Re: [ccp4bb] Protein rapidly precipitates when off ice

[Briggs, David C]

Hi Chris,

What is the theoretical pI of your protein? If it is around pH 7.5, you might 
try gel filtering your protein into a different buffer/pH combination. Try 
changing by at least 1 pH unit in either direction.

If the pI isn't a problem, then you might try try solubility screening as 
outlined...

http://scripts.iucr.org/cgi-bin/paper?dz5020

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of Chris Fage 

Sent: Thursday, July 13, 2017 11:40:34 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Protein rapidly precipitates when off ice

Dear CCP4BB Community,

This week, I purified a nicely overexpressing protein by Ni-NTA followed by gel 
filtration. In a 4 C centrifuge, I concentrated my gel filtration fractions to 
~1 mL, transferred the spin filter to ice, and then collected 2 uL for 
measurement on the Nanodrop. Sadly, the protein precipitated heavily in the 
pipet tip before I could dispense it onto the Nanodrop pedestal, directly 
adjacent to my ice box. This effect seems to be abated at 4 C, as the protein 
remained stable in cold room-chilled pipet tips. However, the protein also 
precipitated heavily when overnight at 4 C in 1 mL gel filtration buffer (150 
mM NaCl, 10 mM HEPES pH 7.5), but not overnight at 4 C in 10 mL Ni-NTA buffer 
(500 mM NaCl, 30 mM HEPES pH 7.5, 10% glycerol) prior to gel filtration. Has 
anyone experienced and resolved a similar issue before? Do any useful additives 
come to mind?

Things I have tried with the gel filtration sample:
-Exchanging buffer to restore the salt concentration to Ni-NTA levels (e.g. 500 
mM).
-Exchanging buffer to add 10% glycerol.
-Simply diluting the protein in gel filtration buffer to rule out concentration 
dependence.

In each case, the protein precipitates to a milky solution within about a 
minute of removal from ice (I am working with 20-50 uL volumes in PCR tubes).

Many thanks for any suggestions!

Best,
Chris

[ccp4bb] Stable Refinement as Low(ish) resolution - Follow up

[Rhys Grinter]

Hi All,

Thanks for all the useful comments. Buster gave me quite stable refinement
and nice maps, so I've been working with that so far. I'll work through the
other suggestions however, and see what gives the best results.

Cheers,

Rhys

-- 
Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213
+61 (0)403 896 767

Re: [ccp4bb] Fine Phi Slicing

[Graeme Winter]

Jacob

If you have a complete 360 deg data set and your sample is still alive, and you 
have a multi-axis gonio, I would recommend rotating the crystal about the beam 
(ideally by ~ maximum scattering 2-theta angle) and collecting again. This 
would record your blind region as well as moving the reflections to different 
pixels, and (as a bonus) also will move reflections out from the tile join 
regions into somewhere they can be measured, which would not happen for small 
2-theta shift.

See http://scripts.iucr.org/cgi-bin/paper?BA0020 Figure 16 as excellent 
illustration of this.

Biggest risk with this is getting *moving* shadows on the data on the second 
run, as an effective 45-50 degree chi shift (say) will usually be a pretty wide 
opening angle for a kappa gonio. XDS and DIALS both have mechanisms to deal 
with this, and automated processing packages are able to apply these given a 
reasonable understanding of the beamline.

Also saves building 2-theta axes which can handle 92 kg ;o)

Cheers Graeme

On 13 Jul 2017, at 21:00, Keller, Jacob 
> wrote:

I thought there was a new paper from the Pilatus people saying fine slicing is 
worth it even beyond the original 1/2 mosaicity rule?

I would think, actually, more gains would made by doing light exposures at, 
say, 1/3 mosaicity, collecting 360 deg, then shifting the detector in 2theta by 
a degree or two to shift uniformly the spots to new pixels, maybe accompanied 
by a kappa change. One would have to remember about the two-theta when 
processing, however!

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerd 
Rosenbaum
Sent: Thursday, July 13, 2017 3:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] weird diffraction pattern

Dear Gerard,

   my "sound like a sales person" was meant as poking a little fun - nothing 
serious, of course.

I and our users like our not-so-new-anymore Pilatus3 6M. It's a great detector 
in many ways. But, there is a lot of hype that this detector solves 
all-problem, for instance fine slicing that is claimed to be only possible with 
a pixel array detector. People get carried away and use
0.01 degree slices even as the mosaicity of their sample is, say, 0.3 degree. 
Slicing beyond 1/3 of the mosaicity will gain you very little - only more 
frames, more processing time.

This discourse is already drifting away from the original topic of the thread 
so I will comment on the other arguments  you made like resolution in a private 
e-mail.

Best regards,

Gerd

On 13.07.2017 14:00, Gerard Bricogne wrote:
Dear Gerd,

 I can assure you that I have no shares in Dectris nor any
commecial connections with them. What I do have is a lot of still
vivid memories of CCD images, with their wooly point-spread function
that was affected by fine-grained spatial variability as well as by
irredicible inaccuracies in the geometric corrections required to try
and undo the distortions introduced by the fiber-optic taper. By
comparison the pixel-array detectors have a very regular structure, so
that slight deviations from exact registering of the modules can be
calibrated with high accuracy, making it possible to get very small
residuals between calculated and observed spot positions. That, I
certainly never saw with CCD images.

 I do think that asking for the image width was a highly
pertinent question in this case, that had not been asked. As a
specialist you might know how to use a CCD to good effect in
fine-slicing mode, but it is amazing how many people there are still
out there who are told to use 0.5 or even 1.0 degree image widths.

 Compensating the poor PSF of a CCD by fine slicing in the
angular dimension is a tall order. With a Pilatus at 350mm from the
crystal, the angular separation between 174-micron pixels is 0.5 milliradian.
To achieve that separation in the angular (rotation) dimension, the
equivalent image width would have to be 0.03 degree. For an EIGER the
numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.

 Hence my advice, untainted by any commercial agenda :-) .


 With best wishes,

  Gerard.

--
On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:
Dear Gerard,

you sound like a sales person for Dectris. Fine slicing is perfectly
fine with CCD detectors - it takes a bit longer because of the step
scan instead of continuous scan. The read noise issue is often
overstated compared to the sample induced scatter background. If for
fine slicing at 0.05 degree or less the diffraction peaks go too
close to the read noise make a longer exposure - signal goes up,
ratio signal to sample-induced-BG less, as for any fine slicing, same read 
noise.

It would be helpful to analyze the dense spot packing along layer
lines if we knew the wavelength and the sample-to-detector distance
(assuming this is a 300 mm detector) and the 

[ccp4bb] Protein rapidly precipitates when off ice

[Chris Fage]

Dear CCP4BB Community,

This week, I purified a nicely overexpressing protein by Ni-NTA followed by
gel filtration. In a 4 C centrifuge, I concentrated my gel filtration
fractions to ~1 mL, transferred the spin filter to ice, and then collected
2 uL for measurement on the Nanodrop. Sadly, the protein precipitated
heavily in the pipet tip before I could dispense it onto the Nanodrop
pedestal, directly adjacent to my ice box. This effect seems to be abated
at 4 C, as the protein remained stable in cold room-chilled pipet tips.
However, the protein also precipitated heavily when overnight at 4 C in 1
mL gel filtration buffer (150 mM NaCl, 10 mM HEPES pH 7.5), but not
overnight at 4 C in 10 mL Ni-NTA buffer (500 mM NaCl, 30 mM HEPES pH 7.5,
10% glycerol) prior to gel filtration. Has anyone experienced and resolved
a similar issue before? Do any useful additives come to mind?

Things I have tried with the gel filtration sample:
-Exchanging buffer to restore the salt concentration to Ni-NTA levels (e.g.
500 mM).
-Exchanging buffer to add 10% glycerol.
-Simply diluting the protein in gel filtration buffer to rule out
concentration dependence.

In each case, the protein precipitates to a milky solution within about a
minute of removal from ice (I am working with 20-50 uL volumes in PCR
tubes).

Many thanks for any suggestions!

Best,
Chris

Re: [ccp4bb] Fine Phi Slicing

[Gerd Rosenbaum]

Hi Fred,

fine slicing does not alleviate the count RATE limitation of photon 
counting detectors because fine slicing does not reduce the 
instantaneous photon flux on the detector when you cross the diffraction 
maximum. Fine slicing does help if you push the maximum counts per pixel 
per frame to the counter register limit. On integrating detectors, like 
CCDs, there is practically no count RATE limit. They do have a charge 
(~photons) per pixel per frame limit, as well, which is mostly much 
lower than for the photon counting detectors - about 1/10 even after 
taking into account the diffraction spot covers many more pixels.


Different from integrating detectors where you only have to watch the 
overflow, for counting detectors you have to watch for exceeding either 
the count rate limit or the total count limit. The former is not an easy 
task because you will see only the count per exposure. Divide that by 
the exposure time you get the AVERAGE rate, not the peak rate.


The count rate limit, very short exposure time (using high flux) and the 
1-pixel point spread function work against each other. Exposure time = 
0.01 s, count rate limit 1e6 /sec (Pilatus2), 1-2 pixel per spot => 
10-20k counts per spot maximum for the strongest peak.


Dectris has come up with an ingenious hardware and software in the 
Pilatus3 pushing the rate for reasonable dead time correction to over 
1e7 counts/sec so even with 10 ms exposures weak reflections can be well 
recorded besides strong reflections.


Gerd

On 13.07.2017 15:34, Dyda wrote:

I could be wrong here, but isn't the case that fine slicing is an option
with a CCD and a necessity on a PAD b/c of dead time and/or counter dynamic 
range
issues?

