cryo-electron microscopy at leeds approaches and ... · induction - service lab 001 induction -...
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Cryo-electron microscopy at Leeds
Approaches and challenges for high resolution data acquisition
Dr Rebecca Thompson
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Officially opened January 2017..
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Transmission electron microscopes• JEOL 1400- Negative stain/ambient sections
• FEI T12- Negative stain/ambient sections
• FEI F20- Negative stain/ambient sections/ cryo-EM screening/tomography
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Sample preparation of macromolecular complexes
• Leica EM-GP- adherent cells/macromolecular complexes
• Vitrobot Mk 4- macromolecular complexes
• Housed in < 20 % RH sample preparation room
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Cryo-EM data collection Spraying approaches and time resolved EM
Stephen Muench, Howard White
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Sample preparation of cells, tissues and organisms
• New this year!
• Leica EM ICE High pressure freezer- for vitrification of cells, tissues and organisms like C. Elegans
• Leica AFS2 for freeze substitution of high pressure frozen specimens
• Leica UC7 cryo/ultramicrotome for sectioning of ambient/Tokyasu/vitreous (CEMOVIS) samples
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Glow discharge
Quorum GloQube Cressington 208 PELCO easiGlow
Considering purchase of a plasma cleaner- opinions welcome!
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Cryo-Fluorescence
Leica CryoCLEM system Linkam cryo fluorescence stage (Can be fitted onto Zeiss LSM 880 with Airyscan)
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Facility operates at BSL2
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Rebecca ThompsonFacility Manager/ Senior cryo-EM scientist
Dan Maskell Cryo-EM support scientistTraining Lead
Emma HeskethCryo-EM support scientistIT lead
Martin FullerEM technicianJEOL1400/T12 and Ultramicrotomy lead
The Team
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The User Base
• 134 ‘active’ users from across the University
• ~75 % internal users Faculty Biological Sciences, with remainder from Chemistry, Physics, Medicine, Food Science, Engineering/Materials Science, and external collaborators.
• ~ 25-30 use cryo-EM as primary tool of research
• On Titan Krioses, ~ 20 % available time used by external service users
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External Users
• Mix of industry and academia
• Shortly to offer access through Instruct-ERIC
• ~ 75 % attend site for collection (repeat customers most likely not to attend
• Remote customers can see files for inspection during collection, other feedback done via skype and email. Plans to implement remove viewing.
• Do not offer remote control (and no plans for users)
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Equipment Access
• Managed through Instruct’s ‘Aria’ booking system
• Titan Krios bookings managed by facility staff
• Balance of internal/external, projects and unfunded currently managed by Facility staff with oversight from Director
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Standard Single Particle Pipelines
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Preferred orientation at
air water interface
Affinity for the support
Well distributed
Thin ice in center of hole
pushing particles to
the hole edge
• Sample optimization and screening is a massive bottleneck
• We end up using Krioses for (most) screening
• Aim to make this as efficient as possible-2/3 loads per screening day (22-33 grids) plus ‘overnight’ short runs for borderline projects
Drulyte et al 2018
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Data collection
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E Plubirus Paucioribus?(Out of Many, Fewer)
Sarkar et al, NSBM 2017
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Data collection- Facility staff
• How can we as Support Scientists provide best possible service?
• Monitoring of equipment and environment (26 parameters measured and alarmed)
• Work to an SOP (but when providing service will deviate if requested)
• Checklists
• How can we best monitor performance of the microscopes over time?
• Periodic collection of ‘standard’ samples?
• How do you (continually) train cryo-EM support staff?
