Re: [ccp4bb] Difficult Molecular replacement

[Artem Evdokimov]

Hi Marco,

To follow up: if your protein is something like WRN or BLM helicase - you
mention recA like fold after all - (I worked on these in the past hehe)
then the split into domains is almost mandatory - these proteins are very
plastic and doing MR on them is equivalent to doing antibody MR where you
absolutely have to treat domains as separately mobile rigid bodies.

I would love to help more but it would require more sharing that you might
be justifiably unwilling or unable to do :)

Best of luck,

Artem



On Mon, Feb 19, 2024 at 6:51 PM Artem Evdokimov 
wrote:

> Hello Marco,
>
> First: compare your unit cell parameters with RCSB (there is an option for
> that in the search, very helpful). Give it say 5% error margin.
>
> Worst case scenario - cry a little, having found that your protein is
> something else...
>
> Next, check in depth for space group oddities, twinning, or general data
> misbehavior.
>
> Next, split your helicase into two lobes (if this is the right kind of
> helicase) and do MR with both halves, like it was a complex of two proteins
> (requires a bit of skill with MR - but nothing terrifyingly hard tbh). If
> there are more domains - split into more (RQC, HRDC, etc.)
>
> Hope this helps.
>
> Artem
>
>
>
>
> On Mon, Feb 19, 2024, 5:54 PM Marco Bravo  wrote:
>
>> Hello all,
>> I recently collected data on some plate crystals for a previously
>> uncharacterized protein at the ALS light source. The XDS auto-data
>> processing log output indicates that my resolution is 2.8 angstroms. The
>> protein is a helicase with homologs already in the protein data bank making
>> it a suitable target for molecular replacement which I thought initially.
>> However after trying molecular replacements with all known homologs in the
>> protein data bank the R values remain high after MR >0.5. After an initial
>> round of Rigid body or restrained refinement. The R values still remain
>> very high at around >.5. I have tried MR with Rosetta and alphaphold models
>> but the problem of high R values persists. The best solution I get is from
>> the CCP4 cloud automatic molecular replacement and model building pipeline
>> which gives me a free R value of 0.46.. However the solution is only for
>> residues ~100-326 out of a 543 amino acid long protein. And even then the
>> model still has a lot of missing residues and truncated sidechains and
>> overall fits the map quite poorly. Does anyone have any suggestions about
>> how I can solve my structure if at all possible at this point? I ran the
>> crystals and pre-crystalized samples on a gel and it appears that the
>> protein remains stable during crystallization as the molecular weight did
>> not change or any degradation does not appear.
>>
>> 
>>
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Re: [ccp4bb] Difficult Molecular replacement

[Artem Evdokimov]

Hello Marco,

First: compare your unit cell parameters with RCSB (there is an option for
that in the search, very helpful). Give it say 5% error margin.

Worst case scenario - cry a little, having found that your protein is
something else...

Next, check in depth for space group oddities, twinning, or general data
misbehavior.

Next, split your helicase into two lobes (if this is the right kind of
helicase) and do MR with both halves, like it was a complex of two proteins
(requires a bit of skill with MR - but nothing terrifyingly hard tbh). If
there are more domains - split into more (RQC, HRDC, etc.)

Hope this helps.

Artem




On Mon, Feb 19, 2024, 5:54 PM Marco Bravo  wrote:

> Hello all,
> I recently collected data on some plate crystals for a previously
> uncharacterized protein at the ALS light source. The XDS auto-data
> processing log output indicates that my resolution is 2.8 angstroms. The
> protein is a helicase with homologs already in the protein data bank making
> it a suitable target for molecular replacement which I thought initially.
> However after trying molecular replacements with all known homologs in the
> protein data bank the R values remain high after MR >0.5. After an initial
> round of Rigid body or restrained refinement. The R values still remain
> very high at around >.5. I have tried MR with Rosetta and alphaphold models
> but the problem of high R values persists. The best solution I get is from
> the CCP4 cloud automatic molecular replacement and model building pipeline
> which gives me a free R value of 0.46.. However the solution is only for
> residues ~100-326 out of a 543 amino acid long protein. And even then the
> model still has a lot of missing residues and truncated sidechains and
> overall fits the map quite poorly. Does anyone have any suggestions about
> how I can solve my structure if at all possible at this point? I ran the
> crystals and pre-crystalized samples on a gel and it appears that the
> protein remains stable during crystallization as the molecular weight did
> not change or any degradation does not appear.
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>
> This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a
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Re: [ccp4bb] Äktaxpress uninet connector : Off topic

[Artem Evdokimov]

Ebay to the rescue:

https://www.ebay.com/itm/185248167872

is this the correct connector?

Once you have a single connector, you can pin-trace it to your hearts'
delight and make new ones for pennies, since the cable and the plugs are
standard...

Artem
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On Thu, Feb 8, 2024 at 11:17 AM Deepak Deepak 
wrote:

> Dear All,
>
> Apologies for the off-topic post.
>
> We recently acquired some old Äktaxpress systems and learned that they
> require a specific USB to Uninet cable in order to connect to a PC. For
> now, we only find it on Cytiva for 2700 USD.
>
> Could you please guide me to some other seller that might have a similar
> product for cheaper or if your lab might have a spare connector?
>
> Kind regards,
> Deepak
>
> Here is the link from cytiva
>
> https://www.cytivalifesciences.com/en/us/shop/chromatography/tools-and-accessories/cables/akta-usb-uninet-cable-p-05948
>
> --
>
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Re: [ccp4bb] What could these crystals be?

[Artem Evdokimov]

Could be dna crystals. They often do not diffract.

Artem

On Wed, Nov 8, 2023, 11:18 AM careinaedgo...@yahoo.com <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

>
> The reservior solution is 0.2 M NaCl2, 0.1 M HEPES pH 7.5, and 25% 3350 PEG
>
> Protein buffer is 300 mM NaCl, 20 mM HEPES pH 7.5, 3mM TCEP
>
> The drop contains 1.5ul protein-DNA complex and 1.5 ul reservior solution
>
>
> On Wednesday, November 8, 2023 at 05:12:25 PM GMT+2,
> stephen.c...@rc-harwell.ac.uk  wrote:
>
>
> Hi Careina,
>
> Without knowing what's in your protein buffer or crystallisation condition
> it's hard to comment.
>
> Best wishes,
> Steve
>
> Dr Stephen Carr
> Research Complex at Harwell (RCaH)
> Rutherford Appleton Laboratory
> Harwell Oxford
> Didcot
> Oxon OX11 0FA
> United Kingdom
> Email stephen.c...@rc-harwell.ac.uk
> tel 01235 567717
> --
> *From:* CCP4 bulletin board  on behalf of
> careinaedgo...@yahoo.com <02531c126adf-dmarc-requ...@jiscmail.ac.uk>
> *Sent:* 08 November 2023 15:00
> *To:* ccp4bb 
> *Subject:* [ccp4bb] What could these crystals be?
>
> Hi all
> We have been trying with no success to crystalize a protein. Recently we
> got these strange shape "crystals". They are hard and flat but they do not
> diffract at all. Any ideas as to what could cause this?
> Careina
>
> --
>
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Re: [ccp4bb] What could these crystals be?

[Artem Evdokimov]

Dear Careina

These look like crystals indeed. Without more information, I can make some
educated guesses but the ultimate proof is in your hands :)

1. they don't have to be spherulites. Note that low-symmetry crystals can
have edge-less habits, especially P1 are notorious for that - I've had
crystals looking like grape seeds or even spheres/eggs (i.e. not a single
straight edge) and they diffracted well and weren't twinned. So these can
be just crystals (but not diffracting ones, sadly)

2. when you write that these objects are 'hard' - did you actually crush
them with a metal needle? Do they 'pop' or 'squish'? If they make cracking
noises, they're salt or small molecule crystals.

It might not hurt to try and set them up in oil drops without cryo, to see
if this 'cheap room temperature' diffraction experiment reveals protein
like lattice. Of course this requires hands on access to a diffractometer,
which these days isn't a common thing.

All the best,

Artem


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On Wed, Nov 8, 2023 at 10:02 AM careinaedgo...@yahoo.com <
02531c126adf-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi all
> We have been trying with no success to crystalize a protein. Recently we
> got these strange shape "crystals". They are hard and flat but they do not
> diffract at all. Any ideas as to what could cause this?
> Careina
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] [OFF TOPIC] On Ni-NTA resin TEV cleavage

[Artem Evdokimov]

Dear Rafael

In addition to the excellent suggestions already offered by previous
responders, I can attest that Ni-penta resin (sold among others by a
company with an odd name Marvelgent) is resistant to EDTA and DTT, and it
leaks very little Ni (almost none). Plus, it elutes with low inidazole,
owing to the single available chelation point offered by the immobilized Ni
ions.

Depending on the resin and other conditions tou use, there generally is no
reason to worry about the small amount of DTT and EDTA that may be present
in TEV preparations. On the other hand, there is a reason to be slightly
concerned that the leaky Ni ions may inhibit your TEV.

In summary, you have to try it on small scale, the odds are good that
cleavage on resin will work for you. Notably it even works when TEV itself
is his-tagged, presumably owing ro the dynamic nature of His/Ni
interactions (strictly speaking, resin affinity is only about 1 uM, but
avidity and intra resin exchange and recapture keep the bulk of protein
trapped in the resin).

Good luck!

Artem

On Tue, Oct 31, 2023, 3:21 PM Rafael Marques 
wrote:

> Hi everyone,
>
> I have been looking on this bb and other websites as well but I could not
> find a veredict. We are suspecting that when I elute my sample from my
> Ni-NTA column, the imidazole concentration (250 mM) is making it to
> precipitate. Once my sample has a cleavable TEV site, I was planning to
> incubate my loaded resin overnight with TEV and get my sample back simply
> using my lysis buffer. And here lies the problem. Most of the TEVs are kept
> in EDTA and DTT and I wonder if they are essential for its protease
> activity or if I could use another reducing agent more compatible with my
> resin (or maybe do not add both). I saw that someone did not have EDTA and
> used b-mercap. instead of DTT. May I have your comments if you guys already
> faced a similar situation?
>
> Best wishes
>
> __
>
> Rafael Marques da Silva
>
> PhD Student – Structural Biology
>
> University of Leicester
>
> Mestrando em Física Biomolecular
> Universidade de São Paulo
>
> Bacharel em Ciências Biológicas
> Universidade Federal de São Carlos
>
> phone: +55 16 99766-0021
>
> *   "A sorte acompanha uma mente bem treinada"*
> **
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] Autoinduction Medium

[Artem Evdokimov]

These are the ones I've used in the past (I mostly use home-made, since it
gives one more control over the process). For folks who prefer convenience,
please see below (Thermo's $146/liter, though... LOL). Also, in fairness,
at least the Millipore/Sigma offers you isotopically defined media
preparations.

https://formedium.com/product-category/formedium-media/escherichia-coli-media/aim-auto-induction-medium/
https://bocascientific.com/gcm20-0500-506-detail
Overnight Express (VWR or Millipore)
Magic Media (Thermo)

I do NOT recommend autoclaving complete formulations (like the Boca Sci.
one above) because sugars in the medium react with amino acids resulting in
Brown Evil. Look it up if you're curious...

In recognition of the original author, references to Studier papers:

2005:   https://pubmed.ncbi.nlm.nih.gov/15915565/
2014:   https://pubmed.ncbi.nlm.nih.gov/24203322/

Artem

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On Mon, Oct 2, 2023 at 10:15 AM Artem Evdokimov 
wrote:

> Dear  Jinhui Dong
>
> You can simply make swimming-pool size amounts of it, as long as you have
> basic lab equipment and supplies including an autoclave, and access to ~8
> chemicals. The recipe is in the Studier papers, but I am happy to share it
> with you here if you prefer.
>
> Artem
>
> - Cosmic Cats approve of this message
>
>
> On Mon, Oct 2, 2023 at 9:39 AM Dong, Jinhui  wrote:
>
>> Hi,
>> Could you please post the following message on ccp4bb?
>>
>> Autoinduction medium
>> We need autoinduction medium for protein expression in E coli. Could
>> anyone provide information on USA suppliers with trustworthy products and
>> reasonable prices?  Thank you!
>> Jamse
>> Brown University
>>
>> Thanks!
>>
>> Jinhui Dong
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>>
>



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Re: [ccp4bb] Autoinduction Medium

[Artem Evdokimov]

Dear  Jinhui Dong

You can simply make swimming-pool size amounts of it, as long as you have
basic lab equipment and supplies including an autoclave, and access to ~8
chemicals. The recipe is in the Studier papers, but I am happy to share it
with you here if you prefer.

Artem

- Cosmic Cats approve of this message


On Mon, Oct 2, 2023 at 9:39 AM Dong, Jinhui  wrote:

> Hi,
> Could you please post the following message on ccp4bb?
>
> Autoinduction medium
> We need autoinduction medium for protein expression in E coli. Could
> anyone provide information on USA suppliers with trustworthy products and
> reasonable prices?  Thank you!
> Jamse
> Brown University
>
> Thanks!
>
> Jinhui Dong
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
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Re: [ccp4bb] Adding metal ions

[Artem Evdokimov]

Take a protein structure with waters, replace surface waters with Cu++ and
run some very basic MD to optimize distances. Many of the atoms will move
since Cu++ will find itself in an 'unfriendly' environment, as always
caveat emptor :)

Artem

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On Fri, Jun 16, 2023 at 12:00 AM Veerendra Kumar  wrote:

> Dear All,
> I want to add about 200 copper ions on protein surface randomly. Is there
> any program available to serve the purpose?
>
> Thank you
> Best regards
> Veerendra
>
> Get Outlook for Android 
>
> --
>
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Re: [ccp4bb] Regarding the diffraction image

[Artem Evdokimov]

With 50 mM Zn++ in solution, whatever it is will probably have a Zn salt in
it. So if you wanted to solve it by direct methods or via SAD - that should
do well. Sadly (hur hur) it's probably quite small, whatever it is.

Artem

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On Fri, Feb 3, 2023 at 6:20 AM Mark J. van Raaij 
wrote:

> like others mentioned, looks like something in between a salt and a
> protein, perhaps TCEP, the ligand, a peptide cleaved from your protein by
> trace protease.
> If possible, I would move the detector closer, collect an atomic
> resolution dataset and try to solve the structure by direct methods. You
> never know, it could be something interesting.
>
> Mark J van Raaij
> Dpto de Estructura de Macromoleculas, lab 20B
> Centro Nacional de Biotecnologia - CSIC
> calle Darwin 3
> E-28049 Madrid, Spain
> tel. +34 91 585 4616 (internal 432092)
> Section Editor Acta Crystallographica F
> https://journals.iucr.org/f/
> https://namedrop.io/markvanraaij
>
> On 3 Feb 2023, at 09:22, kavyashreem  wrote:
>
> Dear all,
>
> We crystallized a protein (30kDa) + ligand (by cocrystallization), in the
> condition 10%PEG3350, 50mM Zinc acetate.
>
> Protein was in the buffer 20mM HEPES, 150mM NaCl, 1uM ZnCl2, 4mM TCEP, pH
> 8.
>
> Crystal: Crystal:
> crystal under UV m
>
> <8ef9453e.png>
>
> When we collected the data at an in-house facility, it looked something
> like this:
>
> 
>
> The minimum resolution spot is around 9Ang and maximum ~2.2Ang.
>
> I have not come across a protein diffraction like this, nor of a salt.
> When I ran the gel for the incubated protein (protein+ligand), there was no
> degradation.
>
> Although, I was sure there is some problem with this image I tried
> processing, which could not be, But indexing showed a unit cell  of 11Ang,
> 11Ang, 46Ang in P3. which was quite expected for two of the axes but not
> the third.
>
> Can anyone please shed some light on this diffraction image?
>
> How can it happen?
>
>
> Thank you
>
> Regards
>
> Kavya
>
>
> --
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Re: [ccp4bb] small scale -15C chiller

[Artem Evdokimov]

Hi Tim

One last option that's possibly even better, but requires rudimentary
electronics: if you find the logic gate that flips from 0 to 1 (or
backwards) upon thermocouple triggering below threshold temperature, you
can simply supply +5V to the appropriate spot on the PCB, and Bob's your
uncle.

To find the spot one has to trace the connection a bit, and have a handy
voltmeter with a circuit probe, so when you put the TC into cold, it will
register the signal. This assumes a fairly simple operation of the
circuit - it may be that something much more sophisticated is taking place,
like a signal on a CAN bus or god knows what else.

All the best,

Artem
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On Fri, Feb 3, 2023 at 2:16 AM Tim Gruene  wrote:

> Hi Artem,
>
> the simulator is exactly what I was looking for - many thanks!
> We did build a small circuit to generate the voltage (-0.586mV for
> -15C), but this didn't work - probably, our circuit was too simple. 12V
> input looks energetically better to me than using a peltier chiller (I
> meant peliter, not piezo originally...)
>
> Second to that I like Mark's idea of a long cable for the couple and
> stick it in the next -20C cooler.
>
> And yes - I already confirmed with cooling by liquid nitrogen, that the
> concept works: when the thermocouple indicates cold enough, I can
> operate the diffractometer, and hence install our new alien detector. I
> was indeed looking for a long-term solution to make overnight
> measurements.
>
> Thanks to everyone. I feel overwhelmed with the large number of quick
> responses.
>
> Best,
> Tim
>
> On Thu, 2 Feb 2023 20:19:32 -0500
> Artem Evdokimov  wrote:
>
> > A basic Peltier element will likely work (may need a stack of two to
> > reach -20) however the simpler option indeed would be to 'fake out'
> > thermocouple input using a voltage, as described by Ivan.
> >
> > https://us.flukecal.com/Thermocouple-Temperature-Calculator
> >
> > And for $38 one can apparently purchase a thermocouple simulator
> >
> >
> https://www.brightwinelectronics.com/product/temperature-calibrator-k-n-thermocouple-generator-simulator
> >
> > Artem
> >
> > On Thu, Feb 2, 2023, 3:42 PM Rajkovic, Ivan <
> > 95c2dc0d4fa4-dmarc-requ...@jiscmail.ac.uk> wrote:
> >
> > > Hi Tim,
> > >
> > > Not sure if this would work, but can you get a voltage supply and
> > > connect it instead of the thermocouple? You would need something to
> > > provide a few mV:
> > > https://www.thermocoupleinfo.com/type-k-thermocouple.htm
> > >
> > >
> > > Ivan
> > >
> > >
> > > > -Original Message-
> > > > From: CCP4 bulletin board  On Behalf Of Tim
> > > > Gruene
> > > > Sent: Thursday, February 02, 2023 11:57 AM
> > > > To: CCP4BB@JISCMAIL.AC.UK
> > > > Subject: [ccp4bb] small scale -15C chiller
> > > >
> > > > Good day,
> > > >
> > > > I would like to cool a K-type thermocouple down to -15C. The
> > > > temperature
> > > does
> > > > not need to be very accurate, nor exactly -15C, just below that
> > > > level.
> > > >
> > > > I was thinking of using a piezo-chiller, but they don't seem to
> > > > be very
> > > energy
> > > > efficient.
> > > >
> > > > Could anyone make a recommendation for a simple device to cool
> > > > the tiny thermocouple to -15C to -20C?
> > > >
> > > > For the details: we have an inhouse diffractometer with a dead
> > > > detector.
> > > The
> > > > system is blocked, because the temperature of the detector needs
> > > > to be
> > > below -
> > > > 15C. It is measured with a K-type thermocouple. I tested with
> > > > liquid
> > > nitrogen.
> > > > The system show -64C (probably the limit of the thermocoupe) and
> > > > I can
> > > operate
> > > > the system (and mount our new (brand-alien) detector).
> > > >
> > > > Thanks a lot!
> > > >
> > > > Best,
> > > > Tim
> > > >
> > > > --
> > > > --
> > > > Tim Gruene
> > > > Head of the Centre for X-ray Structure Analysis Faculty of
> > > > Chemistry
> > > University
> > > > of Vienna
> > > >
> > > > Phone: +43-1-4277-70202
> > > >
> > > > GPG Key ID = A46BEE1A
> > > >
> > > > 

Re: [ccp4bb] small scale -15C chiller

[Artem Evdokimov]

A basic Peltier element will likely work (may need a stack of two to reach
-20) however the simpler option indeed would be to 'fake out' thermocouple
input using a voltage, as described by Ivan.

https://us.flukecal.com/Thermocouple-Temperature-Calculator

And for $38 one can apparently purchase a thermocouple simulator

https://www.brightwinelectronics.com/product/temperature-calibrator-k-n-thermocouple-generator-simulator

Artem

On Thu, Feb 2, 2023, 3:42 PM Rajkovic, Ivan <
95c2dc0d4fa4-dmarc-requ...@jiscmail.ac.uk> wrote:

> Hi Tim,
>
> Not sure if this would work, but can you get a voltage supply and connect
> it instead of the thermocouple? You would need something to provide a few
> mV:
> https://www.thermocoupleinfo.com/type-k-thermocouple.htm
>
>
> Ivan
>
>
> > -Original Message-
> > From: CCP4 bulletin board  On Behalf Of Tim
> > Gruene
> > Sent: Thursday, February 02, 2023 11:57 AM
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [ccp4bb] small scale -15C chiller
> >
> > Good day,
> >
> > I would like to cool a K-type thermocouple down to -15C. The temperature
> does
> > not need to be very accurate, nor exactly -15C, just below that level.
> >
> > I was thinking of using a piezo-chiller, but they don't seem to be very
> energy
> > efficient.
> >
> > Could anyone make a recommendation for a simple device to cool the tiny
> > thermocouple to -15C to -20C?
> >
> > For the details: we have an inhouse diffractometer with a dead detector.
> The
> > system is blocked, because the temperature of the detector needs to be
> below -
> > 15C. It is measured with a K-type thermocouple. I tested with liquid
> nitrogen.
> > The system show -64C (probably the limit of the thermocoupe) and I can
> operate
> > the system (and mount our new (brand-alien) detector).
> >
> > Thanks a lot!
> >
> > Best,
> > Tim
> >
> > --
> > --
> > Tim Gruene
> > Head of the Centre for X-ray Structure Analysis Faculty of Chemistry
> University
> > of Vienna
> >
> > Phone: +43-1-4277-70202
> >
> > GPG Key ID = A46BEE1A
> >
> > #
> > ###
> >
> > To unsubscribe from the CCP4BB list, click the following link:
> > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=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
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>
> 
>
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Re: [ccp4bb] Alternative server for: Surface Entropy Reduction prediction (SERp)

[Artem Evdokimov]

Hi Yong Tang,

I am not sure if there is another SER server out there, but if you can
share the sequence in a private email I can run it through my code that
does a similar type of analysis (it's not user friendly so I never did the
right thing and put it up as a server).