(no current and/or former financial ties to any manufacturer)

Fred
***
Fred Dyda, Ph.D.   Phone:301-402-4496
Laboratory of Molecular BiologyFax: 301-496-0201
DHHS/NIH/NIDDK e-mail:fred.d...@nih.gov
Bldg. 5. Room 303
Bethesda, MD 20892-0560  URGENT message e-mail: 2022476...@mms.att.net
Google maps coords: 39.000597, -77.102102
http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred
***

Re: [ccp4bb] weird diffraction pattern

[Gerard Bricogne]

Dear Gerd,

 I wasn't really giving much attention to the poke between the
ribs ;-) - for me the more serious matter was to see the merits of
pixel detectors over CCDs made light of, as if they didn't really make
much difference.

 If some people get carried away in the way you describe, well, it
doesn't hurt anyone; but if other people have very small, weakly
diffracting crystals and they are told that they will do as well on a
beamline with a CCD as on one with a Pilatus or an Eiger, then that
will hurt someone.

 The original topic was whether certain images presented without
any information about their angular width and recorded on a CCD were
sufficient to diagnose a diffraction quality problem. As they showed
evidene of at least one long axis, distinguishing streakiness from
plain angular overlap caused by too great an image width seemed the
most natural first step.

 The resolution I was talking about was resolving spots within
images and neighbouring images, either thanks to a detector with a
small PSF or thanks to a very fine image width. CCD detectors are poor
in the first option because of their extended PSF, so emphasis has
always been put on the importance of using the second one when pushed.
Pixel array detectors allow both options to be used simultaneously,
which is especially valuable in the investigation of crystals like
Tang's. Hence, again, my advice in the strict context of the initial
thread. 


 With best wishes,
 
  Gerard.

--
On Thu, Jul 13, 2017 at 02:39:58PM -0500, Gerd Rosenbaum wrote:
> Dear Gerard,
> 
>my "sound like a sales person" was meant as poking a little fun - nothing
> serious, of course.
> 
> I and our users like our not-so-new-anymore Pilatus3 6M. It's a great
> detector in many ways. But, there is a lot of hype that this detector solves
> all-problem, for instance fine slicing that is claimed to be only possible
> with a pixel array detector. People get carried away and use 0.01 degree
> slices even as the mosaicity of their sample is, say, 0.3 degree. Slicing
> beyond 1/3 of the mosaicity will gain you very little - only more frames,
> more processing time.
> 
> This discourse is already drifting away from the original topic of the
> thread so I will comment on the other arguments  you made like resolution in
> a private e-mail.
> 
> Best regards,
> 
> Gerd
> 
> On 13.07.2017 14:00, Gerard Bricogne wrote:
> >Dear Gerd,
> >
> >  I can assure you that I have no shares in Dectris nor any
> >commecial connections with them. What I do have is a lot of still
> >vivid memories of CCD images, with their wooly point-spread function
> >that was affected by fine-grained spatial variability as well as by
> >irredicible inaccuracies in the geometric corrections required to try
> >and undo the distortions introduced by the fiber-optic taper. By
> >comparison the pixel-array detectors have a very regular structure, so
> >that slight deviations from exact registering of the modules can be
> >calibrated with high accuracy, making it possible to get very small
> >residuals between calculated and observed spot positions. That, I
> >certainly never saw with CCD images.
> >
> >  I do think that asking for the image width was a highly pertinent
> >question in this case, that had not been asked. As a specialist you
> >might know how to use a CCD to good effect in fine-slicing mode, but
> >it is amazing how many people there are still out there who are told
> >to use 0.5 or even 1.0 degree image widths.
> >
> >  Compensating the poor PSF of a CCD by fine slicing in the angular
> >dimension is a tall order. With a Pilatus at 350mm from the crystal,
> >the angular separation between 174-micron pixels is 0.5 milliradian.
> >To achieve that separation in the angular (rotation) dimension, the
> >equivalent image width would have to be 0.03 degree. For an EIGER the
> >numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.
> >
> >  Hence my advice, untainted by any commercial agenda :-) .
> >  With best wishes,
> >   Gerard.
> >
> >--
> >On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:
> >>Dear Gerard,
> >>
> >>you sound like a sales person for Dectris. Fine slicing is perfectly fine
> >>with CCD detectors - it takes a bit longer because of the step scan instead
> >>of continuous scan. The read noise issue is often overstated compared to the
> >>sample induced scatter background. If for fine slicing at 0.05 degree or
> >>less the diffraction peaks go too close to the read noise make a longer
> >>exposure - signal goes up, ratio signal to sample-induced-BG less, as for
> >>any fine slicing, same read noise.
> >>
> >>It would be helpful to analyze the dense spot packing along layer lines if
> >>we knew the wavelength and the sample-to-detector distance (assuming this is
> >>a 300 mm detector) and the rotation width - as you pointed out. That would
> >>help to distinguish between multiple crystals (my 

Re: [ccp4bb] Fine Phi Slicing

[Dyda]

I could be wrong here, but isn't the case that fine slicing is an option
with a CCD and a necessity on a PAD b/c of dead time and/or counter dynamic 
range 
issues?

(no current and/or former financial ties to any manufacturer)

Fred
***
Fred Dyda, Ph.D.   Phone:301-402-4496
Laboratory of Molecular BiologyFax: 301-496-0201
DHHS/NIH/NIDDK e-mail:fred.d...@nih.gov  
Bldg. 5. Room 303 
Bethesda, MD 20892-0560  URGENT message e-mail: 2022476...@mms.att.net
Google maps coords: 39.000597, -77.102102
http://www2.niddk.nih.gov/NIDDKLabs/IntramuralFaculty/DydaFred
***

[ccp4bb] Fine Phi Slicing

[Keller, Jacob]

I thought there was a new paper from the Pilatus people saying fine slicing is 
worth it even beyond the original 1/2 mosaicity rule?

I would think, actually, more gains would made by doing light exposures at, 
say, 1/3 mosaicity, collecting 360 deg, then shifting the detector in 2theta by 
a degree or two to shift uniformly the spots to new pixels, maybe accompanied 
by a kappa change. One would have to remember about the two-theta when 
processing, however!

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Gerd 
Rosenbaum
Sent: Thursday, July 13, 2017 3:40 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] weird diffraction pattern

Dear Gerard,

my "sound like a sales person" was meant as poking a little fun - nothing 
serious, of course.

I and our users like our not-so-new-anymore Pilatus3 6M. It's a great detector 
in many ways. But, there is a lot of hype that this detector solves 
all-problem, for instance fine slicing that is claimed to be only possible with 
a pixel array detector. People get carried away and use
0.01 degree slices even as the mosaicity of their sample is, say, 0.3 degree. 
Slicing beyond 1/3 of the mosaicity will gain you very little - only more 
frames, more processing time.

This discourse is already drifting away from the original topic of the thread 
so I will comment on the other arguments  you made like resolution in a private 
e-mail.

Best regards,

Gerd

On 13.07.2017 14:00, Gerard Bricogne wrote:
> Dear Gerd,
>
>   I can assure you that I have no shares in Dectris nor any 
> commecial connections with them. What I do have is a lot of still 
> vivid memories of CCD images, with their wooly point-spread function 
> that was affected by fine-grained spatial variability as well as by 
> irredicible inaccuracies in the geometric corrections required to try 
> and undo the distortions introduced by the fiber-optic taper. By 
> comparison the pixel-array detectors have a very regular structure, so 
> that slight deviations from exact registering of the modules can be 
> calibrated with high accuracy, making it possible to get very small 
> residuals between calculated and observed spot positions. That, I 
> certainly never saw with CCD images.
>
>   I do think that asking for the image width was a highly 
> pertinent question in this case, that had not been asked. As a 
> specialist you might know how to use a CCD to good effect in 
> fine-slicing mode, but it is amazing how many people there are still 
> out there who are told to use 0.5 or even 1.0 degree image widths.
>
>   Compensating the poor PSF of a CCD by fine slicing in the 
> angular dimension is a tall order. With a Pilatus at 350mm from the 
> crystal, the angular separation between 174-micron pixels is 0.5 milliradian.
> To achieve that separation in the angular (rotation) dimension, the 
> equivalent image width would have to be 0.03 degree. For an EIGER the 
> numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.
>
>   Hence my advice, untainted by any commercial agenda :-) .
>   
>   
>   With best wishes,
>   
>Gerard.
>
> --
> On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:
>> Dear Gerard,
>>
>> you sound like a sales person for Dectris. Fine slicing is perfectly 
>> fine with CCD detectors - it takes a bit longer because of the step 
>> scan instead of continuous scan. The read noise issue is often 
>> overstated compared to the sample induced scatter background. If for 
>> fine slicing at 0.05 degree or less the diffraction peaks go too 
>> close to the read noise make a longer exposure - signal goes up, 
>> ratio signal to sample-induced-BG less, as for any fine slicing, same read 
>> noise.
>>
>> It would be helpful to analyze the dense spot packing along layer 
>> lines if we knew the wavelength and the sample-to-detector distance 
>> (assuming this is a 300 mm detector) and the rotation width - as you 
>> pointed out. That would help to distinguish between multiple crystals 
>> (my guess) and lattice translocation disorder. Fine slicing is 
>> definitely needed to figure out what the diffraction pattern at 120 
>> degree could tell you in terms of strong anisotropy .
>>
>> Best regard.
>>
>> Gerd
>>
>> On 13.07.2017 08:20, Gerard Bricogne wrote:
>>> Dear Tang,
>>>
>>>   I noticed that your diffraction images seem to have been 
>>> recorded on a 3x3 CCD detector. With this type of detector, fine 
>>> slicing is often discouraged (because of the readout noise), and yet 
>>> with the two long cell axes you have, any form of thick (or only 
>>> semi-fine) slicing would result in spot overlaps.
>>>
>>>   What, then, was your image width? Would you have access to a 
>>> beamline with a Pilatus detector so that you could collect 
>>> fine-sliced data?
>>>
>>>   I would tend to agree with Herman that your crystals might be 
>>> cursed with lattice translocation 

Re: [ccp4bb] weird diffraction pattern

[Gerd Rosenbaum]

Dear Gerard,

   my "sound like a sales person" was meant as poking a little fun - 
nothing serious, of course.


