The Cryo-Electron Microscopy Center
The Cryo-Electron Microscopy Center supports industry, academic, government, and non-profit research institutes, providing single-particle and microcrystal electron diffraction capabilities. We offer rapid and flexible access to cryo-EM capabilities for proprietary and non-proprietary clients together with expert guidance at competitive rates.
Users may choose a combination of these components in order to achieve their scientific goals. A team of personnel will be available to discuss different options and possible deliverables and will offer expert advice on each user’s projects in order to achieve the best results in a timely fashion.
The Cryo-EM Center at HWI provides:
- Expert staff – The Center has experience with multiple users both commercial and academic. The Center staff work with the full research services team at Hauptman-Woodward to ensure the best structural solution is available to our users.
- State of the art instrumentation – A Thermo Fisher Glacios 200 kV microscope with a Volta phase plate, a Falcon 4 Detector for high-throughput grid screening and high-resolution data collection, and a Ceta-D camera for micro-crystal electron diffraction. Thermo Fisher Vitrobot for grid preparation.
- State-of-the-art laboratories – A well-equipped space for biochemical preparation and biophysical characterization of biological samples; a walk-in humidity-controlled environment dedicated for grid preparation.
- Purpose-built facilities – A microscope facility designed to achieve the best performance. Glycol-cooled temperature control, acoustic and electromagnetic isolation, and laminar air flow are implemented into the construction of these spaces. High-capacity data storage and high-performance computational resources with highly tightened data security and user confidentiality are implemented for remote access during and after data collection.
- Timely access -Experimental runs in the Center are scheduled on a weekly basis and can be prearranged ahead of time to suit the special needs of users’ samples when necessary. Users may inspect their grids and data on the fly during data collection and will have remote access to their data for two months after data collection.
The HWI cryo-EM center team can offer advice to help you in sample preparation for successful studies.
For cryo-EM, we broadly follow the same typical protein requirements as for crystallography. Specifically, our recommendations for protein samples to increase the chance of success in cryo-EM are;
- Amount of protein – 50 -100 microliters of protein at 0.5 – 5.0 mg/ml
- Molecular weight of protein – Lower limit: ~50-60 kDa
For the best data we recommend that samples display a single clear band in Coomassie-blue stained SDS-PAGE; and a single band in native blue-gel if feasible, a sharp peak in a suitable SEC column and/or uniformed size in dynamic light scattering or MALS.
We can hold a sample preparation meeting with you to discuss the biochemistry of the sample and re-purification methods, etc. If necessary, re-purification and concentration of the sample is done according to protocols established in the sample preparation meeting.
To ensure consistency and reproducibility, your samples will be vitrified on a grid using a Vitrobot (a semi-automatic plunger) in a walk-in humidity-controlled chamber.
Grids will be prepared under different conditions to assess the best conditions for data collection.
Cryo-EM grid screening
Grids will be screened using a Glacios cryo-electron microscope equipped with a Falcon 4 Direct Electron Detector to ensure that single-particle studies or microcrystal electron diffraction can be conducted optimally. Areas on grids will be prioritized for full high-resolution data collection. For single particle analysis, we will determine grid properties including ice thickness, particle density and particle distribution. Grid screening can be combined with overnight data collection.
Automatic data collection and remote data delivery
Raw images and fully pre-processed micrographs are will be saved on HWI’s data storage system for 2 months and made available for the user upon a mutually agreed upon method.
Data analysis and structural determination
The HWI cryo-EM center team can analyze the data and perform structural determination.
Screening for microcrystals for electron diffraction and structure determination
Our Glacios cryo-electron microscope is equipped with the Thermo Scientific MicroED Package and a CETA-D Camera, plus Thermo Scientific EPU-D™ software for screening and automated data acquisition.
Instrumentation, outcome, and the people who make it happen:
Our facilty is purposely designed to produce the highest quality data with our Glacios 200 kV microscope, Volta phase plate, and Falcon 4 and Ceta-D detectors.
We offer a range of services up to and including complete structural solutions.
Past publications involving members of the Cryo-EM Center
- Loss of SATB1 Induces p21-Dependent Cellular Senescence in Post-mitotic Dopaminergic Neurons.
