Hi Mohit,
I would also recommend mixing your fresh seeds with fresh protein and redoing
the crystallization screenings. Sometimes a different cryoprotectant may
enhance your resolution.
Best wishes
______________________________________________________
Rafael Marques da Silva
PhD Student – Structural Biology
University of Leicester
Mestre em Física Biomolecular
Universidade de São Paulo
Bacharel em Ciências Biológicas
Universidade Federal de São Carlos
phone: +44 07861 273773
"A sorte acompanha uma mente bem treinada"
________________________________________________
________________________________
De: CCP4 bulletin board <CCP4BB@JISCMAIL.AC.UK> em nome de Eric Girard
<eric.gir...@ibs.fr>
Enviado: quarta-feira, 2 de julho de 2025 11:40
Para: CCP4BB@JISCMAIL.AC.UK <CCP4BB@JISCMAIL.AC.UK>
Assunto: Re: [ccp4bb] Crystallization of SARS-CoV-2 3CLpro and Mutants –
Seeking Advice on Crystal Optimization
Dear Mohit,
In addition to all the useful suggestions already mentioned, you might be
interested in testing crystallophore, a nucleating agent, which acts as a
molecular glue to provide new crystallization conditions or improve existing
ones.
See https://doi.org/10.1039/C7SC00758B and
https://doi.org/10.1107/S1600576719006381
The crystallophore can be purchased from Mitegen/Molecular Dimensions/Polyvalan.
Best of luck,
Eric.
Le 30/06/2025 à 09:17, Mohit Bhardwaj a écrit :
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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Eric Girard
Extremophiles and Large Macromolecular Assemblies (ELMA) group
&
Synchrotron group
Institut de Biologie Structurale
UMR 5075 CEA-CNRS-UGA-PSB
71 avenue des Martyrs
CS 10090
38044 Grenoble Cedex 9
Phone: +33 (0)4 57 42 86 45
Fax: +33 (0)4 76 50 18 90
Web site: http://www.ibs.fr/
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