[ccp4bb] Register for the 2021 ACA Fundamentals of Single-Particle Cryo-EM Workshop

[Herzik, Mark]

Please join us at our comprehensive single-particle cryo-EM workshop – 
Fundamentals of Single-Particle Cryo-EM – at the virtual American 
Crystallographic Association 71st annual meeting "Structural Science Awakens".
This cryo-EM focused workshop will be held virtualy on 2 days: 8/17-18 from 
12:15 to 5/6 pm ET and will focus on the fundamentals of cryo-EM, primarily 
benefiting those who are new to the field. In this workshop, we will highlight 
historical advances leading to the current state of the field, provide an 
overview of EM sample preparation and data collection, and detail cryo-EM data 
processing, interpretation and validation. We will monitor a live data 
collection and provide tutorials on “on-the-fly” cryo-EM data processing. This 
workshop is a companion workshop to “Managing and Using National cryo-EM 
Facilities”.
Full workshop schedule is here: https://www.acameeting.com/workshops
Elizabeth (Liz) Kellogg (Cornell), Ed Twomey (Johns Hopkins), and I, Mark 
Herzik (UC San Diego) have assembled a panel of leading cryo-EM experts to 
present practical, technical advice used in their own groups and to answer 
questions from the audience.
Confirmed speakers include:
Eva Nogales (UC Berkeley)
Chris Russo (MRC)
Mimi Ho (Columbia)
Oliver Clarke (Columbia)
Michael Cianfrocco (U. Michigan)
Ed Eng (NCCAT)
Craig Yoshioka (PNCC)
Ali Punjani (Structura Biotechnology, CryoSPARC)
As with past ACA workshops, this will be a highly interactive session with 
frequent interruptions from the audience and discussion concerning best 
practices among the panelists. A live demonstration of a data collection 
session at a national center will be included as well as “on-the-fly” live data 
processing using cryoSPARC Live.
Fundamentals of single-particle cryo-EM
Cryogenic-electron microscopy (cryo-EM) has emerged as an increasingly more 
powerful technique to visualize frozen biological macromolecules in near-native 
states. Recent advances have enabled cryo-EM to deliver high-resolution density 
maps approaching 1.5 Å or better with maps in the 2-4 Å range now routinely 
being attained, informing the construction of atomic models with high 
confidence. This new capacity has led to increasingly more interest in adopting 
cryo-EM and, consequently, there is a large need for training in cryo-EM 
principles and methods.
Registration information for the ACA Meeting can be found here 
https://www.acameeting.com/registration-information. The ACA virtual meeting 
features the ACA M.J. Buerger Award winner, Wah Chiu, as well as sessions 
related to cryo-EM such as microED (1.1.3 Microcrystal electron diffraction), 
software development (2.1.3 Latest software developments in cryo-EM) and 
advances in detector technology (3.1.1)
https://www.acameeting.com/schedule-sessions
Best wishes and we hope to see you there!
 Mark, Liz, and Ed

 Thanks,
-Mark Herzik, Ph.D.

Assistant Professor
Department of Chemistry and Biochemistry
Natural Sciences Building 4105
University of California, San Diego
9500 Gilman Drive MC 0314
La Jolla, CA 92093
P: 858-246-5875
E: mherzikucsd.edu
herziklab.com




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[ccp4bb] Postdoctoral positions at UT Southwestern, Dallas

[Yanhe Zhao]

On behalf of Prof. Nicastro:

The Nicastro lab 
(https://www.utsouthwestern.edu/labs/nicastro/)
 in the Cell Biology Department of UT Southwestern is recruiting a postdoctoral 
fellow with experience in cell biology or structural biology to work in the 
field of cellular cryo-electron tomography (incl. cryo-FIB).


We have a state-of-the-art cryo-EM facility: 
https://www.utsouthwestern.edu/labs/cemf/
 and a wide range of interesting projects (*).

Interested applicants should email 
daniela.nicas...@utsouthwestern.edu 
.



