Re: [ccp4bb] Phasing a difficult RNA heteroduplex structure

2022-05-06 Thread Francis Reyes
No love for RNA!? Ouch !

I was in the similar problem ten years ago when I was solving riboswitch RNAs 
as a graduate student. B12 riboswitch,stuck at 5-6A for derivatives, but had 
pretty reasonable native data. 

It was a combination of experimental phasing using clusters at low resolution 
and then extending the data with smaller derivatives (iridium/cobalt 
hexammine), multiple crystal averaging, SHARP, and major assistance from people 
who have been in a similar resolution. 

You can find the details in my thesis here 

Or you can reach out to me directly. 


> On May 6, 2022, at 8:34 AM, Eleanor Dodson 
> <> wrote:
> How I hate RNA! 
> However, structures have been solved..
> As others say: 
> Look for twinning
> Look for translation non-cryst symmetry.
> It seems likely when you have a doubling of the a axis for the Zn derivative. 
> I use CCP4I2 and the data processing report will do both these tests.
> Then you need to decide whether the SG is P6i or P6i22
> IF there is twinning then the apparent P6i22 symmetry could be due to this..
> Where are your sites? If they are related by crystal symmetry then it is hard 
> to distinguish the hand.
> And low solvent content hampers DM 
> Good luck Eleanor
> On Fri, 6 May 2022 at 12:51, Petr Kolenko  > wrote:
> Dear Charlie Nichols,
> when optimizing your SHELX runs, SHELIXIR may help you in some cases 
> ( 
> ).
> A couple of days ago, I created quick and dirty tutorial to SHELIXIR command 
> line: 
>  and also the same quality 
> tutorial to the GUI: 
> There may be some parameters to further optimize in which SHELIXIR may help 
> you (trying more space groups, optimizing of high or low resolution cut-off, 
> solvent content). However, SHELX C/D/E is generally known to have problems at 
> lower resolution (say 3.5 AA and lower). To say a bit more about your 
> problem, would you share the files off the list?
> Btw, the great thing about SHELX is that you do not even need to know the 
> content of the AU in some cases!
> Best regards,
> Petr
> From: CCP4 bulletin board  > on behalf of Nichols, Charlie 
> Sent: Friday, May 6, 2022 1:17:20 PM
> Subject: [ccp4bb] Phasing a difficult RNA heteroduplex structure
> Dear all,
> I am trying to solve the structure of an RNA heteroduplex + ligand with 
> approximate MW of 6800.
>   *   Structure likely to have a core helical region and a couple of bases of 
> single stranded material at both ends on both strands
> I have datasets from visually similar crystals with different, but related 
> unit cells:
> Form1:
> 32.70 32.70 54.55 90.00 90.00 120.00 – best native ~2.9A
> - different pipelines reported P62 2 2, P62 and P32 from auto-indexing
> - P6222 impossible to fit 1 complete heteroduplex
> - P62, most consistent indexing choice, 37% solvent, low Matthews probability 
> but possible to fit 1 heteroduplex
> However, Xia/Dials report tNCS
>   *   If the ASU only has room for 1 copy the heteroduplex there can’t be 
> tNCS, does this therefore mean we must have twinning?
>   *   There is a reasonable similarity NMR structure in the PDB, this is ~45A 
> long
>   *   I am therefore guessing that the duplexes are most probably making 
> end-end contacts to form long fibres that are ~aligned along the Z-axis and 
> that the crystal either contains fibres bound both ways up, or that the 
> duplex can bind either way up to create the fibres, the twinning then 
> superposes the two orientations to create two identical repeats mimicking 
> tNCS  – does this seem a reasonable interpretation?
> I have a dataset with Zn/Mg exchange and good anomalous signal to ~3.8A, 
> ShelX finds good sites but DM / phase-extension with the 2.9A native data 
> creates a mess and there is little difference between the two hands – can you 
> give any advice on how I might try to proceed with experimental phasing in 
> this case?
> Form2:
> 62.40 62.40 54.82 90.00 90.00 120.00 – best native ~2.5A
> - Auto-indexes as P62 / P64, higher resolution data than Form1
> - Again, Xia/Dials report tNCS
> - Cell has a doubling of a/b dimensions but c is the same
> - Molecular replacement fails completely
> - Cobalt-hexammine soak gave strong anomalous signal but only to ~6A, again 
> ShelX finds good sites, but DM / phase-extension gives a mess
> - From the pathology of Form1, I am concerned that we have exactly the same 

Re: [ccp4bb] Semet derivative dying almost immediately in beam

2019-08-29 Thread Francis Reyes
Ah, low resolution experimental phasing. Fun stuff, seems like a lost art these 

Are the crystals large ? 
Is it possible to do a (continuous helical data) collection?  (Manually or if 
your beamline permits you to). 

