Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Bosch, Juergen]

This is a very interesting topic I have to say.

But what I missed in this discussion is the pain you go through when freezing 
in the cold room. As the name implies it's supposed to be cold (most of the 
times). But that's not too much of an issue as you can dress up accordingly. 
The problem I always had was freezing up of the  Hampton 
Magnetic Wand  and icing up towards your fingertips after some 
time when moisture from the cold room condenses and freezes. I hate wearing 
gloves when handling crystals so there was not much of a skin protection.

How do you guys solve this problem ?

Jürgen

..
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
http://lupo.jhsph.edu

On Jul 13, 2012, at 8:01 PM, 
mailto:mjvdwo...@netscape.net>> 
mailto:mjvdwo...@netscape.net>> wrote:

FYI, I live at 5500 ft elevation and the oxygen content of air is 21.5% here. 
The TOTAL amount of oxygen is less where I am than where you are because there 
is less air (lower density). Therefore my body has to do more work to get the 
same amount of oxygen to my cells.

OSHA has nothing to do with how much air we have and the sensors you can buy 
will tell you that the percentage is 21.5, even at 5500 feet.

No, nitrogen is not toxic. The question is if you can displace enough oxygen so 
you cannot absorb enough of it anymore. Unlike the experience you may have had 
when you hold year breath, you can breathe fine, there is lots of gas around 
you. Just not the right kind. We are not on the cusp of suffocating. On the 
other hand, paradoxically, oxygen is toxic. When you get too much of it, you 
will damage your CNS (not the program) and your eyes.

When premature babies are given oxygen so they can survive, their eyes may get 
damaged.

Too far removed from CCP4. This cannot happen in the cold room while harvesting 
crystals.

Mark







-Original Message-
From: Jacob Keller 
mailto:j-kell...@fsm.northwestern.edu>>
To: mjvdwoerd mailto:mjvdwo...@netscape.net>>
Cc: CCP4BB mailto:CCP4BB@jiscmail.ac.uk>>
Sent: Fri, Jul 13, 2012 5:10 pm
Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

The expansion ratio of liquid to gaseous nitrogen is approximately 1:700, that 
is, 1 liter of liquid becomes 700 liters of gas (at room temperature). When you 
are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide and 10 
(~30ft) meters long and you assume that it is poorly ventilated (i.e. no gas 
replacement at all), then you will have 3x6x10 = 180m3 volume of gas, which is 
180,000 liters. Air consists of 21% oxygen and is considered deficient if it 
goes down to 19.5%. OSHA recommends having monitors present in the case you 
might, in worst case scenario, reach 19.5%. Note: I don't know, but it seems 
unlikely that you are critically injured at 19.5%

How can this OSHA number be right? At fairly high altitude, say 2500 m, the 
partial pressure of O2 will be about 75% of that at sea level, and most are 
okay with it--so how can a drop from 21% to 19.5% have any importance? Is N2 
competing with O2, perhaps? Never heard of that. Can N2 really be a poison, 
such that we are constantly poised at the cusp of suffocation?

JPK











In this hypothetical case, you will have about 37800 liters of oxygen. If you 
displace some of it with 700 liters of nitrogen (you spilled one liter of 
liquid nitrogen), you will be down to 37100 liters, or approximately 20.5%. So, 
no worry.

If you have cryogenic storage for crystals (typically hundreds of liters) or 
one of those large tanks to back-fill your cryo-system, the story changes a 
lot. Large dewars or large tanks for filling do not normally fail, but when 
they do, you will be at risk. Humans cannot sense the lack of oxygen, you just 
feel sleepy and keel over. So in small rooms with large amounts of liquid 
nitrogen, it makes sense to have a monitor (and it does not make sense to be 
scared of the issue when you have a monitor).

Educationally:

For each safety risk in your environment you are supposed to do a calculation 
like the one above and consider how likely (or not) it is that this may happen 
to you and how bad it will be. Likelihood and severity multiply: if it is very 
unlikely (that a large nitrogen tank will rupture) but the consequence is 
severe (you die), then you need to think about how you can make sure that it 
never happens (install sensor).

Conclusion: if you only work with a small open dewar, then even in a small room 
it is highly unlikely to run out of oxygen.

It is an excellent idea to ask questions like you did. It should be expected 
that your institution has experts who can answer such questions, but some (like 
ours) do not and you have to figure it out yourself. It is a good

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[mjvdwoerd]

FYI, I live at 5500 ft elevation and the oxygen content of air is 21.5% here. 
The TOTAL amount of oxygen is less where I am than where you are because there 
is less air (lower density). Therefore my body has to do more work to get the 
same amount of oxygen to my cells.

OSHA has nothing to do with how much air we have and the sensors you can buy 
will tell you that the percentage is 21.5, even at 5500 feet.

No, nitrogen is not toxic. The question is if you can displace enough oxygen so 
you cannot absorb enough of it anymore. Unlike the experience you may have had 
when you hold year breath, you can breathe fine, there is lots of gas around 
you. Just not the right kind. We are not on the cusp of suffocating. On the 
other hand, paradoxically, oxygen is toxic. When you get too much of it, you 
will damage your CNS (not the program) and your eyes.

When premature babies are given oxygen so they can survive, their eyes may get 
damaged. 

Too far removed from CCP4. This cannot happen in the cold room while harvesting 
crystals.

Mark








-Original Message-
From: Jacob Keller 
To: mjvdwoerd 
Cc: CCP4BB 
Sent: Fri, Jul 13, 2012 5:10 pm
Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)



The expansion ratio of liquid to gaseous nitrogen is approximately 1:700, that 
is, 1 liter of liquid becomes 700 liters of gas (at room temperature). When you 
are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide and 10 
(~30ft) meters long and you assume that it is poorly ventilated (i.e. no gas 
replacement at all), then you will have 3x6x10 = 180m3 volume of gas, which is 
180,000 liters. Air consists of 21% oxygen and is considered deficient if it 
goes down to 19.5%. OSHA recommends having monitors present in the case you 
might, in worst case scenario, reach 19.5%. Note: I don't know, but it seems 
unlikely that you are critically injured at 19.5%




How can this OSHA number be right? At fairly high altitude, say 2500 m, the 
partial pressure of O2 will be about 75% of that at sea level, and most are 
okay with it--so how can a drop from 21% to 19.5% have any importance? Is N2 
competing with O2, perhaps? Never heard of that. Can N2 really be a poison, 
such that we are constantly poised at the cusp of suffocation?


JPK




















 

In this hypothetical case, you will have about 37800 liters of oxygen. If you 
displace some of it with 700 liters of nitrogen (you spilled one liter of 
liquid nitrogen), you will be down to 37100 liters, or approximately 20.5%. So, 
no worry.

If you have cryogenic storage for crystals (typically hundreds of liters) or 
one of those large tanks to back-fill your cryo-system, the story changes a 
lot. Large dewars or large tanks for filling do not normally fail, but when 
they do, you will be at risk. Humans cannot sense the lack of oxygen, you just 
feel sleepy and keel over. So in small rooms with large amounts of liquid 
nitrogen, it makes sense to have a monitor (and it does not make sense to be 
scared of the issue when you have a monitor).

Educationally:

For each safety risk in your environment you are supposed to do a calculation 
like the one above and consider how likely (or not) it is that this may happen 
to you and how bad it will be. Likelihood and severity multiply: if it is very 
unlikely (that a large nitrogen tank will rupture) but the consequence is 
severe (you die), then you need to think about how you can make sure that it 
never happens (install sensor). 

Conclusion: if you only work with a small open dewar, then even in a small room 
it is highly unlikely to run out of oxygen.

It is an excellent idea to ask questions like you did. It should be expected 
that your institution has experts who can answer such questions, but some (like 
ours) do not and you have to figure it out yourself. It is a good idea to 
document your concern, calculation and recommendation. 

Hope this helps.

Mark

PS: nirtogen vendors have excellent reference materials about these things.



-Original Message-
From: Radisky, Evette S., Ph.D., Ph.D. 
To: CCP4BB 
Sent: Fri, Jul 13, 2012 3:19 pm
Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)




Several have mentioned harvesting in the cold room to reduce evaporation.  I 
used to do this also as a postdoc, but I worried whether I risked nitrogen gas 
poisoning from liquid N2 boil-off, since the cold room did not seem very 
well-ventilated.  I’ve also hesitated to recommend it to trainees in my current 
lab for the same reason.  Does anyone have solid information on this?  I would 
like to be convinced that such fears are unfounded …
 
Evette S. Radisky, Ph.D. 
Assistant Professor 
Mayo Clinic Cancer Center 
Griffin Cancer Research Building, Rm 310 
4500 San Pablo Road 
Jacksonville, FL 32224 
(904) 953-6372 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
Rowlett
Sent: Thursday, July 12, 2012 2:11 PM
To: 

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Andrew Purkiss-Trew]

Quoting Jacob Keller :



The expansion ratio of liquid to gaseous nitrogen is approximately 1:700,
that is, 1 liter of liquid becomes 700 liters of gas (at room temperature).
When you are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide
and 10 (~30ft) meters long and you assume that it is poorly ventilated
(i.e. no gas replacement at all), then you will have 3x6x10 = 180m3 volume
of gas, which is 180,000 liters. Air consists of 21% oxygen and is
considered deficient if it goes down to 19.5%. OSHA recommends having
monitors present in the case you might, in worst case scenario, reach
19.5%. Note: I don't know, but it seems unlikely that you are critically
injured at 19.5%



How can this OSHA number be right? At fairly high altitude, say 2500 m, the
partial pressure of O2 will be about 75% of that at sea level, and most are
okay with it--so how can a drop from 21% to 19.5% have any importance? Is
N2 competing with O2, perhaps? Never heard of that. Can N2 really be a
poison, such that we are constantly poised at the cusp of suffocation?



