 16
 20139 - Milano
 Italy
 
 tel +39 02 9437 5167
 fax +39 02 9437 5990
 
 
 
 
 
 
 

Re: [ccp4bb] Mercury phenylglyoxal

[Patrick Loll]

I second the mythical conclusion. We also tried to make it and failed 10-15 yrs 
ago (we came up with the di-iodo compound, if I recall). Many of these 
organomercurials are really tough to handle; I suspect that even if we had 
succeeded in making the desired compound, it would have been as soluble as a 
stone.

Pat

On 11 Jul 2012, at 9:18 AM, David Briggs wrote:

 Hi Vijay,
 
 Not an answer to your question, but I tried and failed to source any
 about 10 years ago. I had a chemist friend try to make some following
 the a protocol we found on the web - which didn't work.
 
 The consensus on the ccp4bb back then was that it was a bit of
 mythical beast, and I never progressed beyond this.
 http://www.ysbl.york.ac.uk/ccp4bb/2001/msg00958.html
 
 We made some Iodo-phenylglyoxal though... never got it to work (as a
 derivative) in action.
 
 Cheers,
 
 Dave
 
 
 David C. Briggs PhD
 http://about.me/david_briggs
 
 
 On 11 July 2012 13:37, Vijay Reddy red...@scripps.edu wrote:
 Hello all,
 
 Could someone please advise me on where to purchase Mercury Phenylglyoxal
 from?
 
 Many thanks,
 Vijay
 
 
 Vijay S. Reddy, Ph.D.
 Associate Professor
 Department of Molecular Biology, TPC6
 The Scripps Research Institute,
 10550 North Torrey Pines Road,
 La Jolla, CA 92037
 
 E-mail: red...@scripps.edu
 WWW:http://www.scripps.edu/~reddyv
 VIPERdb:http://viperdb.scripps.edu
 
 Phone:(858) 784-8191
 FAX:(858) 784-8896
 

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

[Patrick Loll]

We have an Avestin C5 that's about 10 yrs old, and pretty much everything that 
Bert said is also true for us. The instrument works great for bacteria; it can 
also break yeast, but the pressures required are at the upper limit of its 
capabilities, so everything needs to be in tip-top condition. The instrument is 
a little finicky--it needs to be cleaned carefully, and requires regular 
lubrication and replacement of seals and general TLC--so it shouldn't be 
considered low-maintenance. On the bright side, the folks at Avestin are very 
good at talking you through maintenance procedures on the phone, and will also 
visit to do maintenance. I like being able to pack the entire apparatus in ice, 
so heating is not an issue.

We are able to do most maintenance ourselves, but we did need to invest in some 
big old wrenches. And it helps if you have access to a bench vise. We went to 
Sears and bought an enormous air compressor to supply high pressure air to the 
homogenizer. The compressor is really loud but wasn't terribly expensive, and 
it works quite well.

If I were shopping for a new homogenizer today, I'd definitely look at the 
competitors, to see if they required less maintenance; but if not, I'd be happy 
to go with another Avestin.

Pat


On 5 Aug 2012, at 3:37 PM, Van Den Berg, Bert wrote:

 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 
 cleaning, so its not great for sharing between different labs (we all knows 
 how that goes...). Cleaning yourself is possible, but to open it you'll need 
 a big wrench to get sufficient torque. But again, for Ecoli it's very good. 
 We've also had good experiences with the avestin people coming in to do 
 maintenance in terms of waiting time etc.
 
 Bert
 
 From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Marcelo Carlos 
 Sousa [marcelo.so...@colorado.edu]
 Sent: Saturday, August 04, 2012 2:18 PM
 To: CCP4BB@JISCMAIL.AC.UK
 Subject: [ccp4bb] OFF TOPIC: recommendations for High Pressure Homogenizers
 
 Sorry for the off topic question, but it is relevant to those that produce 
 proteins for crystallographic purposes:
 
 We are looking to replace an old French Press with a high pressure 
 homogenizer for cell disruption (E. coli and yeast). We are currently looking 
 at the Avestin C3 and the Niro Panda 2000. I would appreciate any feedback 
 (positive or negative) from users of these instruments (or any other 
 competing homogenizer).
 
 Thanks in advance
 
 Marcelo

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Convert cbf to png/tiff?

[Patrick Loll]

adxv reads cbf images, and can save them as postscript (actually, it's supposed 
to be able to save the image as tiff as well, but at least on my version of the 
program that feature doesn't work).
Pat

On 10 Jan 2013, at 3:36 PM, Frank von Delft wrote:

 Hello all - anybody know an easy way to convert CBF images (Pilatus) into 
 something lossless like tiff or png?
 
 Ideally *easy* as in   r e a l l y   e a s y  and not requiring extensive 
 installation of dependencies and stuff.  Because then I might as well write 
 my own stuff using cbflib and PIL in python.
 
 Thanks!
 phx



---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] refinement hanging--what am I missing?

[Patrick Loll]

Hi all,

Here is a problem that's been annoying me, and demanding levels of thought all 
out of proportion with the importance of the project:

I have two related crystal forms of the same small protein. In both cases, the 
data look quite decent, and extend beyond 2 A, but the refinement stalls with 
statistics that are just bad enough to make me deeply uncomfortable. However, 
the maps look pretty good, and there's no obvious path to push the refinement 
further. Xtriage doesn't raise any red flags, nor does running the data through 
the Yeates twinning server.

Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution of 
data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27

Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. Resolution 
of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26

As you would expect, the packing is essentially the same in both crystal forms. 

It's interesting to note (but is it relevant?) that the packing is quite 
dense--solvent content is only 25-30%.

This kind of stalling at high R values smells like a twin problem, but it's not 
clear to me what specific kind of twinning might explain this behavior.

Any thoughts about what I might be missing here?

Thanks,

Pat


---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] refinement hanging--what am I missing?

[Patrick Loll]

Responding to a couple of questions from Ethan, Charlie, and Phil:

Ethan: Using the default bulk solvent modeling in Phenix; no selenium, but I'll 
double-check wavelengths as a sanity check for scattering factors (but several 
other native data sets from the same synchrotron trip refined beautifully, so I 
suspect there's no gross boo-boos of this nature...)

Charlie:  Solvent regions are pretty clean; I haven't tried any flipping (these 
are molecular replacement models, so it didn't occur to me...). I tried 
applying NCS in one case (the smaller cell) and it had no apparent effect on 
the refinement. The Fo-Fc map has no strong features crying out for 
interpretation. Just based on geometry and map appearance, I'd be inclined to 
say the refinement is done, were it not for the crappy R values.

Phil:  I used TLS for refinement in both xtal forms; it gives a small 
improvement (0.01-0.03) in Rfree in both cases, but nothing magic. I simply 
used one monomer/TLS group (these are ubiquitin variants, so the monomer itself 
is pretty much a little rock, without any internal domain motions). There are 
the usual complement of disordered side chains, but nothing unusual, and  98% 
of the main chain is accounted for. Haven't tried Arp/wArp yet...

Excellent thoughts, keep those cards and letters coming. I'm still chewing on 
the substantive comments from Dean and Adrian...

Thanks,

Pat

On 26 Apr 2013, at 6:17 PM, Carter, Charlie wrote:

 Hi Pat,
 
 Your stats aren't all that bad, but I share your discomfort. 
 
 Do the solvent regions retain any significant features? Have you tried 
 flipping those features? Have you applied NCS? What does the Fo - Fc map look 
 like?
 
 Charlie
 
 On Apr 26, 2013, at 5:38 PM, Patrick Loll wrote:
 
 Hi all,
 
 Here is a problem that's been annoying me, and demanding levels of thought 
 all out of proportion with the importance of the project:
 
 I have two related crystal forms of the same small protein. In both cases, 
 the data look quite decent, and extend beyond 2 A, but the refinement stalls 
 with statistics that are just bad enough to make me deeply uncomfortable. 
 However, the maps look pretty good, and there's no obvious path to push the 
 refinement further. Xtriage doesn't raise any red flags, nor does running 
 the data through the Yeates twinning server.
 
 Xtal form 1: P22(1)2(1), a=29.0, b=57.4, c=67.4; 2 molecules/AU. Resolution 
 of data ~ 1.9 Å. Refinement converges with R/Rfree = 0.24/0.27
 
 Xtal form 2: P2(1)2(1)2(1), a=59.50, b=61.1, c=67.2; 4 molecules/AU. 
 Resolution of data ~ 1.7 Å. Refinement converges w/ R/Rfree = 0.21/0.26
 
 As you would expect, the packing is essentially the same in both crystal 
 forms. 
 
 It's interesting to note (but is it relevant?) that the packing is quite 
 dense--solvent content is only 25-30%.
 
 This kind of stalling at high R values smells like a twin problem, but it's 
 not clear to me what specific kind of twinning might explain this behavior.
 
 Any thoughts about what I might be missing here?
 
 Thanks,
 
 Pat
 
 
 ---
 Patrick J. Loll, Ph. D.  
 Professor of Biochemistry  Molecular Biology
 Director, Biochemistry Graduate Program
 Drexel University College of Medicine
 Room 10-102 New College Building
 245 N. 15th St., Mailstop 497
 Philadelphia, PA  19102-1192  USA
 
 (215) 762-7706
 pat.l...@drexelmed.edu
 

Re: [ccp4bb] Advice on img proc software

[Patrick Loll]

Bernhard,

I've used Matlab to calculate 2D FTs (eg, a Cowtan-esque FT of a  
cat).   I'm sure you could come up with a way to do the phase  
coloring without too much sweat--the degree of flexibility available  
to you in this program is quite large.  My university has a campus- 
wide license for Matlab, so it's *free* for me...I have no idea of  
what it would cost to purchase.


Pat

On Feb 18, 2007, at 10:35 PM, Bernhard Rupp wrote:


Dear All:

I am trying to make FFTs of images of assemblies of
spheres and other shapes to explain diffraction, the
usual thing. So far I do this through cumbersome
cludges, and I bet there are better ways, and I
am looking for free or cheap software to do this.

I load the image into basic mathcad, dump it as a grayscale
array, SFT it with a F90 kludge (s=slow), and load the
transform back into MC and convert it into an image. However, what
I also want to do is generate a false color diffraction
pattern where the color is the phase. I believe this
is quite similar to the Fourier duck transforms Kevin Cowtan
made. I also recall a textbook were two crystallographers
were phase exchanged, so there got to be packages
that to what I want.

The mathcad image processing module is too expensive for my taste.

Any suggestions for software - maybe there is some software
out already to simulate diffraction from one, 2, 3 an array of
objects?

Cheers, br

Bernhard Rupp
www.ruppweb.org



 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Associate Professor FAX: (215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

Re: [ccp4bb] Help with reducing crystal mosaicity

[Patrick Loll]

Jeroen brings up a good point.  Back in the old days, around 5 B. C.  
(Before Cryo), we would use a chilled air generator to blow a stream  
of cold air along the capillary axis to keep the crystals just above  
their freezing point--it made a huge difference in crystal lifetime.  
I recall a colleague devising an apparatus from a 50 ml conical tube.  
The bottom was cut off and cold air was blown in from the other end.  
Windows were cut in either side to allow the beam to pass  covered  
in mylar.  This way the entire capillary was contained within the  
cold tube, so no temperature gradients formed along the length of the  
capillary (temp gradient = distillation = dead crystal).  Later, we  
purchased a very clever goniometer head from Nonius that had a  
plastic cylinder attached to goniometer head, with a swivel, so the  
hose supplying cold air didn't get tangled during data collection...


I've often thought duplicating this apparatus when we encounter cryo  
problems, but I'm always stymied when trying to find a cheap and  
simple source of cold air.  Any bright ideas?



On Jul 10, 2007, at 5:00 AM, mesters wrote:


Mary,

freezing habitually increases mosaicity. In your case, the high  
water content adds to the problem.
Try not to freeze the crystal but collect at sub-zero temperature  
(in short glass capillaries or use oil plugs instead).
You have to optimize the close to freezing data-collection  
temperature.


I collected complete synchrotron datasets (of GCPII in buffer with  
PEG1500 and PEG400) at 260-263 Kelvin which resulted in mosaicity  
values of as small as 0.07 degrees! At 277 K, the crystals only  
last for a few images and freezing did not work (for the buffer  
mentioned before).


- J. -


 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Associate Professor FAX: (215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

[ccp4bb] MS for verification of protein constructs

[Patrick Loll]

I wonder if anyone would care to share experiences/ideas/biases that  
relate to the use of mass spectrometry to verify the identity of  
protein constructs used for crystallization.  Our experience with  
different MS facilities has been checquered.


Specifically:

	What's the current thinking on the best approach to get masses for  
intact proteins of moderate size (say, 40 kD)?  ESI-TOF?
	What kind of resolution should one hope to obtain in such cases  
(10E-04?)


Any suggestions as to good facilities offering fee for service MS  
characterization are welcome (but should be shared off line, I think;  
continental US only).


Thanks,

Pat
 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Associate Professor FAX: (215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

[ccp4bb] coot--saving changes

[Patrick Loll]

Posted for my post-doc...for some reason her subscription is slow in  
starting...any off-line replies should go to [EMAIL PROTECTED]



Dear Coot users out there,

I am a new coot user and have tried many methods to save changes  
while rebuilding, short of save coordinates.


