[ccp4bb] Fwd: Re: [ccp4bb] phase behavior (slightly off-topic)

[Aleksandar Bijelic]

Dear Eugene,

the additive is a metal-polyanion with a net charge of 6- ... yes, NaCl 
affects largely the solubility of the anions, however, the LLPS appears 
within a rather large protein concentration range (10-50 mg/ml = 0.7 - 
3.5 mM) in the presence of e.g. 0.1, 0.5, 1.0, 2.5 and 5.0 mM additive 
but only at NaCl conc = 0.25 M. I will set up conditions with higher 
additive conc to check if the LLPS-region changes with the amount of 
additive.


Am 09.12.2018 um 14:23 schrieb Eugene Osipov:

Hi, Alex,
you did not mention exactly what kind of addictive you use. I suggest 
that amorphous precipitation is due to this addictive as proteins more 
likely to precipitate in presence of polyvalent ions.
May I suggest that sodium chloride could affect solubility of you 
anions and thus zone with higher protein solubility-lower addictive 
contentration are responsible for observed LLPS


сб, 8 дек. 2018 г. в 14:52, Aleksandar Bijelic 
>:


Dear CCP4 Community,

First of all, I want to aplogize in advance for this more or less
off-topic request. I am currently investigating the phase behavior of
Lysozyme (HEWL) in the presence of NaCl and an anionic metal cluster
(additive) using the microbatch under oil technique. Before the
experiment I expected that the additive will might lead to a shift of
the phase boundaries in comaprison to the HEWL-NaCl system, or
maybe to
an increase of the phase space, where nucleation or even crystals
occur.
Unfortunately, the HEWL-NaCl-cluster-system did not exhibit a
textbook-example of a phase diagram as at almost every condition
(different protein, salt and cluster conc.) an amorphous
precipitation
was immediately formed, which in most of the cases became crystalline
within 1-5 days (mostly shower of needles, spherulites and sea
urchins
and sometimes crystals). The transformation from amorphous to
crystalline precipitate was accompanied by liquid-liquid-phase
separation (LLPS), i.e. the amorphous precipitates dissovled
within 1-2
days and LLPS was observed before the crystalline precipitate was
formed. The odd thing is that LLPS was always observed at the same
NaCl
concentration (0.25-0.35 M, but mostly 0.25 M) independent of the
protein or cluster concentration. At the beginning I thought that
I was
located at the edge of a very narrow LLPS-region, however, testing at
higher protein conc. did not change or shift the LLPS conditions
as in
the range of 10-50 mg/ml HEWL (0-50 mg/ml was investigated) and
independet of the cluster conc. (0.1 - 5.0 mM), the LLPS occured
always
at 0.25-0.35 M NaCl. As I am far away from being an expert in protein
phase behavior, I cannot explain this "magical" salt conc. that
induces
at every tested protein and cluster conc. LLPS. Thus, I hope that
somebody of you might have observed the same or a similar behavior
and
is able to explain this to me. Thanks in advance!

Regards,

Aleks



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



--
Eugene Osipov
Junior Research Scientist
Laboratory of Enzyme Engineering
Research Center of Biotechnology


--
---

Dr. Aleksandar Bijelic

Institut für Biophysikalische Chemie
Universität Wien
Althanstrasse 14
A-1090 Wien

Tel: +43 1 4277 52533
e-Mail:aleksandar.bije...@univie.ac.at






To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Adding Zinc to Protein

[Raghurama Hegde]

Hi Nicola, 

If all you are looking for is evidence that you have zinc in your structure 
based on the anomalous difference map, then with the data you already have you 
should be able to calculate the anomalous difference map! All you have to do is 
to reprocess the data in anomalous mode or whatever your favourite data 
processing software calls it. That will process the data with Friedel mates 
kept separate and you can get anomalous differences from them. If you have 
collected the data away from the absorption edge of zinc you should still be 
able to get anomalous differences though they might be small. 

What wavelength was used for the data collection? 

HTH 
Raghu

-Original Message-
From: CCP4 bulletin board  On Behalf Of Nicola Evans
Sent: Monday, December 10, 2018 03:02
To: CCP4BB@JISCMAIL.AC.UK
Subject: [ccp4bb] Adding Zinc to Protein

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!



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Adding Zinc to Protein

[Olga Moroz]

Hi Nicola,

One way to do it is to dilute your protein, 10-100 times, and add zinc (also 
diluted), then concentrate.
Here is the procedure we used some time ago for a zinc-binding protein:

“S100A12 was diluted to 0.1 mg/ml-1 (approximately 10 mM) in a buffer 
containing 20 mM Tris-HCl pH 7.5, 200 mM NaCl and 10 mM zinc-acetate and 
concentrated to 10 mg/ml by ultrafiltration with 10 kDa cutoff membrane (Amicon 
Ultra 15, Millipore; Vivaspin 500ul, Sartorius Stedim Biotech). The procedure 
was repeated three times to achieve complete saturation with zinc while 
avoiding aggregation due to higher zinc concentrations”.

It worked, and we got a zinc complex :)

Good luck,

Olga


> On 9 Dec 2018, at 21:31, 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!
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

Re: [ccp4bb] Adding Zinc to Protein

[Sheena McGowan]

Hi Nicola,

We have had success simply soaking zinc into the crystal prior to data 
collection. This has worked very well for a number of proteins. We simply add 
some zinc to the cryo-protectant and leave it to soak for various times.

Hope this helps.

Kind regards
Sheena







Sheena McGowan
Head, Structural Microbiology Laboratory
Monash Biomedicine Discovery Institute and Department of Microbiology
Adjunct Senior Research Fellow | Monash Institute of Pharmaceutical Sciences
Monash University 
Rm 137, Building 76, Wellington Rd, Clayton
Victoria, AUSTRALIA 3800
T + 61 3 9902 9309 | M +61419399454
E sheena.mcgo...@monash.edu  | Skype s.mcgowan

Dept webpage  | 
Bio/Publications/Projects 

 | Lab Facebook Page  | 
ORCID  | Google Scholar 
 |

Lorne Conference for Protein Structure and Function 
10th - 14th February, 2019

I acknowledge the Traditional Owners and Custodians of the lands on which I 
live and work and pay my respects to Elders past, present and future.

> On 10 Dec 2018, at 8:31 am, 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!
> 
> 
> 
> To unsubscribe from the CCP4BB list, click the following link:
> https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1




To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1

[ccp4bb] Adding Zinc to Protein

[Nicola Evans]

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!



To unsubscribe from the CCP4BB list, click the following link:
https://www.jiscmail.ac.uk/cgi-bin/webadmin?SUBED1=CCP4BB=1