Not N2 poisoning, but lack of Oxygen in the blood. At altitude, the  
body adjusts by breathing deeper and faster and people can become  
acclimatised (so giving rise to altitude training for athletes). The  
really dangerous levels, for a healthy adult, are a fair way below the  
19.5%. The UK generally seems to have O2 alarms set at 19% and maybe a  
second alarm at 17%.


More details are given on the OHSA website  
(http://www.osha.gov/pls/oshaweb/owadisp.show_document?p_id=25743&p_table=INTERPRETATIONS  found with a quick google) and on one of the UK Liquid Nitrogen supplier's websites  
(http://www.cryoservice.co.uk/oxygen_depletion.aspx)


Hope this helps,

Andrew Purkiss



This message was sent using IMP, the Internet Messaging Program.

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Jacob Keller]

>
> The expansion ratio of liquid to gaseous nitrogen is approximately 1:700,
> that is, 1 liter of liquid becomes 700 liters of gas (at room temperature).
> When you are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide
> and 10 (~30ft) meters long and you assume that it is poorly ventilated
> (i.e. no gas replacement at all), then you will have 3x6x10 = 180m3 volume
> of gas, which is 180,000 liters. Air consists of 21% oxygen and is
> considered deficient if it goes down to 19.5%. OSHA recommends having
> monitors present in the case you might, in worst case scenario, reach
> 19.5%. Note: I don't know, but it seems unlikely that you are critically
> injured at 19.5%
>

How can this OSHA number be right? At fairly high altitude, say 2500 m, the
partial pressure of O2 will be about 75% of that at sea level, and most are
okay with it--so how can a drop from 21% to 19.5% have any importance? Is
N2 competing with O2, perhaps? Never heard of that. Can N2 really be a
poison, such that we are constantly poised at the cusp of suffocation?

JPK












> In this hypothetical case, you will have about 37800 liters of oxygen. If
> you displace some of it with 700 liters of nitrogen (you spilled one liter
> of liquid nitrogen), you will be down to 37100 liters, or approximately
> 20.5%. So, no worry.
>
> If you have cryogenic storage for crystals (typically hundreds of liters)
> or one of those large tanks to back-fill your cryo-system, the story
> changes a lot. Large dewars or large tanks for filling do not normally
> fail, but when they do, you will be at risk. Humans cannot sense the lack
> of oxygen, you just feel sleepy and keel over. So in small rooms with large
> amounts of liquid nitrogen, it makes sense to have a monitor (and it does
> not make sense to be scared of the issue when you have a monitor).
>
> Educationally:
>
> For each safety risk in your environment you are supposed to do a
> calculation like the one above and consider how likely (or not) it is that
> this may happen to you and how bad it will be. Likelihood and severity
> multiply: if it is very unlikely (that a large nitrogen tank will rupture)
> but the consequence is severe (you die), then you need to think about how
> you can make sure that it never happens (install sensor).
>
> Conclusion: if you only work with a small open dewar, then even in a small
> room it is highly unlikely to run out of oxygen.
>
> It is an excellent idea to ask questions like you did. It should be
> expected that your institution has experts who can answer such questions,
> but some (like ours) do not and you have to figure it out yourself. It is a
> good idea to document your concern, calculation and recommendation.
>
> Hope this helps.
>
> Mark
>
> PS: nirtogen vendors have excellent reference materials about these things.
>
>
> -Original Message-
> From: Radisky, Evette S., Ph.D., Ph.D. 
> To: CCP4BB 
> Sent: Fri, Jul 13, 2012 3:19 pm
> Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt
> crystal)
>
>  Several have mentioned harvesting in the cold room to reduce
> evaporation.  I used to do this also as a postdoc, but I worried whether I
> risked nitrogen gas poisoning from liquid N2 boil-off, since the cold room
> did not seem very well-ventilated.  I’ve also hesitated to recommend it to
> trainees in my current lab for the same reason.  Does anyone have solid
> information on this?  I would like to be convinced that such fears are
> unfounded …
>
> Evette S. Radisky, Ph.D.
> Assistant Professor
> Mayo Clinic Cancer Center
> Griffin Cancer Research Building, Rm 310
> 4500 San Pablo Road
> Jacksonville, FL 32224
> (904) 953-6372
>  *From:* CCP4 bulletin board 
> [mailto:CCP4BB@JISCMAIL.AC.UK]
> *On Behalf Of *Roger Rowlett
> *Sent:* Thursday, July 12, 2012 2:11 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] cryo for high salt crystal
>
> We frequently crystallize one of our proteins and variants of it in
> 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol
> or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M)
> and ammonium sulfate (5.6 M) have enormous solubilities in water, so I
> would not expect cryoprotectant concentrations of glycerol or glucose to
> cause precipitation (We can save cryoprotectant solutions of at least 2 M
> ammonium sulfate indefinitely). How are you introducing cryprotectant? We
> use one of two methods:
>
>1. Fish the crystal out of the mother liquor and place into artificial
>mother liquor with the same composition as the well solution +
>cryoprotectant. For glycerol or other liquids, you have to make this from
>scratch. For glucose, we just weigh out 300 mg of glucose in a
>microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix
>well of course before use. Gentle heating in a block or sonication will
>help dissolve the glucose.
>2. Add 4 volumes of artificial mother liquor + 37.5% 

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[mjvdwoerd]

Hi Evette:

Technically:

The expansion ratio of liquid to gaseous nitrogen is approximately 1:700, that 
is, 1 liter of liquid becomes 700 liters of gas (at room temperature). When you 
are in a room that is 3 (~10ft) meters tall, 6 (~18ft) meters wide and 10 
(~30ft) meters long and you assume that it is poorly ventilated (i.e. no gas 
replacement at all), then you will have 3x6x10 = 180m3 volume of gas, which is 
180,000 liters. Air consists of 21% oxygen and is considered deficient if it 
goes down to 19.5%. OSHA recommends having monitors present in the case you 
might, in worst case scenario, reach 19.5%. Note: I don't know, but it seems 
unlikely that you are critically injured at 19.5%.

In this hypothetical case, you will have about 37800 liters of oxygen. If you 
displace some of it with 700 liters of nitrogen (you spilled one liter of 
liquid nitrogen), you will be down to 37100 liters, or approximately 20.5%. So, 
no worry.

If you have cryogenic storage for crystals (typically hundreds of liters) or 
one of those large tanks to back-fill your cryo-system, the story changes a 
lot. Large dewars or large tanks for filling do not normally fail, but when 
they do, you will be at risk. Humans cannot sense the lack of oxygen, you just 
feel sleepy and keel over. So in small rooms with large amounts of liquid 
nitrogen, it makes sense to have a monitor (and it does not make sense to be 
scared of the issue when you have a monitor).

Educationally:

For each safety risk in your environment you are supposed to do a calculation 
like the one above and consider how likely (or not) it is that this may happen 
to you and how bad it will be. Likelihood and severity multiply: if it is very 
unlikely (that a large nitrogen tank will rupture) but the consequence is 
severe (you die), then you need to think about how you can make sure that it 
never happens (install sensor). 

Conclusion: if you only work with a small open dewar, then even in a small room 
it is highly unlikely to run out of oxygen.

It is an excellent idea to ask questions like you did. It should be expected 
that your institution has experts who can answer such questions, but some (like 
ours) do not and you have to figure it out yourself. It is a good idea to 
document your concern, calculation and recommendation. 

Hope this helps.

Mark

PS: nirtogen vendors have excellent reference materials about these things.



-Original Message-
From: Radisky, Evette S., Ph.D., Ph.D. 
To: CCP4BB 
Sent: Fri, Jul 13, 2012 3:19 pm
Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)



Several have mentioned harvesting in the cold room to reduce evaporation.  I 
used to do this also as a postdoc, but I worried whether I risked nitrogen gas 
poisoning from liquid N2 boil-off, since the cold room did not seem very 
well-ventilated.  I’ve also hesitated to recommend it to trainees in my current 
lab for the same reason.  Does anyone have solid information on this?  I would 
like to be convinced that such fears are unfounded …
 
Evette S. Radisky, Ph.D. 
Assistant Professor 
Mayo Clinic Cancer Center 
Griffin Cancer Research Building, Rm 310 
4500 San Pablo Road 
Jacksonville, FL 32224 
(904) 953-6372 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
Rowlett
Sent: Thursday, July 12, 2012 2:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo for high salt crystal

 
We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M 
ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% 
glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium 
sulfate (5.6 M) have enormous solubilities in water, so I would not expect 
cryoprotectant concentrations of glycerol or glucose to cause precipitation (We 
can save cryoprotectant solutions of at least 2 M ammonium sulfate 
indefinitely). How are you introducing cryprotectant? We use one of two methods:

Fish the crystal out of the mother liquor and place into artificial mother 
liquor with the same composition as the well solution + cryoprotectant. For 
glycerol or other liquids, you have to make this from scratch. For glucose, we 
just weigh out 300 mg of glucose in a microcentrifuge tube and make to the 1.0 
mL mark with well solution. (Mix well of course before use. Gentle heating in a 
block or sonication will help dissolve the glucose.
Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the drop 
the crystals are in. You can do this all at once, or in stages, keeping the 
drop hydrated by placing the hanging drop back in the well between additions.

