News and Events
New Krios Microscopes
The Krios#1 microscope has been operational despite the construction, and has been producing high quality data more or less continuously. The Krios#2 microscope is installed and will become available to users sometime before the end of January 2017. The Krios#3 has arrived, is being built, and should be online in the first quarter 2017.
March 7, 2017
New User Training
Attended by these people in the photo above, New User Training took place on February 21st. The morning session covers sample preparation (negative-staining and plunge freezing), and the afternoon session covers basic TEM use (loading samples and introduction to Leginon). Please email EMG@NYSBC.org to inquire about future training session dates.
February 23, 2017
Appion Workshop: Advanced Techniques - Going from Appion to RELION 2 and FREALIGN
A one-day master class was held February 23rd from 10:00AM to 4:00PM for experienced Appion users interested in using RELION2 and FREALIGN for 3D refinements. The focus of this workshop was how to take advantage of tools within Appion for efficient single particle analysis concurrent with Leginon data collection. A real-time demonstration was given using our GPU workstations including a brief overview of the RELION2 and FREALIGN interfaces. The workshop limited to 10 attendees. If you are interested in learning when the next Appion Advanced Workshop will be held, please e-mail EMG@NYSBC.org
February 6-7, 2017
Workshop on Management of Large CryoEM Facilities
Attended by the group pictured above, a Workshop on Management of Large CryoEM Facilities was held on February 6th and 7th. A major goal of the workshop was to discuss best practices for managing CryoEM facilities. Topics discussed included sample handling, instrument management, computational environments, training, user management and workflow. The agenda, PDFs of slides presented and a link to the YouTube recording is here: http://nramm.nysbc.org/27-282017-management-of-large-cryoem-facilities/.
February 2, 2017
New User Training
Although it is usually offered on the first Tuesday of every month, February's New User Training took place on February 2nd to accommodate workshops being offered next week. The morning session covers sample preparation (negative-staining and plunge freezing), and the afternoon session covers basic TEM use (loading samples and introduction to Leginon). Please email EMG@NYSBC.org to inquire about future training session dates. Pictured above are the most recent attendees.
Appion Part 1 Workshop
A one-day Appion Workshop was held on Tuesday, January 10th from 10:00AM to 4:00PM. The Appion Part 1 Workshop provides hands-on training for the Appion single-particle data processing workflow. Appion is a "pipeline" for processing and analysis of EM images and is integrated with Leginon data acquisition. Topics include analyzing the quality of your data collection, correcting the CTF, generating and cleaning up stacks, and generating 2D class averages. An Appion Part 2 Workshop will be offered in the following month to provide training for making initial models and doing 3D reconstructions. If you are interested in learning when the next Appion Part 1 Workshop will be held, please e-mail EMG@NYSBC.org.
New User Training
Pictured here are the attendees of January's New User Training Session, offered the first Tuesday of every month. The morning session covers sample preparation (negative-staining and plunge freezing), and the afternoon session covers basic TEM use (loading samples and introduction to Leginon). Please email EMG@NYSBC.org to inquire about future training session dates.
Wednesday and Thursday 12/8-9
Pictured above are the attendees of the Dynamo Workshop.
This workshop provides hands-on training in all practical aspects of sub-tomogram averaging using Dynamo. The new Dynamo catalogue system for organizing and centralizing tomograms will be featured along with new semi-automated picking procedures. All stages of the pipeline will be covered, including: tomogram visualization and archiving, particle picking, membrane picking, filament picking, averaging, refinement, and classification.
To learn when the Dynamo Workshop will be held again, please e-mail EMG@NYSBC.org.
Appion Part 3 Workshop
Pictured above are the attendees of the Appion Part 3 Workshop.
Tilt-series alignment is commonly performed by tracking fiducials in order to recover the three-dimensional structure and orientation of specimen in cryo-electron tomography, negative stain tomography, and tomography on plastic sections. However this approach is sometimes fraught with difficulty due to fiducial movement, fiducial aggregation, or the lack of fiducials in specimen such as FIB/SEM lamella.
