ESTABLISHMENT OF A CRYO-ELECTRON MICROSCOPY LABORATORY UNDER BSL-3 CONDITIONS
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1 ESTABLISHMENT OF A CRYO-ELECTRON MICROSCOPY LABORATORY UNDER BSL-3 CONDITIONS Michael Sherman, Alexander Freiberg, Anne-Sophie Brocard, Dee Zimmerman, Stanley Watowich, Scott Weaver UTMB, Galveston, TX
2 Overview Introduction: - W. M. Keck Center for Virus Imaging - Basics on electron microscopy (EM)/cryo-EM W. M. Keck Center for Virus Imaging: - construction plan - sample freezing and storage, waste handling -cryotransfer - decontamination protocol of microscope Summary First results
3 W. M. Keck Center for Virus Imaging W. M.Keck Center for Virus Imaging = BSL-3 cryo-em lab Cryo-Electron Microscopy Center for Macromolecular Systems (UTMB) Manager: Dr. Michael Sherman mbsherma@utmb.edu Funding: W. M. Keck Foundation, Kleburg Foundation, and UTMB First U.S. laboratory of its kind in a BSL-3 containment environment (approved for select agent work and functional since spring 2008) 3
4 Research program Structural imaging of pathogens presenting a threat to the US and other regions of the world Structural studies: assembly of pathogens development of potential vaccines Research program focuses on RNA viruses: 1) encephalitis (e.g. alphaviruses [VEEV, WEEV] and flaviviruses [WNV, JEV]) 2) hemorrhagic fever (e.g. bunyaviruses [RVFV], arenaviruses [Junin virus], flavivirus [Dengue virus]) 3) acute respiratory disease (e.g. hantaviruses, SARS) 4) influenza (avian influenza) 5) Hepatitis C 4
5 Examples of infectious agents solved by cryo-em Influenza H3N2 Harris et al., PNAS 2006 DENV in complex with the carbohydrate recognition domain of DC-SIGN Pokidysheva et al., Cell 2006 WNV in complex with the Fab fragment of a neutralizing monoclonal antibody Kaufmann et al., PNAS 2006 Herpes simplex virus Grunewald et al., Science
6 Background electron microscopy Magnification: up to x2,000,000 (light microscopes: up to x2,000) Need for special rooms, since microscopes are very sensitive to: vibrations, acoustics, air flow, external magnetic fields, pressure, JEM-2200FS TEM temperature fluctuations JEM: JEOL Electron Microscope TEM: Transmission Electron Microscope 6
7 What is cryo-electron Microscopy (cryo-em)? e.g., Rift Valley Fever virus vaccine strain MP-12 Collapsed virus Surface spikes 100 nm 100 nm 100 nm method Negative staining Cryo-EM stain condition heavy-metal stain often distorted due to dehydration vitrified in amorphous ice (non-crystalline ice) native-like hydrated structure structure limited detailed information detailed information 7
8 Plunge freezing of specimen 3mm 8
9 Plunge freezing of specimen 3mm 9
10 Plunge freezing of specimen cryo transfer station 10
11 Plunge freezing of specimen Virus particles embedded in amorphous ice JEM-2200FS TEM 11
12 Flow diagram in Cryo-EM Virus purification Cryo-EM sample preparation Imaging Data Collection BSL-3 W. M. Keck Center BSL-3 Image Processing Reconstruction Structural Analysis Annotation 12 modified from Chiu et al (2006) JEOL News
13 Ground plan Cryo-EM suite BSL-2 Remote control room W. M. Keck Center for Virus Imaging 200 kev JEM 2200FS (BSL3) 200 kev JEM kev JEM
14 Ground plan Cryo-EM suite BSL-2 Remote control room W. M. Keck Center for Virus Imaging 200 kev JEM 2200FS (BSL3) 200 kev JEM kev JEM
15 Ground plan Cryo-EM suite BSL-2 Remote control room W. M. Keck Center for Virus Imaging 200 kev JEM 2200FS (BSL3) 200 kev JEM kev JEM
16 Ground plan Cryo-EM suite BSL-2 Remote control room W. M. Keck Center for Virus Imaging 200 kev JEM 2200FS (BSL3) 200 kev JEM kev JEM
17 Ground plan Cryo-EM suite BSL-2 Remote control room W. M. Keck Center for Virus Imaging 200 kev JEM 2200FS (BSL3) 200 kev JEM kev JEM
18 Standard Operating Procedures (SOP) Basic general BSL-3 procedures apply, exceptions are: Entry Procedures Exit Procedures Personnel Practices General Laboratory Procedures Respiratory Protection Procedures for Centrifugation Biological Safety Cabinet (BSC) Protocol Waste Handling and Disposal Procedures Equipment Repairs/Service Storage, Packaging and Shipping of Infectious Substances Removal of Biological Material from the BSL-3 Suite Removal of non-biological material from the BSL-3 Suite 18
19 Preparation of infectious agents UTMB campus map BSL-3 x x W.M. Keck Center No containment work (growth, purification, and concentration) is performed in W. M. Keck Center Transport of agents according to the UTMB Institutional Biosafety Committee and Federal Guidelines Police escorted transport of select agents from BSL-3 to Keck Center Entry/Exit procedure protocols 19
20 Select agent storage No storage of select agent after an imaging session has ended Specimen volume brought to the facility should be prepared in such a matter that no leftover material is to be removed EM grids containing non-select agent may be stored in liquid nitrogen dewars for further studies 20