If your drops are drying out during crystal harvesting (very possible in dry 
conditions), you might try harvesting in the cold room, where evaporation is 
slower. We often have problems with crystal cracking and drop-drying in the 
winter months when the humidity is very low indoors. The cold room is usually 
humid enough and cold

Re: [ccp4bb] harvesting in cold room

[Zhijie Li]

RE: [ccp4bb] harvesting in cold roomDensity of LN2=807g/L, that's roughly 
807/28*22.4=645.6L gas at RT, 1 atm. At 4C the volume will be less: 
645.6/298*277=600L. An empty 2x2x2m cold room=8000L, containing 8000x21%=1680L 
oxygen. 
We normally won't let the whole liter of LN2 to evaporate when freezing 
crystals. But if someone leaves a liter of LN2 in a small cold room and forgets 
to take it out before it all evaporates, then it can become a potential hazard 
to next user. We have to consider that some labs also use cold room as storage 
place, that might also greatly reduce the volume of air inside.
I guess the case could be even worth with CO2, as CO2 gas is denser. If someone 
has a large bag of dry ice subliming in a closed space, then it can fill the 
space from bottom.
So, lighting a candle when work in cold room? I am not sure if the fire 
department will like it.



From: Green, Todd 
Sent: Friday, July 13, 2012 5:51 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] harvesting in cold room


I've noticed that pretty much every time there is an autofill of the dewars in 
the hutches of SSRL or APS, the oxygen sensors go off. but that is a small 
space and many liters of liquid nitrogen. I've frozen routinely in a small cold 
room with a liter or 2 of liquid nitrogen with no issue.


-Original Message-
From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Fri 7/13/2012 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] harvesting in cold room

How frequently do the sensors go off?

JPK

On Fri, Jul 13, 2012 at 4:37 PM, David Schuller  wrote:

>  On 07/13/12 17:29, Jacob Keller wrote:
>
> You probably already know this, but nitrogen is not at all
> poisonous--about 78% of the air is nitrogen. I guess you were probably
> worried about asphyxiation?
>
>
> We have oxygen sensors in our X-ray hutches for precisely that reason.
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
>


--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


This email was scanned with Mcafee's Anti-Virus appliance, but this
is no guarantee that no virus exists. You are asked to make sure you
have virus protection and that it is up to date.

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Radisky, Evette S., Ph.D.]

My cold room is also too small for comfort, but I think your method could work 
in a pinch. Thanks!

Evette Radisky, PhD
Mayo Clinic Cancer Center
Griffin Cancer Research Building
4500 San Pablo Road
Jacksonville, FL 32224
tel: 904-953-6372
fax: 904-953-0277
 

On Jul 13, 2012, at 5:52 PM, "tom.p...@csiro.au"  wrote:

> Hello Evette,
> 
> It will depend on the circumstances- how big is your cold room, how much 
> liquid N2 you have in there, etc. 
> You can actually calculate the percentage of N2 (or correspondingly, O2) in 
> the air if all of the liquid N2 were to boil off at once. 
> If the O2 goes below 20% it will feel like you are at elevation and if it 
> goes below 19% it isn't good (I believe most oxygen sensors used for this 
> kind of application alarm if it goes below 20% and then alarm strongly below 
> 19%). 
> As you, we generally avoid cryo-cooling crystals in the cold as we have a 
> small cold room and no real ventilation- just a blower. 
> If we use liquid N2 in there, we keep the door open and have someone stand 
> outside.  There is no warning with N2- you just fall unconscious. 
> 
> Best of luck,  tom
> 
> Tom Peat
> Biophysics Group
> CSIRO, CMSE
> 343 Royal Parade
> Parkville, VIC, 3052
> +613 9662 7304
> +614 57 539 419
> tom.p...@csiro.au
> 
> From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] On Behalf Of Radisky, 
> Evette S., Ph.D. [radisky.eve...@mayo.edu]
> Sent: Saturday, July 14, 2012 7:19 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] harvesting in cold room (was: cryo for high salt 
> crystal)
> 
> Several have mentioned harvesting in the cold room to reduce evaporation.  I 
> used to do this also as a postdoc, but I worried whether I risked nitrogen 
> gas poisoning from liquid N2 boil-off, since the cold room did not seem very 
> well-ventilated.  I’ve also hesitated to recommend it to trainees in my 
> current lab for the same reason.  Does anyone have solid information on this? 
>  I would like to be convinced that such fears are unfounded …
> 
> Evette S. Radisky, Ph.D.
> Assistant Professor
> Mayo Clinic Cancer Center
> Griffin Cancer Research Building, Rm 310
> 4500 San Pablo Road
> Jacksonville, FL 32224
> (904) 953-6372
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Roger 
> Rowlett
> Sent: Thursday, July 12, 2012 2:11 PM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] cryo for high salt crystal
> 
> We frequently crystallize one of our proteins and variants of it in 1.6-1.8 M 
> ammonium sulfate solutions. Cryoprotection with 25-30% glycerol or 25-30% 
> glucose does not cause precipitation of salts. Both KCl (4.6 M) and ammonium 
> sulfate (5.6 M) have enormous solubilities in water, so I would not expect 
> cryoprotectant concentrations of glycerol or glucose to cause precipitation 
> (We can save cryoprotectant solutions of at least 2 M ammonium sulfate 
> indefinitely). How are you introducing cryprotectant? We use one of two 
> methods:
> 
> 1.  Fish the crystal out of the mother liquor and place into artificial 
> mother liquor with the same composition as the well solution + 
> cryoprotectant. For glycerol or other liquids, you have to make this from 
> scratch. For glucose, we just weigh out 300 mg of glucose in a 
> microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix 
> well of course before use. Gentle heating in a block or sonication will help 
> dissolve the glucose.
> 2.  Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to the 
> drop the crystals are in. You can do this all at once, or in stages, keeping 
> the drop hydrated by placing the hanging drop back in the well between 
> additions.
> 
> If your drops are drying out during crystal harvesting (very possible in dry 
> conditions), you might try harvesting in the cold room, where evaporation is 
> slower. We often have problems with crystal cracking and drop-drying in the 
> winter months when the humidity is very low indoors. The cold room is usually 
> humid enough and cold enough to slow evaporation to allow crystal harvesting. 
> (I hate working in the meat locker, though.)
> 
> Cheers,
> 
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
> 
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
> 
> On 7/12/2012 12:55 PM, m zhang wrote:
> Hi Jim,
> 
> 25% is w/v. Thanks for the information. Will check the webinar.
> 
> Thanks,
> Min
> 
> From: jim.pflugr...@rigaku.com
> To: mzhang...@hotmail.com; 
> CCP4BB@JISCMAIL.AC.UK
> Subject: RE: [ccp4bb] cryo for high salt crystal
> Date: Tue, 10 Jul 2012 17:39:56 +
> Sucrose, sorbitol, Splenda, trehalose, etc, bu

Re: [ccp4bb] harvesting in cold room

[Edward A. Berry]

How low does O2 have to get to be dangerous?

To reduce the oxygen concentration by a factor of two, you would have to mix 
equal
volumes of air and nitrogen. Now since the nitrogen is coming off at about 70K,
assuming equal heat capacity for N2 and 80% N2, the temperature of that mixture
would be halfway between 4 C and 70 K, which would probably motivate one to
leave the area already.

However when I brought this up I was told that a surprisingly small decrease in
O2 pressure is enough to make you pass out- the 2-fold change I was considering
might be way beyond fatal. I guess the problem is that it is increased CO2,
and not decreased oxygen, that makes you feel breathless and start breathing 
hard.
All that nitrogen would keep the CO2 way down and you would go on calmly
working until you blacked out.

Green, Todd wrote:

I've noticed that pretty much every time there is an autofill of the dewars in 
the hutches
of SSRL or APS, the oxygen sensors go off. but that is a small space and many 
liters of
liquid nitrogen. I've frozen routinely in a small cold room with a liter or 2 
of liquid
nitrogen with no issue.


-Original Message-
From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Fri 7/13/2012 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] harvesting in cold room

How frequently do the sensors go off?

JPK

On Fri, Jul 13, 2012 at 4:37 PM, David Schuller  wrote:

 > On 07/13/12 17:29, Jacob Keller wrote:
 >
 > You probably already know this, but nitrogen is not at all
 > poisonous--about 78% of the air is nitrogen. I guess you were probably
 > worried about asphyxiation?
 >
 >
 > We have oxygen sensors in our X-ray hutches for precisely that reason.
 >
 > --
 > ===
 > All Things Serve the Beam
 > ===
 > David J. Schuller
 > modern man in a post-modern world
 > MacCHESS, Cornell University
 > schul...@cornell.edu
 >
 >


--
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


This email was scanned with Mcafee's Anti-Virus appliance, but this
is no guarantee that no virus exists. You are asked to make sure you
have virus protection and that it is up to date.

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Radisky, Evette S., Ph.D.]

Thanks much.  It helps a lot.

On Jul 13, 2012, at 5:31 PM, "Henry Bellamy"  wrote:

> liquid N2 expands about 600 fold to RT gas.  The minimum O2 concentration  is 
> 19%  (per OSHA I think) so if the amount of vaporized N2 is  greater than 2% 
> of the cold room volume you could have a problem.   One has to account for 
> the possibility that the Dewar will break or be tipped over and all the N2 
> will be vaporized at once. 
> HTH
> Henry Bellamy

Re: [ccp4bb] harvesting in cold room

[Green, Todd]

I've noticed that pretty much every time there is an autofill of the dewars in 
the hutches of SSRL or APS, the oxygen sensors go off. but that is a small 
space and many liters of liquid nitrogen. I've frozen routinely in a small cold 
room with a liter or 2 of liquid nitrogen with no issue.


