Re: [ccp4bb] How to refine this needle crystal of membrane complex?

2017-03-02 Thread Keller, Jacob 
I'll add another favorite to that one (below.) I especially enjoy the tracking 
of the intensities of the Bragg *rods* along their lengths, showing how the 2D 
lattice only chops reciprocal space in two dimensions, leaving the third 
dimension continuous.

JPK

Nature. 1975 Sep 4;257(5521):28-32.
Three-dimensional model of purple membrane obtained by electron microscopy.
Henderson R, Unwin PN.
Abstract
A 7-A resolution map of the purple membrane has been obtained by electron 
microscopy of tilted, unstained specimens. The protein in the membrane contains 
seven, closely packed, alpha-helical segments which extend roughly 
perpendicular to the plane of the membrane for most of its width. Lipid bilayer 
regions fill the spaces between the protein molecules.
PMID: 1161000



-Original Message-
From: Phoebe A. Rice [mailto:pr...@uchicago.edu] 
Sent: Wednesday, March 01, 2017 3:16 PM
To: Keller, Jacob ; CCP4BB@JISCMAIL.AC.UK
Subject: RE: [ccp4bb] How to refine this needle crystal of membrane complex?

For historical perspective: 

Molecular structure determination by electron microscopy of unstained 
crystalline specimens PNT Unwin, R Henderson - Journal of molecular biology, 
1975
http://www.sciencedirect.com/science/article/pii/0022283675902120


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, March 01, 2017 1:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

There are now quite a few structures solved by ED of 3D crystals, esp by Tamir 
Gonen's group here at Janelia, and a quick count in the pdb yields ~15 
3D-crystal ED structures, some of which are not released. They're getting 
better at it all the time. I guess the news has not spread as much as I 
thought. FYI, last I checked they were looking for good samples to try, 
although you'd have to contact Tamir about that to see whether it's still true, 
or whether they've now got their hands full.

All the best,

Jacob Keller





-Original Message-
From: Tim Gruene [mailto:tim.gru...@psi.ch]
Sent: Wednesday, March 01, 2017 2:22 PM
To: Keller, Jacob 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

Dear Jacob, dear TO,

(I have wondered what can be 'micro' about diffraction - would you know?)

it would be very exciting to get the structure by electron diffraction - as 
much as I know it would be the first new structure solved from a 3D crystal 
with electron radiation in the PDB. You would have to contact one of the 3-4 
labs that to electron diffraction of protein samples (Abrahams, Gonen, 
Hovmoeller / Zou (possibly), and Yonekura/Toyoshima in alphabetic order). The 
first and the last group are equipped with sensitive hybrid pixel detectors 
that might help.

A good test to get an idea about the resolution you might get from electron 
diffraction is to scratch up as many of your crystals and analyse the powder 
pattern with a strong X-ray source (synchrotron, or very good home source).

Note that when you can see the crystals with a light microscope, they are most 
likely too big for electron diffraction - you might end up seeing a black 
screen with no electron penetrating your samples.

Best regards,
Tim

On Wednesday, March 1, 2017 1:59:44 PM CET Keller, Jacob wrote:
> Although trying to refine the crystals is perhaps the most 
> straightforward thing, you might consider trying micro electron 
> diffraction (micro-ED) with the existing samples-the technique is 
> working increasingly better, and would work well with your tiny 
> crystals. Look at Tamir Gonen's recent papers for more info.

> All the best,
>
> Jacob Keller
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Wednesday, March 01, 2017 3:53 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] How to refine this needle crystal of membrane complex?
>
> Dear all,
> I am trying to obtain crystals of membrane complex. Now, I have 
> obtained the complex with appropriate stoichiometry after a series of 
> purification in DDM, then I get extremely thin needles (as shown in
> figure) in  some conditions such as 28% MPD, 0.1 M NaAc pH 4.5 or 45%
> MPD,0.1 M Bis-Tris pH 6.5, 0.2 M CaCl2. However, there are not any 
> improvements through changing the concentration of precipitants/salts 
> or adding the additives/detergents to the protein.

> I would be very happy if anybody could give me some advice about this 
> crystal refinement.

> With best regards.
>
>
> [cid:image001.png@01D2926A.28CB0C70]

--
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/104
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] How to refine this needle crystal of membrane complex?