The manual says that coot saves changes automatically after every  
change and writes a new .pdb.gz file in coot-backup. I notice a new  
file appearing in coot-backup after every change alright but it does  
not have the changes I made. I could write out a .pdb every time I  
make a change but isn't there anything akin to File-save in O  
(writing out binary.o as the default file) that coot has? Or, to make  
the coot-backup files actually reflect the change I've made?


I discovered that save state only saves the position in the  
molecule I am walking and not any changes made, much to my horror. Or  
is there something I am missing here too?


Any help anyone, please?

Thanks a million tons in advance,

Sangeetha Vedula

 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Associate Professor FAX: (215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

[ccp4bb] SGI monitor on Mac Pro

[Patrick Loll]

Hi folks,

I just got a new Mac Pro (NVIDIA Quadro FX 4500 graphics card; Apple  
Cinema LCD display primary) and wonder if I can hook up the big ol'  
CRT monitor from a now-defunct SGI as my 2nd display.  The monitor in  
question is a 21 SGI/Sony GDM-5411.  I have to use two adaptors to  
attach it (Sun to VGA and VGA to DVI),  but that's fine.  When I  
attach and power up, the monitor is black except for this information:


Monitor is working
Input 1: 37.7 kHz/300 Hz
Out of scan range
Change signal timing

Has anyone been able to make this work?  Can't find any mention of  
how to proceed, but that could just be Friday afternoon stupidity...


Cheers,

Pat Loll

 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Associate Professor FAX: (215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

[ccp4bb] ftp server at EMBL?

[Patrick Loll]

Does anyone know if the ftp server at EMBL is working?  I'm trying to  
get some of Svergun's SAXS programs, but the ftp connection has just  
been hanging for the last couple of days...

Pat

 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Associate Professor FAX: (215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

Re: [ccp4bb] apologize

[Patrick Loll]

Check out the letters in the Dec 2007 edition of Acta D.  There is  
lively discussion even among the experts (and while I recall no  
mention of angels in this discussion, there IS a reference to Winnie  
the Pooh).


Acta Cryst. (2007). D63, 1274-1281
Acta Cryst. (2007). D63, 1282-1283



Some people believe 0.02 Å deviation from ideality is reasonable,  
based on
the accuracy of the dictionary values of bond lengths and angles;  
others

consider that to be too sloppy and a way to artificially deflate
Rfactors.

I seem to have detected a tendency in the literature to aim for  
about 0.01
Å deviation.  The new refinement program phenix.refine, which is  
supposed
to optimize weighting between X-ray terms and stereochemical  
constraints
automatically, seems to settle in at quite conservative values,  
such as
0.005 Å, whereas with refmac, I can't seem to get the geometry any  
more
ideal than 0.005 Å even if I try to idealize a structure in the  
absence of

X-ray data.

So, like you, I am a bit confused, and wouldn't mind hearing more  
from the

experts.

All the best,

Bill






yang li wrote:

Dear All,
  I am very sorry to involve you into such insignificance  
discussion,

I
have reached agreement
with Prof Gerard, please stop talking about things beyond science,  
thanks!
  I read a book today, which said A refined model should  
exhibit rms

deviations of no more
than 0.02A for bond length and 4 for bond angels, I just wonder  
about the

standard of the
bond length and the bond angel. I think most of you have read similar
words!
But maybe I
didnot express clearly and made some phrasal mistakes.
  At last, happy new year to you all--though very late!


Sincerely!
Yang Li



 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Associate Professor FAX: (215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

[ccp4bb] radiation damage question

[Patrick Loll]

Hi all,

I had an interesting experience, and wonder if others have seen  
similar things.


I was collecting data from a crystal that contains an iodinated  
macromolecule. After 2 days on a copper rotating anode, with the  
crystal at 100 K, we experienced a detector problem, so I put the  
crystal back into the dewar; it was diffracting nicely when I took it  
off. For various reasons, I didn't get back to this crystal until  
about 3 weeks later. When I put it back on the goniostat, the mother  
liquor was milky white in appearance. There were no ice rings, but  
alas the crystal only gave a few anemic spots around the beamstop.  
Annealing didn't help, and I noticed that when I blocked the cold  
stream, the milky white appearance didn't go away when the sample  
thawed. I finally took the crystal off and looked at it under a  
microscope, at which point I discovered that the milky white  
appearance was due to the presence of bubbles in the mother liquor.


I seem to recall some talks on radiation damage in which people  
mention the evolution of a gas (H2?).


So: Does this seem like a radiation damage phenomenon?  And have  
others seen this kind of delay in the manifestation of damage during  
storage at liquid N2 temperatures?


Thanks,

Pat

 
---

Patrick J. Loll, Ph. D. (215) 762-7706
Professor   FAX: 
(215) 762-4452
Department of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

[EMAIL PROTECTED]

Re: [ccp4bb] helix ordering upon metal binding

[Patrick Loll]

Here's an example (although not Fe):

Futterer, K., Ravelli, R. B. G., White, S. A., Nicoll, A. J.   
Allemann, R. K. (2008). Acta Cryst. D64, 264-272.



On 8 Apr 2008, at 9:02 AM, Florian Schmitzberger wrote:


Dear All,

Are there prominent examples of ordering of an alpha-helix within a  
protein upon metal binding (in particular Fe)?


In my case, I observe that a 10 amino acid fragment seems to become  
visible in electron density maps, only upon Fe2+ binding to a  
glutamate (the first amino acid of the respective 10 residues). The  
fragment is part of a larger alpha-helix (which is ordered  
irrespective of metal binding). The 10 residue part (including the  
Glu) is not visible in the iron-free crystal structure in this  
subunit of the homodimer.


I don't think that the helix is necessarily unfolding/folding,  
since in the other subunit in the asymmetric unit it is visible in  
the absence of Fe (probably due to crystal contacts). I am unsure  
about the significance, as it is only in one of the two subunits in  
the asym. unit that I see the ordering.


Thank you very much in advance for any comments!

Florian


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb] heavy atom derivative(s) for tutorial

[Patrick Loll]

Hi,

Does anyone have a foolproof recipe for preparing one or more heavy  
atom derivatives for a nice, easily crystallized protein like  
lysozyme?  I'm looking for something that I can use as a hands-on MIR/ 
SIR tutorial for beginning students. I'd rather the students be able  
to focus their attention on data collection and analysis, rather than  
spending a lot of time screening different soaks.


I have looked at Peter Sun's paper on quick soaking (Acta Cryst D58:  
1092-1098, 2002), and will definitely try what they did; but any  
other recipes would also be welcome.


Thanks,

Pat


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb] heavy atom tutorial--thanks, and a summary

[Patrick Loll]

Thanks to all who responded to my query about cheap, easy, and  
foolproof systems for teaching students how to do MIR/SAD  
experiments.  Below is a summary of responses.


Pat

- Summary follows  
-


Jim Pflugrath:  Lysozyme co-crystallizes with KAu(CN)2
You know its there because you get orthorhombic crystals if gold is  
incorporated.


Jochen Kuper: We have recently tried Dauters Iodine soak with  
lysozyme, though it is not a covalent modification it worked  
foolproof :-)!


 Dauter Z et al. Novel approach to phasing pro...[PMID: 1015]

Fred Vellieux: For HEW lysozyme: precipitant is 2 to 6 % (w/v) NaCl  
in 0.2 M Na acetate pH 4.7 (the recommended procedure is to prepare a  
stock solution of 10% NaCl). For the derivative, use para-chloro  
mercuri benzene sulphonic acid. In 15 ml of the above stock solution,  
dissolve 0.123g of  PCMBS. Then use at 2 to 6% NaCl. Normally the  
crystals should grow within 24 to 48 hours.
	They are not completely isomorphous with the native crystals though  
but this might be sufficient for a course.


Poul Nissen: We use proteinase K that crystallises very well in AmS.  
It is also easy to cryoprotect and mount. Excellent derivatives with  
e.g. lanthanides, and you routinely get data to the edge on the home  
source and stellar experimental maps. On top of that you can also  
work on e.g. PMSF and benzamidine soaks for diff. maps etc. IN my  
experience far better than HEW. Have no ref at hand.


Bernie Santarsiero:  Depending on your time, I'd recommend adding the  
use of a native gel band

shift to try a one that doesn't work and one that does.
		http://www.doe-mbi.ucla.edu/~sawaya/m230d/Crystallization/ 
crystallization.html
	I know you can use NaBr or NaI to grow lysozyme crystals as well,  
and they stick on hydrophobic patches of the enzyme's surface. I do  
recall, however, that the crystal system changes from tetragonal to  
the triclinic or monoclinic forms.


Jose Antonio Cuesta-Seijo: A 0.5M NaI soak for about 20 seconds  
rarely fails for robust, well diffracting crystals.
It is also perfect for SIRAS, but SIR might also work. The only  
question is how many iodines to look for, but the method is  
straightforward with lysozime, elastase, trypsin... (I personally did  
the elastase with I and the trypsin with Br-). And iodines are easy  
to see and detect even in a home source as they have significant  
anomalous signal and s many electrons.


David Roberts: I know you are looking for a recipe for class, but one  
thing that was always my favorite magic bullet was osmium salts (I  
think hexachloride).  Basically, you add some osmium to the dip  
(solid), and watch it diffuse into the crystal.  The crystal will  
turn colors over time, indicating the osmium is in.  Osmium is good  
as it gives a good anomalous signal (if you are collecting data and  
showing that aspect).  It's always a great derivative.  I'm sure with  
lysozyme it would be a good thing.  Osmium crystals diffract well (it  
doesn't seem to kill crystals as mercury can), and so you usually get  
great derivatives from it.
	As for a class, I have students solve a MIR structure (one that I  
did during my post-doc).  I give them a MIR map (that has been  
phased) and have them trace it from scratch.  I give them a few  
starting residues to get them going, and they use coot to build the  
molecule (using batons).  They then convert it to a polyala  
mainchain, add side chains in round 2, and add waters and fix all in  
round 3.  It's amazing how well it all works.  It's a good lesson on  
model building and protein structure.


James Holton: Many of us here at ALS use a hydrophobic gadolinium  
derivative of tetragonal lysozyme as a test crystal.  The neat  
thing about Gd is that it has just as much anomalous signal as Se at  
12660 eV.  The PDB id is 1H87 and all procedures are described in the  
primary reference therein.  You can either co-crystallize or soak.   
The only tricky part is getting the chelated Gd-DO3A compound.  It  
was developed as a medical imaging contrast agent, so they want to  
sell it to you by the pound.  Corrie Ralston here asked the company  
for a free sample and they sent ~1 mL.  That was 5 years ago, and we  
are still using that stock.


Michael Merckel: You might consider Gd-HPDO3A, Acta Cryst. (2002).  
D58, 1-9.

Should also give an anomalous signal with in house source.

Neil Paterson: you can grow lysozyme quite easily in 250-400mM NaI  
and this phases very nicely from an in-house source (8-9 iodines per  
molecule). I can't remember the exact conditions I was using off the  
top of my head (think it was 100mg/ml lysozyme and 20-30% peg4k, 0.1M  
Na acetate pH 4.6 and NaI)...


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology

[ccp4bb] ccp4 install on Leopard

[Patrick Loll]

Accck!

1.  I tried to install Bill Scott's precompiled ccp4 on an intel mac  
running OS X 10.5.  When attempting launch ccp4i, I receive this  
error message:


/sw/share/xtal/ccp4-6.0.2/ccp4i/bin/ccp4i: line 4 /bltwish: no such  
file or directory


Typing which bltwish returns /sw/bin/bltwish, which jibes with the  
definition of $CCP4I_TCLTK, so I'm not sure where the problem is...


2.  Pfui.  So next I tried jettisoning the apt-get installation, and  
instead used the installer downloaded from the ccp4 site (ccp4-6.0.2- 
osx-i386.dmg.gz).  This goes swimmingly, until I actually try to do  
something.   Commands like ccp4i or mtzdump aren't recognized, so  
I go looking for the ccp4.setup file; but it's not there.  Huh?   
Also, there's no folder named ccp4i, even though this install is  
supposed to include it...


It's Friday, and I'm at the nadir of my weekly brain function, so  
perhaps some kind soul will tell me where I'm going astray.


Thanks,

Pat

 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

Re: [ccp4bb] ccp4 install on Leopard

[Patrick Loll]

1.  I was sourcing /sw/bin/init.csh, so that wasn't the problem...but:

2.  I did find the problem (at least for the precompiled version from  
UC Santa Cruz):  For some reason sudo ccp4i gave the error message,  
but just ccp4i (without the sudo) worked OK.  (Although I thought  
the first time you launched ccp4i you were supposed to do it using  
sudo...??).  Anyway, I'm good (although peeved at my continuing  
inability to master sudo.  I miss living on the edge with root).


3. I didn't try to debug the binaries I got from the ccp4 site, since  
I now have a working package.  However, Iain Kerr kindly provided  
this info, which may prove useful to other small brains out there:


ccp4.setup is in ccp4-6.0.2/bin...it used to be in include..is that  
your problem ?


Also, there's no ccp4i folderthe ccp4i binary is in bin/ also.



