Dear Mohit,
I would also try, if possible, the wizard screens and at least one of
the morpheus screens, preferably the Fusion one.
Good luck!
D
On 30/06/2025 13:04, ΕΜΜΑΝΟΥΗΛ ΣΑΡΕΙΔΑΚΗΣ wrote:
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Dear Mohit,
It seems to me that the first steps of your investigation were
performed as they should and in the right order. It is however not
infrequent for the usual conditions' fine-tuning and standard
macro/microseeding to fail to improve on the original hits. So the
question is, where do you take it from there? Two further well-known
optimisation strategies are: (1) using temperature as an additional
optimisation parameter, and (2) random microseed matrix screening,
about which you should search for Patrick Shaw Stewart and Douglas
Instruments. If these fail or at any rate do not take you where you'd
like, I can recommend two other strategies which are detailed in the
following papers:
https://doi.org/10.1016/S0006-3495(03)74936-4
<https://doi.org/10.1016/S0006-3495(03)74936-4> (using phase diagrams
to uncouple the nucleation and growth stages of crystallisation by
diluting the reservoir during incubation - it can also work by
shifting temperature during incubation, or by diluting the
crystallisation mixture itself if working in batch/microbatch mode);
https://doi.org/10.1073/pnas.0504860102 (using porous nucleants,
especially Bioglass, marketed under the name "Naomi's Nucleants", in
order to nucleate crystals at lower supersaturations, where these
crystals may grow with fewer defects)
Please keep us posted if any of these worked, more information should
go around about such things!
Good luck,
Emmanuel
Principal Researcher, Institute of Nanoscience & Nanotechnology
National Centre for Scientific Research "DEMOKRITOS"
Ag. Paraskevi, Athens 15341, Greece
------------------------------------------------------------------------
*From: *Mohit <[email protected]>
*To: *CCP4BB <[email protected]>
*Date: *Monday, 30 June 2025 11:53 AM EEST
*Subject: *[ccp4bb] Crystallization of SARS-CoV-2 3CLpro and
Mutants – Seeking Advice on Crystal Optimization
Dear members,
I am currently working on the crystallization of the main protease
(3CLpro) of SARS-CoV-2, focusing on both the wild-type and several
point mutants.
Initial crystallization trials were conducted using commercial
screens, including PACT, JCSG+, and INDEX. These trials yielded
crystals of various morphologies—predominantly needles, thin
sheets, and flower-like clusters. To improve crystal quality, I
subsequently performed optimization using the hanging-drop vapor
diffusion method by systematically varying PEG concentration, salt
types/concentrations, and pH conditions. Despite these efforts,
the crystals have not shown significant improvement in morphology
or diffraction quality.
I have also attempted microseeding and macroseeding approaches
during optimization, but the results remain largely the same. I
would greatly appreciate any suggestions or experiences from the
community regarding strategies that could help improve crystal
quality in such systems, particularly with challenging
morphologies like needle or sheet-like forms.
Thank you in advance for your insights.
Mohit Bhardwaj
PhD Scholar
Kusuma School of Biological Sciences
IIT Delhi, Hauz Khas
New Delhi- 110016
Mobile No. : +91-8895172936, 8700227218
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