2017-03-01 Thread Phoebe A. Rice 
For historical perspective: 

Molecular structure determination by electron microscopy of unstained 
crystalline specimens
PNT Unwin, R Henderson - Journal of molecular biology, 1975 
http://www.sciencedirect.com/science/article/pii/0022283675902120


From: CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] on behalf of Keller, Jacob 
[kell...@janelia.hhmi.org]
Sent: Wednesday, March 01, 2017 1:50 PM
To: CCP4BB@JISCMAIL.AC.UK
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

There are now quite a few structures solved by ED of 3D crystals, esp by Tamir 
Gonen's group here at Janelia, and a quick count in the pdb yields ~15 
3D-crystal ED structures, some of which are not released. They're getting 
better at it all the time. I guess the news has not spread as much as I 
thought. FYI, last I checked they were looking for good samples to try, 
although you'd have to contact Tamir about that to see whether it's still true, 
or whether they've now got their hands full.

All the best,

Jacob Keller





-Original Message-
From: Tim Gruene [mailto:tim.gru...@psi.ch]
Sent: Wednesday, March 01, 2017 2:22 PM
To: Keller, Jacob 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

Dear Jacob, dear TO,

(I have wondered what can be 'micro' about diffraction - would you know?)

it would be very exciting to get the structure by electron diffraction - as 
much as I know it would be the first new structure solved from a 3D crystal 
with electron radiation in the PDB. You would have to contact one of the 3-4 
labs that to electron diffraction of protein samples (Abrahams, Gonen, 
Hovmoeller / Zou (possibly), and Yonekura/Toyoshima in alphabetic order). The 
first and the last group are equipped with sensitive hybrid pixel detectors 
that might help.

A good test to get an idea about the resolution you might get from electron 
diffraction is to scratch up as many of your crystals and analyse the powder 
pattern with a strong X-ray source (synchrotron, or very good home source).

Note that when you can see the crystals with a light microscope, they are most 
likely too big for electron diffraction - you might end up seeing a black 
screen with no electron penetrating your samples.

Best regards,
Tim

On Wednesday, March 1, 2017 1:59:44 PM CET Keller, Jacob wrote:
> Although trying to refine the crystals is perhaps the most
> straightforward thing, you might consider trying micro electron
> diffraction (micro-ED) with the existing samples—the technique is
> working increasingly better, and would work well with your tiny
> crystals. Look at Tamir Gonen’s recent papers for more info.

> All the best,
>
> Jacob Keller
>
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Wednesday, March 01, 2017 3:53 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] How to refine this needle crystal of membrane complex?
>
> Dear all,
> I am trying to obtain crystals of membrane complex. Now, I have
> obtained the complex with appropriate stoichiometry after a series of
> purification in DDM, then I get extremely thin needles (as shown in
> figure) in  some conditions such as 28% MPD, 0.1 M NaAc pH 4.5 or 45%
> MPD,0.1 M Bis-Tris pH 6.5, 0.2 M CaCl2. However, there are not any
> improvements through changing the concentration of precipitants/salts
> or adding the additives/detergents to the protein.

> I would be very happy if anybody could give me some advice about this
> crystal refinement.

> With best regards.
>
>
> [cid:image001.png@01D2926A.28CB0C70]

--
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/104
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] How to refine this needle crystal of membrane complex?

2017-03-01 Thread Keller, Jacob 
There are now quite a few structures solved by ED of 3D crystals, esp by Tamir 
Gonen's group here at Janelia, and a quick count in the pdb yields ~15 
3D-crystal ED structures, some of which are not released. They're getting 
better at it all the time. I guess the news has not spread as much as I 
thought. FYI, last I checked they were looking for good samples to try, 
although you'd have to contact Tamir about that to see whether it's still true, 
or whether they've now got their hands full.

All the best,

Jacob Keller





-Original Message-
From: Tim Gruene [mailto:tim.gru...@psi.ch] 
Sent: Wednesday, March 01, 2017 2:22 PM
To: Keller, Jacob 
Cc: CCP4BB@jiscmail.ac.uk
Subject: Re: [ccp4bb] How to refine this needle crystal of membrane complex?

Dear Jacob, dear TO,

(I have wondered what can be 'micro' about diffraction - would you know?)

it would be very exciting to get the structure by electron diffraction - as 
much as I know it would be the first new structure solved from a 3D crystal 
with electron radiation in the PDB. You would have to contact one of the 3-4 
labs that to electron diffraction of protein samples (Abrahams, Gonen, 
Hovmoeller / Zou (possibly), and Yonekura/Toyoshima in alphabetic order). The 
first and the last group are equipped with sensitive hybrid pixel detectors 
that might help.

