Hello Interested Researchers, Mark your calendars and register for our next NIH sponsored Joint CryoEM Service Centers Webinar Series “Cryo-EM single-particle analysis of small and dynamic proteins” by Dr. Jennifer Cash on Thursday July 30th, at 12pm (Eastern Time) (see attachment or below for details). Registration is at no-cost, but sign-up is required: https://us02web.zoom.us/webinar/register/WN_W94PT1EiQsCU5buniPwemA For more information regarding the National CryoEM Service Centers can be found at https://www.cryoemcenters.org/cryoem-centers/. ~ ~ ~ ~ ~ ~ ~ ~ ~ Text version of flyer can be found below: CryoEM Current Practices Webinar
Cryo-EM single-particle analysis of small and dynamic proteins Jennifer Cash, PhD Assistant Professor Molecular and Cellular Biology, University of California, Davis 12PM EDT / 9AM PDT Thursday, July 30th, 2026 The Rho guanine-nucleotide exchange factor (RhoGEF) PDZ-RhoGEF (PRG) is activated downstream of G protein-coupled receptors, mediates cell migration, and is implicated in invasiveness and metastasis in various cancers. PRG catalyzes guanine-nucleotide exchange on the GTPase RhoA through the catalytic domain tandem consisting of the Dbl homology (DH) and pleckstrin homology (PH) domains, leading to activation of downstream signaling. While this catalytic core is well-studied in RhoGEFs, the roles of accessory domains outside of the catalytic core in regulating its activity are less clear. The objective of this project is to elucidate these mechanisms. Guanine-nucleotide exchange assays show that the basal activity of the PRG catalytic core is relatively low but is enhanced upon addition of an N-terminal, 17-residue extension of the DH domain called the GEF switch (GS). The mechanism of this enhancing effect is largely unknown. While crystal structures of a PRG fragment comprising the GS and DH/PH in complex with RhoA are available, they do not provide any information on how the complex may be different in the absence of the GS. We hypothesize that the GS stabilizes the DH domain to encourage productive interactions with RhoA by modifying hydrogen bonding networks, enhancing PRG activity. To test this, we are purifying the PRG DH/PH catalytic core with and without the GS and using an integrative structural biology approach, including small angle X-ray scattering (SAXS), hydrogen-deuterium exchange mass spectrometry (HDX-MS), and cryo-electron microscopy (cryo-EM) to determine the effect of the GS on the structure of the PRG core in complex with RhoA. We show that addition of the GS does not induce large-scale structural changes in the complex with RhoA. However, HDX-MS reveals that the GS stabilizes both the catalytic DH domain of PRG and the switch regions of RhoA that are implicated in guanine-nucleotide binding. Surprisingly, one of these RhoA regions is distal from the GS. To dissect the molecular details of this mechanism, we are determining cryo-EM structures of the DH/PH–RhoA complex. Preliminary work on our in-house Glacios (without energy filter) allowed us to achieve ~4 Å cryo-EM maps of these complexes (representing ~45 kDa of protein). Using data collected on a Krios (with energy filter), we have achieved sub 3 Å resolution maps from these samples. Altogether, these experiments will provide a structural basis for the intrinsic activity and regulation of PRG and inform the rational design of specific PRG inhibitors to limit metastatic cancer. All are welcome to attend. Registration is at no-cost, but sign-up is required: https://us02web.zoom.us/webinar/register/WN_W94PT1EiQsCU5buniPwemA Thank you, _________________________ Kjirsten Wheeler PNCC Program Administrator Pacific Northwest Cryo-EM Center a PNNL and OHSU affiliation Website<https://urldefense.com/v3/__https:/pncc.labworks.org/__;!!Mi0JBg!burdB2N3-jSi-4APwohcbkMgXzrhpmrr-Q_XadX2I6g6qfUKLs-MOS_CSrl8z8tr$> LinkedIn<https://ohsuitg.sharepoint.com/sites/PROJ.PNCC/Shared%20Documents/General/PNCC_Workshops%20&%20Training/SerialEM%20Screening%20Workshop/pacific-northwest-cryo-em-center> YouTube<https://www.youtube.com/@pacificnorthwestcryoemcenter/videos> [email protected]<mailto:[email protected]> ######################################################################## 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/