Note to the long-suffering folks at the secret underground world  
headquarters of ccp4:  Small brain or no, I spent a lot of time  
looking around for some clue as to how to complete the install for  
the ccp4 package, to no avail; a README file to accompany the  
installer would not be unwelcome...


Thanks,

Pat


On 25 Apr 2008, at 4:56 PM, William Scott wrote:


You need to run

source /sw/bin/init.sh

if you use bash or zsh

or

source /sw/bin/init.csh

if you use tcsh.

this will automatically set up the environment variables and then  
it will do what you want it to (which is to set up the ccp4  
environment according to whichever shell you might be using).



On Apr 25, 2008, at 1:19 PM, Patrick Loll wrote:


Accck!

1.  I tried to install Bill Scott's precompiled ccp4 on an intel  
mac running OS X 10.5.  When attempting launch ccp4i, I receive  
this error message:


/sw/share/xtal/ccp4-6.0.2/ccp4i/bin/ccp4i: line 4 /bltwish: no  
such file or directory


Typing which bltwish returns /sw/bin/bltwish, which jibes with  
the definition of $CCP4I_TCLTK, so I'm not sure where the problem  
is...


2.  Pfui.  So next I tried jettisoning the apt-get installation,  
and instead used the installer downloaded from the ccp4 site  
(ccp4-6.0.2-osx-i386.dmg.gz).  This goes swimmingly, until I  
actually try to do something.   Commands like ccp4i or mtzdump  
aren't recognized, so I go looking for the ccp4.setup file; but  
it's not there.  Huh?  Also, there's no folder named ccp4i, even  
though this install is supposed to include it...


It's Friday, and I'm at the nadir of my weekly brain function, so  
perhaps some kind soul will tell me where I'm going astray.


Thanks,

Pat

- 
--

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]



 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb] waaay off topic...

[Patrick Loll]

...but I know you'll come through.

I'm looking for recommendations for a good textbook covering  
fluorescence spectroscopy.


Thanks in advance.

Pat

 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb] Fwd: [ccp4bb] crystallisation and mosaicity

[Patrick Loll]

Hi Charlie,

yes you are right, but I assumed if people see a cloud of condensed  
fog over their LN2 bath they should remove that by

a) filling up the bowl completely e.g. some LN2 drips out of the bowl
b) blow the fog away before you dip


True; this has been demonstrated quite rigorously:

A general method for hyperquenching protein crystals
Journal Journal of Structural and Functional Genomics
Volume 8, Number 4 / December, 2007
DOI 10.1007/s10969-007-9029-0
Pages   141-144

AND

J. Appl. Cryst. (2006). 39, 805-811[ doi:10.1107/S0021889806037484 ]
Hyperquenching for protein cryocrystallography
M. Warkentin, V. Berejnov, N. S. Husseini and R. E. Thorne






 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

Re: [ccp4bb] Friedel vs Bijvoet

[Patrick Loll]

I've always thought that a Bijvoet pair is any pair for which an  
anomalous difference could be observed. This includes Friedel pairs  
(h  h-bar), but it also includes pairs of the form h  h', where h'  
is symmetry-related to h-bar. Thus Friedel pairs are a subset of all  
possible Bijvoet pairs.


This is what Ed and I say in our book, at least (shameless plug); and  
you can buy it from Amazon, so it must be right, yes?


Pat

On 26 Jun 2008, at 11:55 AM, Bernhard Rupp wrote:


Dear All,

I wonder about the conventions using Friedel vs Bijvoet pair.

a) there are no differences. As long as h = -h, it's a Friedel
   or a Bijvoet pair. They are the same.

b) A Friedel pair is any reflection h = -h including hR = -h, i.e.
   including centric reflections.
   A Bijvoet pair is an acentric Friedel pair, it can carry
   anomalous amplitude differences, whereas centric Friedel
   pairs invariably cannot. Actually, Bijvoet pairs (acentric
   Friedel pairs) invariably do carry anomalous amplitude differences.
   There is no such thing as no anomalous scattering.
   We may elect to ignore it, only.

c) of course, this all assumes absence of anisotropic AS.

def b) seems to be helpful in discussions and make sense given that  
absolute


configuration that needs AS signal is somehow associated with  
Bijvoet's

work.

Are any authoritative answers/conventions/opinions available on that ?

Thx, BR

-
Bernhard Rupp
001 (925) 209-7429
+43 (676) 571-0536
[EMAIL PROTECTED]
[EMAIL PROTECTED]
http://www.ruppweb.org/
-
The hard part about playing chicken
is to know when to flinch
-


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

http://www.amazon.com/Protein-Crystallography-Eaton-E-Lattman/dp/ 
0801888085/ref=sr_1_10?ie=UTF8s=booksqid=1214496225sr=1-10

Re: [ccp4bb] Weakest protein-protein complex crystallised

[Patrick Loll]

I hope this isn't too much of a foray into philosophy and semantics,  
but can't you argue that the crystals themselves are weak complexes?  
And since the energies of crystal contacts are typically very weak, I  
would further argue that you should be able to crystallize ANY  
complex with an association constant corresponding to energies as low  
as those associated with crystal contacts. Of course, it's not  
guaranteed, any more than getting a crystal is guaranteed--you need  
some luck.


Of course, it's Monday AM, and I haven't approached my asymptote for  
caffeination.  Am I talking through my hat?


Pat


On 29 Jun 2008, at 3:36 PM, Derek Logan wrote:


Hi,

Can anyone advise me what is currently the weakest protein-protein  
complex yet crystallised? Google searching turned up a paper from  
the Tromsø crystallography group (Helland et al. 1999, JMB 287, 923– 
942) in which a complex between beta-trypsin and a P1 mutant of  
BPTI with a Kd of 68 uM was described as belonging to the weakest  
complexes solved to date, but this article was from 1999 and much  
water has passed under the bridge since then.


Thanks
Derek
_
Derek Logan  tel: +46 46 222 1443
Associate professor  fax: +46 46 222 4692
Molecular Biophysics mob: +46 76 8585 707
Centre for Molecular Protein Science
Lund University, Box 124, 221 00 Lund, Sweden








 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb] os x wiki

[Patrick Loll]

Does anyone know if the Crystallography on OS X Wiki is down?  I  
haven't been able to access the site for a few days, but I don't know  
if the site is down, or if some new and insidious stupidness has been  
perpetrated by the rocket scientists who control IT in our  
institution...thanks.


Pat

 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb] applied optics--LCD projectors

[Patrick Loll]

This is way off-topic, but that's never stopped me before. And what  
group is better qualified to pontificate about matters lying at the  
intersection of computers and optics than this one?


The LCD projector in our departmental seminar room was stolen over  
the weekend (!), and I have been asked to look into what we should  
buy to replace it.  The missing projector was a Dell 3300MP, and IMHO  
it sucked. If I had a nickel for every seminar speaker who said,  
Well, you can't see this, but on my laptop it's very clear that...,  
I'd never need to write another grant.


Alas, I know very little about what's available and what performs  
well. Perhaps you can save me hours of careening around the internet  
researching this question.   Do any of you good folks have experience  
with particular projectors that you like/don't like? Or perhaps have  
a reasoned opinion (or even a wild irrational idée fixe) about the  
sorts of specifications a good projector should exhibit? The room in  
question is not large (about 8 x 10 m, seating a max of ca. 40 people  
for a talk).


I should mention that we're NOT looking for any kind of stereographic  
projection here (cool as that would be); just plain, vanilla,  
projection of the image shown on the laptop's screen.


Many thanks for any info you can contribute.

Pat

 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb] pKa for protein C-terminus?

[Patrick Loll]

What value do we expect for the pKa of a protein's C-terminal  
carboxylate?  pKa values for free amino acids are quite low (2-3),  
but it seems to me that this may have something to do with the  
proximity of a free amine group; I'd expect a higher value (4-ish?)  
for the peptide's C-terminus.


A quick  lazy search using the term pka was stymied by a gazillion  
references to protein kinase A...


Thanks,
Pat


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

Re: [ccp4bb] suggestions for UV spectrometer

[Patrick Loll]

At the risk of dragging this discussion even further afield from  
crystallography:


How can you get realistic numbers for concentrated solutions using  
the Nanodrop?  I understand that the instrument reduces absorbance by  
using a very short path length. However, I thought that in order for  
the Beer-Lambert formalism to be applicable, the solution needs to be  
sufficiently dilute so that the chance of molecules shadowing one  
another is negligible. Isn't this condition violated for concentrated  
solutions (even with short path lengths)?


Pat

On 4 Dec 2008, at 1:27 PM, Michael Giffin wrote:


We also like the Nanodrop...


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
[EMAIL PROTECTED]

[ccp4bb]

[Patrick Loll]

Hi all,

I just read an appalling article in the science section of today's NY  
Times that refers to a new magnetic resonance force microscope  
developed at IBM. The story states For the first time, researchers  
at an IBM laboratory have captured a three-dimensional image of a  
virus.


Not to take away from the good folks at IBM and their really cool new  
instrument, but...


Can anyone give me the authoritative reference for the first crystal  
structure of a virus?  Was it tomato bushy stunt virus?


The same article states that this instrument can be used to ...look  
at the proteins that make up the basic DNA structure...[sic].  Sigh.


Pat



 
-

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexel.edu

Re: [ccp4bb] Crystallography plates, hanging drop but templated sealing film.

[Patrick Loll]

This sounds very similar to a nifty little device the folks in  
Buffalo came up with:


J. Appl. Cryst. (1992). 25, 324-325[ doi:10.1107/S0021889891011354 ]
HANGMAN: a macromolecular hanging-drop vapor-diffusion technique
J. R. Luft and G. T. DeTitta


On 15 Jan 2009, at 4:34 PM, Francis E Reyes wrote:

Does such a thing exist? A 24-well microplate configuration where  
in substitution of glass cover slips, you have a roll of tape  
templated such that there are circular areas where you can add your  
protein where there is no adhesive, but there is adhesive  
everywhere else?


This may be a nightmare for plate manufacturers, but to reuse the  
plate, you just throw away the film and tear a new one.


Thanks!
FR

-
Francis Reyes M.Sc.
215 UCB
University of Colorado at Boulder

gpg --keyserver pgp.mit.edu --recv-keys 67BA8D5D

8AE2 F2F4 90F7 9640 28BC  686F 78FD 6669 67BA 8D5D


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] off-topic--refrigerated shakers

[Patrick Loll]

Any recommendations for WELL-MADE refrigerated shakers?  Our 1980-vintage New 
Brunswick Psycrotherm*  is getting creaky, and the repair people claim that 
parts are not available. Hence, we're looking at the cost-effectiveness of 
replacing it with a new one. 

Probably best to reply off-list, and I can assemble a summary if appropriate. 
Thanks,
Pat


* yes, it's that lovely shade of harvest gold

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] refrigerated shakers, redux

[Patrick Loll]

Hi all,

Here is a summary of the responses to my inquiry about refrigerated shakers:

Virtually all respondents advocated shakers manufactured by New Brunswick 
Scientific. I have rarely encountered such unanimity in this forum.

The only exceptions were three votes for shakers of Swiss manufacture (two 
votes for Infors and one for Kuhner). I confess that I had never encountered 
either of these brands before now, but they both appear to be of high quality.

Thanks for the help. Now if anyone has any suggestions for how to convince my 
administration to unbelt and purchase one of these gems, I'm all ears...

Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Beginning crystallography text

[Patrick Loll]

At the risk of appearing immodest:

http://www.amazon.com/Protein-Crystallography-Eaton-E-Lattman/dp/0801888069/ref=sr_1_10?ie=UTF8s=booksqid=1278618335sr=1-10

On 8 Jul 2010, at 3:35 PM, Peter Hsu wrote:

 Hi all,
 
 I haven't gotten past the phase of growing the crystal, but I'd certainly 
 still like to learn the actual theories of crystallography. Can anyone 
 recommend a good beginner to mid-level text on macromolecular crystallography?
 
 Thanks,
 Peter

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] waaay off-topic: P-1 pumps

[Patrick Loll]

Does anyone know how to disassemble a P-1 peristaltic pump from 
Pharmacia/Amersham/GE?

We have a couple that need simple repairs to either a switch or a rheostat on 
the control panel, but I'm stumped as to how to actually get the damn thing 
open.

If you've succeeded in doing this, I'd be grateful for any pointers.

Pat
---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] problem in heavy metal soaking

[Patrick Loll]

On 26 Sep 2010, at 9:00 AM, Seema Nath wrote:

 I'm working with a protein which crystallizes in a mixture of PEG6K with 0.2M 
 AmSO4,my question is if there's any problem if I want to soak heavy metal 
 derivatives in this crystallizing condition? Does AmSO4 interfere in 
 heavy-metal soaking ? if yes, what's the reason?
 Thank you in advance.
 
There's a nice discussion of heavy atom chemistry and why ammonium ions can be 
problematic in Greg Petsko's review from the 80's:

Methods Enzymol. 1985;114:147-56

The bottom line is that amines (and ammonium is an amine) can participate in 
coordination chemistry for certain metals. If memory serves some of this is 
also covered in Blundell and Johnson (? not sure about that, I don't have my 
copy at hand).

Good luck,

Pat
---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] tRNA expression

[Patrick Loll]

Here's an ignorant question: When people express an exogenous tRNA in E coli 
(to overcome rare codon issues, for example, or to supply a cognate tRNA for an 
orthogonal synthetase), what sorts of promoters are used?  