In this workshop users gain hand-on experience with a semi-automated implementation of Protomo into Appion. Protomo allows for fiducial-less alignment of tilt-series and refinement of specimen orientation on the grid through an iterative geometry refinement process. Appion-Protomo, which installs seamlessly with Appion, significantly reduces the effort and expertice required by the user to align fiducial-free tilt-series collected using any software package. All that is required is a stack of tilt images along with knowledge of their imaging conditions. Integrated within Appion-Protomo are pipelines for defocus estimation, CTF correction, and dose compensation, along with multiple reconstruction methods.
To learn when the next Appion Part 3 Workshop will be held, please e-mail EMG@NYSBC.org.
New User Training
Pictured above are the attendees of the December New User Training, offered the first Tuesday of every month. The morning session covers sample preparation (negative-staining and plunge freezing), and the afternoon session covers basic TEM use (loading samples and introduction to Leginon). Please e-mail EMG@NYSBC.org to learn about future new user training sessions.
Friday, October 28th
Computational Methods for CryoEM Workshop
Pictured here are the attendees of the Computational Methods for CryoEM Workshop, led by Sjors Scheres. This workshop focused on computational approaches for cryoEM. The goal was to bridge between the computational science community in NYC and the cryoEM community.
Tuesday, October 25th
Relion Workshop with Sjors Scheres
This one-day workshop focused on the RELION software package and was led by its developer Sjors Scheres (MRC-LMB). The training involved a hands-on tutorial for cryo-EM single-particle analysis.
For details about RELION, click here.
New User Training
Pictured here are the attendees of the October's New User Training, offered the first Tuesday of every month. The morning session covers sample preparation (negative-staining and plunge freezing), and the afternoon session covers basic TEM use (loading samples and introduction to Leginon). Please email EMG@NYSBC.org to inquire about future training session dates.
Phase Plate Workshop
FEI's volta phase plate has been installed on Krios#1. A SEMC Phase Plate Workshop was held on September 21-22 and led by Rado Danev, MPI Martinstried. The workshop included a demonstration of the optimal use of the device and processing of phase plate data.
New User Training
This month's New User Training was attended by (back row, l-r) Alex Noble, Judith Kribelbauer, Micah Rapp, (front row, l-r) Ed Eng (leader)Stephanie Siegmund, Juncheng Wang, led also by Ashleigh Raczkowski and Bill Rice (not pictured above). New users are required to attend a group new user training session before use of the facility. We offer these 1-day training sessions the beginning of each month from 9am to 4:30pm. Please email EMG@NYSBC.org to inquire about future training session dates.
Holey Gold Grid Workshop
The most recent Holey-Gold Grid Workshop was held on August 25th, 2016, from 1:00-3:00PM. Our Holey-gold grid workshop shows users how to make their own holey-gold grids from standard holey carbon gold grids. Please email EMG@NYSBC.org to inquire about future training session dates.
SEMC Forum: Prof. Jose-Maria Carazo, Spanish National Center for Biotechnology, Madrid, Spain
Cryo-EM data processing and information integration over the Web
Cryo EM is nowadays a fundamental analysis tool in Structural Biology, with a very rapid increase rate in terms of new users coming to the field. As a way to minimize software usability barriers to the varied set of available image processing packages, we have developed Scipion (http://scipion.cnb.csic.es/m/home/
), a workflow-oriented software integration platform where flexibility, user experience, traceability and simplicity are key. Besides a classical desktop-oriented setting, Scipion is also cloud-based, providing not only a very simple way to use the software, but also a very clear computational cost model that can be used in multiple situations. Additionally, we also offer Scipion Web Tools (SWT), providing predefined analysis workflows over multiple software suites, with special emphasis on cryo EM map and model quality analysis (http://scipion.cnb.csic.es/m/services/
). Complementing SWT and following its same developmental rational, we link map and model results into an integrative and interactive environment for easy information exploration termed 3DBionotes (https://3dbionotes.cnb.csic.es
), now expanded to start accommodating genomics information.