Granted this is anomalous, is it possible to then merge enough partial datasets 
together? (Very carefully of course). If not there’s always SIRAS. 

With this rapid decay, are you using a strategy? (Another lost art these days)

Given the difficulty of this project, have you pursued other derivatives (via 

> On Aug 29, 2019, at 11:26 AM, Kimberly Stanek  wrote:
> Hi folks,
> We have a protein that we have been trying to solve the structure of for a 
> while now but so far haven't been able to get any diffraction better than 
> ~4.5A. I was able to collect a full 360 degrees of data and index, but MR is 
> failing so we have turned to de novo phasing.
> Recently we prepared crystals of the Semet derivative under the same 
> condition. While these crystals diffracted to about the same resolution, I 
> found they were dying after just one or two snaps, even with increased beam 
> attenuation and decreased exposure time. I am wondering if anyone has 
> experienced anything like this before and had any suggestions on what to do 
> about it.
> Thank you,
> Kimberly Stanek
> Postdoctoral Researcher, Goulding Lab
> Co-chair, UCI Postdoctoral Association
> University of California, Irvine
> Natural Science I, Room 2302
> (949) 824-0014
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Re: [ccp4bb] 3D

2019-03-11 Thread Francis Reyes
I’m crossing my fingers, or should I say my eyes, that they don’t!

> On Mar 11, 2019, at 9:22 AM, David Schiller  wrote:
> NVIDIA will stop supporting 3D glasses in April
> -- 
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
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Re: [ccp4bb] ice build up during collection using 4 axis goniometer

2017-09-13 Thread Francis Reyes
I've had this problem with the four circle goniometers. I imagine your setup 
looks similar to

At high chi values, I would also observe a 'tail' of ice off my pins. 

As you mentioned in the manual, it's because at high angles, the cryo stream 
hits the pin first and *then* hits your crystal. This disrupts laminar flow, 
introduces turbulence, and then that turbulence is mixing humid air from the 
environment and depositing it on your crystal. 

Reposition the cryostream so the laminar flow hits your crystal first.

Very surprised that its enough to blow your crystal off the pin! 



On Sep 13, 2017, at 3:54 PM, Annette Herta Erbse  

> Dear All,
> We have a Rigaku XtalLab with a MM003 generator, a 4 axis goniometer and an 
> Oxford 700 cryostream. I have been running into the problem that if I make 
> full use of the  4 axis goniometer in the collection strategy  I run into 
> geometries where the angles between the pin/Xtal and the Oxford cryo head 
> nozzle are smaller than 90 0 and in these positions I start collecting a lot 
> of ice to a point were it get's blown off by the cold stream and I have lost  
> Xtals. I was wondering if others have experienced the same and if there is a 
> good way to avoid it. At the moment I feel like I simply have to avoid these 
> ranges which is a shame since it restricts  the data collection strategy.
> I appreciate any advice - Annette  

Re: [ccp4bb] Rmergicide Through Programming

2017-07-04 Thread Francis Reyes
I find the lack of reporting statistics of the low resolution bins unfortunate!

Most statistics in Table 1 report  the average across all resolutions or just 
the high resolution reflection shell.  

With respect to Rmerge, the agreement between the most intense (low resolution) 
symmetry related reflections is very telling to the quality of the merged data 
in my opinion (radiation damage, absorption errors, very weakly exposed 
crystal, other strange systematic errors, etc). 


On Jul 4, 2017, at 4:37 PM, Graeme Winter  wrote:

> Unbiased estimate of the true unmerged I/sig(I) of your data (I find this 
> particularly useful at low resolution) i.e. if your inner shell Rmerge is 10% 
> your data agree very poorly; if 2% says your data agree very well provided 
> you have sensible multiplicity… obviously depends on sensible interpretation.

Re: [ccp4bb] Rcrane error, update

2017-06-21 Thread Francis Reyes
I'm thinking a nomenclature issue. 

H5 ? Should be H5' no? 

Have you run your structure through a pdb remediator? is my favorite. 

You will want to play around with the flags to get the right output. I think 
rCrane likes old style nomenclature (asterisks instead of apostrophes). 