My (ignorant) guess is that something as potent as the T7 promoter might be a 
waste, since you don't need huge quantities of your tRNA, and you probably 
don't want to divert resources away from message production for your target 
gene. But I could be wrong.

Any pointers to sequences that successfully direct tRNA production would be 
welcome, so I could use them as design templates. 

Thanks,

Pat
---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Freezing crystals in a contained system

[Patrick Loll]

How low? Back in the old days when we mounted xtals in capillaries, you could 
sometimes see significant reduction in radiation damage by data collection 
temperature from room temp to ca. 0 deg C (zero is generally safe, since the 
PEGS/salts in your mother liquor will depress the freezing point). In this 
case, it's critical that you bathe the entire capillary in a stream of cool 
air; if you just aim the cold stream at the part of the capillary containing 
the crystal, you'll get all sorts of nasty temperature gradients, leading to 
distillation of components from the mother liquor and slow painful death for 
the crystal (OK, sometimes not so slow). We used to fashion cylinders that 
enclosed the capillary on all sides and extended the entire length of the 
capillary, and blew the cool air through these cylinders (cheap/easy way to do 
this was to cut the end off a 15 ml conical tube, then cut two windows at the 
position of the crystal (opposite one another, 180 deg apart), and tape mylar 
over the windows. This way you have an approximately air-tight cylinder, but 
don't put a lot of scattering material in the beam.

I seem to have heard reports of people flash-cooling in capillaries, but I'm 
not sure where to find details.

Pat

On 17 Feb 2011, at 12:03 PM, R Conners, Biochemistry wrote:

 Dear all,
 
 We are working on a Category 3 protein which must be contained so we have our 
 crystals mounted in a loop and then covered with a plastic Mitegen cover 
 which is glued in place. We're currently collecting at room temperature, but 
 wondered if anyone has any experience of using a contained system at low 
 temperatures? Any attempts I've had so far at freezing through either the 
 plastic or a glass capillary have resulted in formation of ice on the surface 
 so it is not even possible to see the crystal to centre it.
 
 Best wishes,
 
 Becky
 
 -
 Dr Becky Conners
 School of Biochemistry
 University of Bristol, UK
 
 http://www.bris.ac.uk/biochemistry/brady
 r.conn...@bristol.ac.uk
 0117 3312149



---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] xds question: inverse beam, lots of wedges

[Patrick Loll]

We've just collected a number of inverse beam data sets. It turns out the 
crystals showed little radiation damage, so we have a lot of data: 2 x 360 deg 
for each crystal, broken up into 30 deg wedges. The collection order went like 
this: 0-30 deg, 180-210, 30-60, 210-240, etc.

Now, assuming no slippage, I could simply integrate the first set of data 
(non-inverse?) in one run: 0-360 deg. However, since the 12 individual wedges 
making up this 360 deg sweep were not collected  immediately one after the 
other, I don't expect the scale factors for individual images to vary smoothly 
(there should be discontinuities at the boundaries between wedges). If I do 
integrate the data in one fell swoop, am I in danger of introducing errors? For 
example, I seem to recall that denzo had built-in restraints to ensure that 
scale factors for adjacent images didn't vary by too much. Is there a similar 
restraint that in XDS that I might run afoul of?

The alternative is to integrate each each wedge separately, but with 24 wedges 
per xtal, this is starting to look a little tedious.

Cheers,
Pat

[ccp4bb] MS sequencing of Fab

[Patrick Loll]

We've crystallized a complex of an Fab bound to a protein. We have the 
hybridomas from which the Fab was prepared, but no protein sequence for the 
antibody. We're trying to plot the easiest course to get the sequence (since 
the crystals, alas, do not diffract to sufficiently high resolution so as to 
allow us to read off the sequence from the map).

Since we have access to a lot of pure protein, I wonder if some clever mass 
spec jock would be able to assemble enough overlapping sequenced fragments so 
as to give complete coverage of the protein. Has anyone done this/had this done 
for them? 

Alternatively, I guess we can reverse transcribe message from the hybridoma 
cells, but I've heard suggestions that this is not necessarily straightforward 
(e.g., hybridomas may contain rogue Ig mRNA that will show up in a PCR 
experiment), so I'm hoping to avoid this particular can of worms.  Any insights 
welcome.

Cheers,

Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] off-topic: Small molecule service MS

[Patrick Loll]

Can anyone recommend a good fee-for-service facility that does routine high-res 
mass spec on small molecules? 

Probably best to reply off-line; and I'd ask you to limit suggestions to 
facilities within the continental US (for reasons of convenience, rather than 
chauvinism).

Thanks,

Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Protein preps become a jelly

[Patrick Loll]

Certainly not unprecedented, or even that unusual (I remember making gels from 
BSA and IgG solutions during grad school rotations).  Gel formation usually 
requires crosslinking, so consider whether you might be getting adventitious 
disulfide bond formation.
Pat

On 30 Aug 2011, at 11:31 AM, aidong wrote:

 Dear Buddies,
 
 Sorry for bothering you with an off-ccp4 question.  We recently are 
 experiencing a very strange phenomena.  A couple of protein preps with 
 reasonably high concentration (10-20mg/ml) become a jelly after storages for 
 overnight or a couple of days at 4C.  All of them have been purified by gel 
 filtration.  Some of these proteins behave like this from very first preps 
 but some of them had been very kind to us previously.  We have googled 
 extensively in CCP4BB and www but it appears this only happens to us.  It 
 would be highly appreciated that you could exchange their experiences or 
 provide your suggestions.
 
 Aidong Han, Ph.D
 
 Department of Biomedical Sciences
 School of Life Sciences
 Xiamen University
 Xiamen, Fujian 361005
 China
 Phone: 0592-218-8172 (O)
  0592-218-8173 (L)
 Web: http://life.xmu.edu.cn/adhanlab/

Re: [ccp4bb] Mac OSX 10.7 Lion

[Patrick Loll]

Still doesn't beat my all-time favorite, an early Microsoft spell-checker that 
changed diffract to defrocked.

 
 I forgot to mention how delightful the spelling auto-correction  feature 
 can be.  (It should have read nothing unusual in and of itself).
 
 That, at least, can be turned off.

Re: [ccp4bb] Rigaku high voltage tank

[Patrick Loll]

Bill,

What do you mean by high voltage tank? When I hear this term, I think of the 
oil- (and PCB-) filled tank housing the transformer on an old generator; but 
there's nothing like that on the R-Axis. Do you mean the blue box housing the 
detector controller? If so, then I can tell you that we've had ours plugged 
into a 1000 VA UPS (APC Back UPS Pro) for 10 years, with never a glitch (and 
I'm told that Philly power is not pretty).

If instead you're referring to the high voltage tank on the generator, then I 
have no idea of what to do (although replacing the power cable doesn't sound 
like a bad idea)...you'd probably need to steal an entire 7-11 to pay for a UPS 
large enough to condition power for that.

Pat

On 26 Sep 2011, at 10:15 PM, William G. Scott wrote:

 Hi Citizens:
 
 We seem to run through high voltage tanks on our Raxis IV like guano goes 
 through a goose.  Has anyone else had this problem, and, more importantly, 
 what is the best way to protect them.  I am assuming it might have something 
 to do with our electrical supply, which is a bit unreliable.
 
 Also, does anyone have an extra used functional one they want to get rid of?  
 We've run out of 7-11s to rob to pay for this, and our friendly and helpful 
 radiation safety staff think the best way to deal with this problem is to 
 hack apart the power cable, so it is a current (so to speak) source of 
 frustration.
 
 
 Thanks in advance.
 
 Bill
 
 
 
 
 
 William G. Scott
 Professor
 Department of Chemistry and Biochemistry
 and The Center for the Molecular Biology of RNA
 228 Sinsheimer Laboratories
 University of California at Santa Cruz
 Santa Cruz, California 95064
 USA
 

[ccp4bb] is codon optimization worth it?

[Patrick Loll]

Has anyone encountered a case in which a construct with the native sequence 
expressed poorly (or not at all?) in Rosetta(DE3), but the corresponding 
construct with a codon-optimized sequence expressed well? (The gene in question 
is from cerevesiae)
Thanks,
Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] sealing slides on VDX plates?

[Patrick Loll]

Haven't done this for a while, but what we used to do was mix Vaseline plus 
mineral oil (both purchased for cheap at the local drugstore), and then apply 
it using a 10 ml syringe with a pipet tip attached.

We used the mixture of Vaseline + mineral oil because Vaseline alone is too 
viscous (unless you want to develop Popeye forearms).

Heat a jar of Vaseline in a hot water bath (NOT OVER A FLAME--it's flammable!) 
until it melts. Mix in about 10-20% mineral oil by volume (you can play with 
this ratio until you find the viscosity you like); then draw the mixture up 
into syringes while still liquid. The procedure a little messy, so fill a lot 
of syringes at once and you're set for years.

Pat


On 18 Nov 2011, at 12:55 PM, Dima Klenchin wrote:

 Hello,
 
 I wonder what everyone is using for sealing hanging drop slides on VDX 
 plates? For the most part, we paraffin oil but I am unhappy with it because 
 it is too think and too frequently there is break in the oil and the drop 
 dries too much. I find vacuum grease to be not terribly practical because it 
 takes too much time - particularly when the well needs to be opened (seeding, 
 modifying well content, etc).
 
 In ideal world I would like to find a much thicker oil that 1) contains as 
 little volatiles as paraffin oil, and 2) allows no more water vapor diffusion 
 through it than paraffin oil. Hampton suggests Cargille immersion oils for 
 this purpose but the MSDS for these oils states that they have slight odor, 
 so I am a bit concerned with unknown volatiles getting into crystallization 
 drop.
 
 So, what do you like to use? Thanks much,
 
 - Dima

[ccp4bb] Beckman Biosys 2000

[Patrick Loll]

I was just contacted by a group looking for install disks for the Biosys 
program used to run the old Biosys2000 FPLC from Beckman. I don't have the 
install disks anymore (we eventually bludgeoned our Biosys to death, and then 
set it on fire, if I recall--it was always very unreliable). 

However, if anyone does, by some miracle, have these disks, and feels like 
being a good Samaritan (just in time for the winter holidays!), then please 
contact this person directly:  Jean-Baptiste Reiser 
jean-baptiste.rei...@ibs.fr.

Cheers,

Pat Loll
---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] DDM crystals

[Patrick Loll]

Does anyone have any experience with formation of crystals of dodecyl maltoside 
in the presence of PEG? 
Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] synchrotron X-ray picture

[Patrick Loll]

Hi all,

I have a vague memory of having a picture in someone's presentation once, 
showing a smoking hot X-ray beam emerging from the beam pipe in a hutch at a 
synchrotron. I think the picture might have been a double exposure, with a long 
exposure that captured air ionization superimposed on a normal photo (or some 
similar contrivance).

In any case, can anyone point to or provide such a picture? I'm preparing a 
seminar, and I want to lend artistic verisimilitude to an otherwise bald and 
unconvincing narrative...

Thanks in advance, 

Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] old-school stereo and Lion

[Patrick Loll]

The imminent replacement of Mobile-Me by iCloud provides an impetus for me to 
upgrade to Lion; but I'm currently happily using old-school stereo (i.e., a CRT 
monitor + Crystal-Eyes LCD glasses) under Snow Leopard. Can anyone attest to 
being able to use such equipment with Coot/PyMol under LIon? I seem to recall 
that the upgrade originally posed problems for stereo, and I'm wondering if 
these problems have been overcome.
Thanks,
Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] one datum many data? [was Re: [ccp4bb] very informative - Trends in Data Fabrication]

[Patrick Loll]

Hear, hear! I'm glad to know I'm not the last grump left standing. When I raise 
this point every year, my students regard me with bemused stares, as though 
they've just seen a coelacanth swim past their window...


On 1 Apr 2012, at 10:18 AM, Gerard Bricogne wrote:

 Dear Paul,
 
 May I join the mostly silent chorus of Greek/Latin-aware grumps who
 wince when seeing data treated as singular when it is plural. Related
 instances are
 
 * a phenomenon (singular) vs. several phenomena (plural),

* a criterion (singular) vs. several criteria (plural)

 and many more.
 
 And then there is the infamous mix-up between principal (adjective)
 and principle (noun, as in Principle of Least Action, or Peter's
 Principle) giving rise to the favourite hero, the Principle Investigator.
 
 This phenomena is now so widespread that perhaps compliance with
 ancient Greek or Latin morphology is no longer a relevant criteria ;-) . 
 
 
 With best wishes,
 
  Gerard.
 
 --
 On Sun, Apr 01, 2012 at 01:05:10PM +0100, Paul Emsley wrote:
 The PDBe page for 3k78 says:
 
 The experimental data has been deposited
 
 the data cif file says:
 
 data is under question
 
 Grump.
 
 Is it to late to refer to data as if there were more than one of them?
 
 Anyway, the data mtz file is here if you want to refine with it:
 
 http://lmb.bioch.ox.ac.uk/emsley/data/r3k78sf.mtz
 
 Paul.
 
