Structural Molecular Biology Resourceat the

Stanford Synchrotron Radiation Lightsource

The SSRL Structural Molecular Biology program operates as an integratedResource with three primary core areas of technological R&D and scientific focus:macromolecular crystallography (MC), small angle x-ray scattering/diffraction(SAXS), and x-ray spectroscopy (XAS/XES) including x-ray microXAS imaging.Technological foci include development and enhancement of beam lines,specialized instrumentation, detectors, methods, and techniques, taking maximumadvantage of the high x-ray brightness of SSRL’s storage ring (SPEAR3). This iscoupled with vigorous R&D in enhanced data collection, data management and dataanalysis to provide remote-access and "user-friendly, real-time and on-line" datainterpretation. The innovations are guided by close coupling to scientific projectsaimed at solving forefront problems in SMB. Significant synergy between the coreareas enable addressing increasingly complex and challenging problems. The SMBprogram seeks to sustain and enhance the user community through excellentsupport, training and dissemination.

The SMB program is supported by the DOE Office of Biology and Environmental Research

and the NIH National Institute of General Medical Sciences.

Contact: Lisa Dunn, lisa@slac.stanford.edu, ph: 650-926-2087

X-ray Spectroscopy & Imagingelectronic & geometric structure of metal sites

http://www-ssrl.slac.stanford.edu/smb/programs/xas/home.html

Contact: Ritimukta Sarangi, ritis@slac.stanford.edu

The Resource supports studies of ultra-dilute metalloprotein

solutions at liq-He temperatures. Specialized instrumentationenable simultaneous single-crystal XAS and crystallography.Anaerobic sample handling facilities are available. Studies offreeze trapped samples and in-situ bio-catalysis provide pathsto pursue the structure-function paradigm.

The SMB Resource has developed one of thelargest dedicated bioXAS activities in the world withoptimized beam lines, specialized instrumentation,including high-performance detector arrays, datacollection software, and data analysis capabilities,for enabling biological applications.

The Resource provides two dedicated beam lines forsolution XAS on metals and access to beam lines forstudies of ligands such as sulfur, chlorine andphosphorous. Integrated facilities for microXASimaging and tomography (μm to mm spatial resolution,over a wide energy range) are provided, as is a facilityfor XES/HERFD based spectroscopies.

The SMB Resource offers robust trainingprograms for the novice and experienced usercommunity, focusing on the experimental andtheoretical aspects of data measurement,analysis and structural understanding.

http://ssrl.slac.stanford.edu/smb/index.html

Macromolecular Crystallographythree-dimensional structural information at the atomic level

Small Angle X-ray Scatteringsolution conformation of macromolecules

All MC stations are fully remote access (run the experimentfrom your home institution) with standard experiments fullyautomated, using a user friendly interface (Blu-Ice) and theSAM robot for crystal mounting and screening. Diffractionimages are automatically analyzed, indexed and scored.BL12-2 and BL9-2 have high-speed Pilatus detectors for fastshutterless data collection and low-dose crystal searches.

Contacts: Aina Cohen: acohen@slac.stanford.edu

Mike Soltis: soltis@slac.stanford.edu Contact: Thomas Weiss: weiss@slac.stanford.edu

Small angle x-ray scattering (SAXS) from solutions ofmacromolecules and their complexes provides 3D structuralinformation at nanometer resolution, enabling interpretationof atomic-resolution structures in a physiological context,providing a structural tool for studying molecularinteractions, complex structures as well as domain foldingproperties and structural flexibilities in solution.

The SMB Resource’s SAXS BL4-2 is dedicated to non-crystalline x-ray scatteringand diffraction studies in biology.

User friendly software with an intuitive graphical interface(Blu-Ice) is used to control all aspects of the instruments andsample handling.

Fully automated solution scattering data collection, with arobotic sample changer and integrated software pipeline fordata reduction and analysis, providing real-time dataprocessing and analysis, make the data collection at thebeam line highly efficient.

http://smb.slac.stanford.edu/

The SMB Resource provides four stations dedicated tomacromolecular crystallography (MC) that enable studies onthe most challenging problems in structural biology. Theseinclude undulator station BL12-2 optimized for micro-beamexperiments, BL9-2, BL7-1 and BL14-1 optimized for high-throughput screening, and MAD or SAD data collection.

A single-crystal UV-Vis microspectrophotometry capability at BL9-2 enablesspectroscopic monitoring of x-ray dose effects on the metal center of metalloenzymes.The system is compatible with the SAM robot and fully remote accessible.

A next-generation microfocus station, BL12-1 isscheduled for completion in 2018. A broad bandpass

capability providing exceptionally bright microbeams and ahigh-speed Eiger16M detector (133 Hz) will support crystalinjectors and other emerging techniques. Similarities ininstrumentation, existing and new sample delivery systems,and software environments form the foundation of asynergistic relationship between SSRL’s BL12-1 and a newMacromolecular Femtosecond crystallography instrument(MFX) at LCLS, through a gateway approach.

MC staff provide strongtraining programs for newand experienced users.

http://www-ssrl.slac.stanford.edu/~saxs

The beam line features state-of-the-art large area siliconpixel array and gap-free CCD detectors with high photondetection efficiency, a widely reconfigurable SAXS camera, avariety of sample handling devices for automated highthroughput and chromatography-coupled solution scattering(SEC-SAXS), time-resolved scattering in the millisecondtime regime, as well as lipid and fiber diffraction studies.

