IGP Core

X-ray and EM Structure Determination

CORE MANAGER: DUILIO CASCIO

Phone: (310) 825-1551

Email:  cascio@mbi.ucla.edu

Boyer Hall 124, 611 Young Drive East, Los Angeles, CA 90095

Staff:  Génesis Falcon (310) 882-2585      genesis@mbi.ucla.edu

Teacher Scientist: Michael Sawaya  (310) 825-1402  sawaya@mbi.ucla.edu

Our purpose

Functional Insights through 3D Structure

The X-ray and EM Structure Determination Core enables access to sophisticated equipment and technologies, offers advice, training and technical assistance in crystallization and sample preparation for CryoEM, MicroED and X-ray diffraction, data collection, processing, atomic refinement, modeling, publishing and data deposition. The core provides services of exceptionally high technical sophistication, with access to consultation and data analysis.  The dedicated staff is trained to accelerate, enrich and educate structural and non-structural biologists to use imaging techniques including electron and X-ray diffraction and cryoEM. The breadth of skills and knowledge of the staff and PIs in the IGP allows us to apply any of our 3 imaging technologies as needed.  The state-of-the-art resources enable the detailed 3-D analysis of biological macromolecules that play essential roles in the plant and microbial systems under investigation.

Fostering innovation

Cross-functional Synergies

The X-ray and EM Structure Determination Core conducts original research in improving crystallographic tools, continuously developing data collection techniques and processing pipelines (indexing, integration, scaling) and have added electron microscopy methods to our existing X-ray capabilities.  Each structure determined using the core facility not only yields biological insights, but also expands databases used in algorithms for fold assignment, structure verification, atomic refinement, potential energy functions, and analysis of protein-protein interactions. Comprehensive databases are key to maintaining and improving these vital tools that enable reliable, high quality structure determination and prediction.

The core plays a major role in driving EM innovation and dissemination of high-resolution imaging methods; EM innovations contribute to all three projects. IGP PIs are important contributors to new EM developments, particularly the projects concerning MicroED and single-particle scaffolding for small proteins, both within the Enabling Capabilities area.

Services Provided

MicroED and Cryo-EM

  • Facilities for high resolution electron diffraction at 300 kV on a Tecnai F30 equipped with an XF416 detector and a Talos equipped with a CetaD detector.
  • Facilities to screen sample quality by negative stain imaging at 120, 200 and 300 kV
  • Facilities for high resolution EM imaging including Tecnai 12, Tecnai F30 and Talos TEM microscopes and sample preparation equipment
  • Low dose cryo electron microscopy at 120, 200 and 300 KeV
  • Computer clusters for data processing
  • Experimental and computational facilities for structure analysis and refinement
  • Assistance with macromolecular model building, structure refinement, and guidance in the interpretation of the resulting 3D structures
  • Workshops introducing the fundamentals of Cryo-EM work and in-person training on the Electron Microscopes.

X-ray Crystallography

  • Experimental and computational facilities for X-ray based structure analysis and refinement
  • Acquisition of diffraction data using in-house high brilliance X-ray generators
  • Acquisition of Multi-wavelength Anomalous Dispersion data using synchrotron radiation
  • X-ray diffraction data processing and manipulation
  • Assistance with macromolecular model building, structure refinement, and guidance in the interpretation of the resulting 3D structures
  • Web-based services that include tutorials and software tools
  • In-person workshops to introduce the fundamentals of single crystal X-ray diffraction and provide training on the X-ray instruments and remote data collection at the APS synchrotron.

Crystal Screening

  • Consultation and technical assistance
  • Evaluation of sample homogeneity and particle size distribution via Dynamic Light Scattering
  • A variety of crystallization screening kits.
  • Robotic setup of crystallization conditions at 4° and 20° C using TECAN, Mosquito and Echo525 liquid handlers
  • UV/vis microscope to determine whether crystals are composed of protein or salt.
  • Optimization of crystallization conditions
  • In-person training on the instruments.
  • 3D Print Prototyping
  • Micro crystallography: rapid screening of micro-crystals with the Korima UV microscope, and manipulation and dissection of micro-crystals with a micro-manipulator

Facilities and Equipment

The X-ray and EM Structure Determination facility occupies 2200 square feet in total and is located in Boyer Hall. Rooms 105 and 136 are dedicated to CryoEM and MicroED, Rooms 106 and 124 are dedicated to  sample preparation, characterization, crystallization screening and X-ray diffraction.