On Jun 21, 2017, at 3:04 PM, Ursula Schulze-Gahmen  

> I am trying to use Rcrane in stand-alone coot version 0.8.8. The Rcrane 
> window opens and I am able to calculate new conformations for my nucleic 
> acid. But when I am trying to accept a conformation, I am getting an error 
> message:
> Traceback (most recent call last):
>   File 
> "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/",
>  line 1041, in __accept
> self.__pseudoMolecule.finalMoleculeCleanup(fixSegids = 
> bool(self.__pseudoMolecule.hasSavedCoordinates()))
>   File 
> "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/",
>  line 1735, in finalMoleculeCleanup
> curRes[3].sort(key = lambda x: 
> self.__reorderAtomsOrder[str(curRes[2])][x[0][0].strip()])
>   File 
> "/home/programs/x86_64-linux/ccp4/7.0/ccp4-7.0/lib/python2.7/site-packages/coot/rcrane/",
>  line 1735, in 
> curRes[3].sort(key = lambda x: 
> self.__reorderAtomsOrder[str(curRes[2])][x[0][0].strip()])
> KeyError: "H5'"
> Any suggestions what might be wrong, and how I can fix this?
> Thanks
> -- 
> Ursula Schulze-Gahmen, Ph.D.
> Project Scientist
> UC Berkeley, QB3
> 360 Stanley Hall #3220
> Berkeley, CA 94720-3220
> (510) 643 9491

Re: [ccp4bb] mystery feature near a PLP substrate

2017-05-24 Thread Francis Reyes
Fidget spinner?


> On May 24, 2017, at 10:16 AM, Eleanor Dodson  
> wrote:
> Any ideas?

Re: [ccp4bb] Estimating the amount of missing electron density for a model

2017-02-22 Thread Francis Reyes
This is a good point, the difference between Fo and Fc can be great if Fc is 
actually missing (an 'incomplete' structure). And of course this wreaks havok 
in defining the maskf for bulk solvent, and refinement, etc. Incomplete can be 
missing whole parts of the protein (say in model building) or it could mean 
looking for density of a surface-bound ligand (the recent discussion of masking 
out the suspect region is implemented in BUSTER and more recently, phenix's 
POLDER maps). 

However, when it comes to the 'refinement' of incomplete structures, I've found 
that low resolution probability distributions for the missing atoms can improve 
the refinement and the resultant map via the  'MissingAtoms' channel of 
BUSTER-TNT during refinement[1]. There was also a phenix.refine 
use_statistical_model_for_missing_atoms from Tom Terwilliger, where the missing 
density is obtained from RESOLVE density modified maps. 

Of course, the best case scenario is to have an external source of phase 
information (say experimental phasing) and the incomplete Fc has less of an 
effect in the refinement and map quality. 

[1] Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2210-21. Epub 
2004 Nov 26. Refinement of severely incomplete structures with maximum 
likelihood in BUSTER-TNT. Blanc E, Roversi P, Vonrhein C, Flensburg C, Lea SM, 
Bricogne G.

Just my 0.02,


On Feb 21, 2017, at 4:53 PM, Hunter Moseley  wrote:

> Is there a straight-forward way to estimate the amount of missing electron 
> density that a particular protein structure is missing based on the 
> difference between Fo and Fc?
> It appears that the normalization of the Fc due to the employing of a maximum 
> entropy method that keeps Fo and Fc comparable to the standard deviation of 
> Fo would make this difficult. 
> Or am I missing something?
> Cheers,
> Hunter
> -- 
> Hunter Moseley, Ph.D. -- Univ. of Kentucky
> Associate Professor, Dept. of Molec. & Cell. Biochemistry / Markey Cancer 
> Center
> / Resource Center for Stable Isotope Resolved Metabolomics
>  Not just a scientist, but a fencer as well.
>My foil is sharp, but my mind sharper still.
> ---
> Email: (work) (personal)   
> Phone: 859-218-2964 (office)   859-218-2965 (lab)   859-257-7715 (fax)
> Web:
> Address: CC434 Roach Building, 800 Rose Street, Lexington, KY 40536-0093

Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder


[ccp4bb] SHELXL refinement with TYR on special position

2017-01-24 Thread Francis Reyes
Hi all

I'm trying to refine a structure with a tyrosine sitting on a special position 
, or maybe it's some disorder.. or  Suggestions?

Using just FLAT, CHIV,DFIX, and DANG from shelxpro doesn't work. 



Re: [ccp4bb] pukka puckers within coot

2015-04-16 Thread Francis Reyes
Sounds like your refinement is failing probably because of the low 
data:parameter ratio at this resolution and/or the composition of your 
asymmetric unit, poor initial phase information (probably a molecular 
replacement solution which is close, but still far from the truth either in 
sequence composition or atomic configuration). 

In these cases, it is useful to use a reference structure containing an 
idealized A-form (RNA) or DNA helix with the *correct* sequence composition and 
ensure that the refinement program matches the residues appropriately upon the 
start of refinement. If you've got some strange tertiary architectures 
(k-turns, gnra tetraloops or some other non canonical motif), include those as 

Then, there is the issue how the restraints are generated for refinement, I've 
found autoBUSTER's LSSR regime (based on interatomic distances  as opposed to 
dihedral angles) to be quite good at restraining the refinement, but that's my 
own opinion on a few particularly challenging cases. 