UT Southwestern is an equal opportunity employer committed to diversity (for 
more information please visit 
https://www.utsouthwestern.edu/about-us/administrative-offices/equity-access/).



*) We study macromolecular assemblies in:

algae 
(https://rupress.org/jcb/article/218/12/4236/132536/Structural-organization-of-the-C1a-e-c),
 single-celled parasites, yeast, worms, flies, sea urchin 
(https://science.sciencemag.org/content/360/6387/eaar1968.long),
 fish, mouse 
(https://www.biorxiv.org/content/10.1101/2021.06.19.448910v1),
 and human cells ... not to forget bacteria and viruses, and methods 
development 
(https://chanzuckerberg.com/science/programs-resources/imaging/frontiers/clonable-in-situ-label-for-high-resolution-cellular-imaging/).


Yanhe Zhao
Postdoctoral Researcher of Prof. Nicastro's Lab
Cell Biology
UT Southwestern Medical Center
NL05110G
5323 Harry Hines Blvd.
Dallas TX 75390-9039
yanhe.z...@utsouthwestern.edu




UT Southwestern

Medical Center

The future of medicine, today.



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Re: [ccp4bb] pictures in emails

[Patrone Marco]

Dear All,
what about linking to any free cloud service for large attachments? Isn't CCP4 
7.1 already such a cloud?

Best,
marco

Marco Patrone – PhD
Biocrystallography Unit
Division of Immunology, Transplantation, and Infectious Disease (DITID)
DIBIT – San Raffaele Scientific Institute
Via Olgettina, 58
20132 Milano
Italy
phone: +39 0226434921
fax: +39 0226434153
http://www.hsr.it/research/organzation/massimo-degano


From: CCP4 bulletin board  on behalf of Nukri Sanishvili 

Sent: Monday, July 19, 2021 4:34:07 PM
To: CCP4BB@JISCMAIL.AC.UK 
Subject: Re: [ccp4bb] pictures in emails

Hi Frank, CCP4bb-ers,
While the progress in the digital world in 2021 over the 90s is undeniable 
(which, unfortunately cannot be said about the analog world), we should keep in 
mind that some institutional accounts may filter out the "suspiciously" large 
emails.
I would not be surprised if some of these accounts end up blacklisting repeat 
offenders - CCP4bb in this case.
Best wishes,
Nukri

On Mon, Jul 19, 2021 at 8:12 AM Frank von Delft 
mailto:frank.vonde...@cmd.ox.ac.uk>> wrote:
Ahem.  Ed, Tim - the 90s called, they want want their disk space and email 
clients back.

As a co-perpertrator of vast volumes of detector data, and religious proponent 
of text formatting as inseparable part part the message, I disagree that in the 
year 2021, your suggestion still conforms to netiquette.

In case anybody was left with the impression that Tim and Ed's view was 
universal.

frank







On 19/07/2021 10:26, Tim Gruene wrote:

Hi Ed, dear ccp4bbs

I fully support your email!

Most email clients have a tick box to include a plain text version in
addition of the html-version in their preferences.

Reading plain text emails instead of the html-version (which is also an
available option for most email clients) is a very effective barrier
against spam. People should take this into account, considering the
global rise of ransomware attacks, with serious consequences to some
large institutions and companies.

It has always been good netiquette to at least include a text-version
of your email.

Cheers,
Tim

On Sun, 18 Jul 2021 11:17:26 -0400 Edward Berry 

wrote:



Two suggestions for people sending pictures of electron density to
the BB:

1. Reduce the size of the pictures-
The two pictures in yesterday's email appear nice and small in my
email client, but still illustrate what is being described. However
they are actually 4032x3024 and 2040x1458 pixels, making for rather
large emails. All that extra resolution is wasted unless the user
opens the image directly ("view image"), and in any case is
completely unnecessary for the point being made. I think about
600x600 pixels is plenty for almost anything you want to show in
electron density. This does not require manipulation in photoshop or
such- just reduce the size of the graphics window and take a
screenshot of that window.

2. send the picture as an attachment rather than inline. That way it
won't be included in all the replies. Or if the people replying could
find some way to exclude pictures or formt the reply as plain-text,
that would help.