-Original Message-
From: CCP4 bulletin board on behalf of Jacob Keller
Sent: Fri 7/13/2012 4:42 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] harvesting in cold room
 
How frequently do the sensors go off?

JPK

On Fri, Jul 13, 2012 at 4:37 PM, David Schuller  wrote:

>  On 07/13/12 17:29, Jacob Keller wrote:
>
> You probably already know this, but nitrogen is not at all
> poisonous--about 78% of the air is nitrogen. I guess you were probably
> worried about asphyxiation?
>
>
> We have oxygen sensors in our X-ray hutches for precisely that reason.
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
>


-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***


This email was scanned with Mcafee's Anti-Virus appliance, but this 
is no guarantee that no virus exists. You are asked to make sure you
have virus protection and that it is up to date.

Re: [ccp4bb] harvesting in cold room

[Jacob Keller]

How frequently do the sensors go off?

JPK

On Fri, Jul 13, 2012 at 4:37 PM, David Schuller  wrote:

>  On 07/13/12 17:29, Jacob Keller wrote:
>
> You probably already know this, but nitrogen is not at all
> poisonous--about 78% of the air is nitrogen. I guess you were probably
> worried about asphyxiation?
>
>
> We have oxygen sensors in our X-ray hutches for precisely that reason.
>
> --
> ===
> All Things Serve the Beam
> ===
>David J. Schuller
>modern man in a post-modern world
>MacCHESS, Cornell University
>schul...@cornell.edu
>
>


-- 
***
Jacob Pearson Keller
Northwestern University
Medical Scientist Training Program
email: j-kell...@northwestern.edu
***

Re: [ccp4bb] harvesting in cold room

[David Schuller]

On 07/13/12 17:29, Jacob Keller wrote:
You probably already know this, but nitrogen is not at all 
poisonous--about 78% of the air is nitrogen. I guess you were probably 
worried about asphyxiation?


We have oxygen sensors in our X-ray hutches for precisely that reason.

--
===
All Things Serve the Beam
===
   David J. Schuller
   modern man in a post-modern world
   MacCHESS, Cornell University
   schul...@cornell.edu

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Nat Echols]

On Fri, Jul 13, 2012 at 2:19 PM, Radisky, Evette S., Ph.D.
 wrote:
> Several have mentioned harvesting in the cold room to reduce evaporation.  I
> used to do this also as a postdoc, but I worried whether I risked nitrogen
> gas poisoning from liquid N2 boil-off, since the cold room did not seem very
> well-ventilated.  I’ve also hesitated to recommend it to trainees in my
> current lab for the same reason.  Does anyone have solid information on
> this?  I would like to be convinced that such fears are unfounded …

Aside from safety concerns, won't this reduce the solubility?  I hated
harvesting high-salt conditions in the cold room for exactly this
reason.

-Nat

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Jacob Keller]

You probably already know this, but nitrogen is not at all poisonous--about
78% of the air is nitrogen. I guess you were probably worried about
asphyxiation?

JPK


On Fri, Jul 13, 2012 at 4:19 PM, Radisky, Evette S., Ph.D. <
radisky.eve...@mayo.edu> wrote:

> Several have mentioned harvesting in the cold room to reduce evaporation.
> I used to do this also as a postdoc, but I worried whether I risked
> nitrogen gas poisoning from liquid N2 boil-off, since the cold room did not
> seem very well-ventilated.  I’ve also hesitated to recommend it to trainees
> in my current lab for the same reason.  Does anyone have solid information
> on this?  I would like to be convinced that such fears are unfounded …
>
> ** **
>
> Evette S. Radisky, Ph.D.
> Assistant Professor
> Mayo Clinic Cancer Center
> Griffin Cancer Research Building, Rm 310
> 4500 San Pablo Road
> Jacksonville, FL 32224
> (904) 953-6372 
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *Roger
> Rowlett
> *Sent:* Thursday, July 12, 2012 2:11 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] cryo for high salt crystal
>
> ** **
>
> We frequently crystallize one of our proteins and variants of it in
> 1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30% glycerol
> or 25-30% glucose does not cause precipitation of salts. Both KCl (4.6 M)
> and ammonium sulfate (5.6 M) have enormous solubilities in water, so I
> would not expect cryoprotectant concentrations of glycerol or glucose to
> cause precipitation (We can save cryoprotectant solutions of at least 2 M
> ammonium sulfate indefinitely). How are you introducing cryprotectant? We
> use one of two methods:
>
>1. Fish the crystal out of the mother liquor and place into artificial
>mother liquor with the same composition as the well solution +
>cryoprotectant. For glycerol or other liquids, you have to make this from
>scratch. For glucose, we just weigh out 300 mg of glucose in a
>microcentrifuge tube and make to the 1.0 mL mark with well solution. (Mix
>well of course before use. Gentle heating in a block or sonication will
>help dissolve the glucose.
>2. Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant to
>the drop the crystals are in. You can do this all at once, or in stages,
>keeping the drop hydrated by placing the hanging drop back in the well
>between additions.
>
> If your drops are drying out during crystal harvesting (very possible in
> dry conditions), you might try harvesting in the cold room, where
> evaporation is slower. We often have problems with crystal cracking and
> drop-drying in the winter months when the humidity is very low indoors. The
> cold room is usually humid enough and cold enough to slow evaporation to
> allow crystal harvesting. (I hate working in the meat locker, though.)
>
> Cheers,
>
> ___
> Roger S. Rowlett
> Gordon & Dorothy Kline Professor
> Department of Chemistry
> Colgate University
> 13 Oak Drive
> Hamilton, NY 13346
>
> tel: (315)-228-7245
> ofc: (315)-228-7395
> fax: (315)-228-7935
> email: rrowl...@colgate.edu
>
> ** **
>
> On 7/12/2012 12:55 PM, m zhang wrote:
>
> Hi Jim, 
>
> ** **
>
> 25% is w/v. Thanks for the information. Will check the webinar.
>
> ** **
>
> Thanks,
>
> Min
> --
>
> From: jim.pflugr...@rigaku.com
> To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK
> Subject: RE: [ccp4bb] cryo for high salt crystal
> Date: Tue, 10 Jul 2012 17:39:56 +
>
> Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25% (is that
> w/v or v/w?), try using 100% saturated in reservoir, 75% saturated in
> reservoir, or 50% saturated in reservoir.  You will have to TEST these.
>  See also this webinar on cryocrystallography which shows how to make these
> solutions: http://www.rigaku.com/node/1388 
>
> ** **
>
> You could also try high salt solutions with similar technique.
>
> ** **
>
> Good luck!
>
> ** **
>
> Jim
>
> ** **
>
> ** **
> --
>
> *From:* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m zhang [
> mzhang...@hotmail.com]
> *Sent:* Tuesday, July 10, 2012 11:28 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* [ccp4bb] cryo for high salt crystal
>
> regaentDear All, 
>
> ** **
>
> I am sure this question was discussed before. But I am wondering if anyone
> got the same experience as I do. 
>
> I got a crystal out of condition with 1M KCl, 1.4M Ammonium sulfate at
> pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, paraton-N oil,
> or ammonium sulfate itself: The problem is that all the cryo plus original
> reagents in the reservoir precipitate the salts out. And more serious
> problem is because of high salt in the condition, while I am trying to loop
> the crystal, both the drop and cryoprotectant drop form salt crystals (not
> sure it is KCl

Re: [ccp4bb] harvesting in cold room (was: cryo for high salt crystal)

[Radisky, Evette S., Ph.D.]

Several have mentioned harvesting in the cold room to reduce
evaporation.  I used to do this also as a postdoc, but I worried whether
I risked nitrogen gas poisoning from liquid N2 boil-off, since the cold
room did not seem very well-ventilated.  I've also hesitated to
recommend it to trainees in my current lab for the same reason.  Does
anyone have solid information on this?  I would like to be convinced
that such fears are unfounded ...

 

Evette S. Radisky, Ph.D. 
Assistant Professor 
Mayo Clinic Cancer Center 
Griffin Cancer Research Building, Rm 310 
4500 San Pablo Road 
Jacksonville, FL 32224 
(904) 953-6372 

From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
Roger Rowlett
Sent: Thursday, July 12, 2012 2:11 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] cryo for high salt crystal

 

We frequently crystallize one of our proteins and variants of it in
1.6-1.8 M ammonium sulfate solutions. Cryoprotection with 25-30%
glycerol or 25-30% glucose does not cause precipitation of salts. Both
KCl (4.6 M) and ammonium sulfate (5.6 M) have enormous solubilities in
water, so I would not expect cryoprotectant concentrations of glycerol
or glucose to cause precipitation (We can save cryoprotectant solutions
of at least 2 M ammonium sulfate indefinitely). How are you introducing
cryprotectant? We use one of two methods:

1.  Fish the crystal out of the mother liquor and place into
artificial mother liquor with the same composition as the well solution
+ cryoprotectant. For glycerol or other liquids, you have to make this
from scratch. For glucose, we just weigh out 300 mg of glucose in a
microcentrifuge tube and make to the 1.0 mL mark with well solution.
(Mix well of course before use. Gentle heating in a block or sonication
will help dissolve the glucose.
2.  Add 4 volumes of artificial mother liquor + 37.5% cryoprotectant
to the drop the crystals are in. You can do this all at once, or in
stages, keeping the drop hydrated by placing the hanging drop back in
the well between additions.