SEMC Forum: Dr. Joel Mancuso, Ph.D., Dr. Mark Riccio, Ph.D., Carl Zeiss Microscopy, LLC
Non-invasive High Resolution 3D X-ray Microscopy for Life Sciences, Materials Science, and Engineering.
The progress of scientific research and technology development greatly depends upon effective imaging solutions for characterizing the properties and behaviors of your materials. Revealing details of microstructure, ideally in three dimensions (3D), is a critical part of your understanding, whether your goal is to develop and confirm models that describe material properties and behaviors or simply to visualize structural details. ZEISS offers 3D X-ray microscopes (XRM): advanced imaging solutions that have removed major hurdles for three-dimensional imaging by achieving high contrast and submicron resolution imaging even for your relatively large samples. These groundbreaking advances in non-destructive, 3D imaging empower a broad range of technical disciplines.
X-Ray microscopy can also increase productivity in EM workflows by identifying the areas of interest within a tissue, non-destructively, before sectioning and preparing for EM work.
The Xradia Versa family of submicron XRM uses patented X-ray detectors within a microscope turret of objectives for easy zooming down past 700nm spatial resolution with minimum achievable voxels of 70nm. The Xradia Ultra family of nanoscale X-ray microscopes is the only commercially available X-ray microscope that utilizes synchrotron quality X-ray optics and provides true spatial resolution down to <50nm and minimum achievable voxels of 16nm.
SEMC Forum: Dr. Andy Stewart, University of Limerick
Electron diffraction tomography, its many flavors, and applications to beam sensitive specimens.
Electron diffraction tomography (EDT) has been a revelation for the structure solution of nano sized crystals across a vast expanse of materials, including most recently protein crystals, since its introduction in 2009. An overview of the different EDT techniques with some analysis of the strengths and weaknesses of each method, including an exploration of how EDT can be developed further, to improve data quality, the speed of data collection, and most importantly data analysis and structure solution. With a brief look at where EDT sits in the tool box of structural biology with regards to other emerging techniques.
Appion Part 2 Workshop
A one-day Appion Workshop was held on July 26, 2016 from 10:00AM to 4:00PM. This Appion Part 2 Workshop was a follow-on to the Part 1 workshop and focused on 3D initial models and refinement. If you are interested in attending an Appion Part 2 Workshop, please register at: https://www.surveymonkey.com/r/5NK7NGQ. The workshop is limited to 10 attendees.
SEMC Forum: Myriam Badr, Ph.D.
Nanotemper Technologies, Cambridge, MA
Exploring protein thermal and colloidal stability by NanoDSF
NanoDSF is an advanced Differential Scanning Fluorimetry (DSF) method, developed by Nanotemper Technologies, for measuring protein stability with ultra-high resolution using intrinsic tryptophan/tyrosine fluorescence via a dual-UV detection technology. Following changes in the fluorescence at 350 and 330nm, chemical and thermal stability is assessed in a truly label-free and dye-free approach. Applications include antibody engineering, membrane protein research, quality control, and formulation screens. A back reflection optical system can also be integrated to measure colloidal stability of proteins, measured at the same time as thermal stability. Advantages of the instrument include small sample consumption (10 l/sample), a large dynamic range (5 g/ml to 250mg/ml), no buffer restrictions (including detergents), and fast and reliable thermal and colloidal measurements (48 samples combined, ~1hr run).
We encourage you to attend the seminar to learn more the Prometheus instrument from Nanotemper technologies. We will also be present before and after the seminar for an on-site demo, and encourage you to bring samples to test on the instrument.
Holey Gold Grid Workshop
A workshop on making Holey Gold grids was held on Thursday, June 16 from 1:00 to 3:00pm. This provides an overview of making holey gold films from holey carbon films supported on gold mesh. Holey gold films provide a stronger support film and reduce beam induced movement of the specimen. If you would like to attend a holey gold workshop, please register at: https://www.surveymonkey.com/r/6M8ZT5V. The workshop is limited to 6 attendees.
