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eBIC user programme updatesApril 2021Julika RadeckeEM ScientistSpring Symposium 2021
eBIC InstrumentsKrios I located in the synchrotron ringTitan Krios I-IVScios & Aquilosdual beamcryoFIB/SEM I14/eBIC buildingVolta Phase Plates (VPP)Falcon III DEDCeta CCD cameraK3 post GIF DED (Krios I, II, IV)Falcon 4 Selectris (Krios 3)FFI (Krios II IV)Zone 1Glacios (in collaboration with RCaH) Falcon IV CetaD EPU, TOMO, EPU-D, MAPS,Inspect3DLeica CryoCLEM DAPI/GFP/TexasRed filtercube Multichannel sCMOS camera Thunder software forcomputational clearingTalos Arctica (in-house use, training andMicroED user program) VPPK3 post GIF DEDCetaD CCD camera (microED)EPU, TOMO, MAPS, serialEM, EPU-D (MicroED)
Access to eBIC Rapid access: 48 hrs allocation (now reviewed by beamline staff) Block Allocation Group (BAG) access: Multiple sessions for research group consortia Deadline every 6 months for a 2 year time period (PRP assess every 6 months) Currently 20 BAGs - 400 days/6 months i.e. 80 % of total time allocated over 4Krios. BAGs vary from 5-15 PIs Acceptance criteria: Based on scientific excellence, work should be published iNEXT Discovery provides funded access to European users eBIC has 1000 users (Diamond 14,000)
EMDB & PDB Entries from eBIC UsersStructure 0162017201820192020Number of entries EMDB annuallyNumber of entries PDB annuallyTotal number of entries EMDBTotal number of entries PDB2021Total entriesNumber of entries annually400
Covid-19 related accesseBIC at Diamond is offering free remote access to our CryoEM instruments for SPA, cryoET and cryoFIB/SEM, in addition to arange of synchrotron instruments (see below) to help research aimed at combatting the COVID-19 outbreak. Proposals aresubject to light touch rapid review. This applies equally to users from all countries.The short rapid access proposal can be accessed here: rapid-access.htmlCryoEM: ryo-Imaging/eBIC.htmlThe following synchrotron-based techniques are also providing remote access for COVID-19 based research:Cryo MX beamlines: : Screening.htmlCryo X-ray Microscopy and Cryo Super resolution microscopy (incl CLXM & logical-Cryo-Imaging/B24.htmlBioSAXS B21: ed-Matter/small-angle/B21.htmlCircular Dichroism B23: ed-Matter/B23.html
JasonRishiAdrianaAsfarulAccess to eBIC for Industry – full service offeringSample freezingGrid screening &Data collectionImage processingModel-building &analyses Access to state-of-the-art equipment and expertise Excellent user-, data-, and sample management Flexible access modes: block access, collaborative setup, time access Integration with UK’s national synchrotron facilities Applications: Small molecule structure-based drug designRational vaccine designBiologics characterisation and epitope mappingGene vector and drug delivery system characterisationKriosTM M
eBIC – CapabilitiesGeneral: SPA, Tomo, MicroED, CryoFIB, CryoCLEMTEM Specifics:Imaging AFIS (250-500 movies/hr or 600 movies/hr insuperresolution binning 2 mode) Fringe free imaging on 2 Krioses (AFIS FFI 450650 movies/hr) Tomography data acquisition software: SerialEMfor on-site use or TFS Tomo 5.5 for remote useraccess K3 CDS mode (on Krios 1,2,4) fortomography data acquisition or Falcon 4Selectris (on Krios 3) Maps (Thermo Fisher software) VPPPipeline Relion pipeline for OFT data quality evaluationwith integrated crYOLO particle pickerDonovan Webb, Markus Gerstel, Yuriy Chaban (eBIC), Scheres group (LMB),Colin Palmer (CCPEM) Batches of particles, run up to 2d/3d classaverages Users can monitor progress via NoMachineRemote control EPU session set up remotely, requires trainedEPU or super-user Coming soon: remote tomography session setupvia TFS Tomo 5.5
Chameleon: Diamond, RFI and STP labtech collaboration Specs Piezo inkjet dispenser Blot-free nanowire technology Plunge speed as fast as 54 ms Installed: November 2020 Commissioning call open 2-day visit: Chameleon session coupled to time on screening microscope Rapid proposal including preliminary data Information on how to apply on eBIC website Contact Daniel Clare (daniel.clare@diamond.ac.uk) or Miriam Weckener(miriam.weckener@rfi.ac.uk) for more information
Cryo FIB-Milling User Programme User program for on-grid lamella preparation of frozen cells andmicrocrystals Rapid access only: Multiple single day sessions Multiple grid shipments Lamellae stored at eBIC Tomography at eBIC via BAG or rapid access or at anotherinstitution (autoloader required) Reviewed and scheduled by eBIC staff Contact James Gilchrist (james.gilchrist@diamond.ac.uk) for moreinformation and if you would like advice on grid preparationTo apply you will need: Two good grids per day of milling TEM atlases showing grids have thin vitreous ice, intact supportfilm and enough milling targetscryoFIB guidance document on the eBIC websiteThe Scios The Aquilos James
MicroED User ProgrammeMicroED of a small molecule Test data acquisition – paracetamolCetaD detector (with beam stop)Continuous rotation200nm thick Commissioning call in summer 2020 4 proposals accepted Proposals cover small molecules, smallprotein crystals and protein crystallamella MicroED proposal can be combined witha cryoFIB proposal Contact Yun Song(yun.song@diamond.ac.uk) for moreinformation about MicroED
MicroED User ProgrammeMicroED of proteinase K crystal lamella200nm thick200nm thick5 µm5 µmCetaD10 µm10 µmEmma Beale (DLS)Bart Buijsse (Thermo Fisher)Corey Hecksel (former eBIC)Jason Van Rooyen (eB4I)David Waterman (DIALS/CCP4)(Unpublished)More time available this year!Collaborative projects welcome