If your drops are drying out during crystal harvesting (very possible in
dry conditions), you might try harvesting in the cold room, where
evaporation is slower. We often have problems with crystal cracking and
drop-drying in the winter months when the humidity is very low indoors.
The cold room is usually humid enough and cold enough to slow
evaporation to allow crystal harvesting. (I hate working in the meat
locker, though.)

Cheers,

___
Roger S. Rowlett
Gordon & Dorothy Kline Professor
Department of Chemistry
Colgate University
13 Oak Drive
Hamilton, NY 13346

tel: (315)-228-7245
ofc: (315)-228-7395
fax: (315)-228-7935
email: rrowl...@colgate.edu

 

On 7/12/2012 12:55 PM, m zhang wrote:

Hi Jim, 

 

25% is w/v. Thanks for the information. Will check the webinar.

 

Thanks,

Min



From: jim.pflugr...@rigaku.com
To: mzhang...@hotmail.com; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] cryo for high salt crystal
Date: Tue, 10 Jul 2012 17:39:56 +

Sucrose, sorbitol, Splenda, trehalose, etc, but instead of 25%
(is that w/v or v/w?), try using 100% saturated in reservoir, 75%
saturated in reservoir, or 50% saturated in reservoir.  You will have to
TEST these.  See also this webinar on cryocrystallography which shows
how to make these solutions: http://www.rigaku.com/node/1388 

 

You could also try high salt solutions with similar technique.

 

Good luck!

 

Jim

 

 



From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of m
zhang [mzhang...@hotmail.com]
Sent: Tuesday, July 10, 2012 11:28 AM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] cryo for high salt crystal

regaentDear All, 

 

I am sure this question was discussed before. But I am wondering
if anyone got the same experience as I do. 

I got a crystal out of condition with 1M KCl, 1.4M Ammonium
sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose,
paraton-N oil, or ammonium sulfate itself: The problem is that all the
cryo plus original reagents in the reservoir precipitate the salts out.
And more serious problem is because of high salt in the condition, while
I am trying to loop the crystal, both the drop and cryoprotectant drop
form salt crystals (not sure it is KCl or ammonia sulfate) significantly
and very quickly, that cause my crystal dissolved. My crystal doesn't
seem to survive paraton-N oil. Does anyone here have similiar case? any
suggestion will be appreciated.

 

Thanks,

Min

 

Re: [ccp4bb] Rigid body refinement with phenix using EM maps.

[Filip Van Petegem]

Hello Wojtek,

a more straightforward approach is to use the rigid body refinement
implemented in Situs. http://situs.biomachina.org/fguide.html

After placing your pdb into the map via the colores program, you can refine
the positions of individual portions using collage, by splitting your pdb
into separate pdb files for each rigid body.   No need to do any
conversion. All you need is the pdb and the EM map downloaded from the
EMDB.  All are very simple to run and the documentation is very good.

Regards,


Filip

-- 
Filip Van Petegem, PhD
Associate Professor
The University of British Columbia
Dept. of Biochemistry and Molecular Biology
2350 Health Sciences Mall - Rm 2.356
Vancouver, V6T 1Z3

phone: +1 604 827 4267
email: filip.vanpete...@gmail.com
http://crg.ubc.ca/VanPetegem/


On Fri, Jul 13, 2012 at 8:28 AM, Wojtek Potrzebowski  wrote:

> Dear all,
> I want to refine a model of the complex that consists of rigid subunits
> using an electron microscopy electron density map.
> I've tried to use phenix for this purpose. Thus I have to back-FT the
> electron density map to a mtz reflection file.
> I've experienced problem using sfall for this purpose, but cinvfft has
> done a job (I can read a resulting file with mtzdmp).
> However phenix doesn't read this file properly (data labels box is empty).
> Can anyone help me on this, please?
> I am looking forward to hearing from you,
> Wojtek
>

Re: [ccp4bb] Rigid body refinement with phenix using EM maps.

[Pavel Afonine]

Hi Wojtek,

you need to convert your EM map into a reflection file (for example, cns or
mtz formatted, format doesn't matter). This reflection file should contain
"Fobs" - the amplitudes of Fourier map coefficients and phases
corresponding to your EM map. The phases should be presented as
Hendrickson-Lattmann coefficients (in this case only A and B will be
non-zero, and C=D=0). Them phenix.refine should deal with this all right.

I think the problem you face stems from the fact that when you convert your
EM map into a reflection file the "Fobs" you get are complex values (or
pairs: amplitude Fobs and phase PHobs), which phenix.refine would not take
as observations.

If you send me the file (off-list !) I may offer more comments.

Pavel

On Fri, Jul 13, 2012 at 8:28 AM, Wojtek Potrzebowski  wrote:

> Dear all,
> I want to refine a model of the complex that consists of rigid subunits
> using an electron microscopy electron density map.
> I've tried to use phenix for this purpose. Thus I have to back-FT the
> electron density map to a mtz reflection file.
> I've experienced problem using sfall for this purpose, but cinvfft has
> done a job (I can read a resulting file with mtzdmp).
> However phenix doesn't read this file properly (data labels box is empty).
> Can anyone help me on this, please?
> I am looking forward to hearing from you,
> Wojtek
>

Re: [ccp4bb] Rigid body refinement with phenix using EM maps.

[Bruno KLAHOLZ]

Dear Wojtek,

sounds like an mtz format issue.
Can you read it in with coot?
Maybe Pavel or others have an idea?

Bruno


-Message d'origine-
De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de Wojtek 
Potrzebowski
Envoyé : Friday, July 13, 2012 6:51 PM
À : CCP4BB@JISCMAIL.AC.UK
Objet : Re: [ccp4bb] Rigid body refinement with phenix using EM maps.

Dear Bruno,
Thank you very much for your quick response!
I've managed to generate mtz file (I've only needed to add "set spacegroup P1" 
while running sftools).
However, the problem remains.
I am not sure if this is the case but Phenix reads columns 1 and 2 as "Map 
coefs".
Do you have any clue?
Best regards,
Wojtek



On 07/13/2012 05:53 PM, Bruno KLAHOLZ wrote:
> Dear Wojtek,
>
> you can try something of this type to get the EM map converted into an hkl 
> list and mtz file with I's and some sigma I's estimated to 1/10 of the I's 
> (the latter is required for running fittings/refinements etc.).
>
> Best,
>
> Bruno
>
>
>
> /usr/local/rave/rave_linux/lx_mapman< re m2 name_40-8A.ccp4 ccp4
> no m2
> uvw
> m2
> 3
> 1
> 2
> write m2 name_40-8A-uvw.ccp4 ccp4
> quit
> EOF
>
> /usr/local/ccp4-version/bin/sftools< MAPIN name_40-8A-uvw.ccp4 map
> MAP2SF
> calc q col sigma = col 1 10 /
> CALC I col test = rfree(0.05)
> WRITE name_40-8A-uvw.fobs col 1 3 2 4
> xpl
> FOBS
> PHASE
> write
> name_40-8A-uvw.mtz
> quit
>
>
>
>
> -Message d'origine-
> De : CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] De la part de 
> Wojtek Potrzebowski Envoyé : Friday, July 13, 2012 5:28 PM À : 
> CCP4BB@JISCMAIL.AC.UK Objet : [ccp4bb] Rigid body refinement with 
> phenix using EM maps.
>
> Dear all,
> I want to refine a model of the complex that consists of rigid subunits using 
> an electron microscopy electron density map.
> I've tried to use phenix for this purpose. Thus I have to back-FT the 
> electron density map to a mtz reflection file.
> I've experienced problem using sfall for this purpose, but cinvfft has done a 
> job (I can read a resulting file with mtzdmp).
> However phenix doesn't read this file properly (data labels box is empty).
> Can anyone help me on this, please?
> I am looking forward to hearing from you, Wojtek
>
> ##
> #
> Dr. Bruno P. Klaholz
> Department of Integrated Structural Biology Institute of Genetics and 
> of Molecular and Cellular Biology IGBMC - UMR 7104 - U 964 1, rue 
> Laurent Fries BP 10142
> 67404 ILLKIRCH CEDEX
> FRANCE
> http://www.igbmc.fr/
> http://igbmc.fr/Klaholz
>
>
>
>

Re: [ccp4bb] Rigid body refinement with phenix using EM maps.

[Wojtek Potrzebowski]

Dear Bruno,
Thank you very much for your quick response!
I've managed to generate mtz file (I've only needed to add "set 
spacegroup P1" while running sftools).

However, the problem remains.
I am not sure if this is the case but Phenix reads columns 1 and 2 as 
"Map coefs".

Do you have any clue?
Best regards,
Wojtek



On 07/13/2012 05:53 PM, Bruno KLAHOLZ wrote:

Dear Wojtek,

you can try something of this type to get the EM map converted into an hkl list 
and mtz file with I's and some sigma I's estimated to 1/10 of the I's (the 
latter is required for running fittings/refinements etc.).

Best,

Bruno



/usr/local/rave/rave_linux/lx_mapman

Re: [ccp4bb] offtopic: AKTA prime

[Phoebe Rice]

If its old and out of warrenty, see if you have a local shop that can do it.  
We found the guys in physics here are great with such things (and a lot cheaper 
than official company repair guys).