As a polishing step (and if you have to revert to real space refinement), the 
rcrane plugin but Kevin Keating is very helpful. It is accessible within coot.  


On Apr 16, 2015, at 8:22 AM, Almudena Ponce Salvatierra 

 Dear all, 
 Despite molprobity analysis tells me my structure contains no puckers that 
 are wrong, the pukka puckers tool within coot lists a number of them with for 
 example inconsistent distance bewteen the phosphates. 
 I would like to ask if any of you have dealt with this before, and how does 
 one make consistent the distance between phosphates with the sugar 
 puckering? Only by direct space refinement? Or is there a way to change 
 something else...
 My structure is a 3 A resolution so I can definitely see the backbone of the 
 nucleic acid chain but for sure I can not tell whether the pucker was 
 assigned properly or not from the electron density.
 Any ideas?
 Thanks a lot in advance. 
 Best wishes, 
 Almudena Ponce-Salvatierra
 Macromolecular crystallography and Nucleic acid chemistry
 Max Planck Institute for Biophysical Chemistry
 Am Fassberg 11 37077 Göttingen

[ccp4bb] collection strategy to collect the intersection with a reference set.

2015-02-24 Thread Francis Reyes
Hi all,

I'm on the lookout for a prediction program that'll tell me the best collection 
angles that match the reflections of a reference set, as opposed to the the 
reflections that'll complete a reference set. 

It seems that mosflm and XDS prescribe the angles for completion rather than 
the intersection. 



Re: [ccp4bb] Sharp: Solomon density modification step

2015-01-28 Thread Francis Reyes
Also, check the quality of the map going into density modification (by either 
solomon or DM) and whether the output solvent mask is consistent with your 
structure. Depending on the quality of the experimental phases, you can 
generally see the outline of the molecule and see NCS in your experimental maps 
(e.g. 6A maps of yeast pol II by Kornberg, yeast FAS, etc).  If you cannot see 
a reasonable outline of the molecule, the solvent mask for DM/solomon is going 
to be poor as well. 


On Jan 26, 2015, at 10:21 AM, Nicolas Soler wrote:

 The documentation seems to suggest to restrict yourself up to the resolution 
 where good phasing information is available (6.5A in my case) and I get 
 excellent indicators only if I do that (they become horrible if I use the 
 full resolution range). How about phase extension ? Which parameters would 
 you then use?

[ccp4bb] Looking for supplier for 'Q' crystallization plates.

2014-12-01 Thread Francis Reyes
Hi all,

This plate used to be the Q plate (HR3-124) from hampton research, but I 
cannot find them there anymore. Does anyone know where these plates can be 
bought from? 
Schematic of the plate and its wells is here : 



Re: [ccp4bb] PyMol and Schrodinger

2014-04-23 Thread Francis Reyes
On Apr 23, 2014, at 11:43 AM, Cygler, Miroslaw wrote:

 They do not offer the option of purchasing the software and using the 
 obtained version without time limitation. This policy is very different from 
 many other software packages, which one can use without continuing licensing 
 fees and additional fees are only when an upgrade is needed. At least I 
 believe that Office, EndNote, Photoshop and others are distributed this way.

Office 365 is $10 a month, Adobe Creative Cloud (what used to be their Creative 
Suite) is $50 a month with an annual commitment. 

Licensing the use of software on a time-limited basis as a business model seems 
like it's going to stick around.  

Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder


Re: [ccp4bb] jiffy for analyzing spot.xds?

2014-04-15 Thread Francis Reyes
Thank you all, 

Once again the ccp4bb delivers. 


On Apr 15, 2014, at 6:38 AM, T. Nakane wrote:

 Dear Francis,
 You can visualize SPOT.XDS with adxv.
 Since column 3 of SPOT.XDS is the image number of spot centroid,
 you can extract spots near frame 4 like this:
 awk '{if (int($3+0.5) == 4) print $1, $2;}' SPOT.XDS  spots_04.adx
 Then you can load spots_04.adx into adxv.
 (you must load the image into adxv before reading the spot list)
 Best regards,
 Takanori Nakane
 On 2014-04-15 10:30, Harry Powell wrote:
 Hi Francis
 At the risk of offending Wolfgang and Kay, why not try using other
 software to index  visualize spots found on the images? Mosflm is the
 one that comes to my mind straight away, but there are others that
 could probably do the job...
 It would certainly be possible to write a jiffy that would read a
 SPOT.XDS and write it in Mosflm .spt format, which could then be read
 directly into iMosflm.
 On 15 Apr 2014, at 02:24, Francis Reyes wrote:
 Hi all,
 I'm trying to diagnose a tricky indexing issue and I suspect the
 spot picking is poor. Any jiffy's for analyzing the spot.xds file
 (prior to running IDXREF) ? (like an overlay onto the actual image)?
 Francis E. Reyes PhD
 215 UCB
 University of Colorado at Boulder
 --** note change of address **
 Dr Harry Powell, MRC Laboratory of Molecular Biology, Francis Crick
 Avenue, Cambridge Biomedical Campus, Cambridge CB2 0QH
 Chairman of European Crystallographic Association SIG9
 (Crystallographic Computing)

[ccp4bb] ipmosflm not connecting to XQuartz on Mac.