(No, I'm not receiving these emails via 1200 baud modem- but I like
to save the messages for future reference. If the trend continues
toward high-resolution inline screenshots, that will take a
significant amount of disk space. And yes, I know there is an
archive.) eab



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Re: [ccp4bb] pictures in emails

[Nukri Sanishvili]

Hi Frank, CCP4bb-ers,
While the progress in the digital world in 2021 over the 90s is undeniable
(which, unfortunately cannot be said about the analog world), we should
keep in mind that some institutional accounts may filter out the
"suspiciously" large emails.
I would not be surprised if some of these accounts end up blacklisting
repeat offenders - CCP4bb in this case.
Best wishes,
Nukri

On Mon, Jul 19, 2021 at 8:12 AM Frank von Delft 
wrote:

> Ahem.  Ed, Tim - the 90s called, they want want their disk space and email
> clients back.
>
> As a co-perpertrator of vast volumes of detector data, and religious
> proponent of text *formatting* as inseparable part part the message, I
> disagree that in the year 2021, your suggestion still conforms to
> netiquette.
>
> In case anybody was left with the impression that Tim and Ed's view was
> universal.
>
> frank
>
>
>
>
>
>
>
> On 19/07/2021 10:26, Tim Gruene wrote:
>
> Hi Ed, dear ccp4bbs
>
> I fully support your email!
>
> Most email clients have a tick box to include a plain text version in
> addition of the html-version in their preferences.
>
> Reading plain text emails instead of the html-version (which is also an
> available option for most email clients) is a very effective barrier
> against spam. People should take this into account, considering the
> global rise of ransomware attacks, with serious consequences to some
> large institutions and companies.
>
> It has always been good netiquette to at least include a text-version
> of your email.
>
> Cheers,
> Tim
>
> On Sun, 18 Jul 2021 11:17:26 -0400 Edward Berry  
> 
> wrote:
>
>
> Two suggestions for people sending pictures of electron density to
> the BB:
>
> 1. Reduce the size of the pictures-
> The two pictures in yesterday's email appear nice and small in my
> email client, but still illustrate what is being described. However
> they are actually 4032x3024 and 2040x1458 pixels, making for rather
> large emails. All that extra resolution is wasted unless the user
> opens the image directly ("view image"), and in any case is
> completely unnecessary for the point being made. I think about
> 600x600 pixels is plenty for almost anything you want to show in
> electron density. This does not require manipulation in photoshop or
> such- just reduce the size of the graphics window and take a
> screenshot of that window.
>
> 2. send the picture as an attachment rather than inline. That way it
> won't be included in all the replies. Or if the people replying could
> find some way to exclude pictures or formt the reply as plain-text,
> that would help.
>
> (No, I'm not receiving these emails via 1200 baud modem- but I like
> to save the messages for future reference. If the trend continues
> toward high-resolution inline screenshots, that will take a
> significant amount of disk space. And yes, I know there is an
> archive.) eab
>
> 
>
> To unsubscribe from the CCP4BB list, click the following 
> link:https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
> available at https://www.jiscmail.ac.uk/policyandsecurity/
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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Re: [ccp4bb] pictures in emails

[Frank von Delft]

Ahem.  Ed, Tim - the 90s called, they want want their disk space and 
email clients back.


As a co-perpertrator of vast volumes of detector data, and religious 
proponent of text /formatting/ as inseparable part part the message, I 
disagree that in the year 2021, your suggestion still conforms to 
netiquette.


In case anybody was left with the impression that Tim and Ed's view was 
universal.


frank







On 19/07/2021 10:26, Tim Gruene wrote:

Hi Ed, dear ccp4bbs

I fully support your email!

Most email clients have a tick box to include a plain text version in
addition of the html-version in their preferences.

Reading plain text emails instead of the html-version (which is also an
available option for most email clients) is a very effective barrier
against spam. People should take this into account, considering the
global rise of ransomware attacks, with serious consequences to some
large institutions and companies.