=
Phoebe A. Rice
Dept. of Biochemistry & Molecular Biology
The University of Chicago
phone 773 834 1723
http://bmb.bsd.uchicago.edu/Faculty_and_Research/01_Faculty/01_Faculty_Alphabetically.php?faculty_id=123
http://www.rsc.org/shop/books/2008/9780854042722.asp


 Original message 
>Date: Thu, 12 Jul 2012 21:28:25 -0700
>From: CCP4 bulletin board  (on behalf of aaleshin 
>)
>Subject: Re: [ccp4bb] offtopic: AKTA prime  
>To: CCP4BB@JISCMAIL.AC.UK
>
> it is not cheap - probably $500
>
>   When we purchased the pump, it was below $300 (a
>   year ago). Replacement took ~4 hours, but you must
>   like doing it. You'll have to disassemble almost
>   entire pump module, so make pictures of each step
>   and mark tubings ends with labels.
>   Alex
>   On Jul 12, 2012, at 6:28 PM, Jacqueline Vitali
>   wrote:
>
> Yes, it has happened to me more than once.  It is
> not a good pump.  Acta Prime Plus has a better
> one.  They still have parts.  Call GE technical
> support and they will tell you what to do. 
>
> The part costs some money (it is not cheap -
> probably $500).  I think the Akta Prime will keep
> on running as long as you maintain it.  They can
> come to your lab to do the job for you but I do
> not know how much it is per hour.  This team is
> called labcrew and they are very good.
>
> I would call them and try to arrange with them. 
>
> Make sure you have your solutions elevated above
> the pump.  Even though it is a pump you need to
> help it.  Also do not use viscous solutions with
> it.  (I do not use glycerol because of the pump). 
> Finally clean it up with 20% ethanol after each
> use to avoid mold. 
>
> On Thu, Jul 12, 2012 at 5:09 PM, Peter Hsu
>  wrote:
>
>   Hi all,
>
>We've got an old Akta prime that I think is on
>   the verge of kicking it. Hearing some high
>   pitched sounds coming from the pump when we're
>   running it. Line A seems clogged and makes a
>   thudding noise when we try to do a pump wash
>   through that line. Does anyone have any
>   experience w/fixing one/replacing the pump? Or
>   any idea how much it's going to set us back to
>   get it fixed?
>
>   Sorry for the off topic question/thanks in
>   advance for any thoughts and ideas.
>   Peter

Re: [ccp4bb] Rigid body refinement with phenix using EM maps.

[Bruno KLAHOLZ]

Dear Wojtek,

you can try something of this type to get the EM map converted into an hkl list 
and mtz file with I's and some sigma I's estimated to 1/10 of the I's (the 
latter is required for running fittings/refinements etc.).

Best,

Bruno



/usr/local/rave/rave_linux/lx_mapman 

[ccp4bb] Rigid body refinement with phenix using EM maps.

[Wojtek Potrzebowski]

Dear all,
I want to refine a model of the complex that consists of rigid subunits 
using an electron microscopy electron density map.
I've tried to use phenix for this purpose. Thus I have to back-FT the 
electron density map to a mtz reflection file.
I've experienced problem using sfall for this purpose, but cinvfft has 
done a job (I can read a resulting file with mtzdmp).

However phenix doesn't read this file properly (data labels box is empty).
Can anyone help me on this, please?
I am looking forward to hearing from you,
Wojtek

Re: [ccp4bb] strict structure based alignment

[David Cobessi]

Dear Christian,
PDBefold superimposes the structures and generates the sequence
alignment in fasta(??) format. You can then read this file in Multialign
for example to get the sequence alignment and then add the secondary
structures to the sequence alignment using ESPript for example.
David

On 07/13/2012 04:30 PM, Christian Roth wrote:
> Dear all,
>
> I want align a couple or protein structures by secondary structure matching 
> to 
> one target and want get a kind of aminoacid alignment file e.g. what residue 
> fit 
> the other, without adjustments due to sequence based alignments. 
> I tried Strap, but as far as I understood it, it takes also the sequence into 
> account. I tried also Rapido, but this does only a pairwise comparison. 
> Superpose does align it nicely (ccp4 based or Coot based) but there seems to 
> be no option to print the sequence alignment in a file and it is again  just 
> a 
> pairwise comparison .
> Is there an other program which does something similar?
>
> Best Regards
>
> Christian 
>


-- 
David Cobessi
Institut de Biologie Structurale
41, Rue Jules Horowitz
38027 Grenoble Cedex-1, France
Tel:33(0)438789613
33(0)608164340
Fax:33(0)438785122 

Re: [ccp4bb] Chiral volume outliers SO4

[Boaz Shaanan]

Hi Robbie,

Interesting! In the article you referred to there isn't a single mention of the 
word "chiral" or its derivatives. They talk more about strained ligands, a 
problem  which is addressed by the Grade operation and similar tools, and wrong 
geometry altogether, which we all agree should be cured. As Ian pointed out 
"computational" chirality (Pasteur must be spinning in his grave  right now) is 
addressed nicely in the cif file (chirality "both").

My 2p.

  Boaz


Boaz Shaanan, Ph.D.
Dept. of Life Sciences
Ben-Gurion University of the Negev
Beer-Sheva 84105
Israel

E-mail: bshaa...@bgu.ac.il
Phone: 972-8-647-2220  Skype: boaz.shaanan
Fax:   972-8-647-2992 or 972-8-646-1710






From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Robbie Joosten 
[robbie_joos...@hotmail.com]
Sent: Friday, July 13, 2012 5:18 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] Chiral volume outliers SO4

Hi Andrew,

Indeed, provided the atom labeling is correct the chiral volume restraint
actually says whether the groups on the ring a axial or equatorial. The cif
files do not define that any other way, so without the restraint the
description of the molecule is ambiguous. Note that the chirality restraint
only describes the hand, the actual chiral volume is calculated from the
angle restraints.

In the more general case I think people are talking different languages.
There is chemical chirality (the real deal) and computational chirality. In
refinement and structure comparison the latter does matter (again, see
Dale's post). Problems with restraints are one of the reasons why there are
relatively many problems with ligands in the PDB (e.g.
http://www.springerlink.com/content/eu28538101v7v885/).

Cheers,
Robbie

> -Original Message-
> From: Andrew Purkiss [mailto:a.purk...@mail.cryst.bbk.ac.uk]
> Sent: Friday, July 13, 2012 14:09
> To: Robbie Joosten
> Cc: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Chiral volume outliers SO4
>
> Dear Robbie and ccp4bb,
>
> Is 1N1 not a different type of problem though, where a chirality restraint
is
> valid and so the atom labelling is important?
>
> Are you saying that we should always use the cif dictionary, even when
there
> are errors? Surely in the SO4 case, as Ian said, it is better to remove
the
> unnecessary restraint altogether. The sulphate cif file seems to have had
this
> bug introduced in the current CCP4 version.
>
> Andrew
>
> On Fri, 2012-07-13 at 12:54 +0200, Robbie Joosten wrote:
> > Dear All,
> >
> > I'm with Dale on this one. It's better to have a standard and roll
> > with it, than allow for ambiguity. The discussion just happened to
> > start with a rather silly example as Tim pointed out. The ligand 1N1
> > (http://ligand-expo.rcsb.org/reports/1/1N1/1N1_D3L1.gif) is a better
> > example:
> > The atoms N5 and N6 can have inverted chirality. If it is just one of
> > the two, then the molecule is distorted (IFF the restraint file is
> > correct!). If both have inverted chirality than the problem can be
> > fixed by label swapping. Hacking the restraint file to allow both
> > positive and negative chirality would allow you to distort the molecule.
> >
> > Cheers,
> > Robbie
> >
>
> --
> Andrew Purkiss
> X-ray Laboratory
> London Research Institute
> Cancer Research UK
>

Re: [ccp4bb] strict structure based alignment

[Paul Emsley]

On 13/07/12 15:30, Christian Roth wrote:

Dear all,

I want align a couple or protein structures by secondary structure matching to
one target and want get a kind of aminoacid alignment file e.g. what residue fit
the other, without adjustments due to sequence based alignments.
I tried Strap, but as far as I understood it, it takes also the sequence into
account. I tried also Rapido, but this does only a pairwise comparison.
Superpose does align it nicely (ccp4 based or Coot based) but there seems to
be no option to print the sequence alignment in a file and it is again  just a
pairwise comparison .


It is not clear to me what you mean by "just" a pairwise comparison.  
Coot outputs the SSM residue alignment to the terminal.


HTH,

Paul.

Re: [ccp4bb] strict structure based alignment

[sujata halder]

Hi,

SSM (via PDBefold) (http://www.ebi.ac.uk/msd-srv/ssm/cgi-bin/ssmserver)
will do a structure based alignment and outputs a table of rmsd but will
not give you a alignment file as such.

-Sujata

On Fri, Jul 13, 2012 at 10:30 AM, Christian Roth <
christian.r...@bbz.uni-leipzig.de> wrote:

> Dear all,
>
> I want align a couple or protein structures by secondary structure
> matching to
> one target and want get a kind of aminoacid alignment file e.g. what
> residue fit
> the other, without adjustments due to sequence based alignments.
> I tried Strap, but as far as I understood it, it takes also the sequence
> into
> account. I tried also Rapido, but this does only a pairwise comparison.
> Superpose does align it nicely (ccp4 based or Coot based) but there seems
> to
> be no option to print the sequence alignment in a file and it is again
>  just a
> pairwise comparison .
> Is there an other program which does something similar?
>
> Best Regards
>
> Christian
>

[ccp4bb] strict structure based alignment

[Christian Roth]

Dear all,

I want align a couple or protein structures by secondary structure matching to 
one target and want get a kind of aminoacid alignment file e.g. what residue 
fit 
the other, without adjustments due to sequence based alignments. 
I tried Strap, but as far as I understood it, it takes also the sequence into 
account. I tried also Rapido, but this does only a pairwise comparison. 
Superpose does align it nicely (ccp4 based or Coot based) but there seems to 
be no option to print the sequence alignment in a file and it is again  just a 
pairwise comparison .
Is there an other program which does something similar?