I and our users like our not-so-new-anymore Pilatus3 6M. It's a great 
detector in many ways. But, there is a lot of hype that this detector 
solves all-problem, for instance fine slicing that is claimed to be only 
possible with a pixel array detector. People get carried away and use 
0.01 degree slices even as the mosaicity of their sample is, say, 0.3 
degree. Slicing beyond 1/3 of the mosaicity will gain you very little - 
only more frames, more processing time.


This discourse is already drifting away from the original topic of the 
thread so I will comment on the other arguments  you made like 
resolution in a private e-mail.


Best regards,

Gerd

On 13.07.2017 14:00, Gerard Bricogne wrote:

Dear Gerd,

  I can assure you that I have no shares in Dectris nor any
commecial connections with them. What I do have is a lot of still
vivid memories of CCD images, with their wooly point-spread function
that was affected by fine-grained spatial variability as well as by
irredicible inaccuracies in the geometric corrections required to try
and undo the distortions introduced by the fiber-optic taper. By
comparison the pixel-array detectors have a very regular structure, so
that slight deviations from exact registering of the modules can be
calibrated with high accuracy, making it possible to get very small
residuals between calculated and observed spot positions. That, I
certainly never saw with CCD images.

  I do think that asking for the image width was a highly pertinent
question in this case, that had not been asked. As a specialist you
might know how to use a CCD to good effect in fine-slicing mode, but
it is amazing how many people there are still out there who are told
to use 0.5 or even 1.0 degree image widths.

  Compensating the poor PSF of a CCD by fine slicing in the angular
dimension is a tall order. With a Pilatus at 350mm from the crystal,
the angular separation between 174-micron pixels is 0.5 milliradian.
To achieve that separation in the angular (rotation) dimension, the
equivalent image width would have to be 0.03 degree. For an EIGER the
numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.

  Hence my advice, untainted by any commercial agenda :-) .
  
  
  With best wishes,
  
   Gerard.


--
On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:

Dear Gerard,

you sound like a sales person for Dectris. Fine slicing is perfectly fine
with CCD detectors - it takes a bit longer because of the step scan instead
of continuous scan. The read noise issue is often overstated compared to the
sample induced scatter background. If for fine slicing at 0.05 degree or
less the diffraction peaks go too close to the read noise make a longer
exposure - signal goes up, ratio signal to sample-induced-BG less, as for
any fine slicing, same read noise.

It would be helpful to analyze the dense spot packing along layer lines if
we knew the wavelength and the sample-to-detector distance (assuming this is
a 300 mm detector) and the rotation width - as you pointed out. That would
help to distinguish between multiple crystals (my guess) and lattice
translocation disorder. Fine slicing is definitely needed to figure out what
the diffraction pattern at 120 degree could tell you in terms of strong
anisotropy .

Best regard.

Gerd

On 13.07.2017 08:20, Gerard Bricogne wrote:

Dear Tang,

  I noticed that your diffraction images seem to have been recorded
on a 3x3 CCD detector. With this type of detector, fine slicing is
often discouraged (because of the readout noise), and yet with the two
long cell axes you have, any form of thick (or only semi-fine) slicing
would result in spot overlaps.

  What, then, was your image width? Would you have access to a
beamline with a Pilatus detector so that you could collect fine-sliced
data?

  I would tend to agree with Herman that your crystals might be
cursed with lattice translocation disorder (LTD), but you might as
well try and put every chance of surviving this on your side by making
sure that you collect fine-sliced data. LTD plus thick slicing would
give you random data along the streaky direction. Use an image width
of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS
to process your images.


  Good luck!
Gerard

--
On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote:

Hi David,
Thanks for your comments. Although the spots become streaky in certain 
directions, I have processed the data in HKL3000 and imosflm, which suggested 
the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
the solved native structure, the molecular replacement solution was poor. So I 
ran Balbes with the split domains of the native structure. Although the 
solutions were also 

Re: [ccp4bb] weird diffraction pattern

[Edward A. Berry]

Thanks for spelling it out!
Would that advice still hold if the mosaicity of the crystal is 0.7 degrees?
(I know, I should go read the paper., but . . .)
eab

On 07/13/2017 03:00 PM, Gerard Bricogne wrote:

Dear Gerd,

  I can assure you that I have no shares in Dectris nor any
commecial connections with them. What I do have is a lot of still
vivid memories of CCD images, with their wooly point-spread function
that was affected by fine-grained spatial variability as well as by
irredicible inaccuracies in the geometric corrections required to try
and undo the distortions introduced by the fiber-optic taper. By
comparison the pixel-array detectors have a very regular structure, so
that slight deviations from exact registering of the modules can be
calibrated with high accuracy, making it possible to get very small
residuals between calculated and observed spot positions. That, I
certainly never saw with CCD images.

  I do think that asking for the image width was a highly pertinent
question in this case, that had not been asked. As a specialist you
might know how to use a CCD to good effect in fine-slicing mode, but
it is amazing how many people there are still out there who are told
to use 0.5 or even 1.0 degree image widths.

  Compensating the poor PSF of a CCD by fine slicing in the angular
dimension is a tall order. With a Pilatus at 350mm from the crystal,
the angular separation between 174-micron pixels is 0.5 milliradian.
To achieve that separation in the angular (rotation) dimension, the
equivalent image width would have to be 0.03 degree. For an EIGER the
numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.

  Hence my advice, untainted by any commercial agenda :-) .


  With best wishes,

   Gerard.

--
On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:

Dear Gerard,

you sound like a sales person for Dectris. Fine slicing is perfectly fine
with CCD detectors - it takes a bit longer because of the step scan instead
of continuous scan. The read noise issue is often overstated compared to the
sample induced scatter background. If for fine slicing at 0.05 degree or
less the diffraction peaks go too close to the read noise make a longer
exposure - signal goes up, ratio signal to sample-induced-BG less, as for
any fine slicing, same read noise.

It would be helpful to analyze the dense spot packing along layer lines if
we knew the wavelength and the sample-to-detector distance (assuming this is
a 300 mm detector) and the rotation width - as you pointed out. That would
help to distinguish between multiple crystals (my guess) and lattice
translocation disorder. Fine slicing is definitely needed to figure out what
the diffraction pattern at 120 degree could tell you in terms of strong
anisotropy .

Best regard.

Gerd

On 13.07.2017 08:20, Gerard Bricogne wrote:

Dear Tang,

  I noticed that your diffraction images seem to have been recorded
on a 3x3 CCD detector. With this type of detector, fine slicing is
often discouraged (because of the readout noise), and yet with the two
long cell axes you have, any form of thick (or only semi-fine) slicing
would result in spot overlaps.

  What, then, was your image width? Would you have access to a
beamline with a Pilatus detector so that you could collect fine-sliced
data?

  I would tend to agree with Herman that your crystals might be
cursed with lattice translocation disorder (LTD), but you might as
well try and put every chance of surviving this on your side by making
sure that you collect fine-sliced data. LTD plus thick slicing would
give you random data along the streaky direction. Use an image width
of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS
to process your images.


  Good luck!
Gerard

--
On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote:

Hi David,
Thanks for your comments. Although the spots become streaky in certain 
directions, I have processed the data in HKL3000 and imosflm, which suggested 
the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
the solved native structure, the molecular replacement solution was poor. So I 
ran Balbes with the split domains of the native structure. Although the 
solutions were also poor, I found the MR score of one solution above 35. On the 
basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. 
Unfortunately, there are four molecules in the asymmetric unit and it is to 
hard for me to reduce the Rfree further.

All best,

Chenjun Tang

Re: [ccp4bb] weird diffraction pattern

[Gerard Bricogne]

Dear Gerd,

 I can assure you that I have no shares in Dectris nor any
commecial connections with them. What I do have is a lot of still
vivid memories of CCD images, with their wooly point-spread function
that was affected by fine-grained spatial variability as well as by 
irredicible inaccuracies in the geometric corrections required to try
and undo the distortions introduced by the fiber-optic taper. By
comparison the pixel-array detectors have a very regular structure, so
that slight deviations from exact registering of the modules can be
calibrated with high accuracy, making it possible to get very small
residuals between calculated and observed spot positions. That, I
certainly never saw with CCD images.

 I do think that asking for the image width was a highly pertinent
question in this case, that had not been asked. As a specialist you
might know how to use a CCD to good effect in fine-slicing mode, but
it is amazing how many people there are still out there who are told
to use 0.5 or even 1.0 degree image widths.

 Compensating the poor PSF of a CCD by fine slicing in the angular
dimension is a tall order. With a Pilatus at 350mm from the crystal,
the angular separation between 174-micron pixels is 0.5 milliradian.
To achieve that separation in the angular (rotation) dimension, the
equivalent image width would have to be 0.03 degree. For an EIGER the
numbers become 75 microns, hence 0.21 milliradian i.e. 0.012 degree.

