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

[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] Cell disruption

[Adam Middleton]

Hi Bernhard,

The Department has an Avestin emusiflex here, and it is kept in the cold
room. Works great as long as your cells are properly resuspended and in a
big enough volume. Highly recommended.

Adam



On Sun, Aug 16, 2020 at 4:48 PM Pascal Egea <
4aa44fc90f38-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear Bernhard ,
> I would recommend the emulsified from Avestin. It is great . Depending on
> your budget you can ask for stainless  steel (probe  to slow tear ) or
> ceramic (longer lasting but a bit more expensive).
> We have the ceramic but I have worked with the stainless steel one and it
> is really good with E. coli cells. Not the best for
> Yeast despite pressurizing more. Yeast are better dealt with bead beaters
> or cryo-mill grinders.
> 3 passes are usually sufficient to process up to 250 ml of extract in a
> decent time (15-20) minutes .
>
> We operate ours at room temperature just cool the serpentine tubing coming
> out of the chamber in ice . There is an option to add a cooling plate but
> we have never considered it. Overheating comes from overpressurizing but
> for E Coli at 15000 psi max  3 passes will do the trick without overhea tu
> bf the sample. I have processEs successfully many membrane proteins And
> protein complexes with it.
>
> I have never seen one in a cold room to be honest but it should be fine
> although I don’t think it would be necessary . I would Ask avestin about
> that .
> Maintenance and cleaning is simple . Once in a while I will replace the
> seal, it s not very difficult to do. The whole assembly is metal so you can
> sterilize the whole thing After complete disassembly in extreme cases .
> This is a bit more involved but not overwhelming.
> The people at Avestin are super nice and responsive ( Canadians) so they
> will guide you if necessary . And we have had their engineers show up
> sometimes to just check the instrument for free . I have had this one for
> 10 years in my lab now. And before that was using one for 7 years during my
> post doc.  I would not lyse bacteria by any other method now.
>
> I hope this helps.
> All the best.
> Pascal
>
> On Sat, Aug 15, 2020 at 9:08 PM Bernhard Lechtenberg <
> lechtenber...@wehi.edu.au> wrote:
>
>> Dear colleagues,
>>
>>
>>
>> We are currently looking to purchase a cell disruptor/homogeniser mainly
>> for routinely processing a few 100 mls of E. coli suspensions. With the
>> current COVID-19 restrictions it is very difficult for us to test any
>> equipment. I thus hope that some of you can share their experiences with
>> the different models. I found a similar thread on the CCP4BB from 2013 but
>> wondered if anybody had had some more up to date information.
>>
>>
>>
>> We are mainly looking at the Avestin Emulsiflex C3 homogeniser and the
>> Microfluidics LM20 Microfluidizer. In particular we are interested to know
>> more about ease of use, maintenance, reliability and if anybody operates
>> these in a cold room (4°C).
>>
>>
>>
>> Thanks in advance,
>>
>>
>>
>> Bernhard
>>
>>
>>
>> 
>>
>>
>>
>> 
>>
>> --
>>
>> Bernhard C. Lechtenberg, Ph.D.
>>
>> Laboratory Head
>>
>> Ubiquitin Signalling Division
>>
>> The Walter and Eliza Hall Institute
>>
>> 1G Royal Parade
>> 
>>
>> Parkville VIC 3052
>> 
>>
>> Australia
>> 
>>
>> Phone: +61 3 9345 2217
>>
>> Email: lechtenber...@wehi.edu.au
>>
>>
>>
>> ___
>>
>> The information in this email is confidential and intended solely for the
>> addressee.
>> You must not disclose, forward, print or use it without the permission of
>> the sender.
>>
>> The Walter and Eliza Hall Institute acknowledges the Wurundjeri people of
>> the Kulin
>> Nation as the traditional owners of the land where our campuses are
>> located and
>> the continuing connection to country and community.
>> ___
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>
> --
> Pascal F. Egea, PhD
> Associate Project Scientist
> UCLA, David Geffen School of Medicine
> Department of Biological Chemistry
> Boyer Hall room 356
> 611 Charles E Young Drive East
> Los Angeles CA 90095
> office (310)-983-3515
> lab  (310)-983-3516
> email pegea at mednet.ucla.edu
>
> --
>
> To unsubscribe from the CCP4BB list, clic