Thank you for your e-mail. I am out of office until July 8th, 2025. I will respond to your e-mail as soon as possible upon my return. For urgent matters please contact: [email protected] Thank you for your understanding.
With kind regards, Elisabeth Laurent >>> CCP4BB automatic digest system <[email protected]> 1.7.25 01:02 >>> There are 8 messages totaling 24543 lines in this issue. Topics of the day: 1. Crystallization of SARS-CoV-2 3CLpro and Mutants – Seeking Advice on Crystal Optimization (4) 2. New multi-crystal features in DIALS/xia2 3. Data uncertainty: Minimising it and characterising the uncertainty that remains 4. CBMS Lecture Series - EXTRA - July 2nd Wednesday - Hessel 5. Obituary Prof. Dr. Dr. h.c. Rolf Hilgenfeld (*03.04.1954 – †19.06.2025) ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ---------------------------------------------------------------------- Date: Mon, 30 Jun 2025 12:47:51 +0530 From: Mohit Bhardwaj <[email protected]> Subject: 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 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ------------------------------ Date: Mon, 30 Jun 2025 08:20:47 +0000 From: "Beilsten-Edmands, James (DLSLtd,RAL,LSCI)" <[email protected]> Subject: New multi-crystal features in DIALS/xia2 Dear CCP4BB, We recently released our latest version of DIALS, v3.25.0, and wanted to draw your attention to a few new features for multi-crystal data processing that we think will be of interest to the wider community. As of v3.25.0, it is now possible to process XDS-integrated data with the xia2.multiplex pipeline, or the DIALS data reduction tools, if the data have first been converted with the dials.import_xds tool (special thanks to Clemens Vonrhein who contributed a lot to fixing this tool). See this page for the usage instructions of dials.import_xds: https://dials.github.io/documentation/programs/dials_import_xds.html. xia2intensity-based clustering using cosym coordinates, in addition to the existing hierarchical clustering methods. We described these new methods in a recent publication in Acta D: https://journals.iucr.org/d/issues/2025/06/00/rr5252/index.html. There is also a shorter description of the relevant multiplex program options at https://xia2.github.io/multiplex-multi-crystal.html. Please feel free to get in touch with us with any questions or issues, you can reach us at [email protected]<mailto:dials-user-group%40jiscmail.net>, post an issue on our GitHub repositories or post to our slack channel at dials-support.slack.com<http://dials-support.slack.com>. Best wishes, James Beilsten-Edmands, senior software scientist, Diamond Light Source, On behalf of the DIALS & xia2 developers. This e-mail and any attachments may contain confidential, copyright and or privileged material, and are for the use of the intended addressee only. If you are not the intended addressee or an authorised recipient of the addressee please notify us of receipt by returning the e-mail and do not use, copy, retain, distribute or disclose the information in or attached to the e-mail. Any opinions expressed within this e-mail are those of the individual and not necessarily of Diamond Light Source Ltd. Diamond Light Source Ltd. cannot guarantee that this e-mail or any attachments are free from viruses and we cannot accept liability for any damage which you may sustain as a result of software viruses which may be transmitted in or with the message. Diamond Light Source Limited (company no. 4375679). Registered in England and Wales with its registered office at Diamond House, Harwell Science and Innovation Campus, Didcot, Oxfordshire, OX11 0DE, United Kingdom. ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ------------------------------ Date: Mon, 30 Jun 2025 12:34:53 +0100 From: John R Helliwell <[email protected]> Subject: Data uncertainty: Minimising it and characterising the uncertainty that remains Dear Colleagues, I imagine you will be interested in my post at the IUCr Committee on Data Public Forum on this topic of data uncertainty:- https://forums.iucr.org/viewtopic.php?f=39&t=6464&sid=7966a823f22e445ad1a3ce5f9c42cdca All best wishes, John -- Professor John R Helliwell DSc ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ------------------------------ Date: Mon, 30 Jun 2025 15:04:22 +0300 From: ΕΜΜΑΝΟΥΗΛ ΣΑΡΕΙΔΑΚΗΣ <[email protected]> Subject: Re: Crystallization of SARS-CoV-2 3CLpro and Mutants – Seeking Advice on Crystal Optimization 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 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 -----Original Message----- 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 To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ------------------------------ Date: Mon, 30 Jun 2025 15:30:22 +0100 From: Dom Bellini <[email protected]> Subject: Re: Crystallization of SARS-CoV-2 3CLpro and Mutants – Seeking Advice on Crystal Optimization 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: > CAUTION: This email originated from outside of the LMB: > *[email protected].* > Do not click links or open attachments unless you recognize the sender > and know the content is safe. > If you think this is a phishing email, please forward it to > [email protected] > > > -- > > 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 > questi> 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 > > ------------------------------------------------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> > > > ------------------------------------------------------------------------ > > To unsubscribe from the CCP4BB list, click the following link: > https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 > <https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1> > ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ------------------------------ Date: Subject: CBMS Lecture Series - EXTRA - July 2nd Wednesday - Hessel PLEASE SHARE You are cordially invited to join us ..... "Shining a light on force production