 Hence my advice, untainted by any commercial agenda :-) .
 
 
 With best wishes,
 
  Gerard.

--
On Thu, Jul 13, 2017 at 01:25:08PM -0500, Gerd Rosenbaum wrote:
> Dear Gerard,
> 
> you sound like a sales person for Dectris. Fine slicing is perfectly fine
> with CCD detectors - it takes a bit longer because of the step scan instead
> of continuous scan. The read noise issue is often overstated compared to the
> sample induced scatter background. If for fine slicing at 0.05 degree or
> less the diffraction peaks go too close to the read noise make a longer
> exposure - signal goes up, ratio signal to sample-induced-BG less, as for
> any fine slicing, same read noise.
> 
> It would be helpful to analyze the dense spot packing along layer lines if
> we knew the wavelength and the sample-to-detector distance (assuming this is
> a 300 mm detector) and the rotation width - as you pointed out. That would
> help to distinguish between multiple crystals (my guess) and lattice
> translocation disorder. Fine slicing is definitely needed to figure out what
> the diffraction pattern at 120 degree could tell you in terms of strong
> anisotropy .
> 
> Best regard.
> 
> Gerd
> 
> On 13.07.2017 08:20, Gerard Bricogne wrote:
> >Dear Tang,
> >
> >  I noticed that your diffraction images seem to have been recorded
> >on a 3x3 CCD detector. With this type of detector, fine slicing is
> >often discouraged (because of the readout noise), and yet with the two
> >long cell axes you have, any form of thick (or only semi-fine) slicing
> >would result in spot overlaps.
> >
> >  What, then, was your image width? Would you have access to a
> >beamline with a Pilatus detector so that you could collect fine-sliced
> >data?
> >
> >  I would tend to agree with Herman that your crystals might be
> >cursed with lattice translocation disorder (LTD), but you might as
> >well try and put every chance of surviving this on your side by making
> >sure that you collect fine-sliced data. LTD plus thick slicing would
> >give you random data along the streaky direction. Use an image width
> >of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS
> >to process your images.
> >
> >
> >  Good luck!
> >Gerard
> >
> >--
> >On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote:
> >>Hi David,
> >>Thanks for your comments. Although the spots become streaky in certain 
> >>directions, I have processed the data in HKL3000 and imosflm, which 
> >>suggested the C2221 space group (66.59, 246.95 and 210.17). The 
> >>Rmerge(0.14), completeness(94.8%), redundancy(4.6) are OK. When I tried to 
> >>run Balbes with the solved native structure, the molecular replacement 
> >>solution was poor. So I ran Balbes with the split domains of the native 
> >>structure. Although the solutions were also poor, I found the MR score of 
> >>one solution above 35. On the basis of this solution, I tried to run 
> >>Buccaneer and the Rfree could be 0.46. Unfortunately, there are four 
> >>molecules in the asymmetric unit and it is to hard for me to reduce the 
> >>Rfree further.
> >>
> >>All best,
> >>
> >>Chenjun Tang

Re: [ccp4bb] weird diffraction pattern

[Gerd Rosenbaum]

Dear Gerard,

you sound like a sales person for Dectris. Fine slicing is perfectly 
fine with CCD detectors - it takes a bit longer because of the step scan 
instead of continuous scan. The read noise issue is often overstated 
compared to the sample induced scatter background. If for fine slicing 
at 0.05 degree or less the diffraction peaks go too close to the read 
noise make a longer exposure - signal goes up, ratio signal to 
sample-induced-BG less, as for any fine slicing, same read noise.


It would be helpful to analyze the dense spot packing along layer lines 
if we knew the wavelength and the sample-to-detector distance (assuming 
this is a 300 mm detector) and the rotation width - as you pointed out. 
That would help to distinguish between multiple crystals (my guess) and 
lattice translocation disorder. Fine slicing is definitely needed to 
figure out what the diffraction pattern at 120 degree could tell you in 
terms of strong anisotropy .


Best regard.

Gerd

On 13.07.2017 08:20, Gerard Bricogne wrote:

Dear Tang,

  I noticed that your diffraction images seem to have been recorded
on a 3x3 CCD detector. With this type of detector, fine slicing is
often discouraged (because of the readout noise), and yet with the two
long cell axes you have, any form of thick (or only semi-fine) slicing
would result in spot overlaps.

  What, then, was your image width? Would you have access to a
beamline with a Pilatus detector so that you could collect fine-sliced
data?

  I would tend to agree with Herman that your crystals might be
cursed with lattice translocation disorder (LTD), but you might as
well try and put every chance of surviving this on your side by making
sure that you collect fine-sliced data. LTD plus thick slicing would
give you random data along the streaky direction. Use an image width
of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS
to process your images.


  Good luck!
  
Gerard


--
On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote:

Hi David,
Thanks for your comments. Although the spots become streaky in certain 
directions, I have processed the data in HKL3000 and imosflm, which suggested 
the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
the solved native structure, the molecular replacement solution was poor. So I 
ran Balbes with the split domains of the native structure. Although the 
solutions were also poor, I found the MR score of one solution above 35. On the 
basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. 
Unfortunately, there are four molecules in the asymmetric unit and it is to 
hard for me to reduce the Rfree further.

All best,

Chenjun Tang

[ccp4bb] 2nd CryoEM sample preparation Workshop 2-4th Oct 2017 @ DLS - deadline for applications 6th august

[Martin Walsh]

Dear all we are pleased to announce a 2nd workshop aimed at newcomers to CryoEM 
for prepration and initial characterization of samples for single particle 
CryoEM experiments– full details in link below

http://www.diamond.ac.uk/Home/Events/2017/cryoEM-Worskhop-October-2017.html

the workshop will provide near one-to-one tuition and hands-on preparation of 
cryoem sample grids taking full advantage of the convocation of titan krios’s 
at eBIC ( http://www.diamond.ac.uk/Science/Integrated-facilities/eBIC.html )

Martin


--
Martin A. Walsh,
Diamond Light Source Ltd
Diamond House,
Harwell Science & Innovation Campus
Didcot OX11 0DE
UK
T:+44 1235 778518
E: martin.wa...@diamond.ac.uk


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Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom

Re: [ccp4bb] weird diffraction pattern

[Rajesh Kumar]

Hi Chenjun,

Few suggestions from my side. Process the data with XDS and look into
acentric intensity distribution (it indicates any twinning possibility).
Run XTRIAGE and SFCHECK to understand any twinning or pseudo translation
possibilities. Twinning can confuse the program and suggest you smaller
unit cell with higher symmetry. Your images indicate longer cell axis.
If you need more help, please send me an email.

Thank you
Rajesh

 ---x
With regards
Rajesh K. Harijan, Ph.D.
Schramm Laboratory
Albert Einstein College of Medicine
1300 Morris Park Ave., Bronx, NY 10461
Tel: 718.430.2777  |  Fax: 718.430.8565




On Thu, Jul 13, 2017 at 3:56 AM, 唐晨骏 <0910010...@cau.edu.cn> wrote:

> hello everyone,
> I would like to seek your opinion on my crystal hits. I am working on a
> helicase
>
> of which the native structure is solved and the all solution statistics are
>
> fine. I am trying to crystallize and solve the structure of the
> protein/ssDNA
>
> complex. I recently got some hits from commercial screens using sitting
> drop
>
> vapor diffusion. After crystallization optimization, these crystals
> diffract
>
> weakly but to 3.2 Angstroms for the longer exposure time. However, when the
>
> crystals rotate between 120 degrees to 180 degrees, the spots become
> streaky
>
> (attached), no matter the crystals are hexagonal or flaky. I have tried to
>
> determine the structure by molecular replacement method, but the
> Rwork/Rfree
>
> values are huge (above 0.5) and can’t be reduced further. I suspect the
>
> obtained crystals quality and resulting processed statistics is the reason
> for
>
> the observed high Rwork/Rfree values. Are there any suggestions?
>
> All comments will be appreciated!
>
> Best,
> Chenjun Tang
>
>
>

Re: [ccp4bb] weird diffraction pattern

[Mark J van Raaij]

- which may well be caused by your cryo-protection or flash-cooling procedure.
I'd try to collect a few images at room temperature to see how good the 
crystals can be and if this procedure can be improved.
To prevent overlaps, it may help to find a way to collect the data with the 
crystal rotating around the most problematic cell axis, which tends to be the 
shortest in the crystal. Bent loops might be helpful.

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 




> On 13 Jul 2017, at 11:13, Keller, Jacob  wrote:
> 
> You've got multiple lattices--try seeding approaches mentioned in a 
> recent/current thread.
> 
> JPK
> 
> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Thursday, July 13, 2017 3:56 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] weird diffraction pattern
> 
> hello everyone, 
> I would like to seek your opinion on my crystal hits. I am working on a 
> helicase 
> 
> of which the native structure is solved and the all solution statistics are 
> 
> fine. I am trying to crystallize and solve the structure of the protein/ssDNA 
> 
> complex. I recently got some hits from commercial screens using sitting drop 
> 
> vapor diffusion. After crystallization optimization, these crystals diffract 
> 
> weakly but to 3.2 Angstroms for the longer exposure time. However, when the 
> 
> crystals rotate between 120 degrees to 180 degrees, the spots become streaky
> 
> (attached), no matter the crystals are hexagonal or flaky. I have tried to 
> 
> determine the structure by molecular replacement method, but the Rwork/Rfree 
> 
> values are huge (above 0.5) and can’t be reduced further. I suspect the 
> 
> obtained crystals quality and resulting processed statistics is the reason 
> for 
> 
> the observed high Rwork/Rfree values. Are there any suggestions?
> 
> All comments will be appreciated!
> 
> Best,
> Chenjun Tang
> 
> 

Re: [ccp4bb] crystallization optimization

[Patrick Shaw Stewart]

Vaheh, one thing you can do is set up a microbatch-under-oil plate, varying
protein against precipitant, with a nice big variation in both parameters.
Then set up exactly the same experiment but with say 20 nl of seed-stock
added to each drop.  Hey presto that gives you the one dotted and two solid
lines that I showed on my diagram, conveniently laid out on two plates.
 (Convenient also that you happen to have good automation at MedImmune  ; )

That kind of thing is very helpful for people who want to grow crystals for
xFEL or neutron diffraction - it may not be absolutely essential for
routine structure solution.  But I always find it helpful to bear in mind,
as Frank suggests.