A good test to get an idea about the resolution you might get from electron 
diffraction is to scratch up as many of your crystals and analyse the powder 
pattern with a strong X-ray source (synchrotron, or very good home source).

Note that when you can see the crystals with a light microscope, they are most 
likely too big for electron diffraction - you might end up seeing a black 
screen with no electron penetrating your samples.

Best regards,
Tim

On Wednesday, March 1, 2017 1:59:44 PM CET Keller, Jacob wrote:
> Although trying to refine the crystals is perhaps the most 
> straightforward thing, you might consider trying micro electron 
> diffraction (micro-ED) with the existing samples—the technique is 
> working increasingly better, and would work well with your tiny 
> crystals. Look at Tamir Gonen’s recent papers for more info.
 
> All the best,
> 
> Jacob Keller
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Wednesday, March 01, 2017 3:53 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] How to refine this needle crystal of membrane complex?
> 
> Dear all,
> I am trying to obtain crystals of membrane complex. Now, I have 
> obtained the complex with appropriate stoichiometry after a series of 
> purification in DDM, then I get extremely thin needles (as shown in 
> figure) in  some conditions such as 28% MPD, 0.1 M NaAc pH 4.5 or 45% 
> MPD,0.1 M Bis-Tris pH 6.5, 0.2 M CaCl2. However, there are not any 
> improvements through changing the concentration of precipitants/salts 
> or adding the additives/detergents to the protein.
 
> I would be very happy if anybody could give me some advice about this 
> crystal refinement.
 
> With best regards.
> 
> 
> [cid:image001.png@01D2926A.28CB0C70]

--
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/104
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A

Re: [ccp4bb] How to refine this needle crystal of membrane complex?

2017-03-01 Thread Tim Gruene 
Dear Jacob, dear TO,

(I have wondered what can be 'micro' about diffraction - would you know?)

it would be very exciting to get the structure by electron diffraction - as 
much as I know it would be the first new structure solved from a 3D crystal 
with electron radiation in the PDB. You would have to contact one of the 3-4 
labs that to electron diffraction of protein samples (Abrahams, Gonen, 
Hovmoeller / Zou (possibly), and Yonekura/Toyoshima in alphabetic order). The 
first and the last group are equipped with sensitive hybrid pixel detectors 
that might help.

A good test to get an idea about the resolution you might get from electron 
diffraction is to scratch up as many of your crystals and analyse the powder 
pattern with a strong X-ray source (synchrotron, or very good home source).

Note that when you can see the crystals with a light microscope, they are most 
likely too big for electron diffraction - you might end up seeing a black 
screen with no electron penetrating your samples.

Best regards,
Tim

On Wednesday, March 1, 2017 1:59:44 PM CET Keller, Jacob wrote:
> Although trying to refine the crystals is perhaps the most straightforward
> thing, you might consider trying micro electron diffraction (micro-ED) with
> the existing samples—the technique is working increasingly better, and
> would work well with your tiny crystals. Look at Tamir Gonen’s recent
> papers for more info.
 
> All the best,
> 
> Jacob Keller
> 
> From: CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] On Behalf Of ???
> Sent: Wednesday, March 01, 2017 3:53 AM
> To: CCP4BB@JISCMAIL.AC.UK
> Subject: [ccp4bb] How to refine this needle crystal of membrane complex?
> 
> Dear all,
> I am trying to obtain crystals of membrane complex. Now, I have obtained the
> complex with appropriate stoichiometry after a series of purification in
> DDM, then I get extremely thin needles (as shown in figure) in  some
> conditions such as 28% MPD, 0.1 M NaAc pH 4.5 or 45% MPD,0.1 M Bis-Tris pH
> 6.5, 0.2 M CaCl2. However, there are not any improvements through changing
> the concentration of precipitants/salts or adding the additives/detergents
> to the protein.
 
> I would be very happy if anybody could give me some advice about this
> crystal refinement.
 
> With best regards.
> 
> 
> [cid:image001.png@01D2926A.28CB0C70]

-- 
--
Paul Scherrer Institut
Tim Gruene
- persoenlich -
OFLC/104
CH-5232 Villigen PSI
phone: +41 (0)56 310 5297

GPG Key ID = A46BEE1A


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