2014-04-15 Thread Francis Reyes
Hi all,

Anyone having issues getting ipmosflm to connect to XQuartz? I'm getting a hang 
when running go just when I expect the old GUI to load. 



Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder


[ccp4bb] jiffy for analyzing spot.xds?

2014-04-14 Thread Francis Reyes
Hi all, 

I'm trying to diagnose a tricky indexing issue and I suspect the spot picking 
is poor. Any jiffy's for analyzing the spot.xds file (prior to running IDXREF) 
? (like an overlay onto the actual image)?



Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder


[ccp4bb] rotating a map with maprot.

2014-03-24 Thread Francis Reyes
Hi all,

I'm having a difficult time rotating a map from crystal A to crystal B. 

I obtained the transformation matrices from lsqkab.  Specifically, the Crowther 
(Euler) Alpha beta gamma angles and the orthogonal A translation vector from 
superpose I supplied directly to maprot. 

However, the rotated map does not overlay with the target structure at all. 

Any ideas?



Here are the log files :


   -0.40473  0.58092  0.70621
   -0.18898  0.70248 -0.68616
   -0.89469 -0.41117 -0.17453
  TRANSLATION VECTOR IN AS -4.9859926.07288   -22.20167

  TRANSLATION VECTOR IN fractions of cell edge 0.130.402062   

-0.405 0.581 0.706
-0.189 0.702-0.686


  trace  0.1232092
  not 10- dc(1),dc(2),dc(3),chk  0.1529777  0.8905933 -0.4283007

 CROWTHER (Euler) ALPHA BETA GAMMA135.82501  -100.05162   155.31825
  SPHERICAL POLARS OMEGA PHI CHI115.3598680.25346   116.00163
  DIRECTION COSINES OF ROTATION AXIS  0.15298 0.89059-0.42830

maprot script:

XYZLIM -32 57 -21 30 -15 41
GRID WORK 100 48 60
CELL WORK 105.9580 49.9510 62.6330 90. 114.7000 90.
ROTATE EULER 135.82501 -100.05162 155.31825
TRANS -4.98599 26.07288 -22.20167

Re: [ccp4bb] High Rwork/Rfree vs. Resolution

2014-02-22 Thread Francis Reyes
 I'm guessing the low completeness of the 1.65 angstrom dataset has to do with 
 obstacles the processing software encountered on a sizable wedge of frames 
 (there were swaths of in red in HKL2000). I'm not sure why this dataset in 
 particular was less complete than the others.

This is bad. Large swaths of red circles during integration is bad. I believe 
(check the Denzo manual) this means overlaps and overlaps get thrown out. Thus 
you are getting lower completeness. Was your oscillation range too large? 
Crystal very mosaic?

However this could be because of a poor crystal orientation matrix by HKL2000 
which in some cases can be alleviated by mosflm and xds. (HKL2000 is much more 
manual, there's a lot of buttons, which means you can shoot yourself in the 
foot if you are not careful).

I would be particularly interested in a resolution bin breakdown in the 
integration and merging statistics. (I/sig and rmerge). You might as well post 
the refinement statistics (r and rfree) by resolution bin as well.

You have a smallish unit cell that shoots to high resolution and getting a 
reasonable completion of the low resolution bins is paramount.  Post the 
completeness of the 20-10A bin. 

Is this molecular replacement? How complete is the model? Aside from the 
completeness of the model, how far is it from the target?

You mentioned that some regions of your crystal had smeary spots. This is also 
bad, particularly if the errors are not random  (I.e anisotropic along one 
axis). This will confuse ML refinement. Let's see a single frame of your data.


Re: [ccp4bb] Symmetry problem

2014-02-20 Thread Francis Reyes
I thought I saw this problem before. Though I wouldn't try it if you had 
solutions from different space groups.