Very best wishes, Patrick


On 13 July 2017 at 14:49, Oganesyan, Vaheh  wrote:

> What I’m about to write should be referred as a question rather than an
> answer. However, it might also help to find the answer to crystallization
> question discussed here.
>
> The good old crystallization diagram so far for me was something that I’d
> look after successful crystallization story and find in which direction my
> optimization went. Each condition in every screen is just a point at the
> diagram. Were on the diagram that point is situated you don’t know because
> the scales of X and Y axes are unknown. You can find those scales by
> deliberately setting up similar screens with diluted (or concentrated, or
> both) of protein sample (Y axis scale) and diluted (mostly) crystallization
> screen. This is the way I can make use of the crystallization diagram.
> Unfortunately, often we cannot spare enough protein to do so. In such cases
> going through different screens and looking for similar conditions sometime
> allows finding horizontal line on which your crystallization position
> should be. After this few optimization attempts at different protein
> concentrations may help finding position on the diagram and clues where to
> go.
>
>
>
> I hope what I just wrote makes sense. If there is a better way of using
> crystallization diagram I’d love to hear.
>
>
>
> *Regards,*
>
>
>
> *Vaheh Oganesyan*
>
> *www.medimmune.com *
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Philippe
> BENAS
> *Sent:* Thursday, July 13, 2017 12:47 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] crystallization optimization
>
>
>
> Dear all,
>
>
>
> I fully agree with all the suggestions, but it seems that no one has
> raised the issue of the solubility curve changes on the pH. If the dilution
> of the protein or precipating agent can indeed modify starting and the
> equilbrium points on the phase diagram, I would also suggest trying various
> pH as they can change a whole lot of the net protein charge, therefore the
> corresponding solubility curve and nucleation zone and hence the entire
> corresponding phase diagram (for more info PubMed search with "Madeleine
> Riess-Kautt" as keywords, a great scientist who dedidacted her career to
> understanding of the so-called Hofmeister series).
>
>
>
> All the best,
>
> Philippe
>
>
> --
>
>
>
> *Philippe BENAS, Ph.D. Dog in the manger "*Un importun survient qui
> trouble l'intimité, qui arrête l'expansion, qui glace le plaisir, -
> probablement comme un étranger tombant au milieu d'enfants en train de
> danser une ronde", Alfred Delvau, Dictionnaire de la langue verte (1866).
>
> *Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS*
> Faculté de Pharmacie, Université Paris Descartes
> Case 48
> Av, de l'Observatoire
> F-75270 PARIS cedex 06
> +33.1.5373.1599 <+33%201%2053%2073%2015%2099>
>
> E-mails: philippe.be...@parisdescartes.fr, philippe_be...@yahoo.fr
> URLs: http://lcrbw.pharmacie.univ-paris5.fr/
> 
> , http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18
> 
> --
>
>
>

Re: [ccp4bb] crystallization optimization

[Keller, Jacob]

I’d be curious whether anyone has ever published an empirical phase diagram 
that looks like the one posted here, since I think real experiments have a lot 
more free parameters than those included in the phase diagram.

JPK

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of 
Oganesyan, Vaheh
Sent: Thursday, July 13, 2017 9:50 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

What I’m about to write should be referred as a question rather than an answer. 
However, it might also help to find the answer to crystallization question 
discussed here.
The good old crystallization diagram so far for me was something that I’d look 
after successful crystallization story and find in which direction my 
optimization went. Each condition in every screen is just a point at the 
diagram. Were on the diagram that point is situated you don’t know because the 
scales of X and Y axes are unknown. You can find those scales by deliberately 
setting up similar screens with diluted (or concentrated, or both) of protein 
sample (Y axis scale) and diluted (mostly) crystallization screen. This is the 
way I can make use of the crystallization diagram. Unfortunately, often we 
cannot spare enough protein to do so. In such cases going through different 
screens and looking for similar conditions sometime allows finding horizontal 
line on which your crystallization position should be. After this few 
optimization attempts at different protein concentrations may help finding 
position on the diagram and clues where to go.

I hope what I just wrote makes sense. If there is a better way of using 
crystallization diagram I’d love to hear.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Philippe 
BENAS
Sent: Thursday, July 13, 2017 12:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

Dear all,

I fully agree with all the suggestions, but it seems that no one has raised the 
issue of the solubility curve changes on the pH. If the dilution of the protein 
or precipating agent can indeed modify starting and the equilbrium points on 
the phase diagram, I would also suggest trying various pH as they can change a 
whole lot of the net protein charge, therefore the corresponding solubility 
curve and nucleation zone and hence the entire corresponding phase diagram (for 
more info PubMed search with "Madeleine Riess-Kautt" as keywords, a great 
scientist who dedidacted her career to understanding of the so-called 
Hofmeister series).

All the best,
Philippe


Philippe BENAS, Ph.D.
Dog in the manger
"Un importun survient qui trouble l'intimité, qui arrête l'expansion, qui glace 
le plaisir, - probablement comme un étranger tombant au milieu d'enfants en 
train de danser une ronde", Alfred Delvau, Dictionnaire de la langue verte 
(1866).

Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
Faculté de Pharmacie, Université Paris Descartes
Case 48
Av, de l'Observatoire
F-75270 PARIS cedex 06
+33.1.5373.1599
E-mails: 
philippe.be...@parisdescartes.fr, 
philippe_be...@yahoo.fr
URLs: 
http://lcrbw.pharmacie.univ-paris5.fr/
 , 
http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18




De : Patrick Shaw Stewart >
À : CCP4BB@JISCMAIL.AC.UK
Envoyé le : Mercredi 12 juillet 2017 17h28
Objet : Re: [ccp4bb] crystallization optimization


Alun

I agree Frank's point is very interesting - and he intriguingly refers us to 
the phase diagram.

Is the point that Line A is longer than Line B ?

Best wishes

Patrick




[Inline images 2]






On 12 July 

Re: [ccp4bb] crystallization optimization

[Oganesyan, Vaheh]

What I’m about to write should be referred as a question rather than an answer. 
However, it might also help to find the answer to crystallization question 
discussed here.
The good old crystallization diagram so far for me was something that I’d look 
after successful crystallization story and find in which direction my 
optimization went. Each condition in every screen is just a point at the 
diagram. Were on the diagram that point is situated you don’t know because the 
scales of X and Y axes are unknown. You can find those scales by deliberately 
setting up similar screens with diluted (or concentrated, or both) of protein 
sample (Y axis scale) and diluted (mostly) crystallization screen. This is the 
way I can make use of the crystallization diagram. Unfortunately, often we 
cannot spare enough protein to do so. In such cases going through different 
screens and looking for similar conditions sometime allows finding horizontal 
line on which your crystallization position should be. After this few 
optimization attempts at different protein concentrations may help finding 
position on the diagram and clues where to go.

I hope what I just wrote makes sense. If there is a better way of using 
crystallization diagram I’d love to hear.

Regards,

Vaheh Oganesyan
www.medimmune.com

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Philippe 
BENAS
Sent: Thursday, July 13, 2017 12:47 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

Dear all,

I fully agree with all the suggestions, but it seems that no one has raised the 
issue of the solubility curve changes on the pH. If the dilution of the protein 
or precipating agent can indeed modify starting and the equilbrium points on 
the phase diagram, I would also suggest trying various pH as they can change a 
whole lot of the net protein charge, therefore the corresponding solubility 
curve and nucleation zone and hence the entire corresponding phase diagram (for 
more info PubMed search with "Madeleine Riess-Kautt" as keywords, a great 
scientist who dedidacted her career to understanding of the so-called 
Hofmeister series).


All the best,
Philippe


Philippe BENAS, Ph.D.
Dog in the manger
"Un importun survient qui trouble l'intimité, qui arrête l'expansion, qui glace 
le plaisir, - probablement comme un étranger tombant au milieu d'enfants en 
train de danser une ronde", Alfred Delvau, Dictionnaire de la langue verte 
(1866).

Laboratoire de Cristallographie et RMN Biologiques, UMR 8015 CNRS
Faculté de Pharmacie, Université Paris Descartes
Case 48
Av, de l'Observatoire
F-75270 PARIS cedex 06
+33.1.5373.1599
E-mails: 
philippe.be...@parisdescartes.fr, 
philippe_be...@yahoo.fr
URLs: 
http://lcrbw.pharmacie.univ-paris5.fr/
 , 
http://lcrbw.pharmacie.univ-paris5.fr/spip.php?article18




De : Patrick Shaw Stewart >
À : CCP4BB@JISCMAIL.AC.UK
Envoyé le : Mercredi 12 juillet 2017 17h28
Objet : Re: [ccp4bb] crystallization optimization


Alun

I agree Frank's point is very interesting - and he intriguingly refers us to 
the phase diagram.