All the best,

Artem

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On Fri, Jan 20, 2023 at 1:38 PM Yong Tang  wrote:

> Dear all, I'm interested in considering the SERp but it seems that the
> server is no longer accepting queries.
>
> https://services.mbi.ucla.edu/SER/
>
> Are you aware of any replacement server/software for such analysis?
>
> Many thanks, -yong
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>



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Re: [ccp4bb] TEV vs HRV3C

[Artem Evdokimov]

One final comment: if you need orthogonal proteases, TEV and TVMV do not
seem to cut each others' sites (at least for me they don't). No idea if 3C
will cut both, or only TEV site.

Artem

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On Thu, Dec 8, 2022 at 5:26 AM Dom Bellini - MRC LMB <
dbell...@mrc-lmb.cam.ac.uk> wrote:

> Hi All,
>
> I agree with everything said and I also find 3C, my first choice, to
> cleave more efficiently/rapidly. But I remember all the folks working with
> membrane proteins were using TEV, so I wonder whether TEV may be more
> resistent to detergent? (but I never read/found any info about this).
>
> BW,
>
> D
>
>
>
> On 08/12/2022 05:40, Nikolay Dobrev wrote:
>
> Dear all,
> I agree with all said so far:
> Here are some points from my experience and discussion on this topic.
>
> Most of the lab's due use TEV or 3C, depending on the cleavage site in
> their constructs and make their purification strategy work with what they
> have.
>
> Here are some points from my side:
>
>
>- 3C protease is much faster in cleaving the fusion protein in the
>1:100 mass ratio (a test we use in cleavage protocols, even though we
>should use a molar ratio. Consider the difference between 20 kDa protein
>and 200 kDa protein, it is 10x in the amount of substrate difference) 3C
>cleaves in 30 mins more than 95% of the protein at 4-8C. TEV does need much
>longer, and as default, people go for overnight dialysis and cleavage
>simultaneously. Sometimes that can be too long for sensitive proteins and
>lead to precipitation due to chance in the buffer salt concentration etc,
>for an extended period. Quite prominently is observed for Nucleic acid
>binding protein, due to lowering the salt for IEX column and precipitation
>surprise overnight.
>- Neither 3C nor TEV requires a reducing agent, which is essential to
>know as many people are working with S-S-containing proteins, and they take
>an aliquot of the protease purified in the lab without knowing there was a
>reducing agent in the storage buffer. Then surprises can be observed.
>Consider the concentration of 1 mM DTT; even 10 or 100 diluted is 10 µM at
>least.
>- Same with a chelating agent like EDTA, no need to be added to the
>storage buffer; the danger comes when you cleave ion-containing proteins
>like Zn-fingers.
>- 3C is much more efficient in on/column cleavage (as was already
>mentioned). This simple trick gives much better purity than Imidazole
>elution, especially when the Ni-column is not saturated and many
>contaminants are bound. Releasing the protein with the protease makes it
>much cleaner. Most of the protease has a His-tag, which allows at the same
>time to bind it to do beads (of course this slows down a bit the cleavage
>therefore mixing is required). Alternatively, GST-(only)-tag protease is
>better used for the cleavage on Ni beads and then captured on the GSH 
> resin.
>- 3C protease is much more efficient for cleaving in the presence of
>detergent, there is an extensive study on that topic (
>https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4836831/)
>- Both proteases are easy to produce, as mentioned, and easily 100-300
>mg can be obtained from 1 L culture (TB or auto-induction with OD= 8-16)
>- 3C protease is not happy at high protein concentrations (>4 mg/ml)
>in low salt buffer (150 mM). Therefore huge loss is observed if, during the
>Ni-elution, low salt buffer is used as the protein comes from the Ni-NTA at
>a concentration 15-20 mg/ml. Please use at least 500 mM NaCl for that step
>or better for any step from lysis to elution.
>- And yes both proteases have cross-activity and can cleave the
>opposite target site with lower activity.
>
> These are the few points coming from the top of my head.
> I hope they are helpful and wish everyone happy cleaving of the fusion
> proteins :)
>
> Kind regards,
> Nikolay
>
> *Nikolay Dobrev *
> Postdoctoral Fellow @ Wilmanns group
> EMBL Hamburg, c/o DESY, Building 25A,
> Notkestraße 85, 22607 Hamburg, Germany
> T +49 40 89902 165 | M +49 173 684 0532
> twitter.com/emblevents | facebook.com/embl.org |
> youtube.com/user/emblmedia
> Visit www.embl.org/events for a complete list of all EMBL events.
>
> On 12/07/2022 11:51 PM Lior Almagor 
>  wrote:
>
>
> In my experience, 3C can partially cut TEV sites as well. If using
> sequentially, it is best to plan to use the TEV first.
>
> On Dec 7, 2022, at 2:42 PM, Lau Kelvin <
> 5aaf8435dbef-dmarc-requ...@jiscmail.ac.uk> wrote:
> I agree. I think it depends on the lab and vectors and personal
> preference.
>
> But David, have you used them sequentially? I have once tried to make a
> His-GST-ENLYFQ-3C construct, and I found that it was self cleaving during
> expression. However I have never tried to replicate the results in vitro.
>
>
>
> --
> Kelvin Lau
> Protein production and structure core 

[ccp4bb] hiring (off-topic but could be of interest)

[Artem Evdokimov]

Dear CCP4-ers,

We're hiring :) In case you're interested, please visit the link below.

http://careers.animol-discovery.com/apply/fUyc5HcbL0/Head-Of-Medicinal-Chemistry?referrer=2022083118532124ZTDOKKYYDQHFBP

Artem
VP@ Animol
Cheesecake aficionado



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[ccp4bb] help request (synchrotron schedules)

[Artem Evdokimov]

Dear CCP4-ers :)

I used to have a handy chart where major synchrotron schedules were all
drawn out as lines (across an entire year) with shutdowns and other
blackout dates marked such that it was pretty easy to find what
synchrotron is open for business at what week/month. This schedule took me
a while to assemble since not every synchrotron handily posts their
shutdown schedule and one has to call friends and ask around.

Needless to say, being a putz that I am, I eventually lost this lovely
document. So, I wonder - does anyone here have something similar that they
won't mind sharing? If enough people share (even individual schedules) I am
happy to post the resulting schedule back here for everyone to enjoy...

Many thanks!

Artem

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Re: [ccp4bb] Off topic - gene toxic for expression strains

[Artem Evdokimov]

Hi there,

Somehow I've missed the original email :) Sorry!

There are options for expressing really toxic genes, some of which have
already been mentioned and others perhaps not:

1. tight regulation of expression (promoter, repressor, other regulatory
elements, or a combination thereof). Beyond using araBAD, xylose promoter,
rhamnose promoter, etc. there is one more thing that can be done, which is
quite old school: phage induction. Yes, it means what it says - you add
actual phage to the otherwise normal E.coli and the phage brings
polymerase. This way there is no tangible expression except leakage which
for T7 is close to non-existent.

2. change expression to a different organism: Bacillus, Pseudomonas or
insect/mammalian cells

3. counter-expression of antisense RNA from a weaker promoter: this method
is not exactly easy, but it does work -- you need a weak promoter (e.g.
unmodified lac or trp) that would drive expression of antisense RNA.
Easiest way to go about this is to put the antisense promoter on the 3' end
of the gene, facing 'backwards'. Then, during induction, the sense-strand
promoter is much more active and the sense RNA will win over the antisense.

Good luck!

Artem

P.S. if you use 2% glucose in the media be prepared to fight acidification
and the accumulation of Acetate in the spent medium, both of which can be a
problem - sometimes severe - for 'weaker' E. coli strains. Strongly
buffered medium is a must.
- Cosmic Cats approve of this message


On Mon, Apr 4, 2022 at 9:16 AM Nikolay Dobrev <
nikolay.dob...@embl-hamburg.de> wrote:

> Hi Andy,
> just to follow up on Christian suggestion, which is exactly the way to go.
>
> In case you are using an already pET based vector, simply try BL21-AI (
> https://www.thermofisher.com/order/catalog/product/C607003), which has
> the T7 RNA polymerase under arabinose promoter should do the trick.
> Also, 2% Glucose is a must in this kind of situation.
> I have been dealing with several toxic proteins (from the family of
> restriction enzymes :) ) and BL21-AI was a way to go.
> Please also have a look if your pET backbone has the extra copy of the
> lacI, which makes a difference in leakage expression.
>
> As a sum up:
> 1) try BL21-AI (for the induction of the target protein you will need both
> Arabinose (T7 RNA pol induction) and IPTG for the T7 promoter)
> 2) or BL21 with pLysS or pLysE
> both simple keep 2 % Glucose and also directly from trafo goto liquid
> culture in parallel of the plating approach.
>
> Let me know if you need any further tips.
>
>
> *Nikolay Dobrev *
> Postdoctoral Fellow @ Wilmanns group
> EMBL Hamburg, c/o DESY, Building 25A,
> Notkestraße 85, 22607 Hamburg, Germany
> T +49 40 89902 165 | M +49 173 684 0532
> twitter.com/emblevents | facebook.com/embl.org |
> youtube.com/user/emblmedia
> Visit www.embl.org/events for a complete list of all EMBL events.
>
>
> On 04/04/2022 10:32 AM Christian Roth  wrote:
>
>
> Hi Andy,
> have you tried another promotor? Arabinose is much tighter, just to be
> sure that it is really not leaking.
>
> Cheers
> Christian
>
>
> On Mon, Apr 4, 2022 at 10:20 AM Andrew Lovering 
> wrote:
>
> Dear Board,
>
>
>
> Perhaps off-topic, but in the wider scope it’s relevant to many on here.
>
>
>
> We have a gene that we are able to clone, and propagate in DH5a etc
> non-expression cells (hence nucleotide sequence is non-toxic)
>
>
>
> But, when we attempt to transfer to an expression strain we get no
> colonies
>
>
>
> We have tried pLemo, Glucose addition, 30 degrees, C41/C43 and still no joy
>
>
>
> We’d welcome any suggestions here – it’s a fun protein
>
>
>
> Thanks
>
> Andy
>
> --
>
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Re: [ccp4bb] Strategies for increasing Trp content of proteins

[Artem Evdokimov]

Yes we chose the sites based on predicted or known structure, to be sure.
No that was not a published thing, curse of commercial work :(

Happy to help on a private channel.

Artem

On Sat, Feb 19, 2022, 1:42 PM Tanner, John J.  wrote:

> Casper: You make a good point about using 205 nm. I checked our system
> this morning and the wavelength is fixed at 280 nm (U9-L).
>
>
>
> Artem: Did you consider secondary structure and/or amino acid residue type
> when choosing sites for mutagenesis? Is your work published?
>
>
>
>
>
> *From: *CCP4 bulletin board  on behalf of Casper
> Wilkens <5d34ab0aef35-dmarc-requ...@jiscmail.ac.uk>
> *Date: *Saturday, February 19, 2022 at 2:19 AM
> *To: *CCP4BB@JISCMAIL.AC.UK 
> *Subject: *[ccp4bb] Sv: [ccp4bb] Strategies for increasing Trp content of
> proteins
>
> *WARNING:* This message has originated from an External Source. This may
> be a phishing expedition that can result in unauthorized access to our IT
> System. Please use proper judgment and caution when opening attachments,
> clicking links, or responding to this email.
>
> Is absorbance at 205 nm not a possibility, Jack?
>
>
>
> *Casper Wilkens *
>
> Asst. Prof.
>
> Structural Enzymology & Biorefining
>
> DTU Bioengineering
>
>
>
> *Technical University of Denmark*
>
> Department of Biotechnology and Biomedicine
>
> Søltofts Plads
> Building 224
> Room 028
>
> 2800  Kgs. Lyngby
>
> c...@dtu.dk 
>
> www.bioengineering.dtu.dk/
> <https://nam02.safelinks.protection.outlook.com/?url=http%3A%2F%2Fwww.bioengineering.dtu.dk%2F=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000=viaDJTf6EgVVTGxk4AGAWEyluluwh9rtnwUCUUULmo0%3D=0>
>
> https://www.cazypedia.org/index.php/User:Casper_Wilkens
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> https://twitter.com/protein_artist
> <https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Ftwitter.com%2Fprotein_artist=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000=bKeX%2FhUL7pnDA3DxL5ZFcTUlHkL5x2Vd7VsrJNPA0H0%3D=0>
> https://www.researchgate.net/profile/Casper_Wilkens
> <https://nam02.safelinks.protection.outlook.com/?url=https%3A%2F%2Fwww.researchgate.net%2Fprofile%2FCasper_Wilkens=04%7C01%7Ctannerjj%40MISSOURI.EDU%7Ccd1aefc8cc284ea9e44d08d9f38074db%7Ce3fefdbef7e9401ba51a355e01b05a89%7C0%7C0%7C637808555459123981%7CUnknown%7CTWFpbGZsb3d8eyJWIjoiMC4wLjAwMDAiLCJQIjoiV2luMzIiLCJBTiI6Ik1haWwiLCJXVCI6Mn0%3D%7C3000=VuBOXy%2BOSmA6usq%2BahiVlN2%2Fm19%2FnlVEqNXDRMRnrkY%3D=0>
> --
>
> *Fra:* CCP4 bulletin board  på vegne af Artem
> Evdokimov 
> *Sendt:* 19. februar 2022 02:12:50
> *Til:* CCP4BB@JISCMAIL.AC.UK
> *Emne:* Re: [ccp4bb] Strategies for increasing Trp content of proteins
>
>
>
> We have had success placing single trp on the outside of proteins, as
> weird as it sounds it works pretty well. If your protein is easy to express
> you can try a few choices.
>
>
>
> If your protein is difficult to express, a fusion with a trp-positive
> domain or with gfp may be a better option.
>
>
>
> There are many other options but their application is best considered with
> more information in hand
>
>
>
> Artem
>
>
>
> On Fri, Feb 18, 2022, 8:02 PM Tanner, John J. 
> wrote:
>
> Dear CCP4BB,
>
> We are working on a protein that has no Trp residues, which makes
> chromatography challenging due to the low absorbance at 280 nm (Abs 0.1% =
> 0.104). Does anyone have experience using mutagenesis to increase the Trp
> content of proteins?
>
> Thanks,
>
> Jack Tanner
>
>
>
> --
>
> John J. Tanner
>
> Professor of Biochemistry and Chemistry
>
> Associate Chair of Biochemistry
>
> Department of Biochemistry
>
> University of Missouri
> 117 Schweitzer Hall
>
> 503 S College Avenue
> Columbia, MO 65211
> Phone: 573-884-1280
>
> Email: tanne...@missouri.edu 
> https://cafnrfaculty.missouri.edu/tannerlab/
>
> Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
>

Re: [ccp4bb] Strategies for increasing Trp content of proteins

[Artem Evdokimov]

We have had success placing single trp on the outside of proteins, as weird
as it sounds it works pretty well. If your protein is easy to express you
can try a few choices.

If your protein is difficult to express, a fusion with a trp-positive
domain or with gfp may be a better option.

There are many other options but their application is best considered with
more information in hand

Artem

On Fri, Feb 18, 2022, 8:02 PM Tanner, John J.  wrote:

> Dear CCP4BB,
>
> We are working on a protein that has no Trp residues, which makes
> chromatography challenging due to the low absorbance at 280 nm (Abs 0.1% =
> 0.104). Does anyone have experience using mutagenesis to increase the Trp
> content of proteins?
>
> Thanks,
>
> Jack Tanner
>
>
>
> --
>
> John J. Tanner
>
> Professor of Biochemistry and Chemistry
>
> Associate Chair of Biochemistry
>
> Department of Biochemistry
>
> University of Missouri
> 117 Schweitzer Hall
>
> 503 S College Avenue
> Columbia, MO 65211
> Phone: 573-884-1280
>
> Email: tanne...@missouri.edu 
> https://cafnrfaculty.missouri.edu/tannerlab/
>
> Lab: Schlundt Annex rooms 3,6,9, 203B, 203C
>
> Office: Schlundt Annex 203A
>
>
>
>
>
> --
>
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Re: [ccp4bb] Baculovirus expression system

[Artem Evdokimov]

There are several but if you discount the transfection based ones (i.e.
direct DNA, no virus at all) then the speed is roughly the same - about
four weeks from clone to process. FlashBac works a bit faster because it
avoids the use of ecoli to recombine the bacmid but the time savings is
about a week only.

As to yield - it is very protein specific and you do get many variables in
play including promoters, cell lines, media additives and so on

Artem

On Mon, Feb 7, 2022, 11:25 AM Srivastava, Dhiraj <
dhiraj-srivast...@uiowa.edu> wrote:

> Hi All
>  sorry for the question not related to crystallography. What is
> the baculovirus expression system that people use these days to get good
> yield in less time?
>
> Thank you
> Dhiraj
>
> --
>
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Re: [ccp4bb] OFF_TOPIC: Should you be worried about BPA from plastics? Yes, if you store alkaline reagents in polycarbonate bottles!

[Artem Evdokimov]

Interesting note - and a reminder that alkaline solutions are tricky :) By
personal preference, HDPE bottles are probably the best for storing
strongly alkaline solutions (XLPE also is very good, but harder to get
lab-grade bottles).

Here's a handy chart for plastic vs stuff resistance. Note that HDPE is "E"
whereas PC (polycarbonate) is "N".

https://www.calpaclab.com/chemical-compatibility-charts/

Glass is not recommended for alkali storage, although there are some glass
products that have A1 or better alkali resistance rating. They can get
pretty esoteric, especially if one requires near-complete passivity towards
strongly alkaline solutions.

Artem

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On Sun, Jan 30, 2022 at 1:02 PM Edward Berry  wrote:

> After using the same reagents for the Lowry assay and seeing the color
> yield in the standard curve gradually decreasing year by year, we decided
> to make new reagents last year. Sure enough the color yield was restored,
> but in the next assay a few weeks later the blank was unusually high. after
> a month the blank read nearly 1 AU.
>
> The problem was, we stored the alkaline reagent (NaOH + Na2CO3)in a
> polycarbonate bottle. I like polycarbonate because it is transparent and
> hard like glass, but lighter and less breakable. But polycarbonate is a
> polyester of Bis-phenol A with carbonic acid. Apparently the high pH slowly
> hydrolyzes the ester linkages, or the plastic retains some monomers that
> slowly leach out, and (duh!) bis-phenol A gives a positive reaction with
> the Folin-Ciocalto phenol reagent.
>
> 
>
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[ccp4bb] multiple openings in my team @ Roivant

[Artem Evdokimov]

Friends,

Sorry for the off-topic message :)

We're advertising three positions, and anticipating a posting on the fourth
shortly. *The three jobs with links should be applied to directly* whereas
for non-posted one please write to me directly. I will do my best to reply
to everyone.

Thank you for reading!

0. Senior Director, Structural Biology (not posted yet, but real)
Looking for 15+ years of experience with multiple structure-determination
and structure-application (e.g. drug discovery) methods. A practical
thinker who is not afraid to roll up sleeves and engage at every level. We
firmly believe in leading from the front.

1. RA in protein science (enthusiasm, work ethic, and learning ability are
key attributes needed, we will train in the rest)

https://www.roivantcareers.com/job/research-associate-senior-research-associate-protein-sciences-research-development-us-ma-boston-451-d-street-33-2057242/

2. Heroic Scientist in CryoEM (cryoEM experience a must, again we are happy
to train in drug discovery etc.)

https://www.roivantcareers.com/job/investigator-senior-principal-investigator-cryo-em-research-development-us-ma-boston-451-d-street-33-2057329/

3. Associate Director, Biochemistry

https://www.roivantcareers.com/job/associate-director-biochemistry-research-development-us-ma-boston-451-d-street-33-2057292/

Artem, the washer of many bottles (and also VP of biochem/biophys/structure
and hit finding)

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[ccp4bb] (off topic) Job posting - Associate Director, Protein Science

[Artem Evdokimov]

https://boards.greenhouse.io/roivantsciences/jobs/3549863

Finally posted! Thank you for looking.

Artem

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[ccp4bb] job opening: Associate Director, Protein Sciences

[Artem Evdokimov]

Dear CCP4-ers

I would like to share a preview of a job opening we're about to post for a
senior Protein Sciences research professional. This is an Associate
Director level position, with some wiggle room if exceptional candidates
materialize.

Rather than posting explicit job description, here are the key features:

- 10-15 years of experience making tough proteins in a variety of
expression systems
- demonstrated success with the above
- deeply passionate about protein science
- very creative with respect to finding ways around/through tough challenges
- capable/experienced small team lead
- experience managing CROs
- membrane protein experience a big plus, but is not a stringent requirement

Please contact me directly for details or with any questions. Please note
that I cannot receive offers from agencies or recruiters via this channel -
if you represent someone other than yourself please use the official
Roivant HR channels to submit your applicant.

Needless to say, Roivant Discovery provides equal employment opportunities
to all employees and applicants for employment and prohibits discrimination
and harassment of any type without regard to race, color, religion, age,
sex, national origin, disability status, genetics, protected veteran
status, sexual orientation, gender identity or expression, or any other
characteristic protected by federal, state or local laws.

Thank you,

Artem
(VP of stuff @ Roivant)



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Re: [ccp4bb] RNA-PROTEIN purification on FPLC

[Artem Evdokimov]

Hi

1. Lysate on fplc is messy :) which is why I always do the first step in
batch.

2. A good scrubbing with NaOH, trypsin, and a strong chaotropic agent (in
series) should take care of the contamination, however it may be easier to
find e.g. an old HPLC never used for protein work in order to purify your
material. If your RNA is single stranded, it will have a hard time
surviving this ordeal. Of it is double stranded, should be no worries.