Appion Part 1 Workshop
A one-day Appion Workshop was held on Tuesday, June 14, 2016 from 10:00AM to 4:00PM. The Appion Part 1 Workshop provides hands-on training for the Appion single-particle data processing workflow. Appion is a “pipeline” for processing and analysis of EM images; it is integrated with Leginon data acquisition, but can also be used stand-alone after uploading images or particle stacks. Topics include analyzing the quality of your data collection, correcting the CTF, generating and cleaning up stacks, and generating 2D class averages. If you are interested in attending an Appion Part 1 Workshop, please register at: https://www.surveymonkey.com/r/6TTP5PT. The workshop is limited to 10 attendees.
SEMC Forum: Professor Michael Elbaum
Department of Materials and Interfaces
Weismann Institute of Science
STEM: a re-emerging modality for cryo-imaging and tomography
Until recently, cryo-electron microscopy has been nearly synonymous with wide-field TEM using phase contrast. The interferometric image formation mechanism provides high contrast but also imposes stringent conditions for reliable interpretation. Point-scanning TEM, or STEM, offers an alternative modality with several important advantages. Specifically for tomography, inelastic scattering does not interfere with image formation so losses to the energy filter are avoided. Moreover, pixel values in the image relate quantitatively to elemental composition and density, while flexible configuration of the detectors can give relative weight to light or heavy elements. We take advantage of this sensitivity to examine polyphosphate bodies in bacteria, calcium-rich granules in mitochondria, and isolated metal atoms in ferritin. These examples demonstrate how STEM can expand the range of studies addressed by cryo-microscopy and tomography.
Tuesday, April 26th
SEMC Workshop: A hands-on workshop on three multi-scale visualization and modeling packages will be held on Tuesday, April 26 by Willy Wriggers, Julio Kovacs, and Manual Wahle. Situs is a script-based program package for the 3-D fitting of atomic resolution structures into low-resolution density maps, e.g., from electron microscopy, tomography, or small angle x-ray scattering. TimeScapes bridges between fast and slow time scales and generates 3-D spatial heat map images from a statistical analysis of 1-D time series (or equivalent independent variable) data. Sculptor is a multi-scale molecular graphics package that serves both as a frontend for Situs algorithms and as a standalone 3-D visualization program. The three packages are freely available at the Wriggers lab website and are also supported by the SBGrid consortium (sbgrid.org) in the upcoming release. Please register at: https://www.surveymonkey.com/r/3MF3P99. The workshop is limited to 15 attendees.
Tuesday, April 26th
SEMC Forum: Dr. Willy Wriggers of the Department of Mechanical and Aerospace Engineering at Old Dominion University will give a talk entitled "Multiscale Biomolecular Modeling in 4-D".
Time: 12:00PM, Location: A-11 seminar room at NYSBC
Monday, April 25th
SEMC EM Course: Fitting Atomic Models.
Time: 3:00PM, Location: CUNY Advanced Science Research Center Auditorium
Wednesday, April 20th
SEMC Forum: Dr. Carolyn Larabell of the Department of Anatomy at the University of California, San Francisco will speak on "Localizing Molecules in Cellular CT Scans".
Time: 3:00PM, Location: CUNY Advanced Science Research Center Auditorium
Monday, April 18th
SEMC EM Course: Tom Walz of Rockefeller University will give a lecture on "Validation Methods"
Time: 3:30-5:00PM. Location: A-11 seminar room at NYSBC from 3:30-5pm.
Friday, April 15th
SEMC Forum: Dr. Radoslaw Nowak will visit from Oxford University to speak about "X-Ray and Activity Fragment Screening Across Subfamilies of Lysine Demethylases".
Time: 12:00PM, Location : A-11 seminar room at NYSBC.
Tuesday, April 12th
Appion Training Workshop:
The Appion Part 1 Workshop provides hands-on training for the Appion single-particle data processing workflow. Appion is a "pipeline" for processing and analysis of EM images; it is integrated with Leginon data acquisition, but can also be used stand-alone after uploading images or particle stacks. Topics include analyzing the quality of your data collection,correcting the CTF, generating and cleaning up stacks, and generating 2D class averages. The workshop is limited to 10 attendees. For more information and registration, click here: https://www.surveymonkey.com/r/FZLPDXF.
