Re: [ccp4bb] Does MicroED (microcrystal electron diffraction) have phase problem ?

2020-08-17 Thread Jessica Bruhn 
Hi Edward,

MicroED as defined by Tamir Gonen does not encompass 2D crystals. This is
to keep it separate from 2D electron crystallography in which the data is
collected in a very different manner. Personally though, I don't see why
you couldn't collect microED style data from a 2D crystal. Less copies of
the unit cell means you would get less signal and you might have to bump up
your dose per frame, which can cause more radiation damage, but these are
(probably) not deal breakers.

For me, the thing that defines microED is the collection of electron
diffraction data using the (continuous) rotation method without precession
of the electron beam. It requires microcrystals/nanocrystals because if the
crystals are too thick, the electron beam cannot penetrate them. The beauty
of collecting data in this manner is that this is the same strategy
employed by most X-ray crystallographers and therefore the data can be
processed with familiar programs (DIALS, XDS, HKL3000, MOSFLM, APEX3, etc).
The reality though, is that data processing is not trivial and there is a
lot of room for improvement in handling this data. It is the early days for
this field and I am hopeful that things will continue to improve. And in
the meantime, we are solving lots of small molecule structures that could
not easily be solved by X-ray crystallography or NMR.

Cheers,
Jessica

On Sun, Aug 16, 2020 at 8:32 PM Edward A. Berry  wrote:

> For my own info,
> Does microED encompass work with 2D crystals, or only micro-3D crystals?
>
> On 08/15/2020 10:40 PM, Jessica Bruhn wrote:
> > Hi Alex,
> >
> > Welcome to the field of microED! From a practical standpoint, microED
> also suffers from the phase problem, and somewhat moreso compared to X-ray
> crystallography because anomalous signal is very limited. It is true that a
> TEM microscope operated in imaging mode produces images that contain both
> phase and amplitude information, which you correctly infer means that
> single particle cryoEM does not suffer from the phase problem. This is why
> a map from cryoEM is generally of higher quality than one determined by
> X-ray crystallography at the same resolution, because we don't have to
> guess at the phases.
> >
> > In classic microED data collection, we don't actually take advantage of
> the phase information that can be measured using imaging mode. We quickly
> find our crystals in imaging mode and then flip the switch to diffraction
> mode and collect a dataset devoid of phase information. It has been
> suggested that we could use imaging mode to get at the phases of the
> diffraction patterns, but I am not aware of anyone actually doing this.
> Practically speaking, this would add significant additional time to the
> data collection and we likely would only be able to reliably use the phases
> for the lower resolution range (though, that might not be a deal breaker).
> Thankfully though, molecular replacement and ab initio methods for small
> molecules work pretty well on these datasets. Plus, most X-ray structures
> are solved this way anyways.
> >
> > There have been efforts to use radiation damage as a means to phase a
> small peptide bound to zinc (
> https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313391/ <
> https://urldefense.com/v3/__https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313391/__;!!GobTDDpD7A!d9GMLVpOCJjYcJ8TK3Sn2QYRR5JPvL8Vb51isRJMME8AssIo499krUEDQ5gHtKOV$>).
> And there is some interesting work being done with dynamical scattering as
> a way to see differences in Friedel pairs (see Tim Gruene's post here:
> https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=CCP4BB;b22eae56.2007 <
> https://urldefense.com/v3/__https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=CCP4BB;b22eae56.2007__;!!GobTDDpD7A!d9GMLVpOCJjYcJ8TK3Sn2QYRR5JPvL8Vb51isRJMME8AssIo499krUEDQx24VbXZ$>),
> but these methods are not "classic microED" and require completely
> different data processing software that is unfamiliar to most X-ray
> crystallographers.
> >
> > For now, we just crank up the cycles in SHELXT/SHELXD or hope for a good
> molecular replacement model.
> >
> > Best of luck,
> > Jessica
> >
> > On Sat, Aug 15, 2020 at 6:03 PM Alex Lee  > wrote:
> >
> > Hi, All,
> >
> > I am new to MicroED (microcrystal electron diffraction). I know that
> X-ray crystallography has phase problem, and I think MicroED has phase
> problem too (it is diffraction of electron instead of x-ray). However, when
> I read the Wikipedia, I could not understand the following description of
> MicroED: One of the main difficulties in X-ray crystallography is
> determining phases in the diffraction pattern. Because of the complexity of
> X-ray lenses, it is difficult to form an image of the crystal being
> diffracted, and hence phase information is lost. Fortunately, electron
> microscopes can resolve atomic structure in real space and the
> crystallographic structure factor phase information can be experimentally
> determined from an image's F