Artem


On Mon, Jun 21, 2021, 8:47 PM WENHE ZHONG 
wrote:

> Dear members,
>
> We are planing to purify RNA only or/and RNA-protein complex on FPLC. The
> application of the purified materials is for crystallization. However, our
> concern is that our FPLC has been frequently used in bacterial expressing
> protein purification including purifying proteins from crude lysate, which
> is contaminated by RNase. Do you have experience of completely cleaning up
> the system before conducting the RNA work? Or it is impossible for 100%
> cleaning and we have to use a separate FPLC for this purpose?
>
> Thank you.
>
> Best regards,
> Wim
>
> --
>
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Re: [ccp4bb] Enzyme Vmax and Km

[Artem Evdokimov]

Is your enzyme monomeric or multimeric?

What time exactly does it mean when you say unprocessed substrate versus
processed?

What are your error margins (no error bars evident on your plots)?
Depending on your errors, your second plot is not a 'steep decline' but
instead could be a constant line.

So, if your affinity for processed substrate is higher, it does not have to
mean that catalytic efficiency will also be higher
 For example RNA polymerase have to eventually let go of the promoter
region in order to elongate, but if the promoter binds too tightly they
will instead abort translation and fall off. Nature is complicated :)

In general it would help us a lot to help you if we understand the details
of your issue.

There is also a common issue that can be encountered with weakly associated
multimeric enzymes that act on polymeric substrate (in your case, DNA) -
namely when you add more substrate than enzyme the monomers of enzyme can
become 'smeared' along the DNA and this results in very odd kinetic
behavior.

Artem


On Fri, Jun 18, 2021, 12:05 AM Prem Prakash  wrote:

> Dear all,
> Sorry for this off topic. I am working on an enzyme that has an
> exonuclease activity. The enzyme preferentially cleaves an unprocessed
> substrate at a faster rate than the processed one (known by qualitative
> analysis). Recently, I calculated the Vmax, Km and kcat of the enzyme for
> unprocessed substrate which are 18.2 pmol/min, 182 nM and 7.1 sec-1
> respectively. However, the Processed substrate has apparently a lower range
> of Km (not calculated) as reflected from the curve (because the same
> increasing concentration range which is used for unprocessed, shows a steep
> decline in the initial velocity of the enzyme with processed substrate.
> The latter suggests that Km is way lower than expected. In this case, the
> question is, if the Km of processed substrate is way lower than the
> Unprocessed, how can we see a faster rate with the former substrate than
> later. i.e lower Km and slower rate of cleavage. If it's possible please
> give some insights. I have attached the plot comparison between two kinetic
> assays.
>
> With kind regards,
>
> Prem
>
>
>
> --
>
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Re: [ccp4bb] Looking for proteins for undergraduate biochemistry lab

[Artem Evdokimov]

All the previously suggested proteins are awesome candidates!

Here are a few more in case you want to add some extra 'zing' to your
curriculum:

- luciferase
- magneto reactive protein(s) that can be isolated with strong magnets
- spy-tag and spycatcher (covalently reactive proteins!)
- split GFP components (react and create fluorescence where there was none)
- dextransucrase (makes gels out of sucrose)
- gelatinase (dissolves gel)
- secreted thermolysin (followed by aspartame synthesis demo)
- ruby-tag (rubredoxin) very dark brown, does not denature on gel
- DHOD converts blue 2,6-dichloroindophenol into colorless reduced form
that spontaneously reoxidizes back to blue in air

And many others :)

Artem




On Wed, Jun 16, 2021, 6:19 PM P. H  wrote:

> Hello All,
>
> We are looking for some candidate proteins for an undergraduate level
> advanced biochemistry lab. They should be expressed in bacteria, simple
> enough to purify and it will be nice to perform some simple
> characterization experiments(binding assays, enzymatic assays).
> Any suggestions?
>
> Thank you in advance.
> Prerna gupta
>
> --
>
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Re: [ccp4bb] [OUT-OF-TOPIC] Compound service management CRO

[Artem Evdokimov]

Frontier
Pretty good service, but definitely not the cheapest.

Artem


On Tue, Jun 1, 2021, 4:54 AM Wim Zhong  wrote:

> Hi Group,
>
> Does anyone have experience with CROs that can provide good compound
> service management in terms of quality and cost? We are looking for a
> partner to manage our compound library.
>
> Sorry again if this topic is inappropriate.
>
> Best regards,
> Wim
>
> --
>
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Re: [ccp4bb] crystallizing fusion proteins

[Artem Evdokimov]

If you have a biophysical way to confirm functionality and/or folding it
would go a long way towards helping you make this choice.

Does your domain bind a ligand of any kind? Is it significantly stable in a
simple thermal melt experiment (of course you have to account for the
melting of the fusion partner)? What are the symptoms of precipitation or
"disappearance" upon cleavage? How far did you explore the buffer
composition for the cleaved material?

I have seen a whole spectrum of situations like yours from a nasty
misfolded protein kept afloat by fusion, to a perfectly folded domain that
simply did not like the buffer conditions and was essentially insoluble
after cleavage due to its bull properties' mismatch with those of the
solvent.

Artem

On Mon, Mar 15, 2021, 5:08 AM Schreuder, Herman /DE <
herman.schreu...@sanofi.com> wrote:

> Dear Bulletin Board,
>
> Sorry for the slightly off-topic question, but we are struggling with a
> receptor domain that expresses well as a fusion protein, but gets lost the
> moment it is cleaved from the fusion partner. It could be that the receptor
> domain is not or misfolded, but it could also be a solubility problem.
>
>
>
> I have seen some crystal structures of fusion proteins with MBP and for
> membrane proteins, T4-lysozyme fusions are often crystallized. What is your
> experience? Would it be worth trying to express and crystallize a fusion
> protein, or would it be better to look for other constructs, e.g. to
> include more receptor domains?
>
>
>
> Thank you very much for your advice!
>
> Herman
>
>
>
>
>
>
>
> --
>
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[ccp4bb] Off-topic: research associate job posting

[Artem Evdokimov]

Dear CCP4 friends

We are looking for fresh talent :) please see the posting below or directly
at MassBio.

https://careers.massbio.org/jobs/?keywords=Research+Associate+Silicon+Therapeutics=Boston%2C+MA%2C+United+States_completion=city%3DBoston%24state%3DMassachusetts%24country%3DUnited+States_type=city_text=Boston%2C+MA%2C+United+States_autocomplete=true=320


Artem


Silicon Therapeutics (“SITX”) is a privately held, physics-driven
integrated drug discovery company. SITX’s discovery efforts as well as
physics-based platform are all completely in-house, with a full wet lab as
well as a team of experienced R professionals spanning biology,
biochemistry, chemistry, biophysics (NMR + X-ray) & preclinical sciences.
SITX’s engine leverages quantum mechanics and molecular dynamics, which are
deployed on its own internal supercomputer composed of over 400 GPU’s and
FPGA’s and allows SITX to accurately simulate the physical motion and
properties of biological targets at an atomistic level resolution. We are
the only company that owns the entire spectrum of physics-driven drug
discovery from chip-to-clinic with a team of over 60 individuals in Boston.

Our diversity and our cross-functional, multi-dimensional teams make us
strong. We foster an open-minded culture where we come to work without
preconceptions about how people think. With all channels open for
communication, we can rapidly fuse information across disciplines and teams.

Job Summary

Silicon Therapeutics is seeking an energetic, passionate practitioner of
the laboratory arts to join the Biochemistry/Biophysics/Structural Biology
platform as a Research Associate . This is a foundational support role for
someone who enjoys technical challenges and is not afraid to get their
hands dirty by helping with core lab functions from cell culture, protein
purification, or biochemical experiments all the way to laboratory
automation, instrument maintenance, etc. The qualified candidate will have
basic laboratory practices under their belt and would be passionate to
learn additional techniques while supporting a growing group dedicated to
Discovery research. The successful candidate will work as part of a
collaborative team of scientists with expertise in protein production,
biochemistry, structure, and biophysics supporting hit-ID, hit-to-lead, and
lead-ID phases of our innate immuno-oncology drug discovery programs and
will have opportunities to learn cutting edge techniques.

Job Responsibilities

Responsible for bacterial and eukaryotic cell culture for the purpose of
recombinant protein expression.
Basic protein purification under supervision of expert practitioners.
Preparation of common biochemical reagents, maintenance of lab readiness.
Biochemical/biophysical characterization of recombinant proteins
Work as part of a cross-functional project team(s) to provide timely,
robust, high-impact results.
Carefully document research workflows, data and protocols using an
electronic laboratory notebook.
Capture experimental results in database.
Organize and present data as needed
Other duties as assigned


Requirements

A minimum of B.Sc. with 2+ years of research experience
Documented experience with recombinant protein production
Propensity towards tinkering with complex equipment
Ability to multitask on the job, combining diligence and tenacity to thrive
in a highly malleable and fluid research environment
Demonstrated ability to execute experiments independently and under the
supervision of experts.
Passion for supporting teams and learning on the job.
Excellent communication skills.
Silicon Therapeutics provides equal employment opportunities to all
employees and applicants for employment and prohibits discrimination and
harassment of any type without regard to race, color, religion, age, sex,
national origin, disability status, genetics, protected veteran status,
sexual orientation, gender identity or expression, or any other
characteristic protected by federal, state or local laws.

Job Information

Job ID: 55846631
Location:
Boston, Massachusetts, United States
Position Title: Research Associate, Biochemistry/Biophysics/Structural
Biology
Company Name: Silicon Therapeutics
Job Function: Scientist
Job Type: Full-Time
Job Duration: Indefinite
Min Education: BA/BS/Undergraduate
Min Experience: 2-3 Years
Required Travel: None


Silicon Therapeutics (“SITX”) is a privately-held, physics-driven
integrated drug discovery company in the preclinical IND stage targeting
innate immunity to start, but with the capability to move into many other
franchise opportunities. The Company’s initial pipeline is focused on
modulation of the innate immune system to “light the spark” within
immunologically cold tumors by using its physics-enabled drug discovery
engine.



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This 

Re: [ccp4bb] OFFTOPIC question "Two plasmids in one host cell"

[Artem Evdokimov]

Hi

An easy fix is to PCR amplify your low copy number plasmid (modern
polymerases have no issues amplifying up to 20 kB if you have a largeish
vector) and then sequence the product. Alternatively you can amplify just a
portion of interest, of course (you said sequence the plasmid so I went
with your exact request). The spectionmycin trick has already been proposed
- that also works well :)

Now, you also mentioned that you would like to quantify the plasmid - for
that a simple qPCR (or even a carefully tuned regular PCR) will do the
trick, or if you want to go seriously old school you can quantify it by
dilution, transformation, and colony counting against a known standard
plasmid.

Happy new year

Artem

On Wed, Dec 30, 2020, 4:35 AM Anamika Singh  wrote:

> Hi All,
>
> I have two constructs having different ori, p15ori and M13 ori, different
> promoters araBAD promoter and LacI, and different antibiotic resistance
> chloramphenicol and Ampicillin respectively.
> I am managed to get the transformants and getting the expected result
> after blunt digestion with the EcoRV enzyme. Since both the plasmids have
> the site for EcoRV. But the p15 ori has a low copy number that's why I am
> seeing the very faint band as compared to other plasmid in the sample.
>
> So I would like to know is there any way that I can quantify the low copy
> number plasmid.
> Because I am not able to sequence it with the specific primers it could be
> due to its low concentration.
>
> Please advise.
>
> Thank you.
> --
> Dr. Anamika Singh
> Post-Doctoral Fellow
> Silberman Institute of Life Sciences
> Hebrew University of Jerusalem, Israel
> No: 054-294-8036
>
> --
>
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Re: [ccp4bb] To solve the problem of an extremely asymmetric peak shape obtained from gel filtration chromatography

[Artem Evdokimov]

Probably a good idea to share an image :) worth many words...

Artem

On Wed, Dec 9, 2020, 9:17 AM   wrote:

> Dear All
> There is a 43kd protein purified via Ni-chelating affinity
> chromatography, anion exchange chromatography and gel filtration
> chromatography in sequence. However the chromatogram obtained showed an
> extremely asymmetric peak shape. The aggregation forms of proteins are
> mainly in the range of monomers and dimers(Hepes and low concentration of
> salt were used as buffers for gel filtration chromatography). 5% glycerinum
> and 1mM Benzamidine hydrochloride had been added in order to maintain the
> stability of the protein and prevent it from degrading. But well, all the
> efforts seem to be useless. We wonder if there are any effective measures
> can be taken to radically solve this problem. We would be much indebted for
> the suggestions you offer.
>
> --
>
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Re: [ccp4bb] AW: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking and less pipetting (?)

[Artem Evdokimov]

Well that is sad, and true, and also very common. I have personally
experienced dozens of cases where methods from literature do not reproduce
because (and this is important) the authors "just slap some generic
boilerplate" instead of the actual methods. My favorite is always to read
stuff like "such and such protein was cloned into bacterial expression
vector, expressed and and purified using standard methods" and then later
find out through considerable effort and twisting hands of original
researchers that the protein can only be expressed when fused with a Spider
Monkey cadherin domain and expressed in minimal medium supplemented with 5%
Pregnant Horse Urine at exactly 13.5 degrees C. And then purified using the
Spider Monkey cadherin monoclonal antibody. And the yield is 1 mg in 24
liters. None of which was ever disclosed in literature...

Sorry for the rant, I guess I am just saying that literature, IMO, has long
ago stopped being generally directly reproducible. Not getting into the
obvious reasons as to why it happened, but still sad that it happened.

Artem

On Tue, Dec 8, 2020, 8:28 AM Hughes, Jonathan <
jon.hug...@bot3.bio.uni-giessen.de> wrote:

> scientific research requires that experimental results must be testable,
> so you have to publish your methods too. if the alphafold2 people don't
> make their code accessible, they are playing a game with different rules.
> maybe it's called capitalism: i gather they're a private company
>
> best
>
> jon
>
>
>
> *Von:* CCP4 bulletin board  *Im Auftrag von *Goldman,
> Adrian
> *Gesendet:* Dienstag, 8. Dezember 2020 12:33
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* Re: [ccp4bb] External: Re: [ccp4bb] AlphaFold: more thinking
> and less pipetting (?)
>
>
>
> My impression is that they haven’t published the code, and it is science
> by press-release.  If one of us tried it, we would - rightly - get hounded
> out of time.
>
>
>
> Adrian
>
>
>
>
>
>
>
> On 4 Dec 2020, at 15:57, Michel Fodje  wrote:
>
>
>
> I think the results from AlphaFold2, although exciting and a breakthrough
> are being exaggerated just a bit.  We know that all the information
> required for the 3D structure is in the sequence. The protein folding
> problem is simply how to go from a sequence to the 3D structure. This is
> not a complex problem in the sense that cells solve it deterministically.
> Thus the problem is due to lack of understanding and not due to
> complexity.  AlphaFold and all the others trying to solve this problem are
> “cheating” in that they are not just using the sequence, they are using
> other sequences like it (multiple-sequence alignments), and they are using
> all the structural information contained in the PDB.  All of this
> information is not used by the cells.   In short, unless AlphaFold2 now
> allows us to understand how exactly a single protein sequence produces a
> particular 3D structure, the protein folding problem is hardly solved in a
> theoretical sense. The only reason we know how well AlphaFold2 did is
> because the structures were solved and we could compare with the
> predictions, which means verification is lacking.
>
>
>
> The protein folding problem will be solved when we understand how to go
> from a sequence to a structure, and can verify a given structure to be
> correct without experimental data. Even if AlphaFold2 got 99% of structures
> right, your next interesting target protein might be the 1%. How would you
> know?   Until then, what AlphaFold2 is telling us right now is that all
> (most) of the information present in the sequence that determines the 3D
> structure can be gleaned in bits and pieces scattered between homologous
> sequences, multiple-sequence alignments, and other protein 3D structures in
> the PDB.  Deep Learning allows a huge amount of data to be thrown at a
> problem and the back-propagation of the networks then allows careful
> fine-tuning of weights which determine how relevant different pieces of
> information are to the prediction.  The networks used here are humongous
> and a detailed look at the weights (if at all feasible) may point us in the
> right direction.
>
>
>
>
>
> *From:* CCP4 bulletin board  *On Behalf Of *Nave,
> Colin (DLSLtd,RAL,LSCI)
> *Sent:* December 4, 2020 9:14 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* External: Re: [ccp4bb] AlphaFold: more thinking and less
> pipetting (?)
>
>
>
> The subject line for Isabel’s email is very good.
>
>
>
> I do have a question (more a request) for the more computer scientist
> oriented people. I think it is relevant for where this technology will be
> going. It comes from trying to understand whether problems addressed by
> Alpha are NP, NP hard, NP complete etc. My understanding is that the
> previous successes of Alpha were for complete information games such as
> Chess and Go. Both the rules and the present position were available to
> both sides. The folding problem might be in a different category. It would
> be nice if someone could explain 

Re: [ccp4bb] Off-topic: Mild cross-linking protocol

[Artem Evdokimov]

If you have hopes for cysteine residues in reasonable proximity, then
bis-iodoacetamide (with a suitable spacer, commercially available is the
ethylenediamine spacer). Any reasonable chemist can make you other spacer
lengths to order.

Homobifunctional PEG with maleimide, N-hydroxy succinimide esters, etc
might work also.

Homobifunctional PEG with amines on ends can be used with a water soluble
coupler e.g. EDC NBT mix.

Notably if you are lucky just treating protein as a dimer with EDC NBT mix
can promote clinking.

Bis aldehydes are an old standby but they are usually quite harsh.

Feel free to write directly for/with additional details.

Artem

On Mon, Oct 19, 2020, 3:18 AM Chiara Bruckmann 
wrote:

> Dear all,
>
> Sorry for the off-topic question. I need to prepare an heterodimeric
> protein complex for an immunisation, and I would like to make sure that the
> dimer will be stable and it won't dissociate after injection.
>
> I am wondering if any of you has a mild cross-linking protocol (and
> suggestions of a suitable reagent) to share.
>
> Thanks and regards,
> Chiara
>
> 
>
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[ccp4bb] offtopic: three job postings @ Silicon Therapeutics

[Artem Evdokimov]

Dear CCP4 friends,

I would like to let you know that we have just posted three jobs open
within my team at Silicon Therapeutics. Qualified inquiries are welcome!

Artem

1. Senior position in Biochemistry

https://silicontx.com/careers/open-positions/senior-investigator-principal-investigator-biochemistry/

2. Mid-to-senior position in Biophysics/Structure

https://silicontx.com/careers/open-positions/investigator-senior-investigator-principal-investigator-biophysics-structural-biology/

3. Research associate in Biochemistry/Biophysics/Structure

https://silicontx.com/careers/open-positions/research-associate-biochemistry-biophysics-structural-biology/



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[ccp4bb] slightly offtopic: antibody and nanobody development providers

[Artem Evdokimov]

Dear CPP4ers

Once again I would like to refill my cup of borrowed wisdom from the
collective fountain that is this excellent list :)

I am looking to make antibodies/nanobodies for crystal-chaperone
(nano/Fab/ScFv etc) kind of a deal. I have a few providers from my past
endeavors, but really would like to gather some new ones especially if
people used them and liked the net result. Would be happy to summarize the
outcome and distribute the summary in this list.

Many thanks,

Artem

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Re: [ccp4bb] opinions about crystal soaking service providers

[Artem Evdokimov]

Good Morning (depending on where you are, I guess)

Sadly, this topic did not receive as many replies as I had hoped for
(actually I am very surprised how few responses I got). So I cannot provide
you with a summary so far. I will be digging into this on my own so if I
learn enough to share, I will post it here.

Cheers,

Artem

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On Fri, Sep 11, 2020 at 10:26 AM Artem Evdokimov 
wrote:

> Dear CCP4ers!
>
> I would like to once again benefit from the communal wisdom and experience
> of our community. Thank you in advance for your advice!
>
> Question: we are considering engaging an external partner to perform
> crystal soaking campaign(s) on our targets. In the past I've successfully
> done this 'myself' (i.e. with teams of internal professionals) and had all
> kinds of results ranging from awesome to bleh. Time has moved on, however,
> and I know that there are several very competent teams out there (Diamond,
> Crystals First, etc.) who excel at providing services of this kind (for
> money).
>
> Could you please comment, privately (since it's going to likely generate
> arguments if posted in an open forum), on what external xtal-fragment
> vendors have you used and what your experiences and outcomes were? In
> return, I am happy to summarize the results and share them back (assuming
> there are enough replies to merit a summary).
>
> Many thanks!
>
> Artem
>
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[ccp4bb] opinions about crystal soaking service providers

[Artem Evdokimov]

Dear CCP4ers!

I would like to once again benefit from the communal wisdom and experience
of our community. Thank you in advance for your advice!

Question: we are considering engaging an external partner to perform
crystal soaking campaign(s) on our targets. In the past I've successfully
done this 'myself' (i.e. with teams of internal professionals) and had all
kinds of results ranging from awesome to bleh. Time has moved on, however,
and I know that there are several very competent teams out there (Diamond,
Crystals First, etc.) who excel at providing services of this kind (for
money).

Could you please comment, privately (since it's going to likely generate
arguments if posted in an open forum), on what external xtal-fragment
vendors have you used and what your experiences and outcomes were? In
return, I am happy to summarize the results and share them back (assuming
there are enough replies to merit a summary).

Many thanks!

Artem

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Re: [ccp4bb] Advice on DNA negative staining

[Artem Evdokimov]

Hiya

*Half an hour in the library saves a month of research.* That's what my
late advisor Dr. Frolow used to say a lot and he was not wrong.