On Feb 20, 2014, at 6:09 AM, Tim Gruene wrote:

 Hash: SHA1
 Dear Monika,
 would you mind summarising how you solved your problem? It might help
 people with similar problems!
 On 02/20/2014 02:45 PM, Monika Coronado wrote:
 Dear users,
 thanks alot for the tips. The problem was solved!
 Warmest regard,
 Monika Coronado
 2014-02-20 0:57 GMT-03:00 Jens Kaiser
 Monika, There are several possible causes for the problem you
 are encountering, but your description is a little too vague to
 discern them. Scenario 1) You ran phaser with the option all
 possible spacegroups, for several different components of your
 crystal, setup individually, and the runs do not agree on the
 best spacegroup? -- In that case, phaser had problems
 determining the correct spacegroup, I'd suggest you search for
 all components, but in separate runs for each possible 
 spacegroup. Scenario 2) You assumed your spacegroup assignment
 was correct, and ran MR for each of your components individually,
 and when you display the solutions, they overlap. In this case,
 you might have your solutions on different origins. The best way
 out is to use the first solution as a fixed solution, which is
 possible in most MR programs, and then search for the next
 component. There might be other scenarios, if you describe your
 situation in more detail (how many components in the crystal
 setup, what program you used, how you used it, and what you mean
 by different symmetries), we might be able to help you better,
 On Tue, 2014-02-18 at 14:59 -0300, Monika Coronado wrote:
 Does anyone know how to merge two molecules with different
 I will explain:
 I have done the molecular replacement using the domains of the 
 molecules separately, now I have to put all together, however
 they have a different symmetry.
 I will appreciate any kind of help.
 -- __o _`\,_ (*)/ (*) -+-+-+-+-+-+- * * * * * * * * * * * * *
 * * * * * * * * * * * * * * * * * * * * * * * * * * ...E tudo
 * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * * *
 * * * * * * *
 - -- 
 - --
 Dr Tim Gruene
 Institut fuer anorganische Chemie
 Tammannstr. 4
 D-37077 Goettingen
 GPG Key ID = A46BEE1A
 Version: GnuPG v1.4.12 (GNU/Linux)
 Comment: Using GnuPG with Icedove -

Re: [ccp4bb] Sister CCPs

2014-02-13 Thread Francis Reyes
The CCP4bb is great.. it truly is. 

The access to experts and their experience  (probably the most valuable) is 

However, mailing lists to organize discussions and disseminate new ideas is 
just so ... 90s. 

Wikis? maybe you've just crossed into the new millenium.

These days, if the questions/answers of the ccp4bb moved into something like 
It would save me a lot of time.


On Feb 13, 2014, at 10:21 AM, Nat Echols wrote:

 One comment (not a complaint) on all this: it seems like the same questions 
 get asked over and over again.  If there is a good place for a general 
 crystallography FAQ list it is well past time for one to be put together - or 
 maybe it just needs to be better advertised?  At a minimum, for instance:
 - what cryoprotectant should I use?
 - how do I get big single crystals?
 - how do I improve diffraction?
 - how can I tell if I've solved my structure?
 - why is my R-free stuck?
 - is pick random statistic suitable for publication?
 Some of the other common queries (name my blob!) still need to be handled 
 on a case-by-case basis, but it would be much more efficient for everyone if 
 the standard answers were collected somewhere permanent.
 On Thu, Feb 13, 2014 at 7:05 AM, Eugene Valkov 
 I absolutely agree with Juergen.
 Leaving aside methods developers, who are a completely different breed, there 
 is no such thing as a crystallographer sitting in a dark room solving 
 structures all day. If there are, these are anachronisms destined for 
 evolutionary demise.
 More and more cell biologists, immunologists and all other kinds of 
 biologists are having a go at doing structural work with their molecules of 
 interest themselves without involving the professionals. Typically, they 
 learn on the job and they need advice with all kinds of things ranging from 
 cloning and protein preps through to issues with tetartohedrally-twinned data 
 and interpreting their structures.
 So, a modern structural biologist is one who is equipped for the wet lab and 
 has some idea of how to go about solving structures. CCP4BB is a wonderful 
 resource that is great for both the quality of the advice offered to those 
 that seek it and for the variety of topics that are addressed in the scope of 
 structural biology. I have learnt greatly from reading posts from very 
 skilled and knowledgeable scientists at this forum and then implemented these 
 insights into my own research. I am very grateful for this.
 In short, please do not discourage your colleagues, particularly very junior 
 ones, from posting to the CCP4BB. Some of the questions may appear quaint or 
 irrelevant but it is easy to simply ignore topics that are of no interest! 
 On 13 February 2014 14:41, Bosch, Juergen wrote:
 Let me pick up Eleanor’s comment:
 is there something like a crystallographer today ? I mean in the true sense ?
 I think as a “crystallographer” you won’t be able to survive the next decade, 
 you need to diversify your toolset of techniques as pointed out in this 
 And I’m not quite sure how software developers see themselves, as I would 
 argue they are typically maybe not doing so much wet lab stuff related to 
 crystallography (I may be wrong here) but rather code these days.
 What “type” of crystallographer is a software developer ?
 I think like our beloved crystals “we” come in different flavors. And we need 
 to train the next generation of students with that perspective in mind.
 Just my two cents on a snowy day (30cm over night)
 Jürgen Bosch
 Johns Hopkins University
 Bloomberg School of Public Health
 Department of Biochemistry  Molecular Biology
 Johns Hopkins Malaria Research Institute
 615 North Wolfe Street, W8708
 Baltimore, MD 21205
 Office: +1-410-614-4742
 Lab:  +1-410-614-4894
 Fax:  +1-410-955-2926
 On Feb 13, 2014, at 6:41 AM, Eleanor Dodson wrote:
 I agree with Frank - it keeps crystallographers modest to know how
 challenging wet lab stuff still is..
 On 12 February 2014 19:23, Robbie Joosten wrote:
 It's not an e-mail bulletin board, but Researchgate seems to be quite
 popular for wet lab questions. IMO the QA section of the social network is
 a bit messy. That said, the quality seems to improve gradually.
 Sent from my Windows Phone
 Van: Paul Emsley
 Verzonden: 12-2-2014 19:23
 Onderwerp: Re: [ccp4bb] Sister CCPs
 On 12/02/14 15:59, George Sheldrick wrote:
 It would be so nice to have a 'sister CCP' for questions aboud wet-lab
 problems that have nothing to do with CCP4 or crystallographic
 computing, The is clearly a big need for it, and 