Riessland M, Kolisnyk B, Kim TW, Cheng J, Ni J, Pearson JA, Park EJ, Dam K, Acehan D, Ramos-Espiritu LS, Wang W, Zhang J, Shim JW, Ciceri G, Brichta L, Studer L, Greengard P. Cell Stem Cell. 2019 Oct 3;25(4):514-530.e8. doi: 10.1016/j.stem.2019.08.013. Epub 2019 Sep 19.
- Cdc42 Regulates Neuronal Polarity during Cerebellar Axon Formation and Glial-Guided Migration. Govek EE, Wu Z, Acehan D, Molina H, Rivera K, Zhu X, Fang Y, Tessier-Lavigne M, Hatten ME.iScience. 2018 Mar 23;1:35-48. doi: 10.1016/j.isci.2018.01.004.
- De novo centriole formation in human cells is error-prone and does not require SAS-6 self-assembly. Wang WJ, Acehan D, Kao CH, Jane WN, Uryu K, Tsou MF. Elife. 2015 Nov 26;4:e10586. doi: 10.7554/eLife.10586.
- Positive feedback between Golgi membranes, microtubules and ER exit sites directs de novo biogenesis of the Golgi. Ronchi P, Tischer C, Acehan D, Pepperkok R. J Cell Sci. 2014 Nov 1;127(Pt 21):4620-33. doi: 10.1242/jcs.150474. Epub 2014 Sep 4.
- A bacterial tubulovesicular network. Acehan D, Santarella-Mellwig R, Devos DP. J Cell Sci. 2014 Jan 15;127(Pt 2):277-80. doi: 10.1242/jcs.137596. Epub 2013 Nov 20.
- Role of fatty-acid synthesis in dendritic cell generation and function. Rehman A, Hemmert KC, Ochi A, Jamal M, Henning JR, Barilla R, Quesada JP, Zambirinis CP, Tang K, Ego-Osuala M, Rao RS, Greco S, Deutsch M, Narayan S, Pachter HL, Graffeo CS, Acehan D, Miller G. J Immunol. 2013 May 1;190(9):4640-9. doi: 10.4049/jimmunol.1202312. Epub 2013 Mar 27.
- Dendritic cells limit fibroinflammatory injury in nonalcoholic steatohepatitis in mice. Henning JR, Graffeo CS, Rehman A, Fallon NC, Zambirinis CP, Ochi A, Barilla R, Jamal M, Deutsch M, Greco S, Ego-Osuala M, Bin-Saeed U, Rao RS, Badar S, Quesada JP, Acehan D, Miller G. Hepatology. 2013 Aug;58(2):589-602. doi: 10.1002/hep.26267. Epub 2013 Jun 24.
- Tafazzin knockdown in mice leads to a developmental cardiomyopathy with early diastolic dysfunction preceding myocardial noncompaction. Phoon CK, Acehan D, Schlame M, Stokes DL, Edelman-Novemsky I, Yu D, Xu Y, Viswanathan N, Ren M. J Am Heart Assoc. 2012 Apr;1(2):jah3-e000455. doi: 10.1161/JAHA.111.000455. Epub 2012 Apr 24.
- The physical state of lipid substrates provides transacylation specificity for tafazzin. Schlame M, Acehan D, Berno B, Xu Y, Valvo S, Ren M, Stokes DL, Epand RM.Nat Chem Biol. 2012 Oct;8(10):862-9. doi: 10.1038/nchembio.1064.
- Dendritic cell populations with different concentrations of lipid regulate tolerance and immunity in mouse and human liver.
Ibrahim J, Nguyen AH, Rehman A, Ochi A, Jamal M, Graffeo CS, Henning JR, Zambirinis CP, Fallon NC, Barilla R, Badar S, Mitchell A, Rao RS, Acehan D, Frey AB, Miller G.
Gastroenterology. 2012 Oct;143(4):1061-72. doi: 10.1053/j.gastro.2012.06.003. Epub 2012 Jun 12.