Erenpreisa J. 1981. Staining of DNA with uranlyacetate in hydrolyzed
ultrathin sections. Acta histochem 68(1): 22-26

there are several very clever methods, using depurination and various
chemical treatments (e.g. phenylhydrazine) followed by staining with
various heavy atoms (tungsten derivatives)

In general, shadowing with tungsten is a highly adequate method, see for
example gorgeous images of replicaiton forks here:

https://www.jbc.org/content/285/18/13349.full.pdf

Artem

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On Tue, Aug 4, 2020 at 7:11 AM Panne, Daniel (Prof.) <
daniel.pa...@leicester.ac.uk> wrote:

> Hi Meytal,
>
> 1. The concentration seems way too high at 254mg/ml. Typically 0.1-5mg/ml
> are used.
> 2. Uranyl ions react with phosphate groups resulting in positive stain.
> The resulting contrast is frequently poor.
> 3. Rotary shadowing can be used to enhance contrast.
>
> Best,
> Daniel
>
>
> On 4 Aug 2020, at 11:20, Marin van Heel <
> 057a89ab08a1-dmarc-requ...@jiscmail.ac.uk> wrote:
>
>
> Dear Meytal,
>
> Suggestion: Apply a strong high-pass filter to the images before visually
> judging them!
>
> Marin
>
> On Tue, Aug 4, 2020 at 3:50 AM Meytal Galilee 
> wrote:
>
>> Hi Arunabh Athreya,
>> Sure (this is the linear 2200 b.p. at 175uM)
>> 
>>
>>
>>
>> Get Outlook for Android
>> 
>>
>>
>> --
>> *From:* Arunabh Athreya 
>> *Sent:* Tuesday, August 4, 2020, 12:54
>> *To:* CCP4BB@JISCMAIL.AC.UK; Meytal Galilee
>> *Subject:* Re: Advice on DNA negative staining
>>
>> Hi Meytal
>>
>> Do you have an EM image snapped from your negative staining sample?
>>
>> Get Outlook for Android
>> 
>>
>>
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB=1
>> 
>>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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> 
>
>
>
> --
>
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Re: [ccp4bb] Protocol for bacmid transformation in E.coli

[Artem Evdokimov]

Yes

It requires electroporation and very careful handling of the bacmid. Other
than that it is a fairly simple process.

Here is an example reference:

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5067234/

Artem


On Sat, Jul 18, 2020, 9:12 PM Digant Nayak  wrote:

> Dear all,
>
> Sorry for the off topic question, but i assure you that the long term
> output of the project has structural biology application. I want to
> transform a bacmid into E.coli cell and I was intrigued to find that there
> is no protocol for this available on the internet (i would really
> appreciate it if somebody proves me wrong and directs me to a link). Is it
> similar to transforming a plasmid in E.coli? I would really like to hear
> your experiences on this topic.
>
> Thanks,
> Digant
>
>
>
>
> --
>
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Re: [ccp4bb] Looking for suggestions with protein expression

[Artem Evdokimov]

Hi

It is hard to give specific advice without knowing details about your
protein. There are multiple reasons that may be responsible for it's
behavior (fundamentally the simplest reason is that proteins by and large
are not designed by Nature to be purified and handled) so the more you
share with us the more we may be able to help you.

Artem

On Sat, Jun 27, 2020, 3:15 AM Umar Farook  wrote:

> Dear All,
>
> Sorry for an offtopic question, your suggestions are highly appreciated.
>
> We have been working on iron sulfur cluster binding protein, which is
> usually expressed as a nice soluble protein expressed in BL21 cells but
> aggregated in the affinity column itself and unable to recover from it. We
> had made n number of truncations and fused to soluble tags such as MBP, but
> always ended up in large aggregates. Anyone has experience in working with
> iron-sulfur cluster binding protein before, please let us know the critical
> steps in purification of such proteins, whether you have completely done
> the expression, purification and crystallization in anaerobic conditions?
> or else changing the expression system to eukaryotic system such as Baculo
> or HEK 293T would help?
>
> Please share your valuable experience, thank you.
>
>
>
> --
> Best Regards,
> Umar Farook
>
> --
>
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Re: [ccp4bb] Highest resolution of X-ray / neutron / electron crystallography, cryo EM

[Artem Evdokimov]

Recent cryoEM resolution in apoferritin (yes, the Lysozyme of cryoEM, but
still) seems to be 1.2 A by two groups in the UK and Germany.

Notably (yes, I am kidding, please don't think that I really believe this)
the resolution of MODEL structures is ZERO angstroms :) It's perfectly
good... as long as you don't apply any ugly experiments to the beautiful
theories :)

>From the Evdokimov Unified Dictionary of Science:



*Experiment*

ex·per·i·ment (/ikˈsperəmənt/)

*Noun*

“A scientific procedure undertaken to inflict pain and suffering on the
performing scientist and their colleagues.”

*Verb*

“To discredit or invalidate an otherwise beautiful theory”



Artem


Artem

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On Tue, Jun 9, 2020 at 9:36 AM Tobias Beck  wrote:

> Dear all,
>
> I was asked by a student what the highest resolution is, for each of the
> four methods listed above. Maybe someone has researched the current numbers
> previously and would like to share them? For X-ray, I found 0.48 A in the
> PDB. For EM method details, the PDB gives me 0.6 A, but it is actually for
> electron diffraction. I found a structure with 1.8 A for Cryo EM.
>
> I am aware that resolution is only one parameter and that high resolution
> may not correspond to high data quality. However, maybe someone knows the
> record holders, either for biomacromolecules or small molecules or for
> both.
>
> Thanks!
>
> Best, Tobias.
>
>
>
>
> --
>
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Re: [ccp4bb] Question about small molecule crystallography

[Artem Evdokimov]

Hi

A small organic molecule is typically crystallized from organic solvents
(or water, if soluble) by means of at least three main techniques:

1. slow evaporation of solvent leading to supersaturation and eventual
crystallization
2. supersaturation at higher temperature followed by gradual drop in
temperature causing crystallization
3. counter-diffusion of an incompatible solvent to drop solubility of the
substance and cause crystallization

Many times, just leaving an NMR tube with a tiny hole in the plastic cap
for a week or so will cause crystals to form.

Schnobviously, some substances will not crystallize easily - some form
oils, amorphous precipitates, etc. and others will form liquid hydrated
forms or just plain decompose. If you have any specific questions please
don't hesitate to contact me in person. I've spent half of my PhD
crystallizing weird small molecules for fun and profit.

As to how to solve structures of small molecules - any synchrotron is a
massive overkill. Just get in touch with a University X-ray lab, many of
which still have functional small molecule instruments. SHELX is the
software of choice - of course! (I still have the blue/white polka dot
SHELX cup, it's one of my more treasured curios).

Artem
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On Mon, Jun 1, 2020 at 6:01 PM Jiyuan Ke 
wrote:

> Hi Everyone,
>
> I want to crystallize a small organic molecule. I have very limited
> experience in small molecule crystallography. I found that the Crystal
> Screen HT from the Hampton research is good for both small molecule and
> macromolecule crystallization. Plan to set up a sitting drop screen just
> like setting up protein crystallization. I don’t know if this is the proper
> way to do it. Is the MRC sitting drop 2-well plate (HR3-083) used for
> protein crystallization good for small molecule crystallization? Are there
> any special plates used for small molecule crystallization? Is room
> temperature ok or not?
>
> For data collection, can I use the beamline for protein crystals to
> collect data on small molecule crystals? Larger oscillation angle, shorter
> exposure, reduced beam intensity?
>
> For structure determination, is SHELXL the preferred software for solving
> small molecule structures?
>
> If anyone has experience in small molecule crystallography, please help.
> Thanks!
>
> Best Regards,
>
> --
>
> *Jiyuan Ke, Ph.D.*
>
>
> Research Investigator
>
> H3 Biomedicine Inc.
>
> 300 Technology Square, Floor 5
>
> Cambridge, MA 02139
>
> Phone: 617-252-3923
>
> Email: jiyuan...@h3biomedicine.com
>
> Website: www.h3biomedicine.com
>
>
>
>
>
>
> [This e-mail message may contain privileged, confidential and/or
> proprietary information of H3 Biomedicine. If you believe that it has been
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> message including any attachments, without copying, using, or distributing
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Re: [ccp4bb] Fusion proteins to solubilize your protein

[Artem Evdokimov]

Probably the easiest would be blasting sequences all by all and extracting
peaks above certain height with a filter of some sorts. Not entirely
trivial.

I can add to your list, although not all of these proteins are in the PDB -
some are from my own collection :)

Lysozyme (s) several are used
Artificial helix bundles
GS
SUMO
Ubq
NusA
Fc and other mAb domains
Nanobodies (as fusions)
Cyt b562
Rubredoxin
Flavodoxin
VLR
Barnase
MHC proteins


Bostjan Kobe wrote a paper in 2015 which you probably saw already.

Artem




On Tue, May 5, 2020, 2:45 PM Murpholino Peligro 
wrote:

> I was wondering how many proteins that help to crystallize the protein of
> interest are out there...
>
> and how effective is the phase extension method (i.e. use MR to get the
> structure of the fusion protein into its density and the density of the
> protein of interest...)
>
> Is there a way to get this data from the PDB?
>
> Here is my list so far:
> - MBP
> - GFP
> - TRX
> - Lysozyme
> - GST
>
> Thanks
>
>
>
>
> --
>
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Re: [ccp4bb] server to predict structural stability

[Artem Evdokimov]

Rosetta will do this.

But it's not a server, and there is a fairly steep learning curve.

Artem

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On Mon, May 4, 2020 at 6:04 AM Firdous Tarique 
wrote:

> Hi
>
>  I am trying to incorporate novel disulfide bond by introducing Cys
> mutants at the interface of a protein complex (A+B) and (C+B) whose crystal
> structure are already known. The idea is to introduce novel disulfide bonds
> in these individual complexes which does not dissociate and can be tried
> for cryo-EM studies for a larger complex (A+B+C) .  As discussed
> previously in the bb I am using 'Disulfide by Design" server to introduce
> new Cys residues at the interface of the A+B and C+B The server suggested a
> number of cys residues at the interface and now I am confused from where to
> start with. Is there any known server  which can predict the stability of
> A+B and C+B with different disulfide bonds in it, making it easier to
> decide and pick. In silico mutants which makes the complex more stable will
> be chosen for site directed mutagenesis and downstream biochemical
> experiments to check the stability of A+B+C.
>
> Thanks
>
> Kahkashan
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
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Re: [ccp4bb] somewhat off-topic :)

[Artem Evdokimov]

Thank you

Artem

On Tue, Apr 28, 2020, 7:22 PM Thomas Cleveland 
wrote:

> Hi,
>
> There are tons of these listed on eBay, many in very good condition or
> even unused. I used to get them there all the time.
>
> Best
>
> On Tue, Apr 28, 2020 at 8:51 AM Artem Evdokimov 
> wrote:
>
>> CCP4 friends,
>>
>> Sorry for the somewhat off-topic post!
>>
>> For many years I've been a fan of the Pharmacia XK columns and heavily
>> relied on them for the bulk of my work. Lately, however, GE has increased
>> the prices so much that I am reluctant to buy new columns from them. And I
>> don't mean filled columns - packing columns is a relatively trivial process
>> if you know how to do it right.
>>
>> So - before I spend a day searching and making calls - do you have an
>> alternative suggestion that's more reasonable? Clearly the fittings etc.
>> may need to be adapted, but that's not a big deal. I was already looking at
>> Bio-Rad and found them almost as expensive, so I am actually looking for
>> some sort of 'knockoff' manufacturer :)
>>
>> Thank you
>>
>> Artem
>>
>> - Cosmic Cats approve of this message
>>
>> --
>>
>> To unsubscribe from the CCP4BB list, click the following link:
>> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>>
>



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Re: [ccp4bb] somewhat off-topic :)

[Artem Evdokimov]

Thank you very much!

Artem

On Tue, Apr 28, 2020, 1:03 PM Guenter Fritz <
guenter.fritz.phenix.c...@gmail.com> wrote:

> Dear Artem,
>
> I am a fan of the former "Superformance" column from Merck. The production
> / selling  of these columns is sourced out to a small company called
> Goetec. The columns are not really cheap, but good quality, last forever,
> suited also for higher pressures, only glas and Teflon in contact with your
> sample. Packing is easy.
> I just had a look at their website, https://www.goetec-labortechnik.de
> unfortunately only in German and scarce info.
> Here is a link to a pdf with the now so-called Supercompact system.
>
> https://cdn.website-editor.net/844eacb718df4813ad43c226fccdb915/files/uploaded/Artikelliste_SuperCompact_de.pdf
>
> Best, Guenter
>
> CCP4 friends,
>
> Sorry for the somewhat off-topic post!
>
> For many years I've been a fan of the Pharmacia XK columns and heavily
> relied on them for the bulk of my work. Lately, however, GE has increased
> the prices so much that I am reluctant to buy new columns from them. And I
> don't mean filled columns - packing columns is a relatively trivial process
> if you know how to do it right.
>
> So - before I spend a day searching and making calls - do you have an
> alternative suggestion that's more reasonable? Clearly the fittings etc.
> may need to be adapted, but that's not a big deal. I was already looking at
> Bio-Rad and found them almost as expensive, so I am actually looking for
> some sort of 'knockoff' manufacturer :)
>
> Thank you
>
> Artem
>
> - Cosmic Cats approve of this message
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
>
>
>



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[ccp4bb] somewhat off-topic :)

[Artem Evdokimov]

CCP4 friends,

Sorry for the somewhat off-topic post!

For many years I've been a fan of the Pharmacia XK columns and heavily
relied on them for the bulk of my work. Lately, however, GE has increased
the prices so much that I am reluctant to buy new columns from them. And I
don't mean filled columns - packing columns is a relatively trivial process
if you know how to do it right.

So - before I spend a day searching and making calls - do you have an
alternative suggestion that's more reasonable? Clearly the fittings etc.
may need to be adapted, but that's not a big deal. I was already looking at
Bio-Rad and found them almost as expensive, so I am actually looking for
some sort of 'knockoff' manufacturer :)

Thank you

Artem

- Cosmic Cats approve of this message



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Re: [ccp4bb] insect secretion recommendations?

[Artem Evdokimov]

Gentle spin followed by TFF - a cheap peristaltic pump plus $200 filter
cartridge will do the trick.

Artem

On Mon, Apr 20, 2020, 11:56 AM Gloria Borgstahl 
wrote:

> Hi Friends,  We are secreting Spike Ecto domain into the media from insect
> cells for purification.  As we scale up I am wondering what is recommended
> for collecting the media from large volumes of culture.  Centrifugation?
> Filtration of some kind?  I imagine we need to be gentle to not lyse the
> insect cells.  Thank you, Gloria
>
> --
>
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Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!

[Artem Evdokimov]

Sadly, I agree: it is pretty clear that the authors did not want
coordinates revealed. Therefore asking them for information is not likely
to lead to getting anything. To be completely fair, it's likely not even
the authors themselves, but their corporate policy that's responsible.

In cases like this, I normally choose not to publish, but maybe their
bosses insisted. Anyway, many options to find excuses, but it looks like
Artem has to go solve this structure himself.

Thank you all very much, many good responses :)

Arte,

- Cosmic Cats approve of this message


On Tue, Apr 7, 2020 at 12:06 PM Schreuder, Herman /DE <
herman.schreu...@sanofi.com> wrote:

> Dear Frank,
>
>
>
> here I disagree. I think it is bad practice to complain to the editors or
> start naming and shaming before asking the authors first. Only if they do
> not want to cooperate, it would be time to bring the flame-throwers in
> position.
>
>
>
> However, I think the situation is more subtle and that that is the reason
> Artem wrote his email: He wants the data, but does not want to reveal his
> identity to his competitors, who apparently made a significant effort not
> to reveal any useful details.
>
>
>
> Here I would let me guide by the (perceived) commercial interest: if it is
> not gigantic, then I would contact the authors to prevent a wasteful
> duplication of research efforts.
>
> If there is a significant commercial interest, it would probably be better
> not to contact them and go the hard way of solving the structure yourself.
> A thing to consider is also what would happen if the authors still would
> refuse: they know the identity of the competitor and you still do not have
> the data.
>
>
>
> An alternative may be to ask an academic friend who also works on sausage
> esterases to inquire with the authors…
>
>
>
> Good luck with your decision!
>
> Herman
>
>
>
> *Von:* CCP4 bulletin board  *Im Auftrag von *Frank
> von Delft
> *Gesendet:* Dienstag, 7. April 2020 17:19
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* [EXTERNAL] Re: [ccp4bb] on-topic: your opinions requested!
>
>
>
> *EXTERNAL : *Real sender is owner-ccp...@jiscmail.ac.uk
>
>
>
> Write to the editor - that's their job.
>
> Though they may also see it as their job to ignore emails like yours
> because that's far easier than dealing with them.
>
> Alternatively, go the Rupp Route:  Name and Shame! ;)
>
> On 07/04/2020 16:08, Artem Evdokimov wrote:
>
> Dear CCP4ers,
>
>
>
> I would like to solicit your thoughts on the following (this is a real
> situation, but salient details are changed):
>
>
>
> Imagine that you're an industrial scientist in a small company, working on
> the Bavarian Sausage (Weisswurst) Esterase project. The overall structure
> is previously unknown, with no good homologs in the PDB, trying to model
> is "OK not great" so the structure is really needed...
>
>
>
> Then, you find an article from a large commercial competitor, that somehow
> managed to solve the Stadtwurst (Saxony Sausage) Esterase structure (which
> is a very close homolog to the one you need!).
>
>
>
> Sounds good - but as you read the paper you realize that the authors
> managed to find a journal that allowed them to publish their work without
> disclosing neither the coordinates of the model, nor even the
> crystallization conditions of the protein - all that's available is a
> tantalizing still picture of the active site in surface mode, with a
> ball-and-stick ligand positioned such that it is impossible to say what it
> interacts with.
>
>
>
> So you sit and ponder - whether to write to the Editor, or maybe to
> contact the authors directly (but then they would know that you're working
> on this, which is not necessarily great since you're competing), or to just
> buck up and do the structure on your own (which feels a bit wasteful).
> Then, you realize that your friends at CCP4 have a lot of wisdom to offer,
> so you sit down and pen an email...
>
>
>
> Any thoughts?
>
>
>
> Artem
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1
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>
>
>
>
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>
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[ccp4bb] on-topic: your opinions requested!

[Artem Evdokimov]

Dear CCP4ers,

I would like to solicit your thoughts on the following (this is a real
situation, but salient details are changed):

Imagine that you're an industrial scientist in a small company, working on
the Bavarian Sausage (Weisswurst) Esterase project. The overall structure
is previously unknown, with no good homologs in the PDB, trying to model
is "OK not great" so the structure is really needed...

Then, you find an article from a large commercial competitor, that somehow
managed to solve the Stadtwurst (Saxony Sausage) Esterase structure (which
is a very close homolog to the one you need!).

Sounds good - but as you read the paper you realize that the authors
managed to find a journal that allowed them to publish their work without
disclosing neither the coordinates of the model, nor even the
crystallization conditions of the protein - all that's available is a
tantalizing still picture of the active site in surface mode, with a
ball-and-stick ligand positioned such that it is impossible to say what it
interacts with.

So you sit and ponder - whether to write to the Editor, or maybe to contact
the authors directly (but then they would know that you're working on this,
which is not necessarily great since you're competing), or to just buck up
and do the structure on your own (which feels a bit wasteful). Then, you
realize that your friends at CCP4 have a lot of wisdom to offer, so you sit
down and pen an email...

Any thoughts?

Artem



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[ccp4bb] off-topic: coronavirus test summaries

[Artem Evdokimov]

Just FYI, here are two useful links for those who would like to see what's
being used to test for coronavirus:

https://www.who.int/docs/default-source/coronaviruse/whoinhouseassays.pdf


https://www.finddx.org/covid-19/pipeline/

May be this saves someone twenty minutes of searching online.

Artem

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Re: [ccp4bb] SARS-CoV-2 test on a smartphone

[Artem Evdokimov]

To be honest I would set up reasonably equipped test centers - yes I
realize that sample shipping is a drag - but probably still cheaper than in
other countries. There are foundations with good budgets for this and the
machinery is much more affordable these days. Again there would be no need
for expensive stuff.

The antibody option is even cheaper once the bait protein is determined.
And those tests are inexpensive in bulk...

This is not to say that the original idea is off, I love the idea. My
comment was more for the sake of people who want to give this a whirl
themselves, with the equipment that may be available in good (sadly, not
all) high school labs.

Artem



On Thu, Apr 2, 2020, 3:17 PM Jurgen Bosch  wrote:

> Hi Artem,
>
> How would you do this in resource limited countries?
> I think James idea is pointing toward that direction, deliver an easy to
> apply reagent, take your phone and detect. It still is challenging in most
> African countries but more doable, there are more smartphones e.g. older
> Android & iPhones available than one might anticipate.
>
> Jürgen
>
> On Apr 2, 2020, at 2:52 PM, Artem Evdokimov 
> wrote:
>
> For DIY fanatics out there:
>
> Step 1. Express reverse polymerase (single subunit one for simplicity,
> like the one from swine retrovirus). For hardcore fans also express Taq.
> That is easy. Purify both with minimal RNAse contamination. Less easy but
> we know how to do that.
>
> Step 2. Design and order a couple of nice primers to clean regions of
> viral RNA - for the RT part. Ditto for the PCR part
>
> Step 3. RT PCR the snot (literally) out if it.
>
> For single experiments there isn't really a need to use dye labeled
> primers, quenchers, scorpions and so forth - either RT PCR product appears
> on the gel, or not.
>
> Doing this at home is a little tricky but anyone with molbio lab access
> can do it. Not sexy, no fancy tools involved - but will likely work. Need
> good positive control, ideally.
>
> Alternatively, deconstruct an antibody based kit and express whatever it
> uses as bait, then buy some anti-human IgG and IgM antibodies, label them a
> bit and do Homebrew ELISA on serum.
>
> Artem
>
> Artem
>
> On Thu, Apr 2, 2020, 11:28 AM James Holton  wrote:
>
>> Personally, if I were infected with SARS-CoV-1 instead of SARS-CoV-2 I'd
>> still like to know that.
>>
>> It is most certainly true that the primer design must be done right:
>> checking for self-annealing, low genomic variability, cross-reactivity to
>> potential contaminants etc.  Fortunately, we have tools for this that can
>> be used at home.
>>
>> I agree the CRISPR-based tests are very exciting, as are many of the
>> other new tests being rolled out.  Assay times of 15 minutes, 5 minutes,
>> and now 2 minutes have been claimed.  The problem I see is they all rely on
>> specialized equipment, skilled technicians and expensive reagents.  Ramping
>> up production to the billion-test scale may not be feasible.  Even if it
>> were, all the PPE needed to extract those samples safely would be
>> prohibitive, as would be the sample-tracking logistics.
>>
>> For reasons such as this, I am curious to see if an at-home
>> do-it-yourself test is possible.  It may serve no purpose other than to
>> satisfy indiviual curiosity, but I think it would go a long way to defusing
>> the fear that comes from not knowing.  This would not just be for sputum,
>> but possibly doorknobs, packages, and, yes, mobile phones.
>>
>> And for those wondering about those nasal swabs:  I've done a little
>> research on them and I think the reason for going full "Total Recall" and
>> sticking it way up inside your head is not because the virus is more
>> concentrated there (we don't even know what the concentration is), but
>> rather because potential contaminants are minimized.  Think about it: PCR
>> is a very sensitive technique, and you want to make sure the sample came
>> from the intended patient, not the other patient who walked through the
>> door just before you did after sneezing in their hand and touching the
>> doorknob.  If you touched that same doorknob and then  "scratched"
>> your nose, then a swab of your nostrils might pick up a virus or two.  That
>> would be a false positive.
>>
>> I expect there are many aspects of current test that don't have to be the
>> way they are, but nonetheless are "required" because they were inherited
>> from previous tests.  I expect we all have learned the hard way that in
>> biological science when you are handed a protocol you follow that protocol
>> to the letter.  How many times have you had

Re: [ccp4bb] SARS-CoV-2 test on a smartphone

[Artem Evdokimov]

For DIY fanatics out there:

Step 1. Express reverse polymerase (single subunit one for simplicity, like
the one from swine retrovirus). For hardcore fans also express Taq. That is
easy. Purify both with minimal RNAse contamination. Less easy but we know
how to do that.