Is the point that Line A is longer than Line B ?

Best wishes

Patrick




[Inline images 2]






On 12 July 2017 at 14:40, Alun R Coker 
> wrote:
Hi Everyone,
Franks point is really interesting. We routinely reduce the protein 
concentration when we see too many precipitated wells, but we never dilute the 
screen. Has anyone tried this?
All the best,
Alun

On 12/07/17 08:48, Frank von Delft wrote:
The point I was failing to make:  reducing either protein or precipitant 
concentration will indeed reduce 

[ccp4bb] Major PDB FTP archive update to V5.0

[John Berrisford]

*The model files in the PDB FTP archive have been updated to V5.0 of 
thePDBx/mmCIF dictionary 
.Both 
mmCIF and 
XML formats have been updated. These files were providedpreviously 
for 
the community to review and test. There is no change to PDB format files 
as PDB format is alegacy format 
. These, 
therefore, do not contain all of the remediated information. *


*

The changes to V5.0 include:


 *

   Improvedaudit
   
categories
   to capture details of changes to files down to the category level
   for entry revisions.

 *

   Better organized data content
   
and
   much more extensive metadata in model files for electron microscopy
   derived models.

 *

   Corrected source organism and sequence references for each sequence
   fragment in chimeric proteins.

 *

   Standardized data in several categories, including software name,
   detector name and detector type.

The complete list of changes can be found at thewwPDB website 
.


Questions regarding V5.0 data should be sent to deposit-h...@mail.wwpdb.org.

Regards

John

*

--
John Berrisford
PDBe
European Bioinformatics Institute (EMBL-EBI)
European Molecular Biology Laboratory
Wellcome Trust Genome Campus
Hinxton
Cambridge CB10 1SD UK
Tel: +44 1223 492529

http://www.pdbe.org
http://www.facebook.com/proteindatabank
http://twitter.com/PDBeurope

Re: [ccp4bb] Granada Crystallization Boxes?

[Diana Tomchick]

?If it weren't for the fact that trying to 3-D print translucent plastic is 
still problematic, you would think that this would be a perfect application of 
that technology.


Diana


**
Diana R. Tomchick
Professor
Departments of Biophysics and Biochemistry
University of Texas Southwestern Medical Center
5323 Harry Hines Blvd.
Rm. ND10.214A
Dallas, TX 75390-8816
diana.tomch...@utsouthwestern.edu
(214) 645-6383 (phone)
(214) 645-6353 (fax)

From: CCP4 bulletin board  on behalf of Gloria Borgstahl 

Sent: Thursday, July 13, 2017 7:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Granada Crystallization Boxes?

We probably would be interested in used ones.

On Thu, Jul 13, 2017 at 4:18 AM, Patrick Shaw Stewart 
> wrote:

Gloria, would you be interested in used ones?  I don't actually have any - we 
threw them out a few months ago, I've just checked - but someone might have 
some.

Best wishes, Patrick


On 11 July 2017 at 19:04, Gloria Borgstahl 
> wrote:
I have recently found out that these are no longer being manufactured or sold 
commercially.  But, as fortune has it, we have just been funded to fly some 
large quartz capillaries crystallization experimente up to the International 
Space Station for neutron crystallography.  Our experimental design is to fly 
the experiments in the Granada Crystallation Boxes!  NASA has already approved 
the 3x10x0.5 cm plastic Granada boxes for flights.   Does anyone have any in 
their lab supplies that they do not plan to use?  We would be willing to buy 
them from you!  Thanks, Gloria



--
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36




UT Southwestern


Medical Center



The future of medicine, today.

Re: [ccp4bb] weird diffraction pattern

[Gerard Bricogne]

Dear Tang,

 I noticed that your diffraction images seem to have been recorded
on a 3x3 CCD detector. With this type of detector, fine slicing is
often discouraged (because of the readout noise), and yet with the two
long cell axes you have, any form of thick (or only semi-fine) slicing
would result in spot overlaps.

 What, then, was your image width? Would you have access to a
beamline with a Pilatus detector so that you could collect fine-sliced
data?

 I would tend to agree with Herman that your crystals might be
cursed with lattice translocation disorder (LTD), but you might as
well try and put every chance of surviving this on your side by making
sure that you collect fine-sliced data. LTD plus thick slicing would
give you random data along the streaky direction. Use an image width
of at most 0.1 degree (0.05 would be better) on a Pilatus, and use XDS
to process your images.


 Good luck!
 
   Gerard

--
On Thu, Jul 13, 2017 at 01:21:02PM +0100, Tang Chenjun wrote:
> Hi David, 
> Thanks for your comments. Although the spots become streaky in certain 
> directions, I have processed the data in HKL3000 and imosflm, which suggested 
> the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
> completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
> the solved native structure, the molecular replacement solution was poor. So 
> I ran Balbes with the split domains of the native structure. Although the 
> solutions were also poor, I found the MR score of one solution above 35. On 
> the basis of this solution, I tried to run Buccaneer and the Rfree could be 
> 0.46. Unfortunately, there are four molecules in the asymmetric unit and it 
> is to hard for me to reduce the Rfree further.
> 
> All best,
> 
> Chenjun Tang

-- 

 ===
 * *
 * 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] 2nd CryoEM sample preparation Workshop 2-4th Oct 2017 @ DLS - deadline for applications 6th august

[Martin Walsh]

Dear all we are pleased to announce a 2nd workshop aimed at newcomers to CryoEM 
for prepration and initial characterization of samples for single particle 
CryoEM experiments– full details in link below

http://www.diamond.ac.uk/Home/Events/2017/cryoEM-Worskhop-October-2017.html

the workshop will provide near one-to-one tuition and hands-on preparation of 
cryoem sample grids taking full advantage of the convocation of titan krios’s 
at eBIC ( http://www.diamond.ac.uk/Science/Integrated-facilities/eBIC.html )

Martin

--
Martin A. Walsh,
Diamond Light Source Ltd
Diamond House,
Harwell Science & Innovation Campus
Didcot OX11 0DE
UK
T:+44 1235 778518
E: martin.wa...@diamond.ac.uk


-- 
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are not the intended addressee or an authorised recipient of the addressee 
please notify us of receipt by returning the e-mail and do not use, copy, 
retain, distribute or disclose the information in or attached to the e-mail.
Any opinions expressed within this e-mail are those of the individual and not 
necessarily of Diamond Light Source Ltd. 
Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments 
are free from viruses and we cannot accept liability for any damage which you 
may sustain as a result of software viruses which may be transmitted in or with 
the message.
Diamond Light Source Limited (company no. 4375679). Registered in England and 
Wales with its registered office at Diamond House, Harwell Science and 
Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom

Re: [ccp4bb] Granada Crystallization Boxes?

[Gloria Borgstahl]

We probably would be interested in used ones.

On Thu, Jul 13, 2017 at 4:18 AM, Patrick Shaw Stewart  wrote:

>
> Gloria, would you be interested in used ones?  I don't actually have any -
> we threw them out a few months ago, I've just checked - but someone might
> have some.
>
> Best wishes, Patrick
>
>
> On 11 July 2017 at 19:04, Gloria Borgstahl  wrote:
>
>> I have recently found out that these are no longer being manufactured or
>> sold commercially.  But, as fortune has it, we have just been funded to fly
>> some large quartz capillaries crystallization experimente up to the
>> International Space Station for neutron crystallography.  Our experimental
>> design is to fly the experiments in the Granada Crystallation Boxes!  NASA
>> has already approved the 3x10x0.5 cm plastic Granada boxes for flights.
>> Does anyone have any in their lab supplies that they do not plan to use?
>> We would be willing to buy them from you!  Thanks, Gloria
>>
>
>
>
> --
>  patr...@douglas.co.ukDouglas Instruments Ltd.
>  Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
>  Directors: Peter Baldock, Patrick Shaw Stewart
>
>  http://www.douglas.co.uk
>  Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
> <(877)%20225-2034>
>  Regd. England 2177994, VAT Reg. GB 480 7371 36
>

[ccp4bb] AW: [ccp4bb] weird diffraction pattern

[Herman . Schreuder]

Hi Chenjun Tang,

From the images you sent, it looks like your crystal suffers from lattice 
translocation disorder. See e.g.
http://onlinelibrary.wiley.com/doi/10.1107/S0907444909025153/epdf

Calculating a native Patterson and looking for strange peaks may give some 
hints what is going on. Depending on the nature of the disorder, you may or may 
not correct for it.

Best,
Herman


-Ursprüngliche Nachricht-
Von: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] Im Auftrag von Tang 
Chenjun
Gesendet: Donnerstag, 13. Juli 2017 14:21
An: CCP4BB@JISCMAIL.AC.UK
Betreff: Re: [ccp4bb] weird diffraction pattern

Hi David, 
Thanks for your comments. Although the spots become streaky in certain 
directions, I have processed the data in HKL3000 and imosflm, which suggested 
the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
the solved native structure, the molecular replacement solution was poor. So I 
ran Balbes with the split domains of the native structure. Although the 
solutions were also poor, I found the MR score of one solution above 35. On the 
basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. 
Unfortunately, there are four molecules in the asymmetric unit and it is to 
hard for me to reduce the Rfree further.

All best,

Chenjun Tang

Re: [ccp4bb] weird diffraction pattern

[Tang Chenjun]

Hi Jacob,
I have tried seeding approaches but it didn't help.