 -- 
 
 ===
 * *
 * Gerard Bricogne g...@globalphasing.com  *
 * *
 * Global Phasing Ltd. *
 * Sheraton House, Castle Park Tel: +44-(0)1223-353033 *
 * Cambridge CB3 0AX, UK   Fax: +44-(0)1223-366889 *
 * *
 ===

[ccp4bb] Postdoctoral position available: Protein recognition of anesthetics

[Patrick Loll]

A postdoctoral position will be available Fall 2009 in Philadelphia,  
PA, USA. The successful candidate will join a collaborative research  
effort between the Eckenhoff laboratory at the University of  
Pennsylvania and the Loll laboratory at the Drexel University College  
of Medicine. This collaboration is aimed at studying the structural  
basis of anesthetic recognition by proteins (see for example  FASEB  
J.,19: 567-576 (2005)  ACS Chem. Biol. 1: 377-384 (2006)). Ideally,  
applicants will have experience in macromolecular crystallography, as  
well as one or more of the following areas: preparative protein  
biochemistry, experimental assessment of ligand binding by proteins,  
and/or the biophysical analysis of proteins.


 The Departments of Anesthesiology and Critical Care at Penn and  
Biochemistry and Molecular Biology at Drexel feature excellent  
resources, including instrumentation for fluorescence and circular  
dichroism spectroscopy, calorimetry, and mass spectrometry, and a  
complete X-ray diffraction facility furnished with state-of-the-art  
equipment.
The collaborating laboratories are conveniently located in downtown  
Philadelphia, and offer collegial and stimulating environments with  
many opportunities for collaboration.  Philadelphia is a vibrant and  
eminently livable city with a large biomedical research community.   
The city offers outstanding cultural and recreational activities, and  
is close to New York City and Washington DC, as well as Atlantic  
Ocean beaches and the Pocono mountains.


Please send a CV and a cover letter explaining your research  
interests to Dr. Roderic Eckenhoff: roderic.eckenh...@uphs.upenn.edu.



 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] anode lifetime?

[Patrick Loll]

Does anyone have a reasonable estimate for the expected working  
lifetime of a copper anode on a MicroMax007? (Yes, I'm thinking about  
stimulus funding...).


Thanks,
Pat

 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

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

[Patrick Loll]

Greg Petsko's group did something like this about a billion years ago  
(yet, strangely, I remember the paper, even though I'd be stumped if  
you asked me what I had for breakfast...)


They covered the range from room temp down to very cold, using  
different cryoprotectants (importantly, they were not vitrifying  
their samples).  I recall a plot of ADPs vs. temp that showed an  
essentially linear decrease down to some temp (maybe around 150 K or  
so?), after it plateaued, with no further reductions being seen at  
even very low temp. They rationalized this by saying (I think) that  
the decrease represented the dynamic disorder, which was damped at  
low temperatures, and the plateau represented the point where static  
disorder became the predominant contributor.


I remember thinking at the time that this made great intuitive sense.  
I have no idea if people still buy this.


I can't put my finger on the reference, but if you start here you can  
probably find your way: Ringe D, Petsko GA. Study of protein dynamics  
by X-ray diffraction. Methods Enzymol. 1986;131:389-433.


On 13 May 2009, at 12:30 PM, Jacob Keller wrote:

The reason is that you've missed out one important term: the  
atomic displacement parameters (B-factors), which describe a  
combination of thermal motion and positional disorder between unit  
cells.


A somewhat niggling point: isn't it true that the thermal motion is  
insignificant at 100K? Does anybody know of a paper which  
systematically measures B-factors as a function of temperature? The  
asymptote of the resulting curve would represent all of the non- 
thermal elements, right?


JPK


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] coupling between occupancy and b-values in refinement

[Patrick Loll]

Hi all,

I'm looking for a reference to bolster my response to a referee, in  
which I defend my decision not to refine the occupancy of a ligand in  
structure refined at around 2 A resolution (note the ligand binding  
slte lies on a two-fold crystallographic axis, so the maximum  
occupancy is 0.5)


I recall reading a paper a LONG time ago (decades) in which someone  
described some careful refinement experiments, and concluded that   
the correlation between occupancy and B-value is so strong that it  
simply makes no sense to independently refine both parameters (at  
least for light atoms, and in the absence of super high resolution  
data).


Alas, all that I recall is this take-home message. I have no idea of  
where the paper appeared, or the names of the authors (or indeed, if  
I'm even remembering the paper's message correctly). I've tried  
trolling through Acta, without success.  Does anyone have a better  
idea of where I might find this paper, or one espousing a similar  
message?


Thanks,

Pat


 
-

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexel.edu

Re: [ccp4bb] TEV nucleotude sequence with restriction site

[Patrick Loll]

You seem to be describing the MCS found in many TEV-site-containing  
expression plasmids (am I missing something?)  E.g., look at the  
sequences in Sheffield et al., Protein Expression and Purification  
15, 34 –39 (1999) (let me know if you want a PDF, I don't want to  
send it to the whole bb)



On 5 Jun 2009, at 5:41 PM, Jacob Keller wrote:


Dear Crystallographers,

Does anybody have a TEV-protease-site-coding nucleotide sequence  
with a commonly-used restriction site in it, preferably right at  
the end? Alternatively, does some somebody know of a program to  
determine all equivalent codon permutations for a small coding  
region, filtered for resulting restriction site possibilities? It  
seems like it would be an easy enough script to write...


(I have already done some googling around for such a program, with  
not much luck.)


Jacob

***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
Dallos Laboratory
F. Searle 1-240
2240 Campus Drive
Evanston IL 60208
lab: 847.491.2438
cel: 773.608.9185
email: j-kell...@northwestern.edu
***


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] fortran format descriptor help

[Patrick Loll]

I get this:

read(5,100)xinten,siginten,fobs,sigfobs,itest
100 format(6x,3i5,3(6x,f10.3),/,25x,f10.3,6x,i10)


On 17 Jun 2009, at 4:18 PM, Francis E Reyes wrote:


 INDE 00   -9 IOBS=  9062.000 SIGI=   200.300 FOBS=95.190
   SIGMA= 1.060 TEST= 0


 
---

Patrick J. Loll, Ph. D. 
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] low UV reading on AKTA prime

[Patrick Loll]

I second Scott's post.  About the only problem we've had with our  
Akta instruments is this type of degradation of the filter. I'm not  
sure what the mechanism is, but the filters do seem to crap out after  
a while, at least in the cold room (oxidation? I have no idea of what  
the filter is made of...).


Pat

On 1 Jul 2009, at 5:07 PM, Scott Walsh wrote:


Hi Matt,

Check to make sure the A280 filter is clean.  Ours was filthy.  You  
might need to replace this (~$350).  We were experiencing the same  
thing you mentioned and just had the GE technician out today.   
Also, make sure the A280 dial is set correctly on the lamp.  Please  
check the manual for guidance.


Best,

Scott

- Original Message -
From: Matt Colins matt.colins2...@yahoo.com
Date: Wednesday, July 1, 2009 4:40 pm
Subject: [ccp4bb] low UV reading on AKTA prime
To: CCP4BB@JISCMAIL.AC.UK

 Hi all,

 We recently got low UV reading on our AKTA prime. We contacted
 GE healthcare, but was told that the UV reading on AKTA prime
 should be 20% of the spectrometer reading, because the path
 length of the flow cell of AKTA prime is only 2 mm (20% of the
 pathlength of a spectrometer cuvette).

 I am not sure whether that is the case, but we used to get much
 higher UV readings on the same AKTA prime (almost the same as
 the spectrometer reading). The UV reading just keeps dropping
 over time in the past several months.

 Here I have two questions. First,  should the UV reading on
 AKTA prime be 20% of the spectrometer reading? Second, what
 could go wrong with our AKTA prime? (I know it is not the lamp,
 because we put in a new lamp and it didn't solve the problem)

 Thanks a lot!
 Matt






 
---

Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] cheap/useful alternatives to bloatware

[Patrick Loll]

Hi,

This is off-topic in the sense that it's not about reciprocal space,  
but graphics ARE an integral part of publishing and teaching...


I just upgraded to Leopard*, and find that my versions of Photoshop   
CorelDraw no longer work.  I can't stomach spending big bucks for the  
new version of Photoshop (Adobe is No. 2 behind the Voldemort of  
Seattle on my list of software giants who piss me off), and Corel has  
cold-heartedly decided to bail on their small but devoted OS X users.


SO:  Can anyone suggest OS  X alternatives to these two programs that  
don't require a huge effort to master?


Please don't suggest Illustrator. Bonus points for cheap or open  
source (Note: Gimpshop 2.2.11 (most recent version I could find)  
doesn't seem to work under 10.5).


Respond to me and I'll post a summary.


Cheers,

Pat

_

*  Yup, that's me, the early adopter.
---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] alternative graphics programs for OS X--summary

[Patrick Loll]

Hi,

Thanks to the many replies to my queries about cheap/easy alternatives  
to Photoshop and CorelDraw on OS X. In a rare display of unanimity,  
the bulletin board spoke with essentially one voice:


ALTERNATIVE TO PHOTOSHOP:   

Gimp  (not gimpshop)
http://www.gimp.org/

As one sage respondent said, Gimp is no less intuitive than  
Photoshop.  I could certainly easily figure out how to do the types  
of trivial manipulations for which I previously used Photoshop; power  
users may have a different experience.


ALTERNATIVE TO CORELDRAW/ILLUSTRATOR:

Inkscape
http://www.inkscape.org/

Again, straightforward to use, and I sense that I will find it a  
completely satisfactory replacement for CorelDraw (if only it could  
read .cdr files, sigh).


Notes:

1.  At least one of these programs requires updating the version of  
X11 that comes with 10.5 (e.g., XQuartz 2.3 something, http://xquartz.macosforge.org/trac/wiki) 
. If you are using crystallographic software you've probably already  
done this.


2.  One kind soul pointed out that I already have GraphicConvertor  
installed as part of the OS, and this program can do simple  
manipulations (brightness, contrast, cropping, etc.).  Hidden in plain  
sight.


3.  There was one vote (out of dozens total) for ImageMagick, but as  
far as I know this is command line only. It also occurs to me that  
ImageJ would do at least some of the simple things that I need (e.g.,   
getting an image of a gel ready for publication).


4.  There was one vote for OmniGraffle (but this is commercial, and  
costs a few dollars).


Thanks again. The ccp4bb rules.

Pat


---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Sumo protease (Ulp) expression vector

[Patrick Loll]

Try PubMed, rather than Google:

Weeks SD, Drinker M, Loll PJ (2007) Ligation independent cloning  
vectors for expression of SUMO fusions.  Protein Expr Purif. 2007 May; 
53(1):40-50.
We express the Ud1 domain of the yeast hydrolase. Contact me off-line  
if you're interested.


Pat





On 12 Aug 2009, at 4:51 PM, P Hubbard wrote:


Hi all,

I've had a Google around, but can't find anywhere on the net that  
sells a sumo protease (Ulp) expression vector. Does anyone know any  
different?


As an alternative, does anyone recommend a company that sells  
inexpensive sumo protease.


Thanks!

Express your personality in color! Preview and select themes for  
Hotmail®. Try it now.




---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] off topic--Torrey Pines equipment

[Patrick Loll]

Does anyone have any experience with equipment from Torrey Pines  
Scientific (incubators, constant temperature circulators)?


Probably best to reply off-line; if there's a groundswell of interest  
I can post a summary.


Thanks,

Pat

---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] Postdoctoral position in DUB structural biology

[Patrick Loll]

A postdoctoral position is available to study the structures and  
functions of a novel class of human deubiquitinating enzymes involved  
in neurodegeneration.


An NIH-funded position is available immediately to study the  
structure, catalytic function, and ligand-binding properties of the  
human Josephin domain-containing proteins. These proteins include  
ataxin-3, a polyglutamine protein that is the causative agent of  
Machado-Joseph disease, the most common inherited ataxia. Well- 
diffracting crystals are in hand for several protein-substrate  
complexes. We seek a talented protein chemist/structural biologist to  
assist in studies of these interesting and important molecules.  
Experience in X-ray crystallography is a plus, but we are also happy  
to entertain applications from biochemists/biophysicists who seek to  
extend their training to diffraction analysis.


Drexel University has been ranked among the top 50 private  
universities in the United States, and its College of Medicine is the  
largest private medical school in the nation. The Department of  
Biochemistry  Molecular Biology offers a stimulating and supportive  
environment for biomedical research. The department is located in the  
heart of downtown Philadelphia, which is a cosmopolitan and highly  
livable city.

Please direct inquiries to Pat Loll: pat.l...@drexel.edu.

---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] Postdoctoral position in anesthetic discovery

[Patrick Loll]

We seek a structurally-oriented post-doctoral fellow to participate in  
a general anesthetic discovery program consisting of high through-put  
approaches, coupled to medium through-put secondary screens, to  
include x-ray crystallography, isothermal titration calorimetry and in  
vivo assays.  This project is funded by NIH and represents a  
collaboration between the University of Pennsylvania Department of  
Anesthesiology  Critical Care, Drexel University Department of  
Biochemistry and Molecular Biology and the National Chemical Genomics  
Center.  The successful candidate will have a Ph.D., M.D., or both,  
with training biochemistry, biophysics and/or structural biology.  
Successful candidates will be able to work independently but will also  
have excellent collaborative skills.  Please contact Rod Eckenhoff at roderic.eckenh...@uphs.upenn.edu 
, or Pat Loll at pl...@drexelmed.edu.