Step 2. Design and order a couple of nice primers to clean regions of viral
RNA - for the RT part. Ditto for the PCR part

Step 3. RT PCR the snot (literally) out if it.

For single experiments there isn't really a need to use dye labeled
primers, quenchers, scorpions and so forth - either RT PCR product appears
on the gel, or not.

Doing this at home is a little tricky but anyone with molbio lab access can
do it. Not sexy, no fancy tools involved - but will likely work. Need good
positive control, ideally.

Alternatively, deconstruct an antibody based kit and express whatever it
uses as bait, then buy some anti-human IgG and IgM antibodies, label them a
bit and do Homebrew ELISA on serum.

Artem

Artem

On Thu, Apr 2, 2020, 11:28 AM James Holton  wrote:

> Personally, if I were infected with SARS-CoV-1 instead of SARS-CoV-2 I'd
> still like to know that.
>
> It is most certainly true that the primer design must be done right:
> checking for self-annealing, low genomic variability, cross-reactivity to
> potential contaminants etc.  Fortunately, we have tools for this that can
> be used at home.
>
> I agree the CRISPR-based tests are very exciting, as are many of the other
> new tests being rolled out.  Assay times of 15 minutes, 5 minutes, and now
> 2 minutes have been claimed.  The problem I see is they all rely on
> specialized equipment, skilled technicians and expensive reagents.  Ramping
> up production to the billion-test scale may not be feasible.  Even if it
> were, all the PPE needed to extract those samples safely would be
> prohibitive, as would be the sample-tracking logistics.
>
> For reasons such as this, I am curious to see if an at-home do-it-yourself
> test is possible.  It may serve no purpose other than to satisfy indiviual
> curiosity, but I think it would go a long way to defusing the fear that
> comes from not knowing.  This would not just be for sputum, but possibly
> doorknobs, packages, and, yes, mobile phones.
>
> And for those wondering about those nasal swabs:  I've done a little
> research on them and I think the reason for going full "Total Recall" and
> sticking it way up inside your head is not because the virus is more
> concentrated there (we don't even know what the concentration is), but
> rather because potential contaminants are minimized.  Think about it: PCR
> is a very sensitive technique, and you want to make sure the sample came
> from the intended patient, not the other patient who walked through the
> door just before you did after sneezing in their hand and touching the
> doorknob.  If you touched that same doorknob and then  "scratched"
> your nose, then a swab of your nostrils might pick up a virus or two.  That
> would be a false positive.
>
> I expect there are many aspects of current test that don't have to be the
> way they are, but nonetheless are "required" because they were inherited
> from previous tests.  I expect we all have learned the hard way that in
> biological science when you are handed a protocol you follow that protocol
> to the letter.  How many times have you had to teach a student that?  It is
> not a bad policy, but eventually there comes a time for "assay
> development".  This is when you start asking "why do we do it that way,
> again?"
>
>  For example, swabs with calcium alginate are not allowed becuase they can
> "kill the virus".  If all we want is genomic RNA, then why do we care?
> Possibly because the traditional method of identifying most pathogens is to
> culture them.  The CDC protocol also recommends against cotton swabs with
> wood handles.  Why?  Perhaps because they contain DNA, and for PCR you
> always worry about contamination.  Is there any chance the probes will
> anneal to something in the cotton or pine genomes?  I doubt it, but I also
> doubt that anyone has checked.
>
> Thank you for the suggestions so far!  Very interesting and helpful!
>
> -James Holton
> MAD Scientist
>
>
> On 3/31/2020 11:46 PM, Sahil Batra wrote:
>
> Dear Prof. Holton,
>
> An innovative idea; however all of the 30 kb genome may not be useful for
> specific detection - SARS-CoV1 and SARS-CoV2 share 80% identity.
>
> A similar fluorescent detection approach for SARS Cov2 -- using the
> indiscriminate collateral activity of Cas12 nuclease -- has been reported
> here: https://www.biorxiv.org/content/10.1101/2020.02.29.971127v1.full.pdf
> Although not tested on samples from patients.
>
> Regards,
> Sahil Batra
> PhD candidate, IIT Kanpur
>
> On Wed, Apr 1, 2020 at 12:07 PM Jurgen Bosch  wrote:
>
>> One problem I see is the sputum, there’s a reason why swabs are made to
>> get sufficient viral material.
>>
>> Since stool samples test PCR positive that might be an easier approach 

[ccp4bb] off-topic: work opportunities at Enko

[Artem Evdokimov]

Hello!

Sorry (not sorry) for the off-topic post. We have a few jobs posted and I'd
like to re-post them here.

Please see the link for full descriptions >>>
https://www.enkochem.com/careers-enko

-Scientist, Structural Biology & Protein Chemistry
-Senior Scientist, Protein Target Discovery
-Scientist, Enzymology & Protein Chemistry
-Enzymology Research Associate

(may be less relevant to this crowd - below)

-Regulatory Toxicologist
-Analytical Scientist, Biology
-Greenhouse Technician, Biology
-Greenhouse Manager, Biology

Key points:

(1) if you'd like to learn more, informally - please feel free to write me
directly
(2) if you'd like to apply, please write to *care...@enkochem.com*


Artem

VP of Biochemistry/Molecular Biology
Enko



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Re: [ccp4bb] Expi 293 (Met-) expression medium for selenomethionine labeling

[Artem Evdokimov]

Hi Kelvin

We never tried with ExpiCHO, but the Expi-293 cells in a generic medium
performed like a geriatric gerbil compared to the Old Faithful in the
actual medium. Transfection was still close to 90% but yield suffered
considerably.

I am sure that with enough diligence the secret sauce can be reproduced. We
focused more on vector design, instead.

Notably, there were batches of Expifectamine that did not perform
consistently. Also fresh Expi293 cells are not always the same, in my
experience. But this was ~2 years ago so maybe Thermo made improvements in
their production lines (I bet they did, this is a nice product and it ought
to be a good money-maker for them)

Artem
- Cosmic Cats approve of this message


On Sun, Mar 15, 2020 at 5:27 PM Lau Kelvin  wrote:

> Dear Artem,
>
>
> By work poorly do you mean, not as well and are you referring to all Expi
> cells?
>
> We have adapted ExpiCHO cells to another medium and although we see
> drastic decrease from using the complete Expi system (x15 volumetric
> yield), it was still considerably higher than our previous cells in the
> same medium (x5 volumetric yields).
>
> Will soon try with ExpiSf9 and baculovirus to see if they also perform
> similarly.
>
> Best,
>
> Kelvin
>
> On Mar 11, 2020, at 5:49 PM, Artem Evdokimov 
> wrote:
>
> Hello
>
> Bad news: Expi cells work poorly in non-Expi medium :(
> Good news: you can add SelenoMet and make partially seleniated protein,
> which is usually enough. Note that too much SeMet is pretty toxic, and that
> the toxicity is associated with BOTH enantiomers of the amino acid, so if
> you have the ability to use the L-SeMet pure as opposed to the more common
> L,D-mix then you can put in more. Either way, it works.
>
> Expect your yield to suffer.
>
> Question: did you try iodine incorporation? You can do it on the level of
> pure protein, or crystals...
>
> Artem
>
> - Cosmic Cats approve of this message
>
>
> On Tue, Mar 10, 2020 at 1:51 PM LAURA DEL AMO MAESTRO 
> wrote:
>
>> Dear ccp4 community,
>>
>> I am in my last 3 months of my PhD, working at the Structural Biology
>> Unit at IBMB-CSIC in Barcelona. As my experimental phasing trials using
>> various heavy metals failed so far, we ordered two months ago Expi 293
>> (Met-) expression medium for selenomethionine labeling (see the reference:
>> https://www.thermofisher.com/order/catalog/product/A4096701#/A4096701.
>> But we got now the notice that it will not be delivered until end of April.
>>
>> I had been working so hard on this project, and this medium is essential
>> to finished with the experimental part of my thesis. Thus, I was wondering
>> if someone in the community has a bottle (1L) of this expression medium
>> without methionine that I could borrow, and which I would return
>> immediately once our medium arrives.
>>
>> Also in case you have recommendations or other sources of a defined
>> Se-Met medium for Expi293 cells, please let me know. With kind regards from
>> Barcelona, and hope someone can help,
>>
>> Laura
>>
>> ---
>> Laura del Amo Maestro - Proteolysis Lab
>> SBU-Structural Biology Unit, IBMB CSIC
>> Barcelona, Spain, Europe
>>
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>>
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Re: [ccp4bb] Expi 293 (Met-) expression medium for selenomethionine labeling

[Artem Evdokimov]

Hello

Bad news: Expi cells work poorly in non-Expi medium :(
Good news: you can add SelenoMet and make partially seleniated protein,
which is usually enough. Note that too much SeMet is pretty toxic, and that
the toxicity is associated with BOTH enantiomers of the amino acid, so if
you have the ability to use the L-SeMet pure as opposed to the more common
L,D-mix then you can put in more. Either way, it works.

Expect your yield to suffer.

Question: did you try iodine incorporation? You can do it on the level of
pure protein, or crystals...

Artem

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On Tue, Mar 10, 2020 at 1:51 PM LAURA DEL AMO MAESTRO 
wrote:

> Dear ccp4 community,
>
> I am in my last 3 months of my PhD, working at the Structural Biology Unit
> at IBMB-CSIC in Barcelona. As my experimental phasing trials using various
> heavy metals failed so far, we ordered two months ago Expi 293 (Met-)
> expression medium for selenomethionine labeling (see the reference:
> https://www.thermofisher.com/order/catalog/product/A4096701#/A4096701.
> But we got now the notice that it will not be delivered until end of April.
>
> I had been working so hard on this project, and this medium is essential
> to finished with the experimental part of my thesis. Thus, I was wondering
> if someone in the community has a bottle (1L) of this expression medium
> without methionine that I could borrow, and which I would return
> immediately once our medium arrives.
>
> Also in case you have recommendations or other sources of a defined Se-Met
> medium for Expi293 cells, please let me know. With kind regards from
> Barcelona, and hope someone can help,
>
>
> Laura
>
> ---
>
> Laura del Amo Maestro - Proteolysis Lab
> SBU-Structural Biology Unit, IBMB CSIC
> Barcelona, Spain, Europe
>
> --
>
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Re: [ccp4bb] Mixed oligomeric states in crystallo

[Artem Evdokimov]

5FRT

We got scooped on this one a long time ago - but back in 2015 it was very
gratifying to see that the academic group who published this structure have
also obtained an ASU with two dimers and one monomer - same exact story as
what we saw. The difference can be attributed to the movement of a short
helix.

Nature is cruel, but interesting.

Artem

On Fri, Jan 31, 2020, 10:33 AM Kluenemann, Thomas <
thomas.kluenem...@helmholtz-hzi.de> wrote:

> Dear all,
>
>
>
> We recently solved a the structure of a small c-type cytochrome. We
> observed, that of the eleven chains in the asymmetric unit ten form 3D
> domain swapped dimers by exchanging an α-helix. The eleventh  chain is
> present as a monomer. Based on the anomalous iron signal and the chain
> tracing we are sure that no chain was missed.
>
> I tried to find other examples in the PDB, were one crystal is made of
> different homo- or heterooligomers.  I only found proteins with partial
> occupied peptide binding sites, which is not what I am looking for. Does
> anyone know of a case were the presence of different homo- or
> heterooligomers is required to form the crystal?
>
>
>
> Best regards,
>
> Thomas Klünemann
>
>
>
>
>
> --
>
> Helmholtz-Zentrum für Infektionsforschung GmbH | Inhoffenstraße 7 | 38124
> Braunschweig | www.helmholtz-hzi.de
>
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Re: [ccp4bb] AW: [ccp4bb] Urea vs. Guanidinium/HCl

[Artem Evdokimov]

Thank you for the clarification!

First of all, many proteins will exhibit this sort of behavior when stored
in liquid form at moderately low temperature.

One solution is to store in glycerol (35 percent and up) at -20C. Or in
deep freeze at -80C or in LN2.

The one remaining question that would be useful to address is the degree of
homogeneity in your sample. Is 100% of your protein folded and functional?
We have seen cases where a small population of poorly folded protein would
give rise to aggregates that eventually seed the entire sample into
aggregation. Removing these seeds early in the game was key to keeping the
whole sample happy. In a few very curious cases we were able to achieve
this by controlled heating (e.g. 55C for 5 minutes) of the sample followed
by ultracentrifugation or some other separative technique. The idea is to
accelerate aggregation of the busted protein and then to crash the
aggregate out. This is of course somewhat extreme and cannot be recommended
as a general solution to every issue of this sort.

Addition of the right detergent can also help. Separation via another
orthogonal matrix is often helpful as well.

Artem


On Sun, Feb 2, 2020, 4:22 AM Jon Hughes 
wrote:

> Thanks for you interest. Ok, here are some more details.
>
> The protein is Cph1 (uniprot Q55168) as a full-length (ca. 80 kDa)
> holophytochrome, produced with a C-terminal His6 tag together with its
> co-factor in E. coli, purified via NiNTA and SEC. It is red/far-red
> photochromic (that is, photoactive) such that, as a 2 component sensory
> histidine autokinase / phosphotransferase, its kinase activity can be
> switched on and off by appropriate light pulses. Thus it is unambiguously
> functional. It is also highly soluble (10 mg/ml is no problem) – but
> subsequently (over days and weeks) it aggregates (irrespective of the
> photostate) to form a fluffy precipitate.
>
> Incidentally, I believe that most SHPK's and indeed most phytochromes have
> aggregation problems like this.
>
> Beyond urea being a less potent chaotrope than guanidinium/HCl, the
> different chemical actions of the two might give a hint as to what causes
> the aggregation.
>
> Cheers
>
> jon
>
>
>
> *Von:* CCP4 bulletin board  *Im Auftrag von *Artem
> Evdokimov
> *Gesendet:* Sonntag, 2. Februar 2020 00:46
> *An:* CCP4BB@JISCMAIL.AC.UK
> *Betreff:* Re: [ccp4bb] Urea vs. Guanidinium/HCl
>
>
>
> More details would be helpful. Do you know whether your protein is folded
> and active to begin with? Many partially folded proteins behave in a way
> that resembles your experience... Urea is a less potent denaturant mole for
> mole than GuHCl so it is not super surprising that it behaves differently.
>
>
>
> Artem
>
>
>
> On Sat, Feb 1, 2020, 6:22 PM Jon Hughes <
> jon.hug...@bot3.bio.uni-giessen.de> wrote:
>
> Hello everyone,
> We work on a protein that tends to aggregate. The process is slowed but not
> stopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCl
> dissolves the aggregate readily, urea just turns it into an amorphous
> chewing-gum-like mass. Does that info provide anyone with a clue as to why
> the aggregation occurs and maybe suggest how to stop it in a way that would
> not thwart crystal formation?
> Best,
> jon
>
> 
>
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Re: [ccp4bb] Urea vs. Guanidinium/HCl

[Artem Evdokimov]

More details would be helpful. Do you know whether your protein is folded
and active to begin with? Many partially folded proteins behave in a way
that resembles your experience... Urea is a less potent denaturant mole for
mole than GuHCl so it is not super surprising that it behaves differently.

Artem

On Sat, Feb 1, 2020, 6:22 PM Jon Hughes 
wrote:

> Hello everyone,
> We work on a protein that tends to aggregate. The process is slowed but not
> stopped by glycerol and NDSB201. Interestingly, whereas guanidinium/HCl
> dissolves the aggregate readily, urea just turns it into an amorphous
> chewing-gum-like mass. Does that info provide anyone with a clue as to why
> the aggregation occurs and maybe suggest how to stop it in a way that would
> not thwart crystal formation?
> Best,
> jon
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>



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Re: [ccp4bb] microscope camera

[Artem Evdokimov]

I bought a bunch of cheap cameras from Amscope. They were OK for the price.

Artem

On Wed, Nov 27, 2019, 12:46 Dean Derbyshire 
wrote:

> Forgive the off topic subject:
>
>
>
> Has anyone got any experience with moticam microscope cameras?
>
> We are looking into cheap cameras for record keeping etc and this supplier
> looks good but…
>
>
>
> Cheers in advance
>
>
>
> Sent from Mail  for
> Windows 10
>
>
>
> --
>
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Re: [ccp4bb] Fwd: [ccp4bb] suggestions on small proteins that for a complex

[Artem Evdokimov]

FliS and FliC

Artem

On Sun, Nov 10, 2019, 03:42 Gianluca Cioci  wrote:

> ps: the 3D structure
>   of the complex should
> be solved at high resolution
>
> ;o)
>
>
>
>
> Dear All,
>
> I am looking for examples of two small proteins A and B that can form a
> tight complex AB.
> Each protein should be straightforward to express in E.coli, well folded
> and stable in the isolated form.
> Also, if possible, I would prefer monomeric proteins (although not
> really mandatory).
> Is anybody able to give me some suggestions ?
>
> Thank you,
>
> GIA
>
> --
> 1st French Congress on Integrative Structural Biology
> Please check: http://bsi-2019.ipbs.fr
>
> Dr. Gianluca CIOCI
> Toulouse Biotechnology Institute (TBI)
> BioCatalysis Team
>
> http://www.toulouse-biotechnology-institute.fr/en/research/enzyme-molecular-engineering-and-catalysis/cimes.html
> PICT - Plateforme Intégrée de Criblage de Toulouse
> http://cribligand.ipbs.fr/index.html
>
> Tel: +33 (0)5 61 55 97 68
> E-mail: ci...@insa-toulouse.fr
>
> TBI - INSA Toulouse
> 135 avenue de Rangueil
> 31077 Toulouse CEDEX 04
> http://www.toulouse-biotechnology-institute.fr
>
> 
>
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[ccp4bb] Off topic: 384 well stamper

[Artem Evdokimov]

Colleagues

I have a quick question for you: we are in need of a small footprint 384
well plate stamper with dilution (optional but desired) capability. We are
already evaluating Integra, Jena Analytic, and Apricot models but we are
curious if there are other companies who sell this sort of machinery (any
personal impressions of these and any other similar LH devices are very
welcome!). I would be happy to post a condensed summary of replies.

Thank you

Artem



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Re: [ccp4bb] AW: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping electron density of a residue and ligand

[Artem Evdokimov]

I am willing to bet that the ketone is covalently reacting with the
arginine and the result is what you see :)

Artem

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On Thu, Nov 7, 2019 at 3:33 AM  wrote:

> Dear HK,
>
> In your case mass spec would be extremely valuable. Not only could it
> prove or rule out a covalent link, it will also convince referees that the
> link is real.
> There is clearly more density than the degraded product you showed. I
> would also ask an experienced chemist if a link between your ligand and the
> arginine would be possible. Finally, I would also do a quality check on the
> ligand you used. Might it be that some reactive intermediate from the
> synthesis still is present?
>
> Best,
> Herman
>
> -Ursprüngliche Nachricht-
> Von: t...@em.uni-frankfurt.de 
> Gesendet: Donnerstag, 7. November 2019 07:58
> An: Schreuder, Herman /DE 
> Cc: CCP4BB@jiscmail.ac.uk
> Betreff: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping
> electron density of a residue and ligand
>
> EXTERNAL : Real sender is  prvs=1214477c0d=t...@em.uni-frankfurt.de
>
>
>
> Hi Herman,
>
> Thanks for the suggestion. In principle, I have refined the structure
> without Arg and ligand, as well as the connected residues to the Arg.
> However, I still see the connected density of Arg and the ligand (Figure -
> unmodel1.png - only removal of Arg and ligand).
>
> I have tried to refine the degraded product but the positive density in
> between Arg and the ligand is still there, indicating something should be
> modeled there (Figure - degraded_product.png and degraded_product2.png -
> both figures are the same ligand but with different side view).
>
> I also tried to model the ligand in different conformation but still the
> same as shown in degraded product. The positive density is there between
> Arg and the ligand.
>
> For both cases, I refined Arg with full occupancy but ligand with
> occupancy refinement. Now, Arg did not flip away from the density.
>
> I might have a look at the mass-spec to check whether it is covalently
> linked although I am not aware of the reactivity of the moiety of the
> ligand and we never see this compound to covalent link with protein in our
> experience.
>
> Thanks for the advice and suggestion. It is very helpful.
>
> Thanks.
>
> Best
> HK
>
>
>
> Quoting herman.schreu...@sanofi.com:
>
> > Hi Heng-Keat,
> >
> > I had again a look at your electron density pictures and these were my
> > impressions:
> > 1) the Arginine as fitted looks fully occupied. There are no hints for
> > an alternative conformations.
> > 2) The fit of the ligand, although reasonable, is not extremely good.
> >
> > It seems that the only reason to invoke partial occupancies for the
> > Arginine and ligand are problems to fit both the ligand and the
> > arginine in the available density. This means that for the overlapping
> > parts, to sum of the occupancies is maximal 1.0.
> > However, the ligand and the Arginine do not look half occupied.
> >
> > So I would look at other explanations:
> > - would it be possible that the ligand reacts covalently with the
> > Arginine? The density looks pretty continuous to me.
> > - could it be that instead of the intended ligand, some side- or
> > degradation product has bound?
> >
> > What I would also do, but what you probably already did, is to delete
> > the ligand and the Arginine side chain atoms and run several rounds of
> > refinement, to get an electron density map with as much bias as
> > possible removed.
> >
> > Good luck!
> > Herman
> >
> > -Original Message-
> > From: CCP4 bulletin board  On Behalf Of
> > Heng-Keat Tam
> > Sent: Mittwoch, 6. November 2019 07:30
> > To: CCP4BB@JISCMAIL.AC.UK
> > Subject: [EXTERNAL] Re: [ccp4bb] Occupancy refinement of overlapping
> > electron density of a residue and ligand
> >
> > EXTERNAL : Real sender is  owner-ccp...@jiscmail.ac.uk
> >
> >
> >
> > Hi Nestor,
> >
> > I think the reason for Arg side chain to curl up is because I refined
> > ligand and Arg side chain with occupancy refinement, and the Arg moved
> > away from the density, most likely due to 'repulsion' from ligand.
> >
> > The other question is: Is it possible that the H-bonds stay very
> > close? As I tried to 'real space refine' in coot, and the Arg side
> > chain flipped away from the density.
> >
> > Thanks for the suggestion.
> >
> > Best
> > HK
> >
> >
> > Quoting Nestor Concha :
> >
> >> Hi Tam,
> >> The density looks very strong and therefore I'm going to guess that
> >> the Arg guanidimium stays in contact/interacts with the ligand and
> >> with the phosphate/sulfate next to it. Perhaps it is one of those
> >> close interactions with shorter H-bonds that usual given the
> >> arrangement of ligand-phosphate/sulfate-Arg.  I'd try to find a
> >> rotamer for the Arg that leaves the interactions intact rather than
> >> refine occupancies. Seems that the Arg side chain is curled up 
> >> Nestor
> >>
> >> -Original Message-
> >> From: 

Re: [ccp4bb] lysine methylation in E. coli?