It has always been good netiquette to at least include a text-version
of your email.

Cheers,
Tim

On Sun, 18 Jul 2021 11:17:26 -0400 Edward Berry 
wrote:


Two suggestions for people sending pictures of electron density to
the BB:

1. Reduce the size of the pictures-
The two pictures in yesterday's email appear nice and small in my
email client, but still illustrate what is being described. However
they are actually 4032x3024 and 2040x1458 pixels, making for rather
large emails. All that extra resolution is wasted unless the user
opens the image directly ("view image"), and in any case is
completely unnecessary for the point being made. I think about
600x600 pixels is plenty for almost anything you want to show in
electron density. This does not require manipulation in photoshop or
such- just reduce the size of the graphics window and take a
screenshot of that window.

2. send the picture as an attachment rather than inline. That way it
won't be included in all the replies. Or if the people replying could
find some way to exclude pictures or formt the reply as plain-text,
that would help.

(No, I'm not receiving these emails via 1200 baud modem- but I like
to save the messages for future reference. If the trend continues
toward high-resolution inline screenshots, that will take a
significant amount of disk space. And yes, I know there is an
archive.) eab



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Re: [ccp4bb] Negative electron density

[Clemens Vonrhein]

Just for completeness: BUSTER includes an automatic so-called "void
correction" that tries to detect those incorrectly bulk-solvent filled
cavities and correct for it. Nothing for you to do extra, since that
correction is on by default ... and works as expected in a lot of cases
as far as we can tell. And yes, it is also approaching this in a "all
or nothing" manner, so not very subtle.

Cheers

Clemens

PS: see also

  https://www.globalphasing.com/buster/
  https://www.globalphasing.com/buster/wiki/index.cgi?BusterFAQ#abruptRchange
  
https://www.ccp4.ac.uk/schools/DLS-2019/course_material/day7/Vonrhein_GlobalPhasing_BUSTER_CCP4-DLS2019.pdf


On Sun, Jul 18, 2021 at 10:57:59AM -0700, James Holton wrote:
> There are two ways to test the "too much bulk solvent" hypothesis.
> 
> 1) examine the bulk solvent electron density map.
>   This is (IMHO) a lot harder than it should be, but here are two recipes:
>   refmac5 : on the command line, put "mskout rawmask.map" somewhere among
> the hklin xyzin, etc directives.  This will create a map you can load into
> coot with the "File:Open map..." menu item.
>   phenix: use phenix.fmodel twice.  Once with k_sol=1, and again with
> k_sol=0.  Load the two resulting mtz files into coot with "File:Open MTZ,
> mmCIF, fcf or phs..." and then create a difference map between them using
> "Calculate:Map Tools...:Make a Difference Map...".  This difference map will
> be the bulk solvent component.
> 
> 2) suppress the bulk solvent.
> The easiest way to do this is fill the region with water atoms at low
> occupancy, such as 0.01.  These will contribute essentially no electron
> density of their own, but will also prevent the refinement program from
> putting bulk solvent anywhere near them.  At least with current algorithms,
> atoms at any occupancy exclude 100% of bulk solvent.  Since they will be
> poorly restrained, you might want to fix these waters in place with harmonic
> restraints, or otherwise tell your favorite refinement program to not move
> them.  Your documentation may vary.

-- 

*--
* Clemens Vonrhein, Ph.D. vonrhein AT GlobalPhasing DOT com
* Global Phasing Ltd., Sheraton House, Castle Park 
* Cambridge CB3 0AX, UK   www.globalphasing.com
*--



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Re: [ccp4bb] 60 kDa contamination in Rosetta cells

[PiscoScience]

Hvvxbxhxxhhfjxjdjdhhdydfyhat the 60 kDa is not your biggest concern.
Optimize soluble protein expression and pay attention to cell lysis.  Is
the sample remaining cold throughout the process?  Are you using enough
volume of buffer relative to solid cell pellet?