Re: [ccp4bb] Does MicroED (microcrystal electron diffraction) have phase problem ?

2020-08-16 Thread Edward A. Berry 

For my own info,
Does microED encompass work with 2D crystals, or only micro-3D crystals?

On 08/15/2020 10:40 PM, Jessica Bruhn wrote:

Hi Alex,

Welcome to the field of microED! From a practical standpoint, microED also 
suffers from the phase problem, and somewhat moreso compared to X-ray 
crystallography because anomalous signal is very limited. It is true that a TEM 
microscope operated in imaging mode produces images that contain both phase and 
amplitude information, which you correctly infer means that single particle 
cryoEM does not suffer from the phase problem. This is why a map from cryoEM is 
generally of higher quality than one determined by X-ray crystallography at the 
same resolution, because we don't have to guess at the phases.

In classic microED data collection, we don't actually take advantage of the 
phase information that can be measured using imaging mode. We quickly find our 
crystals in imaging mode and then flip the switch to diffraction mode and 
collect a dataset devoid of phase information. It has been suggested that we 
could use imaging mode to get at the phases of the diffraction patterns, but I 
am not aware of anyone actually doing this. Practically speaking, this would 
add significant additional time to the data collection and we likely would only 
be able to reliably use the phases for the lower resolution range (though, that 
might not be a deal breaker). Thankfully though, molecular replacement and ab 
initio methods for small molecules work pretty well on these datasets. Plus, 
most X-ray structures are solved this way anyways.

There have been efforts to use radiation damage as a means to phase a small peptide bound to zinc 
(https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313391/ 
).
 And there is some interesting work being done with dynamical scattering as a way to see differences 
in Friedel pairs (see Tim Gruene's post here: 
https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=CCP4BB;b22eae56.2007 
),
 but these methods are not "classic microED" and require completely different data 
processing software that is unfamiliar to most X-ray crystallographers.

For now, we just crank up the cycles in SHELXT/SHELXD or hope for a good 
molecular replacement model.

Best of luck,
Jessica

On Sat, Aug 15, 2020 at 6:03 PM Alex Lee mailto:alexlee198...@gmail.com>> wrote:

Hi, All,

I am new to MicroED (microcrystal electron diffraction). I know that X-ray 
crystallography has phase problem, and I think MicroED has phase problem too 
(it is diffraction of electron instead of x-ray). However, when I read the 
Wikipedia, I could not understand the following description of MicroED: One of 
the main difficulties in X-ray crystallography is determining phases in the 
diffraction pattern. Because of the complexity of X-ray lenses, it is difficult 
to form an image of the crystal being diffracted, and hence phase information 
is lost. Fortunately, electron microscopes can resolve atomic structure in real 
space and the crystallographic structure factor phase information can be 
experimentally determined from an image's Fourier transform. The Fourier 
transform of an atomic resolution image is similar, but different, to a 
diffraction pattern—with reciprocal lattice spots reflecting the symmetry and 
spacing of a crystal.

Does the above description mean that MicroED, or more broadly electron 
crystallogaphy does NOT suffer from phase problem?  How about single particle 
cryo electron microscopy, it should NOT have phase problem, right?

Thanks for any input in it.

Best,
Alex


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Re: [ccp4bb] Does MicroED (microcrystal electron diffraction) have phase problem ?

2020-08-15 Thread Jessica Bruhn 
Hi Alex,

Welcome to the field of microED! From a practical standpoint, microED also
suffers from the phase problem, and somewhat moreso compared to X-ray
crystallography because anomalous signal is very limited. It is true that a
TEM microscope operated in imaging mode produces images that contain both
phase and amplitude information, which you correctly infer means that
single particle cryoEM does not suffer from the phase problem. This is why
a map from cryoEM is generally of higher quality than one determined by
X-ray crystallography at the same resolution, because we don't have to
guess at the phases.