[Artem Evdokimov]

It happens.

Ef-Tu is methylated on a single lysine

Also LafV is a putative methylase in E. Coli but its effects were not
studied in detail.

Salmonella methylates flagellin which is how lysine methylation was
discovered - and E.coli is a kissing cousin so...

Artem


On Tue, Nov 5, 2019, 12:31 Jennifer Fleming  wrote:

>
> Dear CCP4 community,
>
>
>
> I am hoping that someone here might know the answer to this as I cannot
> find anything concrete in my literature search.
>
>
>
> I have recently received a mass spec result indicating that a recombinant
> protein expressed in *E. coli* is heavily lysine methylated. I would like
> to know is this an artefact and is it even possible for *E. coli* to
> methylate lysines?
>
>
>
> Thank you for any help,
>
>
>
> Jen
>
>
> --
>
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Re: [ccp4bb] Sodium Ion Binding?

[Artem Evdokimov]

Hi Jacob

Not the easiest task... Based on past experience your major issue will be
the incredible abundance of sodium ions in everything.

So assuming you have high quality sodium free solutions and are willing to
work exclusively in plastic, quartz or fused silica - here are a few
thoughts:

1. Na-22 isotope binding. An oldie but goodie.
2. Sodium-reactive dye equilibrium (see e.g. reference I put at the end)
3. Flame or ion coupled plasma spectroscopy. Very nice to do given the
marvellous sodium band.
4. Sodium selective glass electrode (requires more solution of your analyte
than the other methods)

Overall the key component to these methods is your ability to displace the
sodium with something else prior to measuring the effect of titration the
ion back. Isotopic Na is easier in this regard - but at a cost...

Hope this helps.

Artem

https://bmcresnotes.biomedcentral.com/articles/10.1186/1756-0500-6-556

https://www.google.com/url?sa=t=web=j=http://clinchem.aaccjnls.org/content/clinchem/24/4/580.full.pdf=2ahUKEwis5KL_vM_lAhXCmuAKHY2cAVQQFjAFegQIARAB=AOvVaw0Yhz23Yr_ulB85MDQspIFl=1572833723049


On Sun, Nov 3, 2019, 20:41 Keller, Jacob  wrote:

> Dear Crystallographers,
>
>
>
> Does anyone know of a good biophysical way to identify or quantify sodium
> ion binding to a protein, besides crystallography and ITC? Is this possible
> with SPR, perhaps? Mass spec? Gel shifts? Examples would be greatly
> appreciated!
>
>
>
> All the best,
>
>
>
> Jacob Keller
>
>
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> Desk: (571)209-4000 x3159
>
> Cell: (301)592-7004
>
> +
>
>
>
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Re: [ccp4bb] off topic: microscope in glove box

[Artem Evdokimov]

Short version: It should be OK, especially since the vacuum is transient
and not particularly 'strong*' :)

Long version: if this is an older microscope there may be further
delamination of optically bonded components if air is already admitted
between glass planes (i.e. the optical cement is worn and old). Also some
of the fancier models may have pneumatic balance elements for gross motion
of the optical column - those may experience pressure differentials above
their maximum tolerances. Finally, and very unlikely you may have a
situation where multiple optical elements are sealed together in a single
tube with air trapped between them - if the 'vent' is blocked (by e.g. old
grease or something) then these may pop.

https://www.olympus-lifescience.com/en/microscope-resource/primer/anatomy/oculars/


But the short version is right 99% of the time.

Artem

*"Professor Hubert Farnsworth
: Well, it's a space ship,
so I'd say anywhere between zero and one."
https://www.imdb.com/title/tt0584455/characters/nm0921942

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On Mon, Oct 21, 2019 at 10:00 AM Guenter Fritz <
guenter.fritz.phenix.c...@gmail.com> wrote:

> Dear all,
>
> I want to put one of our microscopes into the glove box. Does anybody
> know whether some parts of the microscope optics do not like vacuum in
> the air lock ?
>
> Thanks and best regards, Guenter
>
> 
>
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[ccp4bb] off topic: job posting

[Artem Evdokimov]

Good morning,

We have two relevant job postingings that I would like to share.

Thank you for your attention :)

Artem

https://careers.massbio.org/job/sr-scientistprincipal-scientist-computational-chemistry/49455467/


and


https://careers.massbio.org/job/scientist-enzymology-protein-chemistry/49690257/

Sr Scientist/Principal Scientist - Computational Chemistry

Enko discovers and develops sustainable solutions for farmers to protect
their crops from pests and disease. It’s proprietary discovery platform
represents a revolutionary approach to rapidly discover novel, safe and
economical solutions that are needed to ensure efficient food production.
Led by a team of proven scientists, entrepreneurs and ag-industry veterans,
Enko uses innovative science and agile design principles to rapidly develop
field-ready crop protection solutions that overcome the rising resistance
barriers traditional pesticides face.

Enko team members will be entitled to participate in Health, Dental,
Vision, Life and Disability insurance, 401(k) plan, and other similar
fringe benefit plans as may be provided by the Company.

We are looking to add a Computational Chemist to join the Chemistry team.
This person will enable the discovery of small molecules for the modulation
of novel biological targets for use in the control of agriculturally
important weeds and pests.

Major responsibilities will include:

Build the computational chemistry project strategy to enhance the
advancement of targets from DNA-encoded library hits to candidate compound
selection
Build the requisite infrastructure to conduct this research, including
identifying tools and technologies needed to support and accelerate
pipeline development and champion their acquisition or the establishment of
collaborations
Lead the structure-based drug design (SBDD) efforts utilizing the suite of
computational chemistry tools including docking/scoring, molecular dynamics
simulations, homology modeling, watermap, and FEP+
Collaborate with medicinal chemists and biologists to create a rich and
diverse pipeline that will address the company’s goals
The ideal candidate will have demonstrated an ability to apply their
expertise in computational chemistry to advance multiple, small molecule
discovery campaigns from screening to development candidates. They will
have demonstrated the ability to thrive in a dynamic, fast-paced,
innovative environment. Excellent written and oral communication skills are
required, as is the desire and ability to excel in a multidisciplinary
environment both as a leader and member across multiple project teams. As
an early addition to the scientific team, this person will have an
opportunity to help us establish a culture that nurtures scientific
excellence, a sense of urgency to achieve project success, and success
built on collaboration and personal accountability.

Qualifications and Skills

D. in Chemistry or a related discipline with 3-5 years of experience in
application of computational chemistry to drug or crop protection chemistry
discovery
Demonstrated expertise in all aspects of modern computational chemistry
including receptor- and structure-based design, conformational analysis,
molecular dynamics, QSAR, binding free energy calculations and
cheminformatics
Track record of accomplishment in small molecule discovery and/or molecular
modelling fields
Demonstration of project leadership and data-driven decision making
Excellent written and oral communication
Strong background in medicinal chemistry, biology, statistics and computer
programming is a plus
No Employment Agencies please, the Company is not responsible for any fees
related to candidates that are unsolicited.

Job Code – PGP13 (Use Job Code in subject line of all communications)

Company: Enko Chem

Contact Name: John Gilroy

Email: john.gil...@enkochem.com

Position Location: Boston, MA

Minimum Required Education: Doctorate

Minimum Years’ Experience: 3-5 years

Scientist, Enzymology & Protein Chemistry

Enko discovers and develops sustainable solutions for farmers to protect
their crops from pests and disease. It’s proprietary discovery platform
represents a revolutionary approach to rapidly discover novel, safe and
economical solutions that are needed to ensure efficient food production.
Led by a team of proven scientists, entrepreneurs and ag-industry veterans,
Enko uses innovative science and agile design principles to rapidly develop
field-ready crop protection solutions that overcome the rising resistance
barriers traditional pesticides face.

Enko team members will be entitled to participate in Health, Dental,
Vision, Life and Disability Insurance, 401(k) Plan and other similar fringe
benefit plans.

We are looking for a creative and highly skilled protein
chemist/enzymologist to join our multidisciplinary team. This scientist
will be responsible for designing, validating, and conducting
enzymatic/biochemical assays and for supporting the overall activities of
the team 

Re: [ccp4bb] challenges in structural biology

[Artem Evdokimov]

Dear Kay


I disagree that 'magic bullet' is impossible. I think the definition is
wrong here - magic bullet to me is a rational set of methods that (when
executed with precision and care) enable crystallization to the maximum
possible benefit. This includes everything - constructs, crystallization
design, etc. Part of the magic bullet is also a precise knowledge when
crystallization is unlikely (i.e. an actual proven predictor that
consistently discriminates between "you're going to succeed if you work
hard" and "it's doomed to fail, don't bother" scenarios in crystallization.

The above is not sexy. It does not present itself as a lovely subject on
which to have international cocktail parties with politicians delivering
fancy speeches. But that is what is needed, and no one is funding that to
the best of my knowledge.

What needs to be done is a significant amount of testing, standardization,
and methods development from the perspective of holistic outcome (i.e.
crystals that work) - and none of the previously advertised 'magic bullets'
work the way I just described.

Having written this, I think you're right - this is a bit of a distraction
from James' original point. However it's a valid opportunity for a lively
discussion on its own :)

Artem

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On Sun, Jul 21, 2019 at 4:52 PM Kay Diederichs <
kay.diederi...@uni-konstanz.de> wrote:

> Dear Artem,
>
> black or white is not my way of thinking, which is why I don't believe in
> Hannibal's approach when it comes to crystallization.
>
> None of the magic bullets that were advertised over the past decades have
> proven generally applicable.  I believe more in incremental improvement
> which in this case includes a few biophysical characterization methods,
> possibly improved microfluidics or other apparatus, and expanded screens.
> And a lot of hard work, perseverance, intuition, frustration
>  tolerance. Nothing that really needs huge funding - of course it does
> need money, but just a  share of what is anyway needed for the usual lab
> work including expression, purification, functional characterization,
> binding studies and the like.
>
> One area where a huge amount of money was burnt is crystallization in
> space, on board of e.g. the spacelab and ISS. This is for me an example of
> a mis-led approach to throw money at a difficult problem, with the
> expectation of a solution. Science does not work like that, and money in
> this case seems more to be the problem than the solution.
>
> This example may illustrate a certain failure of us scientists to resist
> the temptation to promise unrealistic outcomes when confronted with money
> provided for political reasons, which ultimately undermines our
> credibility. But this takes us away from James' points.
>
> best,
>
> Kay
>
> On Sun, 21 Jul 2019 16:06:48 -0400, Artem Evdokimov <
> artem.evdoki...@gmail.com> wrote:
>
> >Dear Kay,
> >
> >Even the small, badly diffracting and 'messed up' crystals are still
> >crystals. There is literally a phase transition (pun very much intended)
> >between growing *usable crystals* versus *having no crystals* (or having
> >crystals that do not qualify as 'diffraction quality' even under the most
> >favorable light). Points 2-9 fall into the 'I have crystals' bucket and
> >everything else is in the 'I have no crystals' bucket.
> >
> >I am being deliberately black and white of course.
> >
> >As to whether huge funding would help to bridge the 'phase gap' - to me
> >this is a purely theoretical question since to the best of my knowledge
> >there never was a 'huge funding' for this particular problem :) And if it
> >is true that the general belief in the art is that crystallization is not
> >worth investing into because there's no hope in it then of course it is a
> >self-fulfilling prophesy.
> >
> >There is an unresolved dichotomy buried in the sentiment above: it seems
> >that we (the community of structural biologists) more or less believe that
> >crystallization research is not fundamentally fruitful (hence the
> >no-funding situation). However, anyone who undertakes significant efforts
> >to determine an actual structure using crystallography inevitably *has to*
> >crystallize their target of interest - and therefore by definition has
> hope
> >that their particular target will work out, against the overall gloomy
> >outlook on the crystallization science as a whole. So we either are a
> >collective of self-induced schizophrenics, or the general sentiment is
> >wrong and systematic crystallization research is meaningful and
> >fruitful - *just
> >very very hard*.
> >
> >In ~200 BC Hannibal reportedly said "

Re: [ccp4bb] challenges in structural biology

[Artem Evdokimov]

Dear Kay,

Even the small, badly diffracting and 'messed up' crystals are still
crystals. There is literally a phase transition (pun very much intended)
between growing *usable crystals* versus *having no crystals* (or having
crystals that do not qualify as 'diffraction quality' even under the most
favorable light). Points 2-9 fall into the 'I have crystals' bucket and
everything else is in the 'I have no crystals' bucket.

I am being deliberately black and white of course.

As to whether huge funding would help to bridge the 'phase gap' - to me
this is a purely theoretical question since to the best of my knowledge
there never was a 'huge funding' for this particular problem :) And if it
is true that the general belief in the art is that crystallization is not
worth investing into because there's no hope in it then of course it is a
self-fulfilling prophesy.

There is an unresolved dichotomy buried in the sentiment above: it seems
that we (the community of structural biologists) more or less believe that
crystallization research is not fundamentally fruitful (hence the
no-funding situation). However, anyone who undertakes significant efforts
to determine an actual structure using crystallography inevitably *has to*
crystallize their target of interest - and therefore by definition has hope
that their particular target will work out, against the overall gloomy
outlook on the crystallization science as a whole. So we either are a
collective of self-induced schizophrenics, or the general sentiment is
wrong and systematic crystallization research is meaningful and
fruitful - *just
very very hard*.

In ~200 BC Hannibal reportedly said "I will find a way or make one". I
think that if we approach problem #1 with this attitude (and an equivalent
of a very large army's worth in funding) then it can be solved.

Artem

- Cosmic Cats approve of this message


On Sun, Jul 21, 2019 at 1:55 PM Kay Diederichs <
kay.diederi...@uni-konstanz.de> wrote:

> Hi Artem,
>
> you are certainly correct in that James' points 2-9 would be moot if his
> point 1 were solved. But as long as this is not the case, we resort to work
> with few and/or small and/or badly diffracting and/or non-isomorphous
> crystals, which makes points 2-9 very relevant.
>
> Maybe the reason why crystallization research is not well funded is that
> it is not expected to yield significant improvements. Personally, I think
> that even huge funding would not result in methods that succeed in
> crystallizing all molecules.
>
> best,
> Kay
>
> On Sun, 21 Jul 2019 11:28:14 -0400, Artem Evdokimov <
> artem.evdoki...@gmail.com> wrote:
>
> >Excellent question :)
> >
> >First of all, thank you for putting this out to the community!
> >
> >Secondly, I agree with several of us who've written that a single
> >conference is not enough to discuss all the possible topics.
> >
> >Thirdly, in my opinion all the other problems are secondary to the main
> >(and only remaining!) problem in crystallography: getting
> >diffraction-quality protein crystals reproducibly and quickly
> >
> >The amount of funding for serious crystallization research seems to be
> >close to non-existent. In general methodology funding is hard to get, but
> >crystallization seems to me like the absolute underdog of the method pool
> -
> >the true 'red headed stepchild' of the methods development funders.
> >
> >At risk of repeating myself - the other problems (worthy, significant, and
> >urgent as they are!) are subservient to the main issue at hand - namely
> >that crystallization remains an unpredictable and artful phenomenon while
> >literally all other aspects of structure determination process (the gene
> to
> >structure pipeline, whatever you might call it)have made astronomic leaps
> >forward.
> >
> >Artem
> >- Cosmic Cats approve of this message
> >
> >
> >On Mon, Jul 15, 2019 at 3:44 PM Holton, James M <
> >270165b9f4cf-dmarc-requ...@jiscmail.ac.uk> wrote:
> >
> >> Hello folks,
> >>
> >> I have the distinct honor of chairing the next Gordon Research
> >> Conference on Diffraction Methods in Structural Biology (July 26-31
> >> 2020).  This meeting will focus on the biggest challenges currently
> >> faced by structural biologists, and I mean actual real-world
> >> challenges.  As much as possible, these challenges will take the form of
> >> friendly competitions with defined parameters, data, a scoring system,
> >> and "winners", to be established along with other unpublished results
> >> only at the meeting, as is tradition at GRCs.
> >>
> >> But what are the principle challenges in biological structure
> >

[ccp4bb] multiple openings at Enko

[Artem Evdokimov]

Sorry for the off-topic post; only the first two out of the four positions
below are related to structural work, I've included the other two for
completeness' sake.

We have several openings at Enko (a small crop protection company in
Woburn, MA).


   - Principal Scientist, Discovery Chemistry
   

   - Research Associate, Enzymology
   
   - Plant Biology Research Assistant
   
   - Patent Liaison
   


(all the links above are from MassBio website and can be separately located
by searching for 'Enko')

Please direct inquiries/submissions to care...@enkochem.com

Thank you,

Artem

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Re: [ccp4bb] Interesting pattern on a crystallization drop

[Artem Evdokimov]

Neat!

Looks like multiple adjacent bubbles that were initially touching but
eventually shrunk down to the central cores - the connectors are protein
filaments (skin on the bubbles) left over from when bubbles had contact
points.

Artem

On Wed, Mar 27, 2019, 19:39 Marshall, Bevan (Manufacturing, Parkville)
 wrote:

> Looked up the condition on C6 (https://c6.csiro.au/C6.asp) and that
> condition is found in both Index and JCSG screens as well as Classics II.
>
>
>
>
>
> *Bevan Marshall*
> Staff Scientist | Collaborative Crystallisation Centre
> *Manufacturing*
> CSIRO
>
> E bevan.marsh...@csiro.aut +61 3 9662 7492
> 343-351 Royal Parade, Parkville, VIC 3052
> www.csiro.au | https://c3.csiro.au
> CSIRO acknowledges the Traditional Owners of the lands that we live and
> work on across Australia and pays its respect to Elders past and present.
>
> *PLEASE NOTE*
> The information contained in this email may be confidential or privileged.
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>
> *Please consider the environment before printing this email.*
>
>
>
> *From:* CCP4 bulletin board [mailto:CCP4BB@JISCMAIL.AC.UK] *On Behalf Of 
> *LEGRAND
> Pierre
> *Sent:* Thursday, 28 March 2019 9:13 AM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] Interesting pattern on a crystallization drop
>
>
>
> Dear Beatriz,
>
>
>
> Nice drops :-))
>
> Could it be that there is a reaction going on in these drops ?
>
> The conditions are quite "exotic" with possibilities of coordination or
> oxydoreduction (Co2+/Co3+) or polymerization...
>
> Do you have reductants with the protein buffer ?
>
> Is the protein an enzyme or a metalloprotein ?
>
> Just some ideas.
>
>
>
> Best wishes,
>
> Pierre
>
>
> --
>
> *De :* CCP4 bulletin board [CCP4BB@JISCMAIL.AC.UK] de la part de Beatriz
> Gomes Guimaraes [beatriz.guimar...@fiocruz.br]
> *Envoyé :* mercredi 27 mars 2019 19:44
> *À :* CCP4BB@JISCMAIL.AC.UK
> *Objet :* [ccp4bb] Interesting pattern on a crystallization drop
>
> Dear all,
>
>
>
> I would like to share with you a surprising pattern I found when examining
> some crystallization plates (attached figures).
>
>
>
> It is less obvious looking the photos, but apparently the "lines" are
> formed by precipitated protein and there are some "bubbles" with small
> drops inside. I wish they were microcrystals but I do not think this is
> the case.
>
> I was suprised by the symmetry !
>
>
>
> And it is not completely random because for the same condition the
> difference between the two drops are : protein alone ("hexagon") and
> protein + ligand ("rhombus")
>
>
>
> crystallization condition is:
>
> 0.01 M Cobalt(II) chloride hexahydrate
>
> 0.1 M Tris pH 8.5
>
> 20% w/v Polyvinylpyrrolidone K 15
>
>
>
> Have you seen anything similar before?
>
>
>
> Thank you for your comments!
>
> Beatriz
>
>
>
>
>
> --
> Beatriz Guimarães
> Laboratory of Structural Biology and Protein Engineering
> Instituto Carlos Chagas - ICC / FIOCRUZ Paraná
> Rua Prof. Algacyr Munhoz Mader, 3775   Bloco C
> CIC 81350-010
> Curitiba - PR, Brasil
> Tel.:+55(41)3316-3225/2104-3438
>
>
> --
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
>
> --
>
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> --
>
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Re: [ccp4bb] KCl in SDS-PAGE workarounds?

[Artem Evdokimov]

K+ and NH4+ ions precipitate SDS - forming the lovely cheese that makes it
very difficult to load and run those samples.

Sometimes using LDS can help with the potassium issue. Heating the sample
right before loading might help as well, or using a lot larger ratio of
buffer to sample.

Precipitation of protein prior to the gel is certainly an option, although
some proteins have difficultues with recovering after that (membrane
proteins in particular).