---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] 3D search for peptide conformers?

[Patrick Loll]

I have a 10-residue stretch of a protein that adopts an interesting  
conformation; I'd like to know if this conformation occurs in other  
proteins. I'd welcome suggestions for tools that will allow me to to  
search for this peptide conformation in the PDB.


I naturally thought of DALI, but it seems to require a minimum length  
of 30 residues.


Thanks,

Pat
---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] python install issues?

[Patrick Loll]

Hi,

I'm simultaneously installing ccp4 on a MacBook Pro running Snow  
Leopard and on a MacPro running Leopard. On both machines, the command:


source /sw*/bin/init.csh   (where /sw* is either /sw64 on the  
machine running 10.6, or /sw on the machine running 10.5)


gives the error:

   ***
   Fatal Error: Incomplete libtbx environment!
   ***
   Please re-run the libtbx/configure.py command.


I've seen this phenomenon mentioned on the ccp4bb before, but I'm not  
clear on how those earlier posts relate to my current situation  
(alright, to be honest, I didn't really understand the previous posts).


Details:Installing ccp4-6.1.2
		using either fink or precompiled binaries from UC SantaCruz (thanks  
again, a thousand times, to Bill Scott!)

One machine has python 2.5.1, the other has 2.6

Any help welcome.

Thanks,
Pat

---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] chiral volumes--losing it

[Patrick Loll]

Friends,

A question about the definition of chiral volumes:

I'm looking for the definition of the SIGN of a chiral volume. The  
only ccp4 reference I can find (readily) is this:


   http://www.ysbl.york.ac.uk/~garib/refmac/docs/theory/chiral.html

This page gives an algorithm for determining the sign, which I  
paraphrase here:


*  Call the central (chiral) carbon a, and its three non-hydrogen  
substituents b, c, and d.
*  Arrange things so that as you look from b to c to d, your eye is  
moving clockwise.
*  If atom a is below the plane formed by b, c,  d, then the chiral  
volume is positive (otherwise negative)


Clear enough.  However, when I start to apply this rule to basically  
every library I have ever used, I get the opposite result.  Try it,  
for example, with the ALA.cif file distributed with ccp4:


_chem_comp_chir.comp_id
_chem_comp_chir.id
_chem_comp_chir.atom_id_centre
_chem_comp_chir.atom_id_1
_chem_comp_chir.atom_id_2
_chem_comp_chir.atom_id_3
_chem_comp_chir.volume_sign
 ALA  chir_01  CA N  CB C negativ

Assigning a as the centre atom and atoms b-d as numbers 1-3, when  
I apply the rule from the refmac page I get a positive chiral volume,  
not the negative one found in the cif file. Ditto for every other  
example that I have tried.


Am I mis-reading what is meant by above and below?  I'm assuming  
that if atoms b, c, and d all lie in a vertical plane, and you are  
facing that plane, then below means on the far side of that plane  
and above means between you and the plane.


When I use the definition V1 * (V2 x V3), where V1 is vector FROM  
chiral atom to 1st substituent (e.g., CA to N in alanine example  
above), V2 = vector from chiral atom to 2nd substituent, etc., then I  
get the expected sign for the chiral volume.


So is the refmac page wrong, or am I falling prey to some roaringly  
obvious stupidity?


Having a rough Monday--starting to question my sanity.

Thanks,

Pat

ps It appears that the term _chem_comp_chir.volume_sign is not  
defined in either the core or mmCIF dictionaries. Can this be right?


---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] chiral volumes--2nd try

[Patrick Loll]

Sorry, the original post looks garbled (mirroring my internal state,  
no doubt).  I'm trying again, sending as plain text:


Friends,

A question about the definition of chiral volumes:

I'm looking for the definition of the SIGN of a chiral volume. The  
only ccp4 reference I can find (readily) is this:


   http://www.ysbl.york.ac.uk/~garib/refmac/docs/theory/chiral.html

This page gives an algorithm for determining the sign, which I  
paraphrase here:


*  Call the central (chiral) carbon a, and its three non-hydrogen  
substituents b, c, and d.
*  Arrange things so that as you look from b to c to d, your eye is  
moving clockwise.
*  If atom a is below the plane formed by b, c,  d, then the chiral  
volume is positive (otherwise negative)


Clear enough.  However, when I start to apply this rule to basically  
every library I have ever used, I get the opposite result.  Try it,  
for example, with the ALA.cif file distributed with ccp4:


_chem_comp_chir.comp_id
_chem_comp_chir.id
_chem_comp_chir.atom_id_centre
_chem_comp_chir.atom_id_1
_chem_comp_chir.atom_id_2
_chem_comp_chir.atom_id_3
_chem_comp_chir.volume_sign
 ALA  chir_01  CA N  CB C negativ

Assigning a as the centre atom and atoms b-d as numbers 1-3, when  
I apply the rule from the refmac page I get a positive chiral volume,  
not the negative one found in the cif file. Ditto for every other  
example that I have tried.


Am I mis-reading what is meant by above and below?  I'm assuming  
that if atoms b, c, and d all lie in a vertical plane, and you are  
facing that plane, then below means on the far side of that plane  
and above means between you and the plane.


When I use the definition V1 * (V2 x V3), where V1 is vector FROM  
chiral atom to 1st substituent (e.g., CA to N in alanine example  
above), V2 = vector from chiral atom to 2nd substituent, etc., then I  
get the expected sign for the chiral volume.


So is the refmac page wrong, or am I falling prey to some roaringly  
obvious stupidity?


Having a rough Monday--starting to question my sanity.

Thanks,

Pat

ps It appears that the term _chem_comp_chir.volume_sign is not  
defined in either the core or mmCIF dictionaries. Can this be right?


---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] chiral volumes--2nd try

[Patrick Loll]

Joel,

Agh.  I can honestly say that this explanation never occurred to  
me, even though it is consistent with the data (But come on, any  
introductory organic chem text explains the R/S rules by moving from  
atom 2 to 3 to 4, and not by jumping from 2 to 4...surely you would  
follow the same convention in the refmac documentation, right?)


What amazes me is my inability to find ANY documentation that actually  
explains the meaning of terms in the CIF used to define chiral  
volumes. OK, some of the terms ARE found in the mmCIF dictionary, but  
others seem made-up, like _chem_comp_chir.atom_id_1. Given that both  
refmac and phenix rely upon upon these libraries for determining  
geometries, you'd think that somewhere the terms would be defined  
explicitly.


As several posters have pointed out, the triple scalar product is of  
course the correct way to define the chiral volume; but my point is  
that if you don't know which atoms the program is assigning to which  
vector, you're still in a pickle...


Pat


On 20 Apr 2010, at 3:51 PM, Bard, Joel wrote:


Hi Patrick-

I feel your pain having gone through exactly the same problem.  It all
has to do with the definition of When the eye goes from atom 2 to  
atom

4.  I think we both assumed that this meant from 2 to 4 via 3 but I
guess it doesn't.  The ful text of my 2004 post:

I think that two of the numbers are reversed in Figure 3 of the chiral
center documentation for refmac5:

http://www.ccp4.ac.uk/dist/html/refmac5/theory/chiral.html

If one follows the Procedure to find the sign of a chiral centre  
with
reference to the figure the eye would move from atom 2 through atom  
3 to

atom 4 as it traveled clockwise.   This would generate a left handed
coordinate system if atom 1 was behind the plane of the web browser so
the sign of the chiral volume would be negative rather than positive  
as

the text says.  Switching the labels of atoms 2 and 3 (or 2 and 4 or 3
and 4 but 2 and 3 make it easier to visualize the right-hand-rule)  
would

make it work.

It seems like a very little thing but I'm feeble-minded enough to have
spent more time than I'd like to admit trying to figure out why a  
little

program I'd written was coming up with the wrong sign for the chiral
volume when it had been correct the whole time.  Of course I should  
have

realized that it would be absurd for the statement: When the eye goes
from atom2 to atom4 it should travel clockwise, to mean When the eye
goes from atom2 to atom4 by passing through atom3 it should travel
clockwise.  It might be worth fixing, though, since I know for a fact
that there are other people out there who are almost as easily  
confused

as I am.

Cheers,

Joel





---
Patrick J. Loll, Ph. D.
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] software/web server to determine ligand volume

[Patrick Loll]

VOIDOO will do this


On 13 Aug 2014, at 2:06 AM, sreetama das wrote:

 Dear all,
 Is there any software or web server available to calculate the volume of a 
 ligand if the ligand coordinates are provided?
 Google seems to come up only with options to calculate protein cavity volume.
 
 Thanks in advance,
 Sreetama Das,
 phd student,
 Physics, IISc




---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pl...@drexelmed.edu

[ccp4bb] XDS.INP for X25

[Patrick Loll]

Hi,
I'm trying to help a colleague process some data collected at beamline X25 of 
the late, lamented NSLS. Does anyone have an XDS.INP file that they know works 
for such data (this is for the Pilatus detector)? I have kludged together a 
file that looks right, but the processing still doesn't feel quite right...
Thanks,
Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry  Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

[ccp4bb] Off-topic x 2: Chaperone co-expression AND Bac-Mam contract work

[Patrick Loll]

Hello everyone,

Please allow me to kill two off-topic birds with one email ‘stone’:  

1) Can anyone suggest where I can lay hands on the pREP4-GroESL plasmid for 
bacterial co-expression of GroEL and GroES? I’ve checked with the original 
author (Philip Cole), but it’s been decades since he published on this, and 
he’s not sure he can find the plasmid. However, I see recurrent (and recent) 
references to the plasmid in the literature, so I guess that it’s loose in the 
wild…

2) We’re looking for a contract research organization to do some Bac-Mam 
expression for us (we’re in a hurry to get some results, so we’d like to farm 
it out rather than get the technology going in-house). Can anyone recommend a 
group (ideally in the US, for logistical ease)?

Thanks for any help.

Cheers,

Pat



---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Director, Biochemistry Graduate Program
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pl...@drexelmed.edu

Re: [ccp4bb] Structural biology software that does not run on Windows or gives important Windows-specific problems

[Patrick Loll]

How about the ability to compile code? Are there decent compilers readily 
available for Windows? I like being able to write & compile the occasional 
fortran program {hic sunt dinosaurs}, and it’s easy to do this on a unix-based 
platform like OSX.

If your reasoned arguments fail, I have found that many institutions' 
information technology administrators are easily intimidated by a few random 
references to unix (Rsync! Grep! Bash!). Such words seem to function as charms 
that keep evil spirits (i.e. IT administrators) at bay.

> On 14 Oct 2016, at 11:14 AM, Mark J van Raaij  wrote:
> 
> Dear All,
> 
> our institution requires me to provide a reasoning not to buy a Windows 
> computer (I want to buy a new MacOSX system), so I am looking for software 
> that does not run or is limited on Windows.
> 
> Not available:
> (Auto)SHARP
> ARPWARP
> 
> Available on Windows but with significant limitations
> Phenix (no MR-Rosetta, no parallelization)
> CCP4 (limitations on file-names)
> 
> Please correct me if pertinent and provide additional examples if possible.
> 
> Gratefully yours,
> 
> Mark
> 
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. (+34) 91 585 4616
> http://wwwuser.cnb.csic.es/~mjvanraaij



---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Message from the Uppsala EDS: "Morituri te salutant"

[Patrick Loll]

Ave atque vale. 

The EDS was hugely useful (and will continue to be so in its new manifestation, 
we hope)—thanks to everyone who made it happen!

Pat

 
> On 13 Dec 2016, at 12:51 PM, Gerard DVD Kleywegt  
> wrote:
> 
> Hi all,
> 
> After tirelessly serving the scientific community with (mostly) beautiful 
> maps for two decades, the Uppsala Electron Density Server (EDS; 
> http://eds.bmc.uu.se/) is now reaching the end of its life (in fact, it has 
> been living on borrowed time for several years already). Some time in 2017 it 
> will therefore be "phased" out and join the choir invisible (despite its 
> beautiful plumage).
> 
> The good news is that much of the EDS functionality (and in particular the 
> delivery of map and mtz files, as well as a much better 3D viewer) is now 
> provided by the Protein Data Bank in Europe (PDBe; http://pdbe.org/).
> 
> There is a short write-up that explains what this means for users who just 
> want to look at maps, for users who want to download files, for users of 
> software that retrieves data from EDS, and for developers of such software 
> (incl. URLs for map, mtz and other relevant files on the PDBe website) at:
> 
>  http://www.ebi.ac.uk/pdbe/eds
> 
> Toodle pip!
> 
> --Gerard
> 
> **
>   Gerard J. Kleywegt
> 
>  http://xray.bmc.uu.se/gerard   mailto:ger...@xray.bmc.uu.se
> **
>   The opinions in this message are fictional.  Any similarity
>   to actual opinions, living or dead, is purely coincidental.
> **
>   Little known gastromathematical curiosity: let "z" be the
>   radius and "a" the thickness of a pizza. Then the volume
>of that pizza is equal to pi*z*z*a !
> **

Re: [ccp4bb] Crystal with ZERO diffraction

[Patrick Loll]

Sadly, I have seen numerous examples of reasonably-sized crystals that give no 
observable ordered diffraction (I shot a few this weekend, in fact). I can’t 
give you evidence for what is happening, but I guess that you can build a 
macroscopic assembly using lattice interactions that are only modestly 
specific, resulting in a structure that is highly disordered internally, even 
though it looks OK visually (the jello model). Alternatively, you may have a 
crystal that was originally well-ordered but subsequently decayed; however, it 
failed to dissolve because proteins at the surface became cross-linked and held 
everything together (the soup dumpling model).