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H&S Induction(1h)
Negative stain/T12(~7h)
F20 training (~2 h ambient, 20 h cryo)
~ 20 h experience
Cryo grid prep (3 h)
Titan Kriosscreening training (8-12 h, 1-1)
Titan KriosEPU training (8-12 h 1-1)
Training ModuleModule
CodeInductions 000Induction - Service lab 001Induction - Cryo lab 002
Grid Prep - Stain 100Carbon coating "Collodion method" 101aCarbon coating "floating" 101bGlow discharge Cresington 102aGlow discharge Ted Pella 102bGlow discharge GloCube 102cNegative staining 103
Grid prep - cryo 200Vitrobot 201aLeica GP 201b
Microscope Training 300JEOL 301T12 - beginners 302T12 - advanced 303F20 304Krios 1 - EPU 305aKrios 2 - EPU 305bKrios 1 - Advanced users 306aKrios 2 - Advanced users 306b
Computing 400Starter pack 401Basic Unix commands 401aGeneral computing guide 401bRelion starters guide 401ccryo sparc 401dUsing Arc3 401eWorking from home 401fAccess screening data - Krios 401gOTF - internal 402
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Data collection- internal users
Total dose?Dose per frame?Which detector?
Mag?
How many images will I
get?
Acquisitions per hole?
Phase plate? How many do I need?
Just HOW do I do it?
Detector mode?
Energyfilter?
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Training ModuleModule
CodeInductions 000Induction - Service lab 001Induction - Cryo lab 002
Grid Prep - Stain 100Carbon coating "Collodion method" 101aCarbon coating "floating" 101bGlow discharge Cresington 102aGlow discharge Ted Pella 102bGlow discharge GloCube 102cNegative staining 103
Grid prep - cryo 200Vitrobot 201aLeica GP 201b
Microscope Training 300JEOL 301T12 - beginners 302T12 - advanced 303F20 304Krios 1 - EPU 305aKrios 2 - EPU 305bKrios 1 - Advanced users 306aKrios 2 - Advanced users 306b
Computing 400Starter pack 401Basic Unix commands 401aGeneral computing guide 401bRelion starters guide 401ccryo sparc 401dUsing Arc3 401eWorking from home 401fAccess screening data - Krios 401gOTF - internal 402
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• Teaching aid
• Resource for external users
• Improves reproducibility
• Reduces sessions ’lost’ for preventable reasons
• Helps with trouble shooting
• Users require less expert knowledge- but still need to understand why they are making choices!
• Reduced appetite for becoming a cryo-EM ‘expert’- learning on a ‘need to know’ basis
In Press Nature Protocols
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• User data sheet provided for every session
• Improves tracking of parameters
• Reduces users errors during processing
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Detector choice
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Which Detector?
• Internal users split pretty evenly across F3EC and K2
• F3EC used mostly in integrating mode
• Tomography/sub ~120-150 kDa single particle projects K2
• Internal users never really use K2 super resolution
• External users preference for K2 counting
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• 48 h data collection• 5619 Micrographs (117/hour)• Autopicked ~300,000• 280,000 particles in final
reconstruction
Emma Hesketh
2.6 Å structure from Falcon III in integrating mode
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Falcon 3 in integrating mode• 200 kDa soluble protein
• 4267 micrographs
• Autopicked 1.6 million particles
• 110 e/A2 so you can see the particles!
• 3.5 Å structure
Ieva Drulyte
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Dealing with the Data Deluge
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‘Current’ OTF image processing
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Assessing Micrograph Quality
Bad dataset ‘Requires investigation’ dataset
Matt Iadanza
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Outlook• Efficient screening continues to be a challenge- Focused screening days and
overnight collections for ‘borderline’ projects. New hardware may help..
• Training and democratization of knowledge- how can we share best practice?
• Single OTF processing changing rapidly- challenge to constantly adapt systems
• Expect tomography work to increase- implementing OTF pipelines for assessing data (especially PP)
• Implementation of other software for routine collection (SerialEM) of tomography data, not just dose-symmetric schemes.
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Acknowledgements
Neil Ranson
Emma HeskethMartin FullerDan Maskell
Matt IadanzaShaun Rawson
Ieva Drylute
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Thank you for your attentionAny questions?
@AstburyBSL @Bex_16