Re: [ccp4bb] How to find the unfound part of a big protein

2014-02-13 Thread Francis Reyes
On Feb 13, 2014, at 4:53 PM, Sun Bingfa wrote:

 Now we have dataset to ~4.2 Angstrom and using extracellular homolog 
 structure we can find a solution for this part(~45% of the whole molecule MW) 
 through molecular replacement, and the molecules are packed as layers, and 
 the other part are presumably between these layers. However, we are having 
 trouble to fit the rest of the protein even though there're some density 
 between the solved part. 

You have an incomplete MR problem.

 Rfree is at 40% now. 

This is fairly typical.

 We're trying to do heavy atom soaking, such as TaBr. We collected data for 
 MIR but it's not helping so far.

This is also fairly typical.

 (Can I combine these MIR data with the native dataset because the MIR set is 
 only at ~6.5 Angstrom)? 

Yes, but you need to be careful in how you do this.  The bad news is that 
jumping from 6.5 - 4A is not trivial. 

The good news is that it can be done. 

See for example,



Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder


[ccp4bb] off-topic Electron density map rendering..

2014-02-11 Thread Francis Reyes
Anyone know the program used to render the electron density maps for the fungal 
FAS in Figure 3c-3h from the paper Mueller, M., Jenni, S.  Ban, N. Strategies 
for crystallization and structure determination of very large macromolecular 
assemblies. Curr Opin Struct Biol 17, 572–579 (2007).. 

The depth cueing is phenomenal. 

I'm thinking O.. I can't seem to get it right in Pymol.



Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

Publications  Citations:

Re: [ccp4bb] Phasing with Many Monomers/AU

2014-01-19 Thread Francis Reyes

 On Jan 19, 2014, at 11:30 AM, Chris Fage wrote:
 Thank you all for your responses. I already have a few ideas about how to 
 approach the problem.
 One of my concerns with so monomers per asymmetric unit at lower resolution 
 was the failure of MR software. Neither PHENIX nor Phaser MR have made 
 progress. I am fairly new to anomalous methods, having solved only two 
 structures by SeMet-based SAD. I've certainly picked up on a number of tricks 
 from the recent messages on heavy atoms, but I thought my case might be a 
 little unusual. I am confident the space group is P1, as it was the only 
 viable option when I indexed four clean albeit low-res datasets. 
 The monomers are ~38 kDa, and the crystals diffracted to 3.4-3.0 at a 

You'll probably get a lot of (good) suggestions on the ccp4bb, but being new to 
structure solving by anomalous methods, you should seek out someone who can 
walk with you through low resolution structure solution. As you may have 
learned, when working at these resolutions, structures just don't 'pop' out. 

I'd recommend the Rapidata course at BNL, which meets in the spring. When I 
last spoke with Bob recently, I had the impression there were open positions.  
You can bring your data and challenge the experts (many of whom are directly 
involved in the crystallography software development). 

Disclaimer: I am not affiliated with the course :) Just an alum. 



 The conditions for both native and SeMet crystals are: 
 8-12% PEG 2000 MME, 0.2 M ammonium sulfate, 0.1 M sodium acetate pH 5.5.
 Macromolecular seeding of native crystals into SeMet drops yields the 
 needle-like crystals.
 Any further input is greatly appreciated!
 On Sat, Jan 18, 2014 at 11:14 AM, Chris Fage wrote:
 Hello Everyone,
 I am currently trying to phase a structure with an asymmetric unit predicted 
 to contain 20-24 monomers (space group P1). The native crystals, while 
 beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms 
 at best, and SeMet-derived crystals grow with poor morphology (small 
 needles). Also, based a fluorescence scan, I know that mercury does not bind 
 appreciably. Other than screening for a new space group, what options might 
 I have for phasing this many monomers at lower resolution? Is there any real 
 chance of solving the structure in this space group?
 Thank you in advance for any suggestions!