So no suggestions as how to proceed (except to follow Eddie’s sage advice to 
grow more/different crystals), but I can assure you that you’re not alone in 
observing this.

Cheers,

Pat Loll
 
> On 21 Dec 2016, at 2:44 PM, Tom Huxford  wrote:
> 
> Dear ccp4b collective mind and experience,
> 
> Greetings from San Diego.
> 
> I have done my fair share of synchrotron data collection on many diverse 
> macromolecular crystal systems.  But this weekend was the first time that I 
> ever shot crystals that failed to diffract entirely.
> 
> Details:  we have purified and crystallized an ~90 kDa proteolytic fragment 
> containing a single point mutant version of a myosin motor domain in complex 
> with a separate light chain polypeptide.  The crystals are relatively small 
> (10-30 microns in each dimension) but clearly crystalline in character (clear 
> faces, edges, and facets).  The crystals tested positive for protein by 
> absorbance at 280 nm.  This weekend we tested more than 40 of them for 
> diffraction in different cryo solvents and did not observe a single 
> identifiable diffracted ray.  It was as if we had only cryo on the end of our 
> loops.  Increasing the time of exposure or annealing did nothing to improve 
> the situation.  Crystals from a different protein system that we also tested 
> this weekend on the same beamline diffracted to beyond 1.3 Å.
> 
> I only post this because, in my experience, crystals of this size and 
> superficial quality always give some signal--even if it is horrifyingly bad.  
> But never complete diffraction silence.  We will work this week to identify 
> what it is that we "crystallized".  But can anybody who has had a similar 
> experience suggest what it is that could be going on here?
> 
> Thanks in advance for any responses.  And happy holidays to us all.
> 
> Tom Huxford.
> ==
> Tom Huxford.
> Structural Biochemistry Laboratory
> Department of Chemistry & Biochemistry
> San Diego State University
> (619) 594-1606
> 

[ccp4bb] ample

[Patrick Loll]

I’m intrigued by the prospect of using AMPLE to test multiple distant homologs 
in a MR problem. I’ve used HHPRED to identify about 20 high-probability 
homologs of known structure, each of which has about 20-25% identity with the 
unknown protein. However, it’s not clear to me from the documentation whether 
the program will use the alignments from HHPRED, and, if so, how I should 
provide that information. 

Or does AMPLE perform its own alignment? I.e., do I simply point the program to 
a directory containing 20 different PDB files and stand back?

Thanks for any insights.

Cheers,

Pat 
---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu

[ccp4bb] comfortable OS X level

[Patrick Loll]

I’m still running Yosemite on my Macs, both because I’m change-averse and 
because folks reported problems with some crystallographic software upon 
upgrading the OS.

These reports have now faded into the haze of the past, and so I ask, have the 
issues been resolved? Is it safe to move to Sierra? 

Thanks as always,

Pat

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pat.l...@drexelmed.edu

Re: [ccp4bb] Fishing crystals from volatile solvent as precipitant

[Patrick Loll]

I second (third?) what Tommi and Kevin said about using an oil to cover the 
drop to slow evaporation (I like paraffin for this—not too viscous). Here’s an 
additional nuance: Saturate the oil with the alcohol first, before using it to 
cover the drop. 

> On 14 Aug 2018, at 2:58 PM, Thomas Krey  wrote:
> 
> Dear crystallization experts,
>  
> We have 3D protein crystals grown from a microseed matrix screening vapor 
> diffusion experiment in either
>  
> 15% (v/v) Reagent alcohol
> HEPES Na pH 7.5
> 0.2 M MgCl2 
>  
> or in 
>  
> 27% Isopropanol
> 0.18 M MgCl2
> 90 mM HEPES Na pH 7.5
> 10% Glycerol
>  
> Upon opening the corresponding wells these crystals move quite a bit – 
> presumably due to the volatility of the alcohols. Does anyone have a good 
> suggestion to stabilize the swirling movements? Does anyone have experience, 
> whether these conditions alone can serve as cryo-protectant (i.e., do we 
> really have to fish, move into cryo solution and fish again)? 
> Any suggestion or input would be highly welcome.
>  
> Thank you very much in advance.
>  
> Thomas
>  
>  
> Prof. Dr. Thomas Krey
> Hannover Medical School  
> Institute of Virology
> Structural Virology Group
> Carl-Neuberg-Str. 1 
> D-30625 Hannover
> phone: +49 (0) 511 - 532 4308
> email: krey.tho...@mh-hannover.de
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
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Re: [ccp4bb] Unknown density

[Patrick Loll]

Calcium likes to form octahedral complexes with water (or other 
oxygen-containing) ligands. This looks like a classic example. 

After you model and refine this, you’ll want to check water-metal distances, to 
make sure they are appropriate for calcium. There is a nice literature on such 
things, which I of course don’t have at my fingertips; but I think Wladek Minor 
has done some data-mining in metal-containing protein structures, and Amy Katz 
and Jenny Glusker have a number of papers that are relevant. There are more, of 
course—a little time in the “library” is warranted.

Cheers,

Pat Loll

> On 6 Mar 2018, at 5:19 PM, Rajesh Kumar  wrote:
> 
> Dear All,
> 
> Have you had experience with this kind of density? I am wandering what this 
> could be?
> 
> Thank you very much for the help.
> 
> -Rajesh
> 
> 
> 
> 

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu

Re: [ccp4bb] help needed with a rabbit-head-shaped blob

[Patrick Loll]

Fig. 2.13 in Gale Rhodes’ “Crystallography Made Crystal Clear” touches on 
rabbit heads and diffraction, and is one of my favorite cartoons (sadly it 
doesn’t directly address your problem).

> On 2 Nov 2018, at 5:14 PM, Deborah Harrus  wrote:
> 
> Dear all,
> 
> I came across an unidentified rabbit-head-shaped blob and would need help for 
> its identification. There are 2 molecules of protein per asymmetric unit but 
> there are some differences between the two chains. The blob is located in 
> between the two chains, and is surrounded by residues Asp, Pro, and Val.
> 
> The protein, a glycosyltransferase, was expressed in E. coli BL21(DE3) and 
> purified on Ni-NTA followed by gel filtration. The purification buffer 
> included Sodium phosphate and NaCl. The crystallization condition had 
> Bis-Tris, ammonium acetate and PEG 2, and glycerol was used as 
> cryoprotectant. From the size, bis-tris was the most probable, but I have 
> tried to fit it and it is not convincing.
> 
> The structure is 1.6 angstrom resolution and this is the only thing left to 
> be done, it's driving me crazy!
> 
> Pictures attached show the face, bottom and top of the head. 2Fo-Fc is at 1.1 
> sigma, Fo-Fc at 3 sigmas.
> 
> Many thanks in advance for your suggestions!
> 
> Regards,
> Deborah.
> 
> 
> =
> Deborah Harrus, PhD.
> 
> University of Oulu / Faculty of Biochemistry and Molecular Medicine
> Aapistie 7 A, 90220 Oulu
> Finland
> 
> office: F123B
> email: deborah.har...@oulu.fi
> phone: +358 50 3502387 / +358 44 2386351
> http://www.oulu.fi/fbmm/node/20603
> =
> 
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> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
> 

---
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Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
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Re: [ccp4bb] nonenzymatic removal of His tag?

[Patrick Loll]

1) Use a different protease, e.g. SUMO protease, which is sufficiently specific 
that it’s unlikely to cause any problems even if a little bit is carried along. 
See, for example (shameless plug #1): DOI: 10.1016/j.pep.2006.12.006

2) Use intein cleavage. NEB sells a vector that lets you fuse an intein & a CBD 
at the C-term end of your POI; we’ve made a similar vector with a His-tag 
(shameless plug #2): DOI: 10.1021/ja208755j

3) Just assure your colleague that you guys are good protein chemists, so don’t 
worry, be happy.

There are chemical cleavage methods (e.g. cyanogen bromide), but they aren’t 
particularly gentle, so if your 110-residue peptide is meant to fold into a 
native structure, I’d approach them with caution (on the other hand, if the 
peptide ISN’T meant to be folded, then just purify via reverse-phase, and 
you’ll for sure get rid of protease contaminants).

Pat


> On 20 Sep 2018, at 4:17 PM, Gloria Borgstahl  wrote:
> 
> Hello, friends in crystallography,
> A colleague just asked me this question.  He is worried about trace
> protease interfering with the receptors he is studying in cell-based
> experiments using a 110 amino acid protein we made for him.  He has
> been unable to make the peptide synthetically.  The company is having
> trouble getting that to happen.  Any ideas?  Happy Thursday, G
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu



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[ccp4bb] Fwd: [ccp4bb] SO4 or PO4

[Patrick Loll]

S has about 0.56 anomalous electrons at 8 keV, whereas P has about 0.44. This 
is a small difference between two weak signals—unlikely to give a clear result. 
If you could get to the sulfur & phosphorus edges, then you could (in 
principle) answer this, but that’s a very hard experiment to accomplish.


> Begin forwarded message:
> 
> From: jlliu20022002 liu 
> Subject: Re: [ccp4bb] SO4 or PO4
> Date: February 16, 2019 at 11:14:07 AM EST
> To: CCP4BB@JISCMAIL.AC.UK
> Reply-To: jlliu20022002 liu 
> 
> How about collect data at sulfur peak. You might see anomalous peak for 
> sulfur.
> 
> Jinyu
> 
> On Sat, Feb 16, 2019 at 4:07 AM 张士军 <21620150150...@stu.xmu.edu.cn 
> > wrote:
> Dear all
> 
> I have got a crystal grown at the condition both have ion of SO4 and PO4, and 
> the diffraction resolution is very well, but the problem is coming: how to 
> tell which is which just from electron density? I think they are exactly 
> same. Thanks a lot !!!
> 
> Beat Regards
> 
> Shijun
> 
> 
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> 
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---
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Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102-1192  USA

(215) 762-7706
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Re: [ccp4bb] A Question about crystal packing

[Patrick Loll]

It is very difficult to demonstrate rigorously that crystal packing does not 
favor a particular conformation. If you can show that the same conformation 
occurs in solution (SAXS, NMR), or that the same conformation is found in a 
different space group with different packing, then you have a pretty conclusive 
answer. Obviously this involves a great deal of extra effort.

> On 28 May 2019, at 9:59 AM, Zizi Tian <18311218...@163.com> wrote:
> 
> Hi All,
> I has recently solved my X-ray structures of the ZnF domains of protein and 
> its complex with dsDNA. A structural comparison of holo-protein and the 
> protein-DNA binary complex reveals that the relative orientation of domains 
> ZnF3 undergoes a rotation of 50.5 degrees Does anyone know how to show there 
> are no effects of the crystal packing .
> 
>  Please help me in this regard. 
> 
>  Best regard
> 
> 
> 
> 
> 
>  
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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Patrick Loll
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Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2

[Patrick Loll]

The idea of contacting the editor (and/or author) is an excellent one, and 
indeed the correct thing to do scientifically. However, I’m disillusioned: I’ve 
been down this path before with a high-profile vanity journal, and while the 
editors paid lip service to the notion that the record should be corrected, in 
reality they led me on for the better part of a year, and got me to write up 
detailed analyses of why the ligand positioning was not justified, before 
eventually saying “no, we don’t see any need to publish a correction.” I 
speculate that the journal prefers not avoid corrections, for fear that too 
many corrections will make the journal a less desirable destination.