Artem

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On Sat, Mar 2, 2019 at 9:53 PM Keller, Jacob 
wrote:

> Dear crystallographers,
>
>
>
> It has been my experience that KCl does nasty things when loading SDS-PAGE
> gels. Does anyone have an easy workaround, perhaps TCA precipitation?
> Ideally this would be something nicely quantitative yet quick and easy….
>
>
>
> Any suggestions appreciated.
>
>
>
> All the best,
>
>
>
> Jacob Keller
>
>
>
> +
>
> Jacob Pearson Keller
>
> Research Scientist / Looger Lab
>
> HHMI Janelia Research Campus
>
> 19700 Helix Dr, Ashburn, VA 20147
>
> Desk: (571)209-4000 x3159
>
> Cell: (301)592-7004
>
> +
>
>
>
> The content of this email is confidential and intended for the recipient
> specified in message only. It is strictly forbidden to share any part of
> this message with any third party, without a written consent of the sender.
> If you received this message by mistake, please reply to this message and
> follow with its deletion, so that we can ensure such a mistake does not
> occur in the future.
>
>
>
> *From:* CCP4 bulletin board  * On Behalf Of *Artem
> Evdokimov
> *Sent:* Saturday, March 2, 2019 8:32 PM
> *To:* CCP4BB@JISCMAIL.AC.UK
> *Subject:* Re: [ccp4bb] ionic strength for extraction buffer of membrane
> proteins
>
>
>
> Hi Alex,
>
>
>
> In my experience you just have to experiment with these parameters (in
> however of a limited space that is afforded by your experimental set-up).
> If you have the right tools you can slog through semifactorial or DoE-based
> scouting of various parameters with relative ease. These parameters
> typically include pH, salinity, detergent(s), and other variables (e.g. the
> nature of the buffer and salt(s) - many cases exist where the 'standard'
> choices do not work well).
>
>
>
> Literature-based analogies do help on occasion. For example,
> membrane-spanning p450-type proteins often prefer high salt, and often
> would do better in sodium acetate as opposed to chloride. I've worked with
> ion channels that preferred 3M NaCl, or 2M MgCl2 and other fairly weird
> conditions...
>
>
>
> Artem
>
> - Cosmic Cats approve of this message
>
>
>
>
>
> On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín <
> aperalva...@gmail.com> wrote:
>
> Dear all,
>
> any reference as a guide for selecting the appropriate salt and
> concentration for membrane proteins extraction buffer?
>
> Best,
>
> Alex
>
> --
> Alex Perálvarez-Marín, Ph.D.
> Centre d'Estudis en Biofísica / Unitat de Biofísica
> Edifici M
> Universitat Autònoma de Barcelona
> 08193 Cerdanyola del Vallés
> Barcelona
> Spain
> Phone: +34 93 581 4504
> FAX:  +34 93 581 1907
> e-mail: aperalva...@gmail.com
> LinkedIn: es.linkedin.com/in/aperalvarez/
>
> 
>
> To unsubscribe from the CCP4BB list, click the following link:
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>
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>
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Re: [ccp4bb] ionic strength for extraction buffer of membrane proteins

[Artem Evdokimov]

Hi Alex,

In my experience you just have to experiment with these parameters (in
however of a limited space that is afforded by your experimental set-up).
If you have the right tools you can slog through semifactorial or DoE-based
scouting of various parameters with relative ease. These parameters
typically include pH, salinity, detergent(s), and other variables (e.g. the
nature of the buffer and salt(s) - many cases exist where the 'standard'
choices do not work well).

Literature-based analogies do help on occasion. For example,
membrane-spanning p450-type proteins often prefer high salt, and often
would do better in sodium acetate as opposed to chloride. I've worked with
ion channels that preferred 3M NaCl, or 2M MgCl2 and other fairly weird
conditions...

Artem
- Cosmic Cats approve of this message


On Fri, Mar 1, 2019 at 7:27 AM Alex Perálvarez Marín 
wrote:

> Dear all,
>
> any reference as a guide for selecting the appropriate salt and
> concentration for membrane proteins extraction buffer?
>
> Best,
>
> Alex
>
> --
> Alex Perálvarez-Marín, Ph.D.
> Centre d'Estudis en Biofísica / Unitat de Biofísica
> Edifici M
> Universitat Autònoma de Barcelona
> 08193 Cerdanyola del Vallés
> Barcelona
> Spain
> Phone: +34 93 581 4504
> FAX:  +34 93 581 1907
> e-mail: aperalva...@gmail.com
> LinkedIn: es.linkedin.com/in/aperalvarez/
>
> 
>
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Re: [ccp4bb] Fwd: dry Shipper made in China

[Artem Evdokimov]

Dry shippers are often featured on auction sites for used equipment and
sometimes can be found for huge discounts...

Artem

- Cosmic Cats approve of this message


On Fri, Jan 25, 2019 at 8:36 AM Muhammad Imran  wrote:

> Dear Community members,
>
> We wanted to buy molecular dimension dry shipper CX100 and universal puck
> starter kit. However due to increase in dollar price compared to Pakistani
> rupee, our budget does not allow to buy these items.
> We tried to search an alternate and found one from Biobase China (details
> attached). I want to know if any one have used it or have experience that
> it can also hold UNIPUCK and be accepted at light sources (Diamond, ESRF,
> SESAME etc).
> Thanks if you can spare time for this.
>
> --
>
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>



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Re: [ccp4bb] looking for 10-20 kDa proteins that express well and migrate appropriately on SDS-PAGE

[Artem Evdokimov]

Off the top of my head, for your mass range with gel behavior that you ask
for:

Human or mouse Synuclein (not folded, very heat stable, incredibly soluble)
- interesting reaction with SDS but runs fine on the gel
T4 Lysozyme with a double mutation (expresses mega-loads per gram of cell
paste)
Ferredoxin
Rubredoxin
cytochrome b562
Ubiquitin

the list goes on and on...

As to polycistronic stuff - it may be easier to mix lysates than to mess
around with multiple expression cassettes - in case you'd want to change
the mix later... just my two cents.

Artem

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On Sun, Jan 6, 2019 at 6:15 PM Tan, Song  wrote:

> Dear Colleagues,
>
> As a followup to my lab’s plasmids for making DNA molecular weight markers
>  for about
> one cent per lane (Henrici et al, Scientific Reports, 7:2438, 2017
> ; plasmids are
> available from Addgene), we are working on a similar project to produce
> protein MW markers in E. coli.  We are having problems finding 10-20 kDa
> proteins that express solubly at high levels in E. coli and that migrate
> appropriately on SDS-PAGE.  We have eliminated multiple proteins including
> thioredoxin and DHFR that express very well but that migrate anomalously
> slowly on SDS-PAGE.
>
> So we are looking for proteins which fulfill the following criteria:
>
> 1.  MW = 8-10 kDa or 18-20 kDa,
> 2.  Express as soluble protein in E. coli,
> 3.  Can be purified from E. coli with yields of greater than 10 mg/L
> culture,
> 3.  Migrate appropriately on SDS-PAGE.
>
> We hope to create a simple and inexpensive system for producing protein MW
> ladders, and to distribute the plasmids through Addgene.  Plan is to
> coexpress multiple proteins using our polycistronic expression system
>  to make
> the production process simpler.
>
> If you know of proteins that fulfill all of the above criteria, please do
> drop me a line.
>
> Regards and thanks,
>
> Song
>
> ---
>
> Dr. Song Tan
> Professor of Biochemistry & Molecular Biology
> Center for Eukaryotic Gene Regulation
> Dept of Biochemistry & Molecular Biology
> 108 Althouse Laboratory   (office in 468A North Frear Lab)
> Penn State University
> University Park, PA   16802
> email:  sx...@psu.edu 
> phone:  814-865-3355 fax: 814-863-7024
> web:  http://www.personal.psu.edu/sxt30
>
>
> --
>
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Re: [ccp4bb] Non-Cysteine modification with heavy atom

[Artem Evdokimov]

Off the top of my head, assuming nothing much about your protein(s) - some
of this is only going to work if you can modify the protein(s) prior to
crystallization. This excludes the 'normal' heavy atom soaking type stuff
which can give rise to binding with covalent-like strength (while still
being noncovalent, technically):

Iodination of tyrosines (and histidines if pH is selected correctly)
Iodination or bromination of DNA/RNA if your complex contains nucleic acid
Introduction of heavy atom into a custom-made peptide (sky is the limit as
to what you can do with this) followed by peptide ligation (i.e. sortase
and the like) - requires modification and re-expression of your protein
construct of course
Reaction of Lys and N-term with suitably modified carboxylic acids (e.g.
para-iodo benzoic acid, para-mercuric derivatives and so on) via the use of
water soluble coupling agents or pre-activated acid derivatives
Reductive amination of Lys residues using aldehydes that bear heavy atom
labels (must be sure that the reducing agent does not knock the HA off,
which it jolly well can do)
Ditto with side chains of Asp and Glu, and the amine is modified with heavy
atom(s)
Introduction of unnatural amino acids (even bearing heavy atoms if desired)
by means of genetic manipulation (stop codon bypass, codon replacement etc.)
Nucleophilic attack on Serine (especially facile if you happen to have a
serine protease or something similar where serine is activated)

Also see: https://www.nature.com/articles/ncomms5740

Even though these methods are not necessarily selective with respect to
amino acids being modified, it is surprisingly often possible to only
modify a few (sometimes even one) specific positions in a protein due to
the selectivity imposed by protein surface environment

I am probably missing another 10-15 methods but gotta run :)

Artem

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On Wed, Jan 2, 2019 at 2:42 PM Chandramohan Kattamuri <
1c5b7cb6c764-dmarc-requ...@jiscmail.ac.uk> wrote:

>
> Dear CCP4 members, Can someone suggest to me a methodology to covalently
> modify (non-cysteine) on component of a multicomponent complex with heavy
> atoms in order to achieve better phase determination?
>
> Thanks in advance
>
>
> Chandra
> kattamuricha...@yahoo.com
>
>
>
>
> --
>
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Re: [ccp4bb] 2019 New Year’s resolutions of a cryo-EM newbie

[Artem Evdokimov]

My apologies for posting this on an open board :) I realize that this is
completely off topic - but it is a pretty painful topic that affects us all
regardless of academic or industrial affiliation, professional preferences
etc.

Artem

--- do not read below unless you want to :) ---

If an average citizen is asked whether they believe in fairies/unicorns or
in atoms (since you cannot see either* it's easy to get confused as to
which ones are real, I guess), they often pick fairies and unicorns. The
nature of belief as a great human fallacy does not cease to amaze me. Its
tendrils affect all of us, without exception.

Fake news is just the latest representation of the (utterly irrational)
power of belief. If one can convince millions that something is real (or
ethically/morally right) then fantasy may become 'real' for the duration of
the shared dream. To be fair, sometimes the dream is that of freedom,
liberty, equality, kindness, etc. but far too often it is the dream of the
dark and disturbing kind that leads to subjugation of self-determination,
oppression, whitewashing of atrocities, and violence.

Recent events in the political sphere simply illuminate some of the
abhorrent fantasies perpetrated by what amounts to nearly 50% of the
population. Mathematically speaking we are pretty close to a 'fake news
country' - especially since the 'dark dreamers' elicit more political power
than the rest of us. This did not begin two years ago. It will not end two
years from now.

Some thoughts on making things better (fairly basic for any practicing
scientist):

- always insist on proof, especially when common sense is at odds with
what's being 'sold'
- exercise passion and don't be afraid to get agitated

"Those who profess to favor freedom and yet depreciate agitation, are
people who want crops without ploughing the ground; they want rain without
thunder and lightning; they want the ocean without the roar of its many
waters." -- Frederick Douglass

* Yes, I realize that we can in fact see atoms with the right equipment.
This makes it even worse, all things considered...

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On Wed, Jan 2, 2019 at 8:07 AM Keller, Jacob 
wrote:

> 6) I most sincerely hope that, if I stick to my five New Year’s
> resolutions and stop wasting the cryo-EM community’s time, my fellow
> Canadians will not extradite me to the fake-news country at our southern
> border.
>
>
>
>
>
> I know this is a joke, but we in the USA are not a fake news people, and
> please don’t punish us for the sins of our leadership--it’s painful for
> many of us.
>
>
>
> JPK
>
>



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Re: [ccp4bb] 2019 New Year’s resolutions of a cryo-EM newbie

[Artem Evdokimov]

https://www.unboundworlds.com/wp-content/uploads/2016/05/takei-oh-my.jpg

oh, my...

Happy new year!

Artem

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On Tue, Jan 1, 2019 at 5:38 PM Marin van Heel <
057a89ab08a1-dmarc-requ...@jiscmail.ac.uk> wrote:

> 1) I shall not make false FSC resolution claims on Wikipedia without
> first reading and understanding the original papers.
>
> (reviewed in: https://www.biorxiv.org/content/early/2017/11/24/224402)
> 2) I shall not anonymously and abusively attack the very inventors of
> the FSC in presumptuous public Wikipedia statements, implying that I
> understand the FSCs better than they ever did.
> 3) I shall explicitly apologize for anonymously posting fake
> information on Wikipedia from an IP address in Ontario Canada, thereby
> discrediting the Ontario cryo-EM community (possibly including my own PI…).
> 4) My special apologies shall go to a Structural Professor at Guelph
> University who is one of the original FSC inventors.
> 5) In my quest to become a real scientist, in 2019 I shall cancel my
> membership of the flat-earth society.
> 6) I most sincerely hope that, if I stick to my five New Year’s
> resolutions and stop wasting the cryo-EM community’s time, my fellow
> Canadians will not extradite me to the fake-news country at our southern
> border.
> Have fun in 2019!
> Marin van Heel
> [image: image.png]
>
> [image: image.png]
>
> --
>
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Re: [ccp4bb] opentrons pipetting robot

[Artem Evdokimov]

Good morning and Happy New Year to you

In brief, it can be used to create screens although programming multiple
ingredients with varying viscosities will initially be a fairly steep
challenge since the software is not specific to crystallization. It can be
done though and provided that you can re-use protocols (I.e. today you set
up PEG 3350 versus salt and pH and tomorrow you set up PEG 6000 versus salt
and pH without changing concentrations) it can even be effective.

You would likely need to write your own code for gradients and suchlike but
maybe you can borrow from existing protocols a bit. It is all in Python
anyway.

I would not use this robot for setting drops unless there is so much
protein available that 1ul + 1ul drops are not wasteful. The pipettors are
essentially the same (in terms of tolerances and volume limits) as your
average wet lab hardware. Now, if you have a willing colleague with
mechanical and coding mojo at your disposal you can probably modify the
robot to use a crystallization friendly operating device, assuming you can
get a hold of an accurate sub microliter dispenser with an open API. The
robot API is open towards change.

In a nutshell this robot is sort of like a large 3D printer chassis with a
deck and pipettors bolted on. If you love to tinker this is a machine for
you. Cannot beat the price.

Artem

On Mon, Dec 31, 2018, 12:46 Doug Juers  Hello All,
>
> I've just learned about the opentrons pipetting robot, which appears to be
> quite affordable relative to other robots. I'm wondering if anyone here on
> ccp4 bb has any experience with it - for creating crystallization screens
> and/or setting drops?
>
> Best,
> Doug
>
>
> -
> Douglas Juers
> Physics Department
> Program in BBMB
> Whitman College
>
> 
>
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Re: [ccp4bb] Adding Zinc to Protein

[Artem Evdokimov]

Dear Nicola,

Happy New Year

It may or may not be possible to re-constitute Zn into purified protein.
Some binding sites are very hard to fill back up once Zn is gone (for
example sites with more than one -SH ligand can and often do undergo
oxidative crosslinking in the absence of a stabilizing ion - we ran into
this plenty of times when working with Zn-finger proteins). Other sites
(e.g. Zn proteases) can easily re-gain Zn that has been stripped away (a
good method for avoiding autoproteolysis when purifying certain
metalloenzymes, but it can also backfire horribly). Note that common
reducing agent DTT is know to strip Zn and other transition elements out of
protein (BME is much less likely to do this and TCEP is virtually safe from
this perspective). Ditto common buffers - citrate, high phosphate
(especially with Iron ions), malonate, imidazole and so on.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1133908/

Please note that Zn complex chemistry in water is "...fundamental, rather
complex, and has important consequences for the role of zinc in biology..."
to quote Krezel & Maret (reference below) - so if reconstitution does not
work in some conditions it very well might work in others.

https://www.sciencedirect.com/science/article/pii/S0003986116301308

So if your protein lost its Zn, there is no really good way to predict the
outcome of an attempted re-fill -- but since you have frozen protein in
spades your best bet is to try a few things, such as dialyzing a small
sample of protein (ideally diluted to ~1mg/ml or so) against low
concentration of Zn in a suitable buffer. A good place to start is ~10uM
(that's micromolar) Zn - which is plenty to saturate the protein with
reasonable binding constant but is (hopefully) not enough to precipitate
the protein out. After dialysis, remove Zn from solution - by briefly
re-dialyzing against Zn-free bufffer - then concentrate the protein and try
it out. You can also attempt to stabilize Zn in solution with an equimolar
(or slightly less than equimolar) amount of a mild chelator like malonate
(not EDTA, its complex with Zn is far too stable) and then add it directly
to your protein - note that adding Zn to protein directly tends to cause
precipitation (but chelating the Zn with a moderate-strength agent tends to
prevent this).

Good luck,

Artem
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On Sun, Dec 9, 2018 at 4:32 PM Nicola Evans <
251ca3b4615e-dmarc-requ...@jiscmail.ac.uk> wrote:

> From a fluorescence scan it would appear a protein I am working on has
> zinc in it. The occupancy is likely to be very low however (a structural
> homologue has several zincs in the x-ray crystal data but at 0.5
> occupancy), as there isn't anything obvious in the electron density map
> (perhaps some of the waters are zinc) and an anomalous difference map
> wasn't possible to obtain on our last beamtime.
>
> Ideally I would want to re-express the protein with zinc added to the
> culture conditions, but I am time-restained, so I was wondering if it is
> possible to add zinc to purified protein instead? I have heard it can cause
> proteins to crash out. I have quite a lot of protein frozen so I can try a
> few things. I would appreciate any advice on how much to add from anyone
> who has had success with this before?
>
> Thanks in advance!
>
> 
>
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[ccp4bb] off-topic: Laboratory RA job posting

[Artem Evdokimov]

Greetings

Sorry for the off-topic post :)

We are looking to fill an RA position in Enzymology/MolBio (junior or
senior, depends on qualifications and experience). Full description follows.

Interested candidates should forward their CV and letter of interest to:

*i...@enkochem.com *
*john.gil...@enkochem.com *

Many thanks, and happy (various) holidays.

Artem

---

TITLE: Enzymology Research Associate



LOCATION: Woburn, MA



Enko Chem is a small, well funded, aggressively developing Crop Protection
research company in Boston/Woburn (MA).



Responsibilities:

We are looking for a motivated scientist to join our multidisciplinary
research group. The Enzymology RA will be responsible for conducting
enzymatic *in vitro* activity assays under the supervision of senior staff
and for supporting the overall activities of the team which may include
molecular biology, protein expression and purification, cell culture, and
other related activities.

*Desired Skills*

· Conducting enzyme assays in microplate format, basic data
analysis and interpretation are a must

· Stringent attention to detail, excellent notebook and
record-keeping practices are a must

· General biochemistry wet-lab skills are a must

· Protein purification/characterization and/or eukaryotic cell
culture are a strong preference

· Experience with cell-based assays in microplate format is
desirable

· Experience with automated/robotic assay technologies, liquid
handler programming, and associated skills are a strong plus





*Qualifications*

   - BS with over 5 years or MS with 2-3 years of professional experience
   running *in vitro* assays in the biotech/pharmaceutical environment, or
   equivalent.



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Re: [ccp4bb] Protein Purification- reg

[Artem Evdokimov]

Hi Amala,

Depending on the resin you used there may be a conflict with BME and also
50 mM TRIS may be too much at the pH 7.5. Easy to test: use HEPES pH 7.5
and TCEP instead of BME, or use 30 mM TRIS at pH 8.0

Alternatively your protein is aggregated and does not bind well...

Artem

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On Mon, Aug 13, 2018 at 9:49 AM, amala mathimaran 
wrote:

> Dear All
>
> I am working with HIS – tag protein in N-terminal (hexa histidine). The
> protein from Prokaryotic origin cloned into pET30a+ vector and expressed in 
> *E.coli
> *BL21 cells. The expression was good. I am trying to purify a protein
> using Ni-affinity column equilibrate with Buffer A 50mM Tris pH7.5, 50mM
> NaCl, 15mM imidazole, 3mM BME, 5%glycerol and eluted with Buffer B ( same
> as buffer A but 250mM imidazole). I eluted the protein in step wise
> gradiant. the protein was less binded in Ni affinity IMAC column because
> eluted fraction contain less amount and the protein remain present in the
> Flow through. Can any one suggest how to increase the binding affinity of
> the protein and how to purify the protein. The protein PI was 6.33
>
> --
>
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Re: [ccp4bb] Zn+2 proteases

[Artem Evdokimov]

 From personal experience, many of them really do need Zn++ and some of
them can get by on Cd++ :)

However, Zn++ can often be used in the micromolar range (so essentially
quasi-stoichiometric with the enzyme) so it's not as much of a PITA as when
you have millimolar quantities.

HTH!

Artem


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On Sun, Jul 29, 2018 at 5:13 PM, chemocev marker 
wrote:

> Dear All
>
> I have a questions about the Zn+2 proteases (Thermolysin based Zn+2 site)
>
> As Zinc is trouble metal when it comes to test the activity.
>
> Which is better substitute for it.
>
> Is MgCl2 or MncCl2
>
> Why one is preferred over the other.
>
> best
>
> Jiri
>
> --
>
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Re: [ccp4bb] Suggestions for deglycosylaiton enzymes

[Artem Evdokimov]

Dear Partha

Treat your culture with Kifunensine prior to transfection (or throughout
growth if you're using stables) and then treat purified protein with EndoH.
Pretty cheap and effective.

PNGase F does not *require* denaturing conditions. It just likes the
protein to be 'loose' - in my experience about half the time the accessible
sites will fall off even under native conditons. However, PNGase F is
somewhat expensive and after treatment certain proteins have fallen out of
solution (presumably because not a single carbohydrate remained, as opposed
to 'shielding' provided by the single remaining sugar left behind by
EndoH). Caveat emptor.