Re: [ccp4bb] Phasing with Many Monomers/AU

2014-01-18 Thread Francis Reyes
You sure about this space group? 24 monomers in P1 is unusual (at least to me)


 On Jan 18, 2014, at 9:14 AM, Chris Fage wrote:
 Hello Everyone,
 I am currently trying to phase a structure with an asymmetric unit predicted 
 to contain 20-24 monomers (space group P1). The native crystals, while 
 beautiful in appearance (see attached), only diffract to ~3.4-3.0 angstroms 
 at best, and SeMet-derived crystals grow with poor morphology (small 
 needles). Also, based a fluorescence scan, I know that mercury does not bind 
 appreciably. Other than screening for a new space group, what options might I 
 have for phasing this many monomers at lower resolution? Is there any real 
 chance of solving the structure in this space group?
 Thank you in advance for any suggestions!

[ccp4bb] Proper format for buccaneer heavy atom file?

2013-12-01 Thread Francis Reyes
Hi all,

Is there a special format (residue naming, atom naming, etc) for the heavy atom 
file to assist buccaneer with sequence assignment? The one I have now seems to 
be returning structures that do not have SeMet at these locations. 



Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

[ccp4bb] I want to dock/align an EM envelope (MRC) into a DM averaging and/or solvent envelope (MSK).

2013-11-14 Thread Francis Reyes
Is there a single tool or suite of tools that addresses this? Or a CCP4 
workflow if need be. 



Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] few questions about resolving new structure through MR

2013-11-07 Thread Francis Reyes
Do you expect more than one molecule in the asymmetric unit?

Determined from the Matthews Coefficient (poor), size exclusion column 
(better), or self RF (best) ? 

On Nov 7, 2013, at 8:36 AM, Zhihong Yu wrote:

 Hi, all
 I'm a rookie in resolving a brand new structure. I have some questions for my 
 current case and look forward to some suggestions.
 Now I’m working on a protein like this: 
 N-ter(55aa)—domainA(110aa)—linker(30aa)—domainB(150aa)—C-ter(20aa), I got a 
 diffraction data just to 3.5Å, and there is no complete homology structure in 
 pdb bank, but only a homology structure (named as structureX later) for 
 domainB with ~30% sequence identity, so I have some questions as following:
 1. Is it possible to find a resolution through MR approach using structureX 
 as a search model? Especially considering that the resolution is only 3.5Å. 
 Currently I just tried once using phaser and refine the structure, I can get 
 a R/Rfree of 0.45/0.55, and it looks like most of backbone in the structureX, 
 especially those within helix or sheet, can be well described by 2Fo-Fc 
 density. Is this primary result promising or not?
 2. If it’s possible, what’s the general optimal procedure I should follow?
 Really thanks for any advice and suggestions!

Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder

Re: [ccp4bb] MacBook Pro graphics card options - what about Mavericks?

2013-10-23 Thread Francis Reyes
I'm currently using CCP4/COOT (from the official installer) and autoPROC  on 
Mavericks without any problems. I imagine the rest of the global phasing tools 
will work nicely. 

I had issues with fink, you have to use a branch from github as well as 
manually install and update the Command Line Tools just to get fink working...


Francis E. Reyes PhD
215 UCB
University of Colorado at Boulder
On Oct 23, 2013, at 3:16 PM, Carlos Kikuti wrote:

 Can I profit from Kristin's question and add one: is it too soon to know if 
 the crystallography software (CCP4, Coot, Autobuster, XDS) will work fine 
 with Mavericks (Mac OS X 10.9)? 
 (I remember a bit of trouble when Lion came off).
 Em 23 oct. 2013, às 22:10, Kristin Low escreveu:
 Hi everyone,
 Sorry for being a bit off topic, but I thought this group would be great for 
 I’m looking at upgrading my current laptop to a newer MacBook Pro. I’m torn 
 as to whether I need integrated vs discrete graphics for structural biology, 
 including molecular modelling, especially since the latest advances by Intel 
 in terms of integrated graphics. Right now with the new releases, the 
 options are between Intel Iris Pro (5200 series) and Intel Iris Pro + Nvidia 
 GT 750M. Thoughts?
 Thanks everyone!
 Kristin Low
 Ph.D. Candidate
 Queen's University
 Department of Biomedical and Molecular Sciences
 Botterell Hall, Room 645
 Kingston, ON
 Canada  K7L 3N6
 tel: +1-613-533-3019