- Dendritic cells promote pancreatic viability in mice with acute pancreatitis. Bedrosian AS, Nguyen AH, Hackman M, Connolly MK, Malhotra A, Ibrahim J, Cieza-Rubio NE, Henning JR, Barilla R, Rehman A, Pachter HL, Medina-Zea MV, Cohen SM, Frey AB, Acehan D, Miller G. Gastroenterology. 2011 Nov;141(5):1915-26.e1-14. doi: 10.1053/j.gastro.2011.07.033. Epub 2011 Jul 27.
- Cardiolipin affects the supramolecular organization of ATP synthase in mitochondria. Acehan D, Malhotra A, Xu Y, Ren M, Stokes DL, Schlame M. Biophys J. 2011 May 4;100(9):2184-92. doi: 10.1016/j.bpj.2011.03.031.
- Cardiac and skeletal muscle defects in a mouse model of human Barth syndrome. Acehan D, Vaz F, Houtkooper RH, James J, Moore V, Tokunaga C, Kulik W, Wansapura J, Toth MJ, Strauss A, Khuchua Z. J Biol Chem. 2011 Jan 14;286(2):899-908. doi: 10.1074/jbc.M110.171439. Epub 2010 Nov 9.
- The dynamics of cardiolipin synthesis post-mitochondrial fusion. Xu FY, McBride H, Acehan D, Vaz FM, Houtkooper RH, Lee RM, Mowat MA, Hatch GM. Biochim Biophys Acta. 2010 Aug;1798(8):1577-85. doi: 10.1016/j.bbamem.2010.04.007. Epub 2010 Apr 29.
- Anisotropic self-assembly of spherical polymer-grafted nanoparticles. Akcora P, Liu H, Kumar SK, Moll J, Li Y, Benicewicz BC, Schadler LS, Acehan D, Panagiotopoulos AZ, Pryamitsyn V, Ganesan V, Ilavsky J, Thiyagarajan P, Colby RH, Douglas JF. Nat Mater. 2009 Apr;8(4):354-9. doi: 10.1038/nmat2404. Epub 2009 Mar 22.
- Distinct effects of tafazzin deletion in differentiated and undifferentiated mitochondria. Acehan D, Khuchua Z, Houtkooper RH, Malhotra A, Kaufman J, Vaz FM, Ren M, Rockman HA, Stokes DL, Schlame M. Mitochondrion. 2009 Apr;9(2):86-95. doi: 10.1016/j.mito.2008.12.001. Epub 2008 Dec 11.
- Plakoglobin is required for effective intermediate filament anchorage to desmosomes. Acehan D, Petzold C, Gumper I, Sabatini DD, Müller EJ, Cowin P, Stokes DL. J Invest Dermatol. 2008 Nov;128(11):2665-2675. doi: 10.1038/jid.2008.141. Epub 2008 May 22.
- Cryoelectron tomography of isolated desmosomes. Owen GR, Acehan D, Derr KD, Rice WJ, Stokes DL. Biochem Soc Trans. 2008 Apr;36(Pt 2):173-9. doi: 10.1042/BST0360173.
- Comparison of lymphoblast mitochondria from normal subjects and patients with Barth syndrome using electron microscopic tomography. Acehan D, Xu Y, Stokes DL, Schlame M. Lab Invest. 2007 Jan;87(1):40-8. doi: 10.1038/labinvest.3700480. Epub 2006 Oct 16.
- Three-dimensional structure of a double apoptosome formed by the Drosophila Apaf-1 related killer. Yu X, Wang L, Acehan D, Wang X, Akey CW. J Mol Biol. 2006 Jan 20;355(3):577-89. doi: 10.1016/j.jmb.2005.10.040. Epub 2005 Nov 8.
- A structure of the human apoptosome at 12.8 A resolution provides insights into this cell death platform. Yu X, Acehan D, Ménétret JF, Booth CR, Ludtke SJ, Riedl SJ, Shi Y, Wang X, Akey CW. Structure. 2005 Nov;13(11):1725-35. doi: 10.1016/j.str.2005.09.006.
- Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW. Mol Cell. 2002 Feb;9(2):423-32. doi: 10.1016/s1097-2765(02)00442-2.
The Cryo-EM Center
Hauptman-Woodward Medical Research Institute
700 Ellicott St., Buffalo,