{"id":3157,"date":"2025-12-02T12:18:16","date_gmt":"2025-12-02T03:18:16","guid":{"rendered":"https:\/\/pf-form.kek.jp\/sbrc\/?p=3157"},"modified":"2025-12-02T17:32:46","modified_gmt":"2025-12-02T08:32:46","slug":"seminar29","status":"publish","type":"post","link":"https:\/\/pf-form.kek.jp\/sbrc\/seminar29\/","title":{"rendered":"SBRC International Cryo-EM Seminar No.29"},"content":{"rendered":"\n
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\u201cUsing phase diagrams with microseeding to prepare crystal samples for advanced data collection techniques\u201d<\/h2>\n<\/blockquote>\n\n\n\n

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Date and Time<\/h2>\n\n\n\n

2:00 PM – 3:00 PM Tuesday, December 9th<\/sup>, 2025<\/p>\n\n\n\n

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Location<\/h2>\n\n\n\n

Hybrid\uff08CryoEM building and Zoom\uff09<\/p>\n\n\n\n

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Speakers: Patrick D. Shaw Stewart<\/h2>\n\n\n\n

Douglas Instruments Ltd\uff08UK\uff09
University of Southampton\uff08UK\uff09 <\/strong><\/p>\n\n\n\n

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Abstract<\/h2>\n\n\n\n

Serial data collection and microED techniques typically require \u201cslurries\u201d of tiny, well-ordered crystals [1]. Neutron diffraction requires very large single crystals. Creating samples for these techniques is often a complex process that requires multiple rounds of optimization. To guide them in this task, protein crystallizers often keep a notional phase diagram in mind, which has four zones: an undersaturated zone where protein always remains in solution, a metastable zone where crystals will grow when seeds are added, a crystal nucleation zone where crystals appear spontaneously, and a protein precipitation zone. However, the shape of real-life phase diagrams can vary, making the interpretation of experimental results difficult. It is therefore very helpful to determine the phase diagrams of individual target proteins experimentally. Douglas Instruments, in collaboration with the University of Southampton, has introduced a rapid and straightforward method for generating custom phase diagrams using just 15 \u2013 60 \u00b5L of protein [Fig. 1]. The most straightforward approach utilizes the microbatch-under-oil method to prevent concentrating the sample drop (as would occur in a vapor diffusion setup). By carrying out the same procedure with and without a seedstock, the metastable zone can be identified [2]. Moreover, advanced methods often require relatively large sample volumes, and microbatch can easily be scaled up to 50 \u00b5L or larger \u201cbatches\u201d using robotics. A new variation of the method eliminates the need for oil by using a sitting drop setup, where solutions are dispensed to the reservoirs that exactly balance the concentrations of the drops. We present case studies where phase diagrams were utilized to enhance control and crystal quality for both routine and advanced data collection.
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Figure 1.\u00a0 The rapid determination of a protein\u2019s phase diagram using\u3000a microbatch-under-oil format.\u00a0 Blue circles indicate the conditions that were tested.\u00a0 Images of the wells are shown in conditions of interest. All points on the accessible phase diagram can be reached by mixing the three ingredients shown: protein stock (red circle), precipitant or cocktail stock (green circle) and a diluent, normally water (cyan circle).\u00a0 To find the border of the metastable zone (dotted line) the experiment was repeated with the addition of a seed-stock (results not shown).<\/p>\n\n\n\n

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[1] Stubbs, J., Hornsey, T., Hanrahan, L.B. Esteban, R. Bolton, M.
Maly, S. Basu, J. Orlans, D. de Sanctis, J. Shim, Shaw Stewart, P. D.,
A.M. Orville, I. Tews and West, J. (2024). IUCrJ  11.<\/p>\n\n\n\n

[2] D’Arcy, A., Villard, F., Marsh, M.  (2007). Acta Cryst. D63(4):550-4.<\/p>\n\n\n\n

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Click here to apply<\/a><\/h2>\n","protected":false},"excerpt":{"rendered":"

\u201cUsing phase diagrams with microseeding to prepare crystal samples for advanced data collection techniques\u201d _ […]<\/p>\n","protected":false},"author":26,"featured_media":0,"comment_status":"closed","ping_status":"closed","sticky":false,"template":"","format":"standard","meta":{"footnotes":""},"categories":[3],"tags":[],"class_list":["post-3157","post","type-post","status-publish","format-standard","hentry","category-kek-cryoem"],"_links":{"self":[{"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/posts\/3157","targetHints":{"allow":["GET"]}}],"collection":[{"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/posts"}],"about":[{"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/types\/post"}],"author":[{"embeddable":true,"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/users\/26"}],"replies":[{"embeddable":true,"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/comments?post=3157"}],"version-history":[{"count":7,"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/posts\/3157\/revisions"}],"predecessor-version":[{"id":3203,"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/posts\/3157\/revisions\/3203"}],"wp:attachment":[{"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/media?parent=3157"}],"wp:term":[{"taxonomy":"category","embeddable":true,"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/categories?post=3157"},{"taxonomy":"post_tag","embeddable":true,"href":"https:\/\/pf-form.kek.jp\/sbrc\/wp-json\/wp\/v2\/tags?post=3157"}],"curies":[{"name":"wp","href":"https:\/\/api.w.org\/{rel}","templated":true}]}}