Time: 10:00AM-4:00PM, Location: SEMC Conference Room at NYSBC
Monday, April 11th
SEMC EM Course: Class in 2D crystallography - Helical (part II)
Time: 3:30-5:00PM, Location: A-11 seminar room at NYSBC
Titan Krios The Krios #1 microscope has been installed, accepted and is now being used to collect data for a first set of member projects. Images and a time lapse movie of the installation can be seen at http://semc.nysbc.org/krios.html. The microscope is currently equipped with a Falcon 2 camera that will be upgraded to the Falcon 3 camera later in the summer. The K2 camera will be moved from the JEM 2100 onto Krios #1 in April. Both the K2 and the Falcon 3 support counting mode, so users will have a choice of cameras for their projects. We will be carrying out characterization tests on both cameras to help users identify the best match of cameras to their projects. A TVIPS F416 CMOS camera will replace the K2 on the JEM2100 and we expect that the JEM2100 will be an excellent cryo screening instrument.
We are delighted to announce that the Simons Foundation has generously provided support to purchase a second Krios microscope. This microscope will replace the JEM3200 and will be equipped with an energy filter, Cs corrector and a Volta phase plate. The JEM3200 will be decommissioned in April and the room will be prepared for expected delivery of Krios #2 in August. The DE20 camera that is on the JEM3200 will be transferred to the F20 microscope in April and we expect that the F20 will be capable of supporting projects for reconstructions of maps in the ~4A and above resolution range.
A major renovation of the HVAC systems for the microscope suite will take place in the spring. The current HVAC system is not capable of maintaining a controlled temperature and humidity environment and this leads to instability in the instruments and impacts their performance. Work on the HVAC upgrade will start in May and we hope to have it all completed by July. Around the same time, work on the room for Krios #2 will also begin and be ongoing for a few weeks. This will include digging a pit in the concrete to accommodate an anti-vibration table. This work will inevitably at times cause some major disruptions in the control room and the microscopes may not be useable for some periods of time. Our intention is to keep the microscopes, especially the Krios, operational as much as possible during the entire period of construction.
October 2015 New Publication: Architecture Formed by Large and Small Terminase Subunits
Packaging of viral genomes inside empty procapsids is driven by a powerful ATP-hydrolyzing motor, formed in many double-stranded DNA viruses by a complex of a small terminase (S-terminase) subunit and a large terminase (L-terminase) subunit, transiently docked at the portal vertex during genome packaging. Despite recent progress in elucidating the structure of individual terminase subunits and their domains, little is known about the architecture of an assembled terminase complex. Here, we describe a bacterial co-expression system that yields milligram quantities of the S-terminase:L-terminase complex of the Salmonella phage P22. In vivo assembled terminase complex was affinity-purified and stabilized by addition of non-hydrolyzable ATP, which binds specifically to the ATPase domain of L-terminase. Mapping studies revealed that the N-terminus of L-terminase ATPase domain (residues 1-58) contains a minimal S-terminase binding domain sufficient for stoichiometric association with residues 140-162 of S-terminase, the L-terminase binding domain. Hydrodynamic analysis by analytical ultracentrifugation sedimentation velocity and native mass spectrometry revealed that the purified terminase complex consists predominantly of one copy of the nonameric S-terminase bound to two equivalents of L-terminase (1S-terminase:2L-terminase). Direct visualization of this molecular assembly in negative-stained micrographs yielded a three-dimensional asymmetric reconstruction that resembles a "nutcracker" with two L-terminase protomers projecting from the C-termini of an S-terminase ring. This is the first direct visualization of a purified viral terminase complex analyzed in the absence of DNA and procapsid.