Leica CryoCLEM (commissioning phase)Leica CryoCLEME.Coli(bacterial chemotaxis)Zhengyi Yang (DLS)U2OS cellsMaud Dumoux (RFI) Halogen lamp LN2 cooled/RT specimen Auto/unclipped grids (2 samples) DAPI/GFP/TexasRed filter cube Multichannel sCMOS camera Data saved in format that can be read by SerialEM and MapsCLEM Correlative Light and Electron Microscopy
Leica CryoCLEM (commissioning phase)Red: Synechocystis sp. PCC 6803Green: e.coli with MCJD transporterCraig MacGregor-ChatwinLook out for a commissioning call!CLEM Correlative Light Electron Microscopy
Fringe Free on Krios IVMagnification(x)Approximate FringeFree Illumination Size(nm)Shots per hole(R2/2 grid)Post FFI pixel size(Å/px)64k1120 nm31.3681k850 nm51.06105k650 nn70.831130k490 nm130.652 FFI allows illuminated areas 40% smaller More shotsper hole Tomography benefits: more positions can be setupclose by more output on lamella FFI has had no effect on the calibrated magnification* Reducing wait times to the minimum after image and stage shiftPeter Harrison & Dan ClareApoF130kx magnification13 shots per hole (R2/2)500 movies per hour (800 movies*)3083 movies, 390568 particlesCurrent resolution 2 Å
Training at eBICTraining Coordinator: Kyle MorrisCourses (Subject to Covid progression): CryoEM sample prep courses (advanced) – planning CryoEM Sample prep workshops (4 courses so far),planed 2 courses/year CryoET workshop – planning Oxford DTP course 26.-29.4.1st MicroED workshop (Nov 2019) Instruct course 10.-14.5. EPU user training:Remote sessions offer users the opportunity to: Collect data in EPU (on-site/remotely) Operate the eBIC Krios (on-site/remotely) Exchange grids via EPU Load more than 4 pre-clipped gridsSuper-user training (Subject to Covid progression): Change grids during a data collection sessionand image multiple samples during a datacollection session (very useful as faster datacollection schemes and cameras become available) Super-users have more ownership of their session We strongly advocate participation insessions especially if you operated similarmicroscopes in your home institution There are different levels of super-user training
Krios User Programme Krios time has been separated into three sessions over the week Mon. 9am–Wed. 9am(48 hrs) Wed. 9am–Fri. 9am(48 hrs) Fri. 9am–Mon. 9am(72 hrs)Cryo-cycle every 3rd Sunday Out of hours call out service - support via Experimental Hall Coordinators: For problemsoutside 9am-5pm (UK time) during the week when the beamline is on, and on weekends(7am-7pm) at 01235 778787 K3/Falcon4 FFI AFIS about 15000 movies/day best used by setting up own session Sessions may be monitored remotely by users with OTF processing enabling assessment andcollection strategy modification if required Remote tomography data collections with TFS Tomo in pilot phase
Remote access to eBIC microscopes Remote connection to support PC viaNoMachine Teamviewer connection from supportto TEM PC Access is via FedID and credentials areonly valid for the duration of bookedsession via Diamond UAS Session setup via TFS EPU software Protocol on eBIC website (& inforthcoming JoVE paper)
Documentlink hereInstructions for remoteaccess and session setup gical-CryoImaging/eBIC.html
Krios User Programme Challenges How do we meet the increasing demands of operating the microscopes for users as detectorsbecome faster Increase in data production Communication with users before and after sessionsWhat can you do to help? Take ownership of your session and be organisedAsk to take steps towards super-user status, let your LC know what you can doIf shipping samples use ISPyBComplete the UAS ERA 2-3 weeks before your sessionBring optimised samples/pre-screened gridsClip your grids before your sessionLet your local contact know what conditions you want to use a week before your sessionMonitor your data collection!
A community based training programme forcryo-electron microscopy Our 5 year program will host training courses in cryoEM samplepreparation and microscopy, hosted at: Astbury Biostructure Laboratory (University of Leeds) eBIC (Diamond Light Source) Scottish Centre for Macromolecular Imaging (University ofGlasgow) Midlands Regional Cryo-EM Facility (University of Leicester) A range of courses ( 5 per year) will be targeted at: Beginner and more experienced users To specific techniques (such as cryoET) Announcements will be made on CCPEM and 3DEM mailing lists in duecourse, but any queries in the meantime contact Rebecca Thompson(r.f.thompson@leeds.ac.uk)
eBIC Computer SystemAdministratorAnnaRackleyData erStudentsEmmaCowanCraig kePeterHarrisonFrederickHawkinsAlistairSiebertYun SongYuewenShengAnnaHorstmannDiamond Support FIB-SEMScientistsEHCsGoods HandlingUser OfficeScientific ComputingSHEeB4I Team Jason van RooyenRishi MatadeenAdriana KlyszejkoAsfarul yMateuszOlek
AcknowledgementseBIC teamScientific computing teamUser officeBuilding and laboratory supportEHCsUAS teamGoods HandlingSHE groupCommunications teamAll usersFormer eBIC membersWe’re here
Thanks for your attention!
microscopes in your home institution There are different levels of super-user training Remote sessions offer users the opportunity to: Operate the eBIC Krios (on-site/remotely) Load more than 4 pre-clipped grids Change grids during a data collection session and image multiple samples during a data