21 Cryo-preparation cryo-plunger sample vial on ice Personnel protection: gown, gloves, respirator cryo-storage box 21
22 Cryo-preparation cont tweezers with EM grids grids are very small (3 mm in diameter) sharp tweezers are needed to manipulate grids liquid ethane cup grid box cryo-storage box liquid nitrogen 22
23 Cryo-preparation cont Ethane (g) C C Ethane (l) Ethane (s) Condensation Freezing gaseous Ethane 1
24 Cryo-preparation cont Ethane (g) C C Ethane (l) Ethane (s) Condensation Freezing gaseous Ethane 2
25 Cryo-preparation cont protecting plastic shield 25
26 Cryo-preparation Sample application 3.5 µl drop of sample 26
27 Cryo preparation Plunge freezing blotting with filter paper guillotine-like motion of plunger plunge freezing (triggered by foot paddel) 27
28 Cryo preparation EM grid transfer transfer of EM grid into cryo-storage box careful removal of tweezer from plunger Note: grid has to be maintained at liquid nitrogen temperatures during all times 28
29 Cryo preparation Grid box transfer transfer dewar with liquid nitrogen storage or imaging Note: frozen grids containing the agent embedded in a solid ice matrix cannot produce any aerosol unless thawed 29
30 Decontamination Once transfer dewar is removed from the BSC: Transfer liquid nitrogen from cryo-box to a styrofoam box and place inside BSC BSC, plunger, tweezers, etc. will be decontaminated using Cavicide All waste and liquids will be autoclaved according to procedures described in BSL-3 cryo-em SOP 30
31 Accidental warm-up / Spill Under no circumstances will transfer boxes be thawed or warmed to a temperature higher than -150 ºC Accidental warm-up: Decontamination of grids and cryo-storage boxes in Cavicide Grid dropped outside of BSC, investigator will : Cover grid and any spilled liquid with absorbent material and flood with 10% bleach or Cavicide Immediately notify EHS and facility director and leave affected area Wait 30 minutes, return to clean the spill 31
32 EM grid transfer into microscope Cryo-holder 32
33 EM grid transfer into microscope liquid nitrogen cryo holder liquid nitrogen cryo transfer station Note: Only manipulation of a frozen sample that will not be done within a BSC, so a respirator will be required of all investigators 33
34 EM grid transfer into microscope cont grid secured with a clip ring grid with frozen agent will be inserted into the holder cryo shield protects specimen against damage 34
35 Cryo-holder transfer into microscope fast but careful holder transfer into the microscope 35
36 Imaging imaging from inside BSL-3 or outside (remote control room) 36
37 Decontamination after imaging session dry-pumping station cryo transfer holder: decontamination at 90ºC for min. of 10 min in drypumping station holder will be held in drypumping station until next use grid will be disposed in Cavicide or a 10% bleach solution 37
38 Safety issues associated with microscope Decontamination of microscope necessary for: shutdown of microscope for service/repair thawed frozen sample loss of grid inside scope (worst-case scenario) Virus inside microscope column and pumping tubes Standard bakeout procedure of microscope column at 60 C Modified to initiate additional bakeout of vaccum lines and valves Full-system bakeout for min. 24 hrs at 60 C 38
39 Some modifications of JEM-2200FS for bakeout insulated pumping tubes temperature controller 39
40 Modifications on pumping equipment Pumping tubes insulated for bakeout HEPA filter on exhaust side of pump HEPA filter insulations 40
41 Heat inactivation curve of BSL-3 agent e.g. RVFV vaccine strain MP-12 No plaques visible in cell-culture plaque assay system 41
42 Summary Modifications in SOP to safely handle BSL-3 agents for preparing EM grids used for structural studies by cryo-em Established heat decontamination protocol for microscope Heat inactivation curves of agents are necessary Equipment modification for usage inside BSL-3 Only functional virus imaging BSL-3 cryo-em facility worldwide Similar facilities under construction: Oxford Particle Imaging Centre, UK (S. Fuller) Purdue University, Indiana, USA (M.G. Rossmann, R.J. Kuhn) 42
43 First results RVFV vaccine strain MP-12 (family Bunyaviridae) a BSL-2 agent 3D reconstruction of RVFV MP Å Frozen-hydrated RVFV MP-12 particles Freiberg et al., JVI,
44 First results Western Equine Encephalitis virus (family Alphaviridae) Frozen-hydrated WEEV particles Preliminary 3D structure of WEEV; first BSL-3 agent studied (MB Sherman, SC Weaver, UTMB, unpublished results) 44
45 Acknowledgments Foundations: W. M. Keck Center for Virus Imaging (Scott Weaver, Stanley Watowich, Wah Chiu) Kleburg Foundation WRCE (David Walker, Kimberley Schuenke) McLaughlin Foundation UTMB departments: Pathology, SCSB, BMB, Neuroscience and Cell Biology UTMB: Misha Sherman, Vincent Hilser, David Gorenstein, Dee Zimmerman, Anne-Sophie Brocard, Linda Roden, Karen Jones 45
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