All best,

Chenjun Tang

Re: [ccp4bb] weird diffraction pattern

[Tang Chenjun]

Hi David, 
Thanks for your comments. Although the spots become streaky in certain 
directions, I have processed the data in HKL3000 and imosflm, which suggested 
the C2221 space group (66.59, 246.95 and 210.17). The Rmerge(0.14), 
completeness(94.8%), redundancy(4.6) are OK. When I tried to run Balbes with 
the solved native structure, the molecular replacement solution was poor. So I 
ran Balbes with the split domains of the native structure. Although the 
solutions were also poor, I found the MR score of one solution above 35. On the 
basis of this solution, I tried to run Buccaneer and the Rfree could be 0.46. 
Unfortunately, there are four molecules in the asymmetric unit and it is to 
hard for me to reduce the Rfree further.

All best,

Chenjun Tang

Re: [ccp4bb] Inquiry - active and non-active state in alternatives

[Dalibor Milic]

Dear Petr,

I observed both open and closed protein conformations discretely disordered in 
the crystals of tyrosine phenol-lyase. In some cases, I could model two ligand 
conformations in the disordered active site, but the polypeptide chain could be 
modeled in the prevalent conformation only (for example see: Milic et al., J. 
Am. Chem. Soc. 2011, 133, 16468-16476, http://dx.doi.org/10.1021/ja203361g). In 
two crystals soaked with two different ligands, there was approximately 50 % 
occupancy of each conformation, so I could model both open and closed 
polypeptide conformations (affecting 129 amino acid residues). Details about 
these two structures can be found in my doctoral thesis (D. Milic, Doctoral 
Thesis, University of Zagreb, 2010; 
https://bib.irb.hr/datoteka/472684.Dalibor_Milic_Doctoral_Thesis.pdf; Section 
4.4 "Discrete disorder of the whole protein domain in the TPL crystals"). Some 
of the interactions between protein subunits observed for the open conformation 
are disrupted after the closure of one of the protein subunits. To compensate 
for these broken interactions, new interactions are formed within the protein 
subunit in the closed conformation. I wrote more about that in my Thesis, page 
106.

Another example of such discrete disorder of a polypetide chain affecting 53 
residues is described in Lehwess-Litzmann et al, Nat. Chem. Biol. 2011, 7, 
678-684 (http://dx.doi.org/10.1038/nchembio.633), PDB ID 3S0C 
(http://dx.doi.org/10.2210/pdb3s0c/pdb), chain D.

I guess there are many more such structures. To find similar examples in PDB, 
one would need to search for entries where there are alternate location 
indicators present for an arbitrary long stretch of neighboring residues.

I hope my answer is helpful.

Best regards,
Dalibor

--
Dr. sc. Dalibor Milić
Heinrich-Heine-Universität Düsseldorf
Institut für Biochemische Pflanzenphysiologie

Gebäude/Raum: 26.02/01.74
Universitätsstraße 1
D-40225 Düsseldorf
Germany

Telephone: +49 211 81-13722
E-mail: dalibor.mi...@hhu.de
http://www.BioChemPlant.hhu.de

Re: [ccp4bb] crystallization optimization

[Anthony Savill]

And the Molecular Dimension s 3D screen.

Tony Savill
Molecular Dimensions Ltd.
Unit 6 Goodwin Business Park
Willie Snaith Road
Newmarket, Suffolk.
UK CB8 7SQ

Tel: +44 1638 561051
Fax: +44 1638 660674

Registered Office
Salisbury House
Station Road
Cambridge
CB1 2LA

Registered in England and Wales:
Registration number 1794026

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Debanu Das
Sent: 12 July 2017 22:38
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] crystallization optimization

Yes, we have done this (addition of water to dilute screen reagents in the 
well) and also try it now in some cases and in fact, this is also the rationale 
behind Hampton's Crystal Screen Lite.
Best,
Debanu
--
Debanu Das

On Jul 12, 2017, at 9:01 AM, Alun R Coker 
> wrote:

So, if we have a commercial 96 well screen where more than around 60% of the 
drops precipitate. It may be worth diluting the whole screen say (30ul screen 
and 20ul water in each well) and repeating . rather than diluting the 
protein.

Has anyone ever tried this?

All the best,

Alun

On 12/07/17 16:54, Frank von Delft wrote:

Yes, exactly.  Thanks for doing the Right Thing and posting the actual diagram.


On 12/07/2017 16:26, Patrick Shaw Stewart wrote:

Alun

I agree Frank's point is very interesting - and he intriguingly refers us to 
the phase diagram.

Is the point that Line A is longer than Line B ?

Best wishes

Patrick











On 12 July 2017 at 14:40, Alun R Coker 
> wrote:


Hi Everyone,

Franks point is really interesting. We routinely reduce the protein 
concentration when we see too many precipitated wells, but we never dilute the 
screen. Has anyone tried this?

All the best,

Alun

On 12/07/17 08:48, Frank von Delft wrote:

The point I was failing to make:  reducing either protein or precipitant 
concentration will indeed reduce nucleation, but often won't get you bigger or 
more single crystals:  it will just make the appearance of crystals less 
reliable.

The way to get big single reliable crystals is to increase protein and greatly 
reduce precipitant.

(Even better:  do seeding.  Like Vicky said.  Incredible how often people don't 
bother to do seeding, yet it solves so many problems.)

phx


On 12/07/2017 07:50, Vicky Tsirkone wrote:
Dear Frank,

I may see in the attached pic several nucleation points and a considerable 
amount of microcrystals. Based to my knowledge decreasing the concentration of 
the precipitant and/or the protein concentration would be a reasonable approach 
to refine the initial hits.
By checking the diagram as you correctly mentioned you may see that the fine 
tuning of protein and precipitant concetration may lead to the desirable result 
without reaching the precipitation zone.

Patrick just check your screens. Just a rule of thumb, if you see precipitation 
in the ~60% of your drops then you should definitely reduce the protein 
concentration.

ps dont forget to try the streak seeding, as well.

Have a nice day and again good luck.

Vicky

On Wed, Jul 12, 2017 at 8:50 AM, Frank von Delft 
> wrote:


Actually, you should try increasing the protein concentration - a lot.  But be 
prepared to drop the precipitant concentration to almost nothing (1 or 2% isn't 
"low").

To understand why, look at the phase diagram and what we assume about vapour 
diffusion.  (Which I'm assuming is what you're doing.)


On 12/07/2017 06:28, Vicky Tsirkone wrote:
Dear Patrick,

You may reduce the protein concentation, as well.
Another option could be the streak seeding by exploiting the drop of your 
initial condition.

Good luck,

V.T.

On Mon, Jul 10, 2017 at 7:17 PM, Patrick Shaw Stewart 
> wrote:


Microseed them into two or three random screens.

Search for MMS and rMMS online.

Good luck

Patrick




On 10 July 2017 at 15:47, Liuqing Chen 
<519198...@163.com> wrote:

hello everyone!
I get a condition (10% w/v PEG 6000, 100mm HEPES PH7.0) in which my protein 
grow small  needle like crystals, how can i optimize it to get bigger crystals? 
 the attach is the crystals  figure.
thanks in advance
sincerely
Liuqing Chen



--
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 
1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36







--

Dr Alun R. Coker

Senior Lecturer

Wolfson Institute for Biomedical Research

University College London

The Cruciform Building

London

WC1E 6BT



Tel: 020 7679 6703 Ext 46703

Web: www.ucl.ac.uk/pxmed

Re: [ccp4bb] Granada Crystallization Boxes?

[Patrick Shaw Stewart]

Gloria, would you be interested in used ones?  I don't actually have any -
we threw them out a few months ago, I've just checked - but someone might
have some.

Best wishes, Patrick


On 11 July 2017 at 19:04, Gloria Borgstahl  wrote:

> I have recently found out that these are no longer being manufactured or
> sold commercially.  But, as fortune has it, we have just been funded to fly
> some large quartz capillaries crystallization experimente up to the
> International Space Station for neutron crystallography.  Our experimental
> design is to fly the experiments in the Granada Crystallation Boxes!  NASA
> has already approved the 3x10x0.5 cm plastic Granada boxes for flights.
> Does anyone have any in their lab supplies that they do not plan to use?
> We would be willing to buy them from you!  Thanks, Gloria
>



-- 
 patr...@douglas.co.ukDouglas Instruments Ltd.
 Douglas House, East Garston, Hungerford, Berkshire, RG17 7HD, UK
 Directors: Peter Baldock, Patrick Shaw Stewart

 http://www.douglas.co.uk
 Tel: 44 (0) 148-864-9090US toll-free 1-877-225-2034
 Regd. England 2177994, VAT Reg. GB 480 7371 36

Re: [ccp4bb] weird diffraction pattern

[Keller, Jacob]

You've got multiple lattices--try seeding approaches mentioned in a 
recent/current thread.

JPK

-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
Sent: Thursday, July 13, 2017 3:56 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] weird diffraction pattern

hello everyone, 
I would like to seek your opinion on my crystal hits. I am working on a 
helicase 

of which the native structure is solved and the all solution statistics are 

fine. I am trying to crystallize and solve the structure of the protein/ssDNA 

complex. I recently got some hits from commercial screens using sitting drop 

vapor diffusion. After crystallization optimization, these crystals diffract 

weakly but to 3.2 Angstroms for the longer exposure time. However, when the 

crystals rotate between 120 degrees to 180 degrees, the spots become streaky

(attached), no matter the crystals are hexagonal or flaky. I have tried to 

determine the structure by molecular replacement method, but the Rwork/Rfree 

values are huge (above 0.5) and can’t be reduced further. I suspect the 

obtained crystals quality and resulting processed statistics is the reason for 

the observed high Rwork/Rfree values. Are there any suggestions?