In classic microED data collection, we don't actually take advantage of the
phase information that can be measured using imaging mode. We quickly find
our crystals in imaging mode and then flip the switch to diffraction mode
and collect a dataset devoid of phase information. It has been suggested
that we could use imaging mode to get at the phases of the diffraction
patterns, but I am not aware of anyone actually doing this. Practically
speaking, this would add significant additional time to the data collection
and we likely would only be able to reliably use the phases for the lower
resolution range (though, that might not be a deal breaker). Thankfully
though, molecular replacement and ab initio methods for small molecules
work pretty well on these datasets. Plus, most X-ray structures are solved
this way anyways.

There have been efforts to use radiation damage as a means to phase a small
peptide bound to zinc (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7313391/).
And there is some interesting work being done with dynamical scattering as
a way to see differences in Friedel pairs (see Tim Gruene's post here:
https://www.jiscmail.ac.uk/cgi-bin/wa-jisc.exe?A2=CCP4BB;b22eae56.2007),
but these methods are not "classic microED" and require completely
different data processing software that is unfamiliar to most X-ray
crystallographers.

For now, we just crank up the cycles in SHELXT/SHELXD or hope for a good
molecular replacement model.

Best of luck,
Jessica

On Sat, Aug 15, 2020 at 6:03 PM Alex Lee  wrote:

> Hi, All,
>
> I am new to MicroED (microcrystal electron diffraction). I know that X-ray
> crystallography has phase problem, and I think MicroED has phase problem
> too (it is diffraction of electron instead of x-ray). However, when I read
> the Wikipedia, I could not understand the following description of MicroED: 
> One
> of the main difficulties in X-ray crystallography is determining phases in
> the diffraction pattern. Because of the complexity of X-ray lenses, it is
> difficult to form an image of the crystal being diffracted, and hence phase
> information is lost. Fortunately, electron microscopes can resolve atomic
> structure in real space and the crystallographic structure factor phase
> information can be experimentally determined from an image's Fourier
> transform. The Fourier transform of an atomic resolution image is similar,
> but different, to a diffraction pattern—with reciprocal lattice spots
> reflecting the symmetry and spacing of a crystal.
>
> Does the above description mean that MicroED, or more broadly electron
> crystallogaphy does NOT suffer from phase problem?  How about single
> particle cryo electron microscopy, it should NOT have phase problem, right?
>
> Thanks for any input in it.
>
> Best,
> Alex
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>


-- 
Jessica Bruhn, Ph.D
Principal Scientist
NanoImaging Services, Inc.
4940 Carroll Canyon Road, Suite 115
San Diego, CA 92121
Phone #: (888) 675-8261
www.nanoimagingservices.com



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[ccp4bb] Does MicroED (microcrystal electron diffraction) have phase problem ?

2020-08-15 Thread Alex Lee 
Hi, All,

I am new to MicroED (microcrystal electron diffraction). I know that X-ray
crystallography has phase problem, and I think MicroED has phase problem
too (it is diffraction of electron instead of x-ray). However, when I read
the Wikipedia, I could not understand the following description of MicroED: One
of the main difficulties in X-ray crystallography is determining phases in
the diffraction pattern. Because of the complexity of X-ray lenses, it is
difficult to form an image of the crystal being diffracted, and hence phase
information is lost. Fortunately, electron microscopes can resolve atomic
structure in real space and the crystallographic structure factor phase
information can be experimentally determined from an image's Fourier
transform. The Fourier transform of an atomic resolution image is similar,
but different, to a diffraction pattern—with reciprocal lattice spots
reflecting the symmetry and spacing of a crystal.

Does the above description mean that MicroED, or more broadly electron
crystallogaphy does NOT suffer from phase problem?  How about single
particle cryo electron microscopy, it should NOT have phase problem, right?

Thanks for any input in it.

Best,
Alex



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