Best Regards

Christian 

Re: [ccp4bb] cryo for high salt crystal

[m zhang]

Hi Anna,
Actually I just tried lithium sulfate: I made a cryo with 1M KCl, 1.3M Lithium 
sulfate to replace the ammonia sulfate totally at pH7. it is not freezing 
clear. I guess I should try to increase the amount of LiSO4. But from all the 
warm responses here, I found sometimes people don't necessary keep the same 
concentration of component in the cryo as in the well solution + 
cryoprotectant. I usually make the artificial mother liquor as Roger or Michael 
mentioned early on. So my question is: will that hurt the crystal if the 
concentration of the component of cryo change?
Want to thanks to all for your warm suggestions. I haven't finish all the 
reading suggested here. but will.
Thanks,Manqing

Date: Thu, 12 Jul 2012 13:40:39 -0700
From: anna.s.gardb...@gmail.com
Subject: Re: [ccp4bb] cryo for high salt crystal
To: CCP4BB@JISCMAIL.AC.UK

Hello, Min.Lithium sulfate is a cryoprotectant at 2.0 M and sometimes even less 
(much lower than the concentrations needed for ammonium sulfate), so I would 
try replacing your ammonium sulfate with lithium sulfate, creating a 
cryoprotectant with 0.5-1.0 M KCl, 1.4-2.0 M LiSO4, at pH 7. You might need to 
transfer your crystals through a couple of intermediate drops. 

Regarding the reservoir precipitation - is there any chance you could control 
the humidity of the area in which you're working? Even filling a couple of 
adjacent reservoirs with water might help buy you a few extra crucial seconds. 
Also, working in a cold room to harvest your crystals will help reduce the 
evaporation rate.

Good luck!
Best,Anna

On Tue, Jul 10, 2012 at 9:28 AM, m zhang  wrote:





regaentDear All,
I am sure this question was discussed before. But I am wondering if anyone got 
the same experience as I do. I got a crystal out of condition with 1M KCl, 1.4M 
Ammonium sulfate at pH7. I tried to use glycerol, ethylene glycol, 25% sucrose, 
paraton-N oil, or ammonium sulfate itself: The problem is that all the cryo 
plus original reagents in the reservoir precipitate the salts out. And more 
serious problem is because of high salt in the condition, while I am trying to 
loop the crystal, both the drop and cryoprotectant drop form salt crystals (not 
sure it is KCl or ammonia sulfate) significantly and very quickly, that cause 
my crystal dissolved. My crystal doesn't seem to survive paraton-N oil. Does 
anyone here have similiar case? any suggestion will be appreciated.

Thanks,Min


  

Re: [ccp4bb] Chiral volume outliers SO4

[Robbie Joosten]

Hi Andrew,

Indeed, provided the atom labeling is correct the chiral volume restraint
actually says whether the groups on the ring a axial or equatorial. The cif
files do not define that any other way, so without the restraint the
description of the molecule is ambiguous. Note that the chirality restraint
only describes the hand, the actual chiral volume is calculated from the
angle restraints. 

In the more general case I think people are talking different languages.
There is chemical chirality (the real deal) and computational chirality. In
refinement and structure comparison the latter does matter (again, see
Dale's post). Problems with restraints are one of the reasons why there are
relatively many problems with ligands in the PDB (e.g.
http://www.springerlink.com/content/eu28538101v7v885/). 

Cheers,
Robbie

> -Original Message-
> From: Andrew Purkiss [mailto:a.purk...@mail.cryst.bbk.ac.uk]
> Sent: Friday, July 13, 2012 14:09
> To: Robbie Joosten
> Cc: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Chiral volume outliers SO4
> 
> Dear Robbie and ccp4bb,
> 
> Is 1N1 not a different type of problem though, where a chirality restraint
is
> valid and so the atom labelling is important?
> 
> Are you saying that we should always use the cif dictionary, even when
there
> are errors? Surely in the SO4 case, as Ian said, it is better to remove
the
> unnecessary restraint altogether. The sulphate cif file seems to have had
this
> bug introduced in the current CCP4 version.
> 
> Andrew
> 
> On Fri, 2012-07-13 at 12:54 +0200, Robbie Joosten wrote:
> > Dear All,
> >
> > I'm with Dale on this one. It's better to have a standard and roll
> > with it, than allow for ambiguity. The discussion just happened to
> > start with a rather silly example as Tim pointed out. The ligand 1N1
> > (http://ligand-expo.rcsb.org/reports/1/1N1/1N1_D3L1.gif) is a better
> > example:
> > The atoms N5 and N6 can have inverted chirality. If it is just one of
> > the two, then the molecule is distorted (IFF the restraint file is
> > correct!). If both have inverted chirality than the problem can be
> > fixed by label swapping. Hacking the restraint file to allow both
> > positive and negative chirality would allow you to distort the molecule.
> >
> > Cheers,
> > Robbie
> >
> 
> --
> Andrew Purkiss
> X-ray Laboratory
> London Research Institute
> Cancer Research UK
> 

Re: [ccp4bb] offtopic: AKTA prime

[Orru, Roberto]

Dear Peter,

This noise could be due to the presence of some air in the pump. Before call 
any service, try to flux about 100 ml of 100% methanol (or ethanol if you don’t 
like to use methanol) at 10 ml/min and 5 ml/min (50:50).
This should be remove any bubble. If the noise persist, the pump probably need 
a cleaning and maintenance.

Best
Roberto

___
Roberto  Orru, PhD
Biochemistry Department, Emory University
1510 Clifton Road
Rollins Research Center Room 4078
Atlanta, GA. 30322 (USA)
Phone: 404.727.5971
Fax: 404.727.2738


-Original Message-
From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Peter Hsu
Sent: Thursday, July 12, 2012 5:09 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] offtopic: AKTA prime

Hi all,

 We've got an old Akta prime that I think is on the verge of kicking it. 
Hearing some high pitched sounds coming from the pump when we're running it. 
Line A seems clogged and makes a thudding noise when we try to do a pump wash 
through that line. Does anyone have any experience w/fixing one/replacing the 
pump? Or any idea how much it's going to set us back to get it fixed?

Sorry for the off topic question/thanks in advance for any thoughts and ideas.

Peter



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[ccp4bb] PhD position in Warsaw

[Grzegorz Chojnowski]

The Bujnicki laboratory at the International Institute of Molecular and 
Cell Biology (IIMCB) in Warsaw, Poland (http://iimcb.gov.pl) offers a 
position for a PhD student with background in experimental structural 
biology and/or biochemistry.


The project aims at structural characterization of RNAs and protein-RNA 
complexes and will be carried out in a multisciplinary environment in 
collaboration with experimentalists and theoreticians. Research will 
involve RNA and protein purification and a lot of various biochemical 
and biophysical experiments (including crystallization and crystal 
structure determination) and will benefit from the cutting-edge 
equipment federated within IIMCB, the top-ranked Polish research 
institute in the field of biology.


More information can be found at http://iimcb.genesilico.pl

Experience in experimental analyses of proteins and DNA/RNA is required.
Experience in crystallography, RNA biochemistry, and enzymology will be 
an advantage.


Applicants must have an MSc degree and be eligible for admission to a 
PhD programme under the supervision of prof. Bujnicki.


The project starts October 1st, 2012. Applications will be collected 
until August 31st, 2012. Interviews will be held with selected 
applicants on September 10th.


Send your application to Prof. Janusz M. Bujnicki i...@genesilico.pl.


--
Grzegorz Chojnowski, PhD
ul. Ks. Trojdena 4
02-109 Warsaw, Poland
Laboratory of Bioinformatics and Protein Engineering
http://genesilico.pl/
International Institute of Molecular and Cell Biology
http://www.iimcb.gov.pl/

Re: [ccp4bb] Chiral volume outliers SO4

[Andrew Purkiss]

Dear Robbie and ccp4bb,

Is 1N1 not a different type of problem though, where a chirality
restraint is valid and so the atom labelling is important? 

Are you saying that we should always use the cif dictionary, even when
there are errors? Surely in the SO4 case, as Ian said, it is better to
remove the unnecessary restraint altogether. The sulphate cif file seems
to have had this bug introduced in the current CCP4 version.

Andrew

On Fri, 2012-07-13 at 12:54 +0200, Robbie Joosten wrote:
> Dear All,
> 
> I'm with Dale on this one. It's better to have a standard and roll with it,
> than allow for ambiguity. The discussion just happened to start with a
> rather silly example as Tim pointed out. The ligand 1N1
> (http://ligand-expo.rcsb.org/reports/1/1N1/1N1_D3L1.gif) is a better
> example:
> The atoms N5 and N6 can have inverted chirality. If it is just one of the
> two, then the molecule is distorted (IFF the restraint file is correct!). If
> both have inverted chirality than the problem can be fixed by label
> swapping. Hacking the restraint file to allow both positive and negative
> chirality would allow you to distort the molecule. 
> 
> Cheers,
> Robbie
> 

-- 
Andrew Purkiss
X-ray Laboratory
London Research Institute
Cancer Research UK

Re: [ccp4bb] Chiral volume outliers SO4

[Robbie Joosten]

Dear All,

I'm with Dale on this one. It's better to have a standard and roll with it,
than allow for ambiguity. The discussion just happened to start with a
rather silly example as Tim pointed out. The ligand 1N1
(http://ligand-expo.rcsb.org/reports/1/1N1/1N1_D3L1.gif) is a better
example:
The atoms N5 and N6 can have inverted chirality. If it is just one of the
two, then the molecule is distorted (IFF the restraint file is correct!). If
both have inverted chirality than the problem can be fixed by label
swapping. Hacking the restraint file to allow both positive and negative
chirality would allow you to distort the molecule. 