>
> Also, from the gel it does not look like your 37 and 15 kDa bands are
> co-eluting.  And your lane labels in the figure are not right.
>
> Good news:  this seems like a solvable problem.  Keep after it!
>
> Tom Huxford.
>
> ==
> Tom Huxford
> Structural Biochemistry Laboratory
> Department of Chemistry & Biochemistry
> San Diego State University
> (619) 594-1606
>
> On Jul 13, 2021, at 5:22 AM, Dilip Badgujar  wrote:
>
> Hello,
>
> Please find the attached gel picture for the reference and some additional
> information.
>
> Lysis buffer-50 mM Tris-pH 8.0, 300 mM NaCl, 1 mM DTT. 1X protease
> inhibitor cocktail.
>
> SEC column – Superdex 200. 16/300
>
> One of the proteins is 37 kDa while the other one is15 kDa. I do not think
> that they are making that strong heterodimer. The total molecular of the
> complex would be ~ 52 kDa but now it is around 60 kDa. I am currently using
> lower temperature (16 ᵒC) but can try around 12 with low IPTG conc.
>
> Regards
>
> Dilip Badgujar
>
> On Mon, Jul 12, 2021 at 11:56 PM zaigham khan 
> wrote:
>
>> Hey Dilip,
>>
>> There are many reasons for this observation. Rafael is right, please do
>> share the image of the gel. Also what are the exact sizes of the two
>> proteins that you co-expressed? I have observed the heterodimeric and
>> pentameric proteins on SDS-PAGE albeit the presence of DTT in the sample
>> buffer, and despite boiling of the samples. Could this be that 60 KD is
>> actually the hetero-dimer? One can perform western blotting, followed by
>> the use of anti-polyhistidine and anti-Streptavidin antibodies on separate
>> blots to confirm the suspected bands. Likewise SEC followed by WB may
>> confirm the identity of the eluted proteins in different fractions after
>> size exclusion chromatography. You may also cleave the tags, and then see
>> the magic!
>>
>> Tom has correctly pointed out that induction at lower temperature is best
>> achieved upon incubation of culture so that the temperature is dropped
>> before the induction.
>>
>> Bon Voyage!
>>
>> -Z
>>
>>
>> Zaigham M Khan, PhD
>> Associate Scientist
>>
>> Icahn School of Medicine at Mount Sinai
>> Department of Oncological Sciences
>> 1470 Madison Avenue
>> 
>> New York
>> 
>> United States
>> 
>>
>>
>> On Mon, Jul 12, 2021 at 2:45 AM Dilip Badgujar 
>> wrote:
>>
>>> Greetings, everyone
>>>
>>> I am trying to co-express two mammalian proteins (less than 50 kDa MW)
>>> in Rosetta cells but getting a contaminating band around 60 kDa. One of the
>>> construct is in pET28a with His tag while the other is in pET21c with Strep
>>> tag and I am adding all the three selection markers during growth of
>>> pre-culture and during induction. Initially, cells were grown at 37 °C
>>> till OD reaches to 0.6 then induced with 0.5mM IPTG and incubated at 16°C
>>> ON. When I do purification using Streptactin resin; I can see proteins of
>>> my interest bound to the resin along with contaminating protein at 60 kDa.
>>> I have tried performing size exclusion as a follow up step but they are
>>> co-eluting in void volume. I have also tried to wash with MgCL2-ATP
>>> solution but it co-elution with contaminant. I am looking for valuable
>>> suggestions to avoid the contamination during or after expression.
>>>
>>> Thanks in advance.
>>>
>>> Regards
>>>
>>> Dilip
>>>
>>> --
>>>
>>> To unsubscribe from the CCP4BB list, click the following link:
>>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>>>
>>
>
> --
> Dilip C. Badgujar, (PhD)
> Post Doctoral Fellow,
> IIT Bomaby
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>
> 
>
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
>



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Re: [ccp4bb] pictures in emails

[Tim Gruene]

Hi Ed, dear ccp4bbs

I fully support your email!

Most email clients have a tick box to include a plain text version in
addition of the html-version in their preferences. 