The facility incorporate emerging technologies, adding  new instruments now available to IGP investigators: 1) The 300Kv Tecnai F30, 2) The 120kV Tecnai T12 TEM microscope, 3) An Acoustic Liquid Handling (Echo Labcyte) for crystallization, synthetic biology and functional screening. 4) An Octet RED96 (ForteBIO) to measure kinetics and ligand binding. 5) A  Wyatt DynaPro Plate Reader to measure Dynamic and Static Light Scattering.  Also we have regular access to a new 200kV Talos instrument that  will further bolster capabilities in MicroED, CryoEM and tomography.

The facility operates an STP LabTech Mosquito nanoliter liquid handler, a Rigaku FRE+  rotating anode X-ray generator  and two Rigaku HTC detectors with varimax confocal optics. Crystals are cooled at 100 K by  X-tream Liquid Nitrogen cryogenic coolers.  All generators are shielded by  leaded glass enclosures. The facility is kept at 20 °C by two independent air conditioning systems.

The cold room also is equipped for crystal growth and mounting of heat-labile crystals. The compressors and water recirculating coolers used to remove heat from the x-ray generators are kept in the small room 116, to isolate the noise from the rest of the facility.

Mode of Operation

The facility operates in two modes:  In the first mode, trained users carry out all aspects of the crystallography experiment from crystallization to data collection and analysis.  In the second mode, a member of the X-ray facility assists the researcher in the steps of structure determination in which he is inexperienced. Every year, approximately thirty graduate students and new postdoctoral fellows are trained through classroom and laboratory instruction. Students grow ProteinaseK crystals and receive introduction on data collection and reduction, cryocrystallography, and model building and atomic refinement.

History

The X-ray Crystallography and EM Structure Determination facility dates to the late 1970s.  It gradually grew over the years with generous support from the National Institutes of Health (1980-2006), the Department of Energy (1993-present), and UCLA. In 1980, the facility operated with two Elliott GX-6 and one GX-21 rotating anode generators, equipped with several precession cameras and an oscillation camera.  In 1981, Richard Dickerson joined the UCLA faculty and brought with him a Nicolet diffractometer.  In 1983, a multiwire area detector was purchased from San Diego Multiwire Systems (Serial Number 001), and in 1986 a second multiwire panel was added and shifted to a new Rigaku RU-200 generator.  In 1987, the Nicolet diffractometer was replaced with a Rigaku AFC-5, and in 1989 a Rigaku RU-200 was purchased for precession camera work.  In 1991, a RAXIS-II imaging plate was installed for use with the newest generator.  Most of this equipment was purchased with NIH funds, with matching funds from UCLA.  In 1993, the UCLA-DOE Lab was refocused within a new Division of Structural Biology and Genetics, and DOE funds permitted purchase of a second RAXIS-II with double collimating mirrors.  In 1996 an RAXIS-IV with mirrors was installed, and the multiwire system was phased out. In 1999 with support from the Keck Foundation, a high flux x-ray FRD generator with confocal mirrors and goniostat and a Quantum-4 ADSC charged coupled device (CCD) with confocal mirrors was installed. In 2009 a High Flux Rigaku FRE+ generator was installed with two Rigaku HTC imaging plates using funds from NIH, DOE and HHMI. In 2000, the Facility recruited a superb teacher-scientist, Dr. Michael Sawaya, whose duties include working with students and postdoctoral fellows in determining crystal structures. In early 2005, the DOE facility pioneered techniques to mount single microcrystals, too small for conventional data collection, but well-suited for newly-developed microfocal X-ray beams at synchrotrons (APS-24-ID-E and ESRF-ID-13). These techniques are still in use today.  In 2010, the advent of X-ray free-electron lasers (XFELs) with their ultra-intense X-ray pulses prompted the facility to develop methods to prepare even smaller (~500 nm), in vivo-produced crystals for diffraction at LCLS (Stanford) and SACLA (Japan).  The facility achieved the first reported success of de-novo phasing at an XFEL by isomorphous replacement and proved it was possible to determine atomic resolution structures from crystals harbored in live bacterial cells.   In mid-2015 the staff at the facility began exploring the use of electron diffraction to determine atomic resolution structures from peptide crystals, so small that they are invisible by conventional light microscopes. The facility developed sample preparation techniques, and produced a pipeline for indexing and integration of electron diffraction data.