Cheers,
Robbie


> -Original Message-
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of
> Tim Gruene
> Sent: Friday, July 13, 2012 10:59
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: Re: [ccp4bb] Chiral volume outliers SO4
> 
> -BEGIN PGP SIGNED MESSAGE-
> Hash: SHA1
> 
> Dear all,
> 
> I am surprised by the discussion about "chiraliy" of an utterly
> centrosymmetric molecule. Shouldn't the four Oxygen atoms be at least
> from a QM point-of-view to indistinguishable? What reason is there to
> maintain a certain 'order' in the human-induced numbering scheme?
> 
> Cheers,
> Tim
> 
> On 07/13/12 00:22, Dale Tronrud wrote:
> > While this change has made your symptom go away it is stretching it a
> > bit to call this a "fix".  You have not corrected the root problem
> > that the names you have given your atoms do not match the convention
> > which is being applied for SO4 groups.  Changing the cif means that
> > you don't have to worry about it, but people who study such details
> > will be forced to deal with the incorrect labels of your model in the
> > future.
> >
> > Wouldn't it just be easier to swap the names of two oxygen atoms in
> > each SO4, leaving the cif alone?  Your difficulties will go away and
> > people using your model in the future will also have a simpler life.
> >
> > This labeling problem is not new.  The fight to standardize the
> > labeling of the methyl groups in Valine and Leucine was raging in the
> > 1980's.  Standardizing the labels on the PO4 groups in DNA/RNA was
> > much more recent.  It helps everyone when you know you can overlay two
> > models and have a logical solution without a "rotation matrix" with a
> > determinate of -1.
> >
> > Besides, you will continue to be bitten by this problem as you use
> > other programs, until you actually swap some labels.
> >
> > Dale Tronrud
> >
> > On 07/12/12 15:00, Joel Tyndall wrote:
> >> Hi all,
> >>
> >> Thanks very much to all who responded so quickly. The fix is a one
> >> liner in the SO4.cif file (last line)
> >>
> >> SO4  chir_01  S  O1 O2 O3both
> >>
> >> which I believe is now in the 6.3.0 release.
> >>
> >> Interestingly the chirality parameters were not in the SO4.cif file
> >> in 6.1.3 but then appeared in 6.2.0.
> >>
> >> Once again I'm very happy to get to the bottom of this and get it
> >> fixed. I do wonder if it had become over parametrised.
> >>
> >> Cheers
> >>
> >> Joel
> >>
> >>
> >>
> >> -Original Message- From: CCP4 bulletin board
> >> [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent:
> >> Thursday, 12 July 2012 12:16 a.m. To: CCP4BB@JISCMAIL.AC.UK
> >> Subject: Re: [ccp4bb] Chiral volume outliers SO4
> >>
> >> Hi Ian,
> >>
> >>
>  @Ian: You'd be surprised how well Refmac can flatten sulfates if
>  you have a chiral volume outlier (see Figure 1d in Acta Cryst. D68:
>  484-496
> >>> (2012)). But this is only because the 'negative' volume sign was
> >>> erroneously used
> >> in
> >>> the chiral restraint instead of 'both' (or better still IMO no
> >>> chiral
> >> restraint at
> >>> all), right?  If so I don't find it surprising at all that Refmac
> >>> tried to
> >> flip the
> >>> sulphate and ended up flattening it. Seems to be a good illustration
> >>> of the GIGO (garbage in - garbage out) principle.
> >>> Just because the garbage input in this case is in the
> >> official
> >>> CCP4 distribution and not (as is of course more commonly the
> >>> case) perpetrated by the user doesn't make it any less garbage.
> >> The problem is that in the creation of chiral volume targets
> >> chemically equivalent (groups of) atoms are not recognized as such.
> >> So any new or recreated restraint files will have either 'positiv' or
> >> 'negativ' and the problem starts all over again.
> >> That is why it is better to stay consistent and choose one chirality
> >> (the same one as in the 'ideal' coordinates in the PDB ligand
> >> descriptions). This will also make it easier compare ligands after
> >> aligning them (this applies to ligands more complex than sulfate).
> >> Obviously, users should not be forced to deal with these things.
> >> Programs like Refmac and COOT should fix chiral volume inversions for
> >> the user, because it is only relevant inside the computer. That is
> >> the idea of chiron, just fix thes

Re: [ccp4bb] Chiral volume outliers SO4

[Tim Gruene]

-BEGIN PGP SIGNED MESSAGE-
Hash: SHA1

Dear all,

I am surprised by the discussion about "chiraliy" of an utterly
centrosymmetric molecule. Shouldn't the four Oxygen atoms be at least
from a QM point-of-view to indistinguishable? What reason is there to
maintain a certain 'order' in the human-induced numbering scheme?

Cheers,
Tim

On 07/13/12 00:22, Dale Tronrud wrote:
> While this change has made your symptom go away it is stretching it
> a bit to call this a "fix".  You have not corrected the root
> problem that the names you have given your atoms do not match the
> convention which is being applied for SO4 groups.  Changing the cif
> means that you don't have to worry about it, but people who study
> such details will be forced to deal with the incorrect labels of
> your model in the future.
> 
> Wouldn't it just be easier to swap the names of two oxygen atoms in
> each SO4, leaving the cif alone?  Your difficulties will go away
> and people using your model in the future will also have a simpler
> life.
> 
> This labeling problem is not new.  The fight to standardize the
> labeling of the methyl groups in Valine and Leucine was raging in
> the 1980's.  Standardizing the labels on the PO4 groups in DNA/RNA
> was much more recent.  It helps everyone when you know you can
> overlay two models and have a logical solution without a "rotation
> matrix" with a determinate of -1.
> 
> Besides, you will continue to be bitten by this problem as you use
> other programs, until you actually swap some labels.
> 
> Dale Tronrud
> 
> On 07/12/12 15:00, Joel Tyndall wrote:
>> Hi all,
>> 
>> Thanks very much to all who responded so quickly. The fix is a
>> one liner in the SO4.cif file (last line)
>> 
>> SO4  chir_01  S  O1 O2 O3both
>> 
>> which I believe is now in the 6.3.0 release.
>> 
>> Interestingly the chirality parameters were not in the SO4.cif
>> file in 6.1.3 but then appeared in 6.2.0.
>> 
>> Once again I'm very happy to get to the bottom of this and get it
>> fixed. I do wonder if it had become over parametrised.
>> 
>> Cheers
>> 
>> Joel
>> 
>> 
>> 
>> -Original Message- From: CCP4 bulletin board
>> [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of Robbie Joosten Sent:
>> Thursday, 12 July 2012 12:16 a.m. To: CCP4BB@JISCMAIL.AC.UK 
>> Subject: Re: [ccp4bb] Chiral volume outliers SO4
>> 
>> Hi Ian,
>> 
>> 
 @Ian: You'd be surprised how well Refmac can flatten sulfates
 if you have a chiral volume outlier (see Figure 1d in Acta
 Cryst. D68: 484-496
>>> (2012)). But this is only because the 'negative' volume sign
>>> was erroneously used
>> in
>>> the chiral restraint instead of 'both' (or better still IMO no
>>> chiral
>> restraint at
>>> all), right?  If so I don't find it surprising at all that
>>> Refmac tried to
>> flip the
>>> sulphate and ended up flattening it. Seems to be a good
>>> illustration of the GIGO (garbage in - garbage out) principle.
>>> Just because the garbage input in this case is in the
>> official
>>> CCP4 distribution and not (as is of course more commonly the 
>>> case) perpetrated by the user doesn't make it any less
>>> garbage.
>> The problem is that in the creation of chiral volume targets
>> chemically equivalent (groups of) atoms are not recognized as
>> such. So any new or recreated restraint files will have either
>> 'positiv' or 'negativ' and the problem starts all over again.
>> That is why it is better to stay consistent and choose one
>> chirality (the same one as in the 'ideal' coordinates in the PDB
>> ligand descriptions). This will also make it easier compare
>> ligands after aligning them (this applies to ligands more complex
>> than sulfate). Obviously, users should not be forced to deal with
>> these things. Programs like Refmac and COOT should fix chiral
>> volume inversions for the user, because it is only relevant
>> inside the computer. That is the idea of chiron, just fix these
>> 'problems' automatically by swapping equivalent atoms whenever
>> Refmac gives a chiral volume inversion warning.  It should make
>> life a bit easier. 
>> 
>>> The point I was making is that in this and similar cases you
>>> don't need a
>> chiral
>>> restraint at all: surely 4 bond lengths and 6 bond angles
>>> define the
>> chiral
>>> volume pretty well already?  Or are there cases where without a
>>> chiral restraint the refinement still tries to flip the
>>> chirality (I would fine
>> that hard to
>>> believe).
>> I agree with you for sulfate, and also for phosphate ;). I don't
>> know what happens in other compounds at poor resolution, when
>> bond and angle targets (and their SDs) are not equivalent. I
>> guess that some angle might 'give way' before others. That is
>> something that should be tested. I have a growing list of chiral
>> centers that have this problem if you are interested.
>> 
>> Cheers, Robbie
> 

- -- 
- --
Dr Tim Gruene
Institut fuer anorganische Chemie
Tammannstr. 4
D-37077 Goettingen

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