Reading plain text emails instead of the html-version (which is also an
available option for most email clients) is a very effective barrier
against spam. People should take this into account, considering the
global rise of ransomware attacks, with serious consequences to some
large institutions and companies.

It has always been good netiquette to at least include a text-version
of your email.

Cheers,
Tim

On Sun, 18 Jul 2021 11:17:26 -0400 Edward Berry 
wrote:

> Two suggestions for people sending pictures of electron density to
> the BB:
> 
> 1. Reduce the size of the pictures-
> The two pictures in yesterday's email appear nice and small in my
> email client, but still illustrate what is being described. However
> they are actually 4032x3024 and 2040x1458 pixels, making for rather
> large emails. All that extra resolution is wasted unless the user
> opens the image directly ("view image"), and in any case is
> completely unnecessary for the point being made. I think about
> 600x600 pixels is plenty for almost anything you want to show in
> electron density. This does not require manipulation in photoshop or
> such- just reduce the size of the graphics window and take a
> screenshot of that window.
> 
> 2. send the picture as an attachment rather than inline. That way it
> won't be included in all the replies. Or if the people replying could
> find some way to exclude pictures or formt the reply as plain-text,
> that would help.
> 
> (No, I'm not receiving these emails via 1200 baud modem- but I like
> to save the messages for future reference. If the trend continues
> toward high-resolution inline screenshots, that will take a
> significant amount of disk space. And yes, I know there is an
> archive.) eab
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1
> 
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
> mailing list hosted by www.jiscmail.ac.uk, terms & conditions are
> available at https://www.jiscmail.ac.uk/policyandsecurity/



-- 
--
Tim Gruene
Head of the Centre for X-ray Structure Analysis
Faculty of Chemistry
University of Vienna

Phone: +43-1-4277-70202

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[ccp4bb] Postdoc positions Leicester, UK

[Panne, Daniel (Prof.)]

Dear all,

We are looking for postdoctoral researchers to join our lab our lab at the 
Leicester Institute for Structural and Chemical Biology, UK. The positions are 
funded by a 5 year Wellcome Trust Investigator award to work on the regulation 
of gene expression and 3D genome organisation. For examples of recent work see 
Nature 578, 472–476 (2020); NSMB 27, 233–239 (2020); Nature 562, 538–544 (2018)

The successful candidates will use a multi-disciplinary approach that includes 
preparative biochemistry of multi-protein complexes, protein engineering and 
state-of-the-art biophysical and structural techniques. The group is part of 
the Leicester Institute of Structural and Chemical Biology, UK. We have 
excellent access to state-of-the-art structural biology technologies including 
a Krios cryo-EM microscope

The advert closes on August 31st.

Please apply at: 
https://jobs.le.ac.uk/vacancies/2872/research-associate-structural-biology-of-genome-regulation.html

If you have questions concerning this role, please contact me directly.
All the best,
Daniel

--

Daniel Panne
Professor of Structural Biology
Department of Molecular and Cell Biology
University of Leicester
Email: daniel.pa...@le.ac.uk



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[ccp4bb] Application deadline approaching - Virtual CCP4 Protein Crystallography Summer School 2021 (6th-10th September)

[David Lawson (JIC)]

Dear colleagues,

We are pleased to announce the Virtual CCP4 Protein Crystallography Summer 
School 2021 to be hosted by the John Innes Centre (Norwich, UK).

The school will take place from Monday 6 September – Friday 10 September 
inclusive and is intended for students and researchers new to crystallography. 
Its dual aims are to provide comprehensive training in crystallography and to 
promote best practice. The lectures cover the range of techniques required for 
protein structure solution, from protein expression through to structure 
validation. Intensively supervised computer tutorials enable students to relate 
the theory presented in the lectures to practice.

The application deadline is Thursday 5 August 2021
(don’t leave it to the last minute – your application needs to be supported by 
your supervisor or line manager)

For further information and to apply please follow this link:
https://www.jic.ac.uk/training-careers/summer-schools/ccp4-protein-crystallography-summer-school/

Best wishes,

Dave, Clare, Julia, Naomi and Sarah (organisers)




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