and regulation in cardiac and skeletal muscles: Leveraging small-angle X-ray diffraction to accelerate translational applications" Anthony Hessel CEO and Co-Founder Accelerated Muscle Biotechnologies Boston (USA) / Hamburg (DE) JULY 2, 13:30(EDT) To register for hybrid attendance: https://bnl.zoomgov.com/j/1601116839?pwd=M0eLem0A9JulosFXWtoEc0DziqGZlX.1 Abstract: Dr. Anthony Hessel is a muscle physiologist with a diverse background in cardiac and skeletal muscle mechanics, assistive devices like prostheses and exoskeletons, and biophysics. His most recent work has focused on the use of the small-angle X-ray diffraction technique - a powerful approach that uses high-powered X-rays generated from synchrotrons (particle accelerators) to track the movement of sarcomere motor proteins under near-physiological conditions. Through this work, his research has helped define the role of the sarcomere proteins titin and myosin-binding protein C (MyBP-C) to cardiac and skeletal muscle performance. As a second research focus, X-ray diffraction science is a niche field requiring advanced laboratories and expertise to perform. To remove some of these access barriers for the broader field, Dr. Hessel formed a contract research organization, Accelerated Muscle Biotechnologies (Boston) with a focus to expand where muscle X-ray diffraction can be conducted, as well as improve the hardware and software technology to make it useful for basic, translational, and clinical applications. ================= Vivian Stojanoff, PhD Education, Training, Outreach User Program p 1(631) 344 8375 e [email protected]<mailto:[email protected]> w https://www.bnl.gov/ps/lifesciences/<https://www.bnl.gov/ps/lsbr/> Address: Center for Biomolecular Structure National Synchrotron Light Source II Building 745 Brookhaven National Laboratory Upton NY 11973 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/ ------------------------------ Date: Mon, 30 Jun 2025 17:34:51 +0000 From: Edward Snell <[email protected]> Subject: Re: Crystallization of SARS-CoV-2 3CLpro and Mutants – Seeking Advice on Crystal Optimization Hi Mohit, I would agree with the temperature and seeding comments by Emmanuel. Although not proven for diffraction characteristics, volume ratio and temperature can be effective optimizing strategies – see Luft et al, 2007 (https://hwi.buffalo.edu/wp-content/uploads/2016/12/dvrt.pdf). I noticed in looking at a number of models in the PDB that dimethyl sulfoxide (DMSO) seems to be significant in the high-resolution models and does seem to form key contacts in packing diagrams. Although it’s probably there to solubilize some of the ligands tested, it does seem to be ordered in the electron density, bridges chains and probably stabilizes them, and could be a useful component to add to your current conditions. It’s not clear from your comments if you had more than one initial crystallization condition that you optimized, but I’d also recommend optimizing ‘crappy’ crystals that appear in initial screens if they are from chemically distinct conditions. A little optimization can go a long way in going from visually poor to diffraction good crystals. I think you are heading in the right direction, and the crystals you do have are beautiful. I presume you have tried a microfocus beamline, but if not, that is also a good approach. Best ofDirector | NSF BioXFEL Science and Technology Center Fellow of the American Crystallographic Society – The Structural Science Society p: +1 716 898 8631 | f: +1 716 898 8660 e: esnell@ buffalo.edu<mailto:[email protected]> The University at Buffalo Hauptman-Woodward Institute 700 Ellicott Street | Buffalo, NY 14203-1102 Website: https://snelllab.website/ [hwi-logo-primary-horizontal] From: CCP4 bulletin board <[email protected]> On Behalf Of Mohit Bhardwaj Sent: Monday, June 30, 2025 3:18 AM To: [email protected] 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 c Warning! This message was sent from outside your organization and we were unable to verify the sender. sophospsmartbannerend 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 ________________________________ To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyands ------------------------------ Date: Mon, 30 Jun 2025 18:58:06 +0000 From: Jeroen Mesters <[email protected]> Subject: Obituary Prof. Dr. Dr. h.c. Rolf Hilgenfeld (*03.04.1954 – †19.06.2025) To whom it may concern It is with great sadness and a heavy heart that we must inform former students, postdocs and colleagues worldwide of the death of Rolf Hilgenfeld at the age of 71. He was a distinguished structural biologist at the Universities of Jena (1995-2002) and Lübeck (2003-2020), where he continued his research as a senior professor. He started his scientific career as a group leader at Hoechst AG (1986-1995), where he was actively involved in the development of long-acting insulin (Lantus®). One of his 3D protease structures served as a model for the bronze sculpture SARS Inhibited in the central square of the Biopolis Science City in Singapore (2006). In 2023, he was awarded the prestigious Carl-Hermann-Medal by the German Society for Crystallography for his life's work. Our condolences go out to his family, close friends and long-time companions. Haifa EL Kilani and Jeroen Mesters (current and former deputy to Rolf) ################################################https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available ------------------------------ End of CCP4BB Digest - 29 Jun 2025 to 30 Jun 2025 (#2025-167) ************************************************************* ######################################################################## To unsubscribe from the CCP4BB list, click the following link: https://www.jiscmail.ac.uk/cgi-bin/WA-JISC.exe?SUBED1=CCP4BB&A=1 This message was issued to members of www.jiscmail.ac.uk/CCP4BB, a mailing list hosted by www.jiscmail.ac.uk, terms & conditions are available at https://www.jiscmail.ac.uk/policyandsecurity/