Titan Krios SEMC is delighted to announce that due to the continued generosity of the Simons Foundation, we will soon install an FEI Titan Krios microscope with a Falcon 2 camera (shortly to be upgraded to a Falcon 3). We are currently renovating space to house the instrument, and we anticipate that it will be ready for users at the end of the first quarter of 2016. The Titan Krios/Falcon 3 instrument will join the current lineup of instrumentation, including the four other transmission electron microscopes (JEOL 3200FSC/DE20, JEOL 2100F/K2, JEOL 1230/CCD, Tecnai F20/CCD), and a dual beam focused ion beam scanning electron microscope (Helios 650).
Renovations As many of you know, we completed the first round of renovations in May 2015. These changes provided our control room and conference room, (which are currently the hub of SEMC), as well as space for computational servers and many improvements to the electrical and computational infrastructure. We now need to begin a second round of renovations to create space for the new Titan Krios. The Krios will be installed in the room currently housing the Helios, and the Helios will be moved to a new space in the wet lab area. Work on this new space will begin very soon, and the move of the Helios is currently scheduled for the end of October. The instrument will be down during the move, and for a short time afterwards as it gets reinstalled and tested. The preparations for the Helios room will also mean that the dehumidified specimen preparation room will be unavailable for use starting next week for a few days. We will move the CP3 into the wet lab area during this time so that grids may still be vitrified. Once the Helios has been moved, the Krios room needs some major work in constructing a vibration isolation platform. This includes removal of concrete to construct a pit, which will inevitably cause disruption to all of the instruments, with the exception of the F20. We will do our best to minimize this disruption, but for some days it will not be possible to acquire data until the construction is complete. SEMC staff will work to adjust the schedule during this time to maximize the use of the instruments and try to best re-accomodate users who lose their booked sessions.
Click here to view an updated Renovation schedule.
New User Training New users are required to attend a group new user training session before use of the facility. We offer these 1-day training sessions the beginning of each month from 9am to 4:30pm. The morning session will cover sample preparation (negative-staining and plunge freezing), and the afternoon session covers basic TEM use (loading samples and introduction to leginon). New users may purchase a starter EM user kit from SEMC. New users are strongly encouraged to do some background reading before their first training session.
Following training users may request time on the on the instruments in consultation with SEMC staff until they gain sufficient experience to receive sign off from SEMC staff to use the instruments independently. Please note that each instrument requires individual training and sign off, which will be provided by SEMC staff on a one-on-one basis.
Please email EMG@NYSBC.org to inquire about future training session dates.
August 2015 On August 5th, 2015 BioTechniques published a piece on the revolution of CryoEM, courtesy of Kelly Chi. Mentioned in the post were Clint Potter and Bridget Carragher, co-directors of the Simons Electron Microscopy Center at the New York Structural Biology Center, and according to the article, it has been an exciting two years for CryoEM. From how CryoEM works, to the challenge of breaking the 3Å resolution barrier, to discussing why CryoEM is useful when x-ray crystallography also garners high resolution imaging, all the way to what lies ahead and the challenges the field is expected to take, this article gives an interesting, concise outlook on the field of Cryo Electron Microscopy.
Read the full text here.
April 2015 New Publication: Mammalian Photoreceptors Ya-Chu Hsu, Jen-Zen Chuang, and Ching-Hwa Sung from Weil Medical College have published a paper concerning the mechanism through which rod photoreceptors in mammals maintain a balance between membrane bound discs that are created and discarded daily.
March 2015: NYSBC Electron Microscopy staff assists users in evaluating the suitability of existing samples for achieving specified goals and in implementing any unusual experimental approaches. Staff members are also available for pilot studies to produce preliminary reconstructions suitable for inclusion in grant proposals. Staff is also available to collaborate with scientists who are not EM users but need EM data for their projects
March 2015: New Publication: 2.8Å Resolution Proteasome Stucture Melody Campbell from the Scripps Research Institute and David Veesler from the University of Washington worked with Bridget Carragher, Clint Potter, and Anchi Cheng from the Electron Microscopy Group at NYSBC using single-particle cryoEM to produce images of protein structure detailed enough to be useful for drug development.