Artem

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On Mon, Jul 16, 2018 at 2:53 PM, Parthasarathy Sampathkumar <
spart...@gmail.com> wrote:

> Dear All,
>
> I am in a situation, almost for the first time within my limited
> experience, that deglycosylation might be necessary to obtain crystal. So,
> I thought of tapping to vast experience of CCP4BBers, while I am searching
> literature.
>
> I have protein that has been expressed in HEK293 cells, secreted into
> media, purified over IMAC and SEC columns. Crystallization-screens with its
> binding partners (they form good complexes based on analytical SEC) have
> not produced any useful hits (whereas complexes with related proteins
> worked well). So, I plan to re-try complex formation and \crystallization
> screen after deglysosylation.
>
> My question is: In practice, Does a kit (for example here: https://www.
> sigmaaldrich.com/catalog/product/SIGMA/NDEGLY?lang=en=US)
> containing Endo F1, F2, F3 be sufficient or should this be tried in
> combination with PNGase (which requires
> desaturating conditions)?!!
>
> Many Thanks in advance for your suggestions, and reference.
>
> Best Wishes,
> Partha
>
> --
>
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Re: [ccp4bb] Glycoprotein expression strategy

[Artem Evdokimov]

Dear Wenhe,

>From my perspective you have quite a few options. Notably, the presence of
glycosylation in the native host does not always (or even often) mean that
you *must* have the protein in the native glyco-state in order to be
'right'. Very often glycosylation sites can be trimmed (by e.g. Kifunensine
and similar compounds) or mutated out. When one mutates glycosylation sites
one runs a significant risk of stopping export - meaning that without
correct glycosylaton some proteins do not secrete. This is very much
protein dependent and host dependent but I have personally experienced
about a dozen cases (out of hundreds which went just fine). So it's not a
#1 item to be scared of.

Options:

1. refold from IB in E. coli
2. secrete into periplasm in E. coli
3. secrete into media from a gram-positive organism (e.g. Bacillus)
4. secrete from yeast
5. secrete from insect cells
6. secrete from mammalian cells

For you it seems that 5 and  6 may be a bit of an overkill, but it all
depends on the nature of the protein you're working with :) I normally
recommend 1 and 6 to 'bracket' the probability spread (1 is very easy but
often not very successful whereas 6 is expensive but is often very
successful, and both are very fast).

Artem

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On Wed, Jul 11, 2018 at 11:54 PM, WENHE ZHONG 
wrote:

> Dear Community,
>
> A little bit out of topic here. An enzyme we like to use in our assay is
> commercially available. However, we found some unfavorable activities that
> probably from the contaminants from this commercial source. We checked the
> company website and found out this enzyme was purified from the original
> organism without tag for enhancing purification. No purity data was shown
> as well.
>
> So we want to see whether we can produce the recombinant protein with tag.
> After checking the literatures, this enzyme is a N-linked glycosylated
> protein (glycan attached to 3 asparagine residues) and is from
> fungi Penicillium citrinum. These 3 glycosylations probably affect protein
> folding, stability and its location in the cell. Currently we have three
> options here:
>
> 1. Express this enzyme in yeast which is a similar system compared to
> Penicillium citrinum. His6 tag will be added to the N- or C- terminal of
> this protein.
> 2. Express this enzyme in the periplasm of E.coli. However, no
> glycosylation system in E.coli. Only one paper published in a not well
> known journal used this method. But the result is not very conclusive to
> us. If their claim is correct, the glycosylation at this enzyme is not
> essential for protein production.
> 3. Express this enzyme in insect cells. More complicated and advanced
> glycosylation system in insect cells compared to fungi.
>
> Anyone has experience with glycoprotein? It will be very helpful if I can
> have your advice. Thank you.
>
> Kind regards,
> Wenhe
>
>
> --
>
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Re: [ccp4bb] disulfate bond ?

[Artem Evdokimov]

It seems that your CoA is one carbon out of reference. You have spotty
difference density over the ligand. Shift it left by one carbon bond and
redune to see if density fits better.

Artem

On Wed, Jul 4, 2018, 02:26 张士军 <21620150150...@stu.xmu.edu.cn> wrote:

> Hi all
>
> I got a structure which has COA in it, and the SH in the tail of COA
> is very close to the SH side chain of Cys in the structure. I don't know
> whether it is disulfate bond or not? I remember they should link together if 
> they are disulfate bond,am I right? so what could this be? Thanks a lot!!!
>
>
> best regards
>
> shijun
>
>
>
> --
>
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Re: [ccp4bb] Please share your experience about "ugly" crystals showing good diffraction

[Artem Evdokimov]

Good evening,

Over the years I've seen great diffraction from any number of hideous
brittle plates with perforated edges, 'Krypton skyscrapers', curved
whiskers, 'hypodermic needles', even from crystals that looked like cat
vomit (hairballs), and so forth.

The structure *ab ovo* story is my favorite, though:

Similar to Debanu's case - we've had crystals (of a novel insecticidal
toxin) that were perfectly egg-shaped, and every one of them was the same
shape - regardless of differences in their size. I mounted them on a bet
with my colleagues and we were cracking all kinds of jokes about making
omelets with X-rays, and so forth - right until the very first egg
diffracted better than 1.6A and produced a lovely structure.

On top of this bit of weirdness, the only way these crystals could be grown
was by reductive methylation followed by proteolysis. Opposite order of
steps did not work, and each step in itself did not work.

This story brings to mind the hellish experience of polishing small
molecule crystals down to little balls in order to minimize absorption
effects (great, now I made myself feel old).

Artem




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On Thu, Jun 28, 2018 at 8:07 PM, Anirban Banerjee  wrote:

>
> Dear all,
>
> Apologies for the non-CCP4 related question.
>
> If you have concrete experience about visually unappealing, i.e. ugly
> crystals ( your own take on ugly is fine)  diffracting better than
> comparably similar sized nicer looking crystals of the same protein, will
> you please share here ? Even better if that led to a published structure.
> Might you also have pictures ?
>
> We have all heard anecdotes about not using visual appearance to judge the
> quality of crystals as far as their ability to give good diffraction data
> is concerned but I am trying to gather some concrete pointers here to
> motivate trainees.
>
> Thanks very much for any help.
>
> Best,
>
> Anirban
>
> P.S. I know that there is probably a lot of thought and wisdom  on this
> this specific topic but I am really looking for actual experience.
>
> --
>
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Re: [ccp4bb] suggestion of crystallization optimization

[Artem Evdokimov]

Janet, Patrick, and others beat me to it :)

We have tremendous luck with MMS in cases like this.

I would also suggest looking at 'atypical' additives - the word atypical is
really not a good choice since the world of additives is pretty much
infinite, but folks too often tend to take the lazy way out and use the few
pre-compiled libraries of 'typical additives' that are available
commercially.

Not to mention that in cases like this a lot of improvement can be achieved
by exploring protein concentration as a factor (not the same as drop ratio,
that's also a lazy way out but it's not the same) and by switching buffers
and salts to other buffers and salts (at the same pH even!).

Crystals, protein crystals, with 8A diffraction - you're 65% done. The last
35% can take a year :)


Artem

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On Tue, Jun 5, 2018 at 12:58 PM, Patrick Shaw Stewart  wrote:

>
> Hi Liuqing Chen
>
> You have a lot of good suggestions, but everyone except for Janet has
> missed out the most important suggestion, and Janet has called it something
> funny - cross seeding often refers to something else.
>
> You may well have a nucleation problem - that's to say many of your
> screening experiments may be in the metastable zone of your protein's phase
> diagram.
>
> Try making a seedstock with your existing crystals and adding it to *random
> *screens.  This can be combined with Janet's suggestions 2 (second part),
> 3, 4, 5, 7, 6 again, and 8.
>
> For more information google MMS crystallization, or rMMS crystallization.
>
> I hope it works - it very often does!
>
> Good luck,
>
> Patrick
>
>
>
>
>
> On 4 June 2018 at 21:41, Janet Newman  wrote:
>
>> Liuqing Chen,
>>
>> Everything that has been said seems reasonable, but there are always
>> infinite possibilities in crystallisation, so it is more a question of
>> priorities. Do the easy (or quick) things first. If you have buckets of
>> prepared protein then what you will try first might be different than if
>> you have to go and make your protein from scratch each time you set up
>> crystal trays.
>>
>> 1. If you have crystals from an additive screen or seeding - try putting
>> them in the beam. If you have access to in-plate screening, you can test
>> the crystals without disturbing them, which will give the best idea of
>> their native diffraction. Perhaps one of the ugly crystals diffracts well
>> enough?
>>
>> 2. Try cross seeding - seed one or more initial screen(s) (rather than an
>> optimisation).  Try initial screening with seeding at different
>> temperatures. If you are currently using vapour diffusion, try microbatch.
>> Or vice versa.
>>
>> 3. Try in-situ proteolysis. Add a very small amount of protease to your
>> protein sample (chymotrypsin is traditional) - say 1:10,000 or 1:1000
>> compared to your protein concentration then set up that mixture in initial
>> screens.
>>
>> 4. Is there a ligand/inhibitor/other small molecule that binds? Add that
>> to your protein before crystallisation. Maybe even try this first!
>>
>> 5. Lysine methylation/cysteine modification/other side chain
>> modifications.
>>
>> 6. Try using DSF or some other technique to look at your protein's
>> stability in the formulation it is in. Maybe you can make happier protein
>> by changing the pH, buffer or salt.
>>
>> 7. If you want to be a little more rigorous, take your protein, and a
>> number of different proteases, and do a time-course experiment with each
>> protease (add 1:1000 protease to your protein, then take samples at
>> timepoints - say 0.5 hours, 1 hour, 5 hours and overnight) then run out on
>> a gel (or analyse by MS) and see if you come down to a stable fragment. If
>> you do - then use that protease, and while you are waiting for the
>> crystallisation trials to do their thing, find out what the end points of
>> the proteolysis fragment are, and make that construct.
>>
>> 6. Try a different expression system (different tag, different position
>> of the tag, cleave/don't cleave the tag). If the protein is produced in a
>> eukaryotic system (and is glycosylated) try a different one to get
>> different glycosylation pattern. Try kifunensine treated cells if you are
>> in a mammalian expression system.
>>
>> 8. Try the same protein from other species
>>
>> Janet Newman
>> Principal Scientist / Director, Collaborative Crystallisation Centre (C3)
>> CSIRO Material Science and Engineering
>> 343 Royal Parade
>> 
>> Parkville.  VIC. 3052
>> Australia
>> Tel +613 9662 7326
>> Email janet.new...@csiro.au
>>
>> 
>> From: CCP4 bulletin board  on behalf of Liuqing
>> Chen <519198...@163.com>
>> Sent: 04 June 2018 20:57
>> To: CCP4BB@JISCMAIL.AC.UK
>> Subject: [ccp4bb] suggestion of crystallization optimization
>>
>> Hello everyone!
>>
>> I get a crystal several months ago, but the crystals diffraction very low
>> 

Re: [ccp4bb] size exclusion columns

[Artem Evdokimov]

Xk10/300 packed with superdex of the appropriate pore size is by far the
most economical and robust option :) pack your own and save.

Artem

On Thu, Apr 26, 2018, 12:06 Markus Heckmann  wrote:

> Dear all,
>
> We are looking for a size exclusion chromatography column
> (silica-based) for protein purification prior to a MALS-detector. We
> looked for (www.waters.com BEH-450), Sepax (Unix-C 300) and Phenomex
> (BioZen SEC-3).  Any 'column' tips or recommendations when dealing
> with large proteins (MDa)?
>
> Many thanks
> Markus
>

Re: [ccp4bb] His-6 versus His-10 tag

[Artem Evdokimov]

Option 1: make two constructs and compare :)

Option 2: put the his10 on the n-term of the protein with a cleavage site

In my experience it matters. I had cases where his4 crystallized and his6
did not. I also had the opposite experience.

Artem


On Sep 19, 2017 6:11 AM,  wrote:

Dear BB,



We are planning the production of a protein for crystallization. From
literature, we know that the construct with a 6-histidine tag crystallizes.
However, for other biophysical measurements, we would prefer to have a
10-histidine tag.



Does anyone has experience with His-6 versus His-10 tags in terms of
crystallization success?



Thanks for your help!

Herman

Re: [ccp4bb] Difficult purification with imac columns

[Artem Evdokimov]

Hi

This is a multidimensional problem, just like any other tricky protein
purification.

1) IMAC columns are also ion exchangers (of both polarities!) so make sure
you have adequate salt.
2) IMAC resins can be polysaccharide-based (agarose) and affinity to sugars
can be an issue. Consider adding a 'mock substrate' (basically a high level
of some inexpensive sugar).
3) Even if you cannot use IMAC there are other options, people purify
native proteins in a 'generic' manner all the time :)
4) definitely a case for trying other resins. Especially consider resins
with pentavalent chelation - they have rather different properties from
resins based on tri (IDA) or tetra (NTA) chelators.
5) as others have mentioned, you may have a problem of the protein as such
- can you assess aggregation state before purification (e.g. SEC of
clarified lysate followed by activity assay).

Artem

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On Fri, Sep 15, 2017 at 7:53 AM, Narayanan Ramasubbu <
ramas...@sdm.rutgers.edu> wrote:

> Hi. We are working on a periplasmic protein that breaks naked glycans in
> peptidoglycans. There is truncated structure available but our target is
> the full length protein. The difficulty us that it strongly binds to the
> resin with or without his.tag. Changing the resin to acrylamide did not
> help.
> Has anyone come across similar problem and how was it resolved.
> The pdb structure is the catalytic domain and mussing a region that, in my
> opinion, binds to the resin.
> Thank you in advance
> Sent from my iPhone

Re: [ccp4bb] Primer design

[Artem Evdokimov]

Have to do primer walking in a cdna library first.

Artem

On Jul 24, 2017 7:26 AM, "syed ibrahim" <
048c02cac012-dmarc-requ...@jiscmail.ac.uk> wrote:

Hello All

I am interested in cloning a gene from a plant. I searched the database
only partial sequence is available, ie: for 160 residues only. The full
length of the protein is around 570 residues. I designed forward primer and
I have no clue to design reverse primer.

Any help

Thank you

Syed

Re: [ccp4bb] Unknown Ligand Density

[Artem Evdokimov]

Flying spaghetti monster. Ramen!

Sorry. Could not resist.

Artem
www.harkerbio.com
"...touched by His Noodly Appendage"

On Jun 14, 2017 12:51 PM, "Nick Thomas"  wrote:

Dear CCP4bb,

I am refining a structure and have come across strong electron density for
an unknown ligand (image attached). Would someone be able to identify this
unknown ligand that somehow was co-purified with the protein.


The electron density shape does not match anything included in the
crystallization conditions.


The crystallization condition is
0.1M MES monohydrate, pH 6.5, 1.6 M Magnesium sulphate heptahydrate

Re: [ccp4bb] Rhodamine in FPLC

[Artem Evdokimov]

30% acetic acid solution should wash it out (exercise some care, it's quite
a strong vinegar).

Hypochlorite and peroxide will bleach the stain, but technically speaking
the leftover oxidized species will still be stuck in there, just colorless.

Artem

www.harkerbio.com
"making lots of *really tiny* drops... for fun and profit"

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On Fri, Jun 9, 2017 at 6:21 PM, Tomas Malinauskas <
tomas.malinaus...@gmail.com> wrote:

> Dear Reza,
> I used hydrogen peroxide (~0.1-2%?) to remove rhodamine stuck on
> Superdex columns. "Pink" columns turned "white" after this kind of
> treatment.
> Hope that helps,
> Tomas
>
> On Fri, Jun 9, 2017 at 10:43 PM, Reza Khayat 
> wrote:
> > Hi,
> >
> > Sorry for the non crystallography question. We're using Rhodamine for
> labeling some material that we need to run through our FPLC. It seems that
> the Rhodamine is getting stuck to the PEEK tubing. Anyone has experience
> with successfully removing the Rhodamine from the tubing? Thanks.
> >
> > Best wishes,
> > Reza
> >
> > Reza Khayat, PhD
> > Assistant Professor
> > City College of New York
> > Department of Chemistry
> > New York, NY 10031
>

Re: [ccp4bb] unknown ligand density

[Artem Evdokimov]

Hi

It helps to see the environment of the blob, and to know what the
resolution is - this *looks* like mid-range resolution (like maybe 2.4A)
but because there is no reference in the image the size can be very
misleading. Also, contour levels are good to know. Basically, more
information.

Artem
www.harkerbio.com
"from blobs to ligands, Gungam-style"


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On Tue, Mar 14, 2017 at 2:03 PM, Dr A.A. Jalan  wrote:

> Dear all,
>
> Could anyone suggest possible ligands to explain the density in attached
> images. The crystals were grown in NaNO3, PEG 3350 and cryoprotected with
> Glycerol.
>
> Thank you
>
> Abhishek
>
>

Re: [ccp4bb] How to determine the concentration of biotinylated peptide?

[Artem Evdokimov]

Hi,

In addition to HABA dye assay (which will work great but will also be
fooled by any biotin that is not conjugated) you can do:

* quantitative MS
* TLC
* HPLC
* elemental analysis
* https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3614710/ biotin catalysis of
the N3- + I3- reaction (also fooled by free biotin of course)
* UV (but beware, biotin only absorbs strongly below 240nm so you're not
super well off there

Artem
www.harkerbio.com
"all of our Biotin comes only from free-range gummy vitamin bears..."

- Cosmic Cats approve of this message

On Mon, Feb 6, 2017 at 2:03 AM, Debasish Kumar Ghosh 
wrote:

> Hi Alex,
>
> In addition to Mirella's suggestion I would like to make an addition which
> might be specifically useful for you. Since your peptide has biotin tag,
> You may use HABA dye assay for the exact quatifiation of biotin (and thus
> biotinylated peptide). As far I recall, Thermo scientific provide a kit for
> this assay. The assay is simple and gives accurate results.
>
> Best !!!
>
>
>
> Debasish
>
> CSIR- Senior Research Fellow (PhD Scholar)
> C/o: Dr. Akash Ranjan
> Computational and Functional Genomics Group
> Centre for DNA Fingerprinting and Diagnostics
> Hyderabad, INDIA
>
> Email(s): dkgh...@cdfd.org.in, dgho...@gmail.com
> Telephone: 0091-9088334375 (M), 0091-40-24749396 (Lab)
> Lab URL: http://www.cdfd.org.in/labpages/computational_
> functional_genomics.html
>
>
>
> - Original Message -
> From: Alex Lee 
> To: CCP4BB@JISCMAIL.AC.UK
> Sent: Mon, 06 Feb 2017 03:02:07 +0530 (IST)
> Subject: [ccp4bb] How to determine the concentration of biotinylated
> peptide?
>
> Dear All,
>
> Sorry for the off-topic question, I'd like to do Biacore SPR assay with
> N-terminal biotinylated peptide as ligand (to Biacore SA chip) and my
> protein as analyte. I have a question of how to determine the concentration
> of biotinylated peptide （synthetic peptide), if the peptide has no Tyr and
> no Trp residue, I guess amino acid analysis may not work because the
> N-terminal of the peptide is biotinylated.
>
> I'd appreciate if anyone share his/her experience on this.
>

Re: [ccp4bb] Unknown blob extended from catalytic serine residue

[Artem Evdokimov]

Hard to see but maybe a transesterified serine like ser-o-AMP

Can you share the nature of the enzyme?

Artem
www.harkerbio.com
"Zoidberg was here"




On Feb 4, 2017 10:14 AM, "sharifah nur hidayah syed mazlan" <
shn.hida...@gmail.com> wrote:

> Dear All,
>
> I am working on a structure with an unknown blob extended from the gamma O
> of the catalytic serine residue. The resolution of the dataset is 1.38 A. I
> have no idea whether the residue is modified or the blob belongs to other
> molecule.
>
> The protein was expressed in Rosettagami (DE3), purified using
> Ni-Sepharose (affinity chromatography) and Q-Sepharose (Anion exchange
> chromatography). The crystallization formulation used contain 15% PEG 8000,
> 0.2 M Ammonium sulphate, 0.1 M sodium cacodylate trihydrate pH 6.5; and the
> buffer composed of 50 mM Sodium chloride and Phosphate buffer pH 8. No
> protease inhibitor was used (eg: PMSF)
>
> I have tried to fit in diethylene glycol as shown in one of the attached
> figures, but as observed, it is not really fit and the molecule is in
> incorrect conformation.
>
> Kindly help me with this matter.
>
>
>
> Thanks and regards,
>
> Sharifah Nur Hidayah
> Universiti Putra Malaysia,
> Malaysia
>
>
>

Re: [ccp4bb] Unknown electron density blob

[Artem Evdokimov]

PEG. It wraps around K or R residues just like you are showing.

Artem
www.harkerbio.com
"where every blob has candy inside"

On Jan 24, 2017 12:19 PM, "Uma Gabale" <
0ebb5dcf3eaa-dmarc-requ...@jiscmail.ac.uk> wrote:

> Dear all,
> While refining a structure at 2.5 A resolution, we observed a
> semi-circular/crescent shaped electron density blob as shown in the
> attached picture. We have been unable to identify it so far, and would
> appreciate any help in identification.
> The protein was expressed in *E. coli* BL21(DE3), purified on Ni-NTA
> followed by gel filtration. The purification buffers included Tris and NaCl
> (no detergent/ other ingredients except for imidazole for Ni-NTA).
> Crystallization condition had HEPES and PEG3350; no cryoprotectant was used.
> The blob is surrounded by residues Trp, Thr, Gln, Arg, and Phe.
> Thanks and regards,
> Uma.
>
> --
> Uma Gabale, PhD
> Research Associate
> Molecular and Cellular Biochemistry
> Indiana University Bloomington
>

Re: [ccp4bb] Off-topic, protein in dye-front (ion front?) on native-PAGE

[Artem Evdokimov]

A) change dye or use pure glycerol to load gel

B) change gel pH or add various substances to it (easy to do with the
buffer)

Artem

On Jan 19, 2017 6:34 AM, "Walt"  wrote:

> Hi,
>
> I have a small protein (~9 kDa) with acidic pI (~4).
> When I run 18% native-PAGE, it appears my protein is in the dye front.
> How can I fix this problem? Changing the pH of separating gel
> might help? How about gradient native-PAGE? Thank you!
>
> Walt
>