Biological Imaging - X-rays and Electrons @ SSRL – SLAC

Biological imaging – using x-rays, electrons or a combination – is available to the scientific community through an integrated facility at SLAC National Accelerator Laboratory. The SSRL Structural Molecular Biology program provides three primary synchrotron x-ray capabilities: macromolecular crystallography (MC), small angle x-ray scattering/diffraction (SAXS), and x-ray spectroscopy (XAS/XES) including x-ray microXAS imaging, with SSRL’s high x-ray brightness beams. The SLAC-Stanford Cryo-EM Center provides state-of-the-art instrumentation, computational capabilities and support facilities for cryo-EM and cryotomography studies. Along with vigorous R&D in enhanced data collection, data management and data analysis, both activities support remote access, and user-friendly, real-time and on-line data interpretation. The innovations are closely coupled to projects aimed at solving forefront science problems. The synergy between the x-ray and cryo-EM facilities enables tackling challenging systems relevant to the DOE-BER and NIH missions. These programs seek to enhance the user community through excellent support, training and dissemination.

Supported by the DOE Office of Biology and Environmental Research, the NIH National Institute of General Medical Sciences, and non-Federal funding

Contact: Lisa Dunn, lisa@slac.stanford.edu, ph: 650-926-2087 SMB / CryoEM User Administration

X-ray Micro-XAS Imaging electronic & geometric structure of metal sites

http://ssrl.slac.stanford.edu/smb/index.html Contact: Sam Webb, samwebb@slac.stanford.edu

The high brightness x-ray beams at SSRL’s beam lines enable x-ray fluorescence-based imaging techniques, over a wide range of length scales, for research projects in fields including biological, environmental, and bio-geological science. Focusing approaches provide flexible beam sizes, ranging from a few microns to a few hundred microns. These techniques, used at beam lines 2-3, 6-2, 10-2 and 14-3, reveal where different chemical elements are located, what elements are present in the sample, the relative amount of each element, and the chemistry of the element at specific locations. They can be applied on a wide variety of samples, e.g. from soils, plants, and tissues to cells. To obtain 3-dimensional images, x-ray fluorescence tomography (μm to mm spatial resolution) is also frequently used.

Quantitative Hg distribution in the outer layer of the eye lens of MeHg-treated zebrafish

Robust training programs for the novice and experienced user community, focus on the experimental and theoretical aspects of data measurement, analysis and chemical/structural understanding, for all techniques and science areas.

X-ray microXAS imaging screening of Arabidopsis sp. seeds for variation in metal phenotypes with genetic variation [Fe (red), Mn (green), Zn (blue)

Cryo Electron Microscopy imaging structure at the near-atomic level

Small Angle X-ray Scattering solution conformation of macromolecules

GroEL is a 14-mer chaperonin required for proper folding of many proteins. Cryo-EM has shown that very particle has a unique configuration of (at least) three different GroEL conformations among its 14 subunits.

Contact: Wah Chiu: wah@slac.stanford.edu Contact: Aina Cohen: acohen@slac.stanford.edu

Small angle x-ray scattering (SAXS) from solutions of macromolecules, their complexes and assemblies provides 3D structural information at nm resolution, enabling interpretation of atomic-resolution structures and complex arrangements in a physiological context, informing molecular interactions, domain folding properties and structural flexibilities in solution.

https://sites.slac.stanford.edu/cryo-em/

The SLAC-Stanford Cryo-EM Center includes 3 high-end Titan Krios and 1 Talos Arctica cryo-EM instruments, computing infrastructure, laboratories for grid and cryo-sample preparation, and tissue culture laboratories with biohazards capabilities.

http://ssrl.slac.stanford.edu/smb/index.html

SSRL’s SAXS BL4-2 provides instrumentation for automated high-throughput chromatography-coupled solution x-ray scattering, time-resolved scattering in the msec time regime, and instrumentation for diffraction studies of partially ordered systems, e.g. lipids, fiber and membranes.

Protein shell surface layer of Caulobacter crescentus; amorphous and crystalline forms

Polyketide Synthase

Bacterial microcompart-ment shell of proteins surrounding an enzyme core; from MC and cryoEM

SSRL’s four MC stations are fully remote access (run the experiment from your home institution) fully automated, using robotics for crystal mounting and screening. Diffraction images are automatically analyzed, indexed and scored. BL12-2 (micro-focus) and BL9-2 have high-speed Pilatus detectors for fast shutterless data collection and low-dose crystal searches. A next-generation microfocus station, BL12-1 will be available in 2018. A broad bandpass capability providing exceptionally bright microbeams and a high-speed Eiger16M detector (133 Hz) will support crystal injectors and other emerging techniques.

Macromolecular Crystallography 3D structural information at the atomic level

Cryo electron tomography (Cryo-ET) using phase plate optics can reveal the 3D structure of a vitrified whole cell volume, without staining or chemical fixation.

A tomogram slice of phage-infected cyanobacterium (left) and its feature annotation (right) showing infecting phages outside, and a variety of newly assembled phage intermediates inside the cell (pink), subcellular structures like ribosomes (magenta), carboxysomes (blue), light harvesting machinery, etc.

Cryo Electron Microscopy (Cryo-EM), uniquely captures individual instances of structures of biological assemblies, in their natural heterogeneity, as they occur in solution. Cryo-EM can average sets of macromolecules whose structures are the same, to derive snapshots of conformational dynamics.

Contact: Thomas Weiss: weiss@slac.stanford.edu

Data from Cryo-EM, SAXS and MC data provide highly complementary structural information at different resolution; the seamless integration of these data and information provide a foundational platform for studies of biological systems.