All comments will be appreciated!

Best,
Chenjun Tang

Re: [ccp4bb] weird diffraction pattern

[Briggs, David C]

Hi,

I'd need to see more processing stats to figure out your data issues. Streaky 
spots in certain directions can be indicative of anisotropy and/or lattice 
disorders.

However, if your Rfree is above 0.5, it is likely that your molecular 
replacement solution is poor/bad/wrong.

HTH,

Dave

--
Dr David C Briggs
Hohenester Lab
Department of Life Sciences
Imperial College London
UK
http://about.me/david_briggs


From: CCP4 bulletin board  on behalf of 唐晨骏 
<0910010...@cau.edu.cn>
Sent: Thursday, July 13, 2017 8:56:03 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] weird diffraction pattern

hello everyone,
I would like to seek your opinion on my crystal hits. I am working on a helicase

of which the native structure is solved and the all solution statistics are

fine. I am trying to crystallize and solve the structure of the protein/ssDNA

complex. I recently got some hits from commercial screens using sitting drop

vapor diffusion. After crystallization optimization, these crystals diffract

weakly but to 3.2 Angstroms for the longer exposure time. However, when the

crystals rotate between 120 degrees to 180 degrees, the spots become streaky

(attached), no matter the crystals are hexagonal or flaky. I have tried to

determine the structure by molecular replacement method, but the Rwork/Rfree

values are huge (above 0.5) and can’t be reduced further. I suspect the

obtained crystals quality and resulting processed statistics is the reason for

the observed high Rwork/Rfree values. Are there any suggestions?

All comments will be appreciated!

Best,
Chenjun Tang

[ccp4bb] weird diffraction pattern

[唐晨骏]

hello everyone, 
I would like to seek your opinion on my crystal hits. I am working on a 
helicase 

of which the native structure is solved and the all solution statistics are 

fine. I am trying to crystallize and solve the structure of the protein/ssDNA 

complex. I recently got some hits from commercial screens using sitting drop 

vapor diffusion. After crystallization optimization, these crystals diffract 

weakly but to 3.2 Angstroms for the longer exposure time. However, when the 

crystals rotate between 120 degrees to 180 degrees, the spots become streaky

(attached), no matter the crystals are hexagonal or flaky. I have tried to 

determine the structure by molecular replacement method, but the Rwork/Rfree 

values are huge (above 0.5) and can’t be reduced further. I suspect the 

obtained crystals quality and resulting processed statistics is the reason for 

the observed high Rwork/Rfree values. Are there any suggestions?

All comments will be appreciated!

Best,
Chenjun Tang

Re: [ccp4bb] Coot: changing scroll map keybinding

[Jan Stransky]

So, thanks to pointing by Paul, I have found the keybinding definition 
in "python/template_key_bindings.py" on coot's github. It is called 
"Step scrollable map number" with suggested key "M"


Jan

On 07/12/2017 10:33 AM, Jan Stransky wrote:

Hi,

I believe, I have seen somewhere a keybinding, which cycle through 
maps attached to scroll wheel (basicaly moving the radio button 
"Scroll" in Display Manager). However, I can't find anywhere what key 
does that... Any ideas? Or do you know a coot function which does that?


Best regards,

Jan

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Tristan Croll]

*Ahem*

*ISOLDE

 
 
Tristan Croll
Research Fellow
Cambridge Institute for Medical Research
University of Cambridge CB2 0XY
 

 

> On 13 Jul 2017, at 08:02, Tristan Croll  wrote:
> 
> This is more-or-less exactly the task I'm building ISODE for. It's early 
> days, still quite rough around the edges and by no means complete, but if you 
> have a machine with a decent GPU running a modern Linux distro, then it's 
> easily available as a plugin to ChimeraX after installing the latest daily 
> build from https://www.rbvi.ucsf.edu/chimerax/download.html. Think of it as 
> an interactive real-space refinement environment, and you won't be far off. 
> This video is getting a little dated now, but should give you some idea of 
> what it can do: 
> https://drive.google.com/open?id=0B6uMjfjuw4k8aUdyTEJWeEJ1UnM. I'm currently 
> working on a longer, narrated demo/tutorial video showing all the latest 
> features (interactive position/rotamer/secondary structure assignment, a very 
> fun tool for shifting stretches in register, ...).
> 
> There are some caveats:
> - It doesn't yet do any building (adding or removing of atoms/residues)
> - It requires all residues to be complete including hydrogens
> - Currently only protein, nucleic acid and metal ions are supported (although 
> the metal ions need more work to be properly useful)
> 
> Let me know if it's of interest, and we can talk more offline.
> 
> Cheers,
> 
> Tristan
> 
>> On 2017-07-13 00:17, Rhys Grinter wrote:
>> Dear All,
>> I'm currently in the process of refining a low(ish) resolution
>> structure at 3.2 Ang, with a fair level of anisotropy. I processed the
>> data through the anisotropy server
>> (https://services.mbi.ucla.edu/anisoscale/ [1]), which elliptically
>> truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved the
>> maps and allowed me to trace the majority of the chain and build most
>> side chains.
>> The R-factors are reasonable (0.29 work and 0.35 free respectively).
>> but I'm having trouble with over fitting in refinement as I continue
>> to refine. What parameters/restraints would the community generally
>> use when refining this kind of structure? Additionally Refmac doesn't
>> seem to read the structure factors from the anisotropy server output
>> file properly, giving vastly inflated R values and strange looking
>> maps.
>> Cheers,
>> Rhys
>> --
>> Dr Rhys Grinter
>> Sir Henry Wellcome Fellow
>> Monash University
>> +61 (0)3 9902 9213
>> +61 (0)403 896 767
>> Links:
>> --
>> [1] https://services.mbi.ucla.edu/anisoscale/

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Tristan Croll]

This is more-or-less exactly the task I'm building ISODE for. It's early 
days, still quite rough around the edges and by no means complete, but 
if you have a machine with a decent GPU running a modern Linux distro, 
then it's easily available as a plugin to ChimeraX after installing the 
latest daily build from 
https://www.rbvi.ucsf.edu/chimerax/download.html. Think of it as an 
interactive real-space refinement environment, and you won't be far off. 
This video is getting a little dated now, but should give you some idea 
of what it can do: 
https://drive.google.com/open?id=0B6uMjfjuw4k8aUdyTEJWeEJ1UnM. I'm 
currently working on a longer, narrated demo/tutorial video showing all 
the latest features (interactive position/rotamer/secondary structure 
assignment, a very fun tool for shifting stretches in register, ...).


There are some caveats:
- It doesn't yet do any building (adding or removing of atoms/residues)
- It requires all residues to be complete including hydrogens
- Currently only protein, nucleic acid and metal ions are supported 
(although the metal ions need more work to be properly useful)


Let me know if it's of interest, and we can talk more offline.

Cheers,

Tristan

On 2017-07-13 00:17, Rhys Grinter wrote:

Dear All,

I'm currently in the process of refining a low(ish) resolution
structure at 3.2 Ang, with a fair level of anisotropy. I processed the
data through the anisotropy server
(https://services.mbi.ucla.edu/anisoscale/ [1]), which elliptically
truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved the
maps and allowed me to trace the majority of the chain and build most
side chains.

The R-factors are reasonable (0.29 work and 0.35 free respectively).
but I'm having trouble with over fitting in refinement as I continue
to refine. What parameters/restraints would the community generally
use when refining this kind of structure? Additionally Refmac doesn't
seem to read the structure factors from the anisotropy server output
file properly, giving vastly inflated R values and strange looking
maps.

Cheers,

Rhys

--

Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213
+61 (0)403 896 767

Links:
--
[1] https://services.mbi.ucla.edu/anisoscale/

Re: [ccp4bb] Stable Refinement as Low(ish) resolution

[Robbie Joosten]

In addition: pdb-redo automatically makes (homology-based) hydrogen bond 
restraints for Refmac at this resolution. You could gibe those a try.

Cheers,
Robbie

Sent from my Windows 10 phone

From: Sudipta Bhattacharyya
Sent: 13 July 2017 06:25
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Stable Refinement as Low(ish) resolution

Hi Rhys,

Along with what others have suggested, you can always try prosmart refinement. 
I found it really helpful in my cases.

Best of luck,
Sudipta.

Sudipta Bhattacharyya
Postdoctoral research fellow
University of Texas at Austin
Texas, USA.

On Jul 12, 2017 6:20 PM, "Rhys Grinter" 
> wrote:
Dear All,

I'm currently in the process of refining a low(ish) resolution structure at 3.2 
Ang, with a fair level of anisotropy. I processed the data through the 
anisotropy server (https://services.mbi.ucla.edu/anisoscale/), which 
elliptically truncated the data to 4.0, 3.8 and 3.2 Ang. This really improved 
the maps and allowed me to trace the majority of the chain and build most side 
chains.

The R-factors are reasonable (0.29 work and 0.35 free respectively). but I'm 
having trouble with over fitting in refinement as I continue to refine. What 
parameters/restraints would the community generally use when refining this kind 
of structure? Additionally Refmac doesn't seem to read the structure factors 
from the anisotropy server output file properly, giving vastly inflated R 
values and strange looking maps.

Cheers,

Rhys

--
Dr Rhys Grinter
Sir Henry Wellcome Fellow
Monash University
+61 (0)3 9902 9213
+61 (0)403 896 767