> On 19 Jul 2019, at 11:23 AM, Bärbel Blaum  
> wrote:
> 
> Hi Rhys,
> the reported B-factors for the “ligands” are all way below the reported 
> B-factors for the protein chains, with the worst of the three complexes 
> reporting unitless numbers 23.2 and 64.8, respectively, just to highlight 
> *one* striking feature of the data collection and refinement table. So even 
> with the limited info normally available to reviewers this table should have 
> raised a red flag. After the re-refinement suggested by others, i.e. your own 
> proper assessment of the crystallographic data, if you do not find noteworthy 
> density you may want to contact the article’s editor with your results. If 
> you work in this field, i.e. really care about this paper scientifically and 
> you are not afraid to confront the authors you could suggest writing a 
> comment/direct response article but in my opinion that would only make sense 
> if you can be sure beforehand that it will be linked visibly to the actual 
> paper, else it will be a waste of time. And don’t forget that just because 
> one or some of the authors did a bad job at the crystallographic end other 
> findings of the paper might still be solid – in collaborations often one 
> author is unable to critically evaluate another author’s contribution and 
> this would not be the first case were good synthetic or biological work is 
> presented along with a bad crystal structure.
> By the way and a bit ironically this protein may have suffered bad 
> crystallography/scientific practice before - I think it was one of the fake 
> Krishna Murthy structures, right? The associated (now retracted) article I 
> mean is here
> https://www.sciencedirect.com/science/article/pii/S002228360093924X?via%3Dihub
> Kind regards, Bärbel
> ---
> Bärbel Blaum, PhD
> Inthera Bioscience AG
> Einsiedlerstrasse 34
> CH-8820 Waedenswil
> Switzerland
> E-Mail: baerbel.bl...@intherabio.com
> Phone: +41 43 477 94 72--
>  
>  
>  
> Von: CCP4 bulletin board  im Auftrag von "Manfred S. 
> Weiss" 
> Antworten an: "Manfred S. Weiss" 
> Datum: Freitag, 19. Juli 2019 um 16:03
> An: 
> Betreff: Re: [ccp4bb] Questionable Ligand Density: 6MO0, 6MO1, 6MO2
>  
> Hi Rhys,
> 
> all three structures are at modest resolution and they don't seem to
> be properly refined. At least they are all below average. I wonder
> how this paper made it past the referees.
> 
> I haven't checked the paper, but there are ways and means how to
> deal with weakly bound ligands in the best possible way. One aspect
> is to improve the phases as much as possible without having the ligand
> present. This was obviously NOT done. Another way is to use the
> PANDDA approach, which relies on having many data sets available. 
> I suppose that this was also not done.
> 
> The best way to check is to delete the ligand and so some extensive 
> refinement in order to remove the phase bias introduced by the
> ligand. Only then you can reliably assess whether something is there
> or not.
> 
> Cheers, Manfred
> 
> Am 19.07.2019 um 15:21 schrieb Rhys Grinter:
>> Hi All, 
>>  
>> I was chatting with a colleague during a recent synchrotron visit and they'd 
>> recently come across some ligand/drug bound structures associated with a 
>> paper recently published in a high impact factor journal.
>>  
>> They had pulled the associated SFs from the PDB and found that the electron 
>> density associated with these ligands didn't match that reported in the 
>> paper and certainly wasn't sufficient to model the alleged ligand.
>>  
>> I also pulled the structure factors and after refinement in the 
>> presence/absence of the alleged ligand I also feel that the density present 
>> does not warrant modelling of the ligand. 
>>  
>> I was hoping that the community might be able to give me an outside opinion 
>> on these datasets (PDB IDs: 6MO0, 6MO1, 6MO2) and if the problem associated 
>> with the data is verified, provide some advice on how to proceed. 
>>  
>> This isn't the first occasion I've seen ligand bound structures with 
>> questionable density deposited in association with papers in well respected 
>> journals. Despite improvements to validation I feel that this problem is 
>> widespread.
>>  
>> Best Regards,
>>  
>> Rhys
>>  
>> -- 
>> Dr Rhys Grinter 
>> NHMRC 

Re: [ccp4bb] Space group/Unit cell

[Patrick Loll]

This reminds me of something that we crystallized a few years back. It indexed 
as I222 (or I2(1)2(1)2(1)) and the cell was quite small; too small for the 
protein of interest. Almost certainly a contaminant, but it didn’t show up in 
Contaminer. Diffracted like gangbusters, but we never figured out what it is. 
Very annoying. Unfortunately I can’t tell you cell constants, because those 
data are locked in my office and my institution is locked down...

> On 22 May 2020, at 6:08 AM, Demou, Maria  wrote:
> 
> Dear all,
> I have a question that may have a straight forward answer, and was wondering 
> if this is a common issue. We have a protein crystallised in I222 space 
> group. This is CRP, but the monomer/pentamer is not predicted to fit in this 
> space group. Is there a possibility of the lipid cubic phase being 
> crystallised on it's own, or is there any other obvious reason?
> 
> Thank you,
> Maria 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> 
__

Patrick J.  Loll, PhD
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St.
Philadelphia, PA 19102-1192 USA

(215) 762-7706
pj...@drexel.edu 
pjl...@gmail.com






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[ccp4bb] CCwork/CCfree

[Patrick Loll]

Hi everyone,

I’d like a little context to assist me as I review a manuscript.

Specifically, I’d like to ask if the hive-mind can provide an estimate of the 
expected range of CCwork and CCfree values for a structure refined at 1.5 Å 
resolution, with “typical” R/Rfree values?

By typical R/Rfree, I mean the peaks of the histograms 
(phenix.r_factor_statistics suggests R ~ 0.15-0.18, Rfree ~ 0.19-0.22)

I realize that I could probably dig up this information on my own, but I’m 
(feeling lazy)/(desirous of the community’s wisdom).

Thanks,

Pat


Patrick Loll
pjl...@gmail.com



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[ccp4bb] Fwd: [ccp4bb] AW: Kevin Denkmann lädt Sie zur Zusammenarbeit auf 'Rechnungen' ein.

[Patrick Loll]

Agreed. In fact, as a matter of course, when die Rechnung arrives I endeavor to 
head for die Tur…

> 
> 
> 
>> On 20 Oct 2020, at 10:23 AM, Schreuder, Herman /DE 
>>  wrote:
>> 
>> Looks more like crowd-phishing to me! 
>> The original message did not make it through my spam filter and it may not 
>> be a good idea to open the shared file.
>> Herman
>> 
>> -Ursprüngliche Nachricht-
>> Von: CCP4 bulletin board  Im Auftrag von Robbie 
>> Joosten
>> Gesendet: Dienstag, 20. Oktober 2020 16:16
>> An: CCP4BB@JISCMAIL.AC.UK
>> Betreff: Re: [ccp4bb] Kevin Denkmann lädt Sie zur Zusammenarbeit auf 
>> 'Rechnungen' ein.
>> 
>> Working on your bills with the entire bulletin board. Is that crowdsourcing 
>> or what? 
>> 
>>> -Original Message-
>>> From: CCP4 bulletin board  On Behalf Of Kevin 
>>> Denkmann
>>> Sent: Tuesday, October 20, 2020 15:27
>>> To: CCP4BB@JISCMAIL.AC.UK
>>> Subject: [ccp4bb] Kevin Denkmann lädt Sie zur Zusammenarbeit auf 
>>> 'Rechnungen' ein.
>>> 
>>> 
>>> Kevin Denkmann shared a file with you
>>> 
>>> Here's the document that Kevin Denkmann shared with you.
>>> 
>>> <https://mtuoued-
>>> my.sharepoint.com:443/:b:/g/personal/kevin_denkmann_dunnlab_de/EZQT
>>> 7ED-bpdJtg5lG-H32GkByQoTQqzWyiKSIRel_DIrFA?e=4%3a9jNtyK=9>
>>> Rechnungen
>>> This link will work for anyone.
>>> Open <https://mtuoued-
>>> my.sharepoint.com:443/:b:/g/personal/kevin_denkmann_dunnlab_de/EZQT
>>> 7ED-bpdJtg5lG-H32GkByQoTQqzWyiKSIRel_DIrFA?e=4%3a9jNtyK=9>
>>> 
>>> Privacy Statement <https://northeuroper-
>>> notifyp.svc.ms:443/api/v2/tracking/method/Click?mi=HgrSi7OfwUKiTn-
>>> 401hPpQ=PrivacyStatement=f97d4ae4336b3342c9a937ee3f36e84e
>>> u=https%3a%2f%2fprivacy.microsoft.com%2fprivacystatement%5c>
>>> <https://northeuroper-
>>> notifyp.svc.ms:443/api/v2/tracking/method/View?mi=HgrSi7OfwUKiTn-
>>> 401hPpQ>
>>> 
>>> 
>>> 
>>> 
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://eur01.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.
>>> jiscmail.ac.uk%2Fcgi-bin%2FWA-JISC.exe%3FSUBED1%3DCCP4BB%26A%3D1d
>>> ata=02%7C01%7CHerman.Schreuder%40SANOFI.COM%7Cf86f5afb3c3d4664586e08d8
>>> 7502dc60%7Caca3c8d6aa714e1aa10e03572fc58c0b%7C0%7C0%7C6373880027010674
>>> 74sdata=fR1KagWSgefPDCTSYiUFKOQKdWgRFUB7ysSaCe8JrTI%3Dreserv
>>> ed=0
>> 
>> 
>> 
>> 
>> To unsubscribe from the CCP4BB list, click the following link:
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>> 
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>> 
>> 
>> 
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> 
> Patrick Loll
> pjl...@gmail.com
> 
> 
> 

Patrick Loll
pjl...@gmail.com



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[ccp4bb] flow rate for cooling stream?

[Patrick Loll]

Sorry, way off topic:

Does anyone have an estimate for the flow rate one would typically use for the 
cold nitrogen stream passing over a protein crystal in a standard data 
collection?

Background: Our nitrogen “generator” has gone belly-up and the vendor no longer 
services it, so I’m testing the feasibility of using the boil-off from a liquid 
nitrogen tank to provide the gas to support a short data collection (this 
nitrogen gas would serve as the feedstock into our helium cryostat). But I 
don’t know the flow rate required, so I can’t calculate if one tank has enough 
nitrogen to support a day or so of data collection. There are flow meters for 
the warm and cold stream on the nitrogen generator, but these flow meters have 
no apparent units anywhere on them, so I have no idea of the rate at which the 
gas would be consumed.

Thanks for any useful tidbits. 

And for those of you in the US, best wishes for a happy “Holy crap, even MORE 
fireworks?!?!!” Day 

Pat


---
Patrick J. Loll, Ph. D.  
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu


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[ccp4bb] dewar horror stories

[Patrick Loll]

Hello community,

We recently had a dry shipping dewar fail catastrophically (while en route to 
the beam line, so, major trauma). I sent it to a company that specializes in 
repair and refurbishing of cryogenic tanks, and they told me it has an internal 
leak, and hence is not reparable. I was expecting that the valve had failed, so 
the internal leak diagnosis came as a surprise.

Has anyone else had a similar experience? Any ideas about how an internal leak 
might come about? The dewar is (was) a Taylor/Wharton CX100, and it was 
traveling in its bespoke shipping case.

Thanks for any insights that might satisfy my curiosity and/or prevent future 
mishaps of this sort.

Cheers,

Pat

__

Patrick J.  Loll, PhD
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St.
Philadelphia, PA 19102-1192 USA

(215) 762-7706
pj...@drexel.edu 



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[ccp4bb] Stabilizing Mitegen reusable bases/mounts

[Patrick Loll]

Hi everyone,

I’ve become very fond of the Mitegen reusable bases for mounting crystals, 
since the reusable aspect savse me from having to discard the base every time I 
break a microloop. However, once the crystals arrive at the synchroteon, I 
observe motions of the loops (some gradual, some sporadic). The the amplitudes 
of these motions are becoming significant as I take data from smaller and 
smaller crystals. I don’t think I’m imagining this, since the good folks at 
NSLS-2/AMX have warned me about this very issue.

I’m writing to ask if anyone has any clever ideas about stabilizing these 
assemblies. Obviously, I can epoxy the pins in place, but then I’ll probably 
need to discard the entire assembly when I break a loop, and I’d prefer not to 
waste more money than necessary. I’ve considered putting a bead of wax at the 
point where the pin enters the base (although I haven’t yet checked to see if 
that will survive immersion in liquid nitrogen). Does anyone have any other 
(better) ideas?

Much obliged in advance,

Pat
__

Patrick J.  Loll, PhD
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St.
Philadelphia, PA 19102-1192 USA

(215) 762-7706
pj...@drexel.edu


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Re: [ccp4bb] The weekly nonsense

[Patrick Loll]

It seems that, for inexplicable reasons, they’ve confused Karl Pearson with 
Brian Matthews? 

> On 22 Jan 2021, at 6:03 PM, Bernhard Rupp  wrote:
> 
> Dear CCP4 Fellows, 
>  
> for the subscribers of my immensely popular show “The weekly nonsense” I 
> recommend
> https://arxiv.org/pdf/2101.06308.pdf <https://arxiv.org/pdf/2101.06308.pdf>
> particularly reference [27] and its contextual environs.
>  
> Am I alone suspecting that this is some AI generated auto-paper-mill product?
>  
> Cheers, BR
>  
> --
> Bernhard Rupp
> The Amt
> http://www.hofkristallamt.org/ <http://www.hofkristallamt.org/>
> b...@hofkristallamt.org <mailto:b...@hofkristallamt.org>
> +1 925 209 7429
> +43 676 571 0536
> --
> Many plausible ideas vanish 
> at the presence of thought
> --
>  
> 
> To unsubscribe from the CCP4BB list, click the following link:
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> <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1>
Patrick Loll
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Re: [ccp4bb] Looking for proteins for undergraduate biochemistry lab

[Patrick Loll]

Transglutaminase, also known in some circles as “meat glue."

> On 17 Jun 2021, at 11:50 AM, Bryan Lepore  wrote:
> 
> Greetings
> 
> This enzyme meets none of the stipulations, but I will point out as it is 
> somewhat unusual to find in a grocery store :
> 
> A food product called Just Egg contains “transglutaminase” as an ingredient.
> 
> Make of that what you will.
> 
> -Bryan W. Lepore
> (No affiliation with Just Egg or anything like that whatsoever)
> 
> PS
> 
> Amylases are readily available through general baking supply - but also meets 
> none of the stipulations.
> 
---
Patrick J. Loll, Ph. D.  (he, him, his)
Professor of Biochemistry & Molecular Biology
Drexel University College of Medicine
Room 10-102 New College Building
245 N. 15th St., Mailstop 497
Philadelphia, PA  19102  USA

(215) 762-7706
pjl...@gmail.com
pj...@drexel.edu


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