Biological small-angle X-ray scattering facility at the Stanford Synchrotron Radiation Laboratory

Smolsky, I. L.; Liu, Ping; Niebuhr, Marc; Ito, Kazuki; Weiß, Thomas; Tsuruta, Hiro

doi:10.1107/s0021889807009624

Cited by 104 publications

(100 citation statements)

References 25 publications

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“…The detector pixel numbers were converted to the momentum transfer (q ϭ 4 ϫ sin/, where 2 ϫ is the scattering angle and is the X-ray wavelength of 1.127 Å, calibrated with silver behenate powder at the capillary position. Protein scattering data were processed by using MarParse, scaled for the transmitted beam intensity integrated for each exposure, and azimuthally averaged (69). A buffer blank was obtained from the average of the first 100 data points (before the void volume) and subtracted from the subsequent protein scattering data.…”

Section: Methodsmentioning

confidence: 99%

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Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Davenport

Guttman

Guo

et al. 2013

J Virol

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dThe HIV-1 envelope glycoprotein (Env) mediates viral entry into host cells and is the sole target of neutralizing antibodies. Much of the sequence diversity in the HIV-1 genome is concentrated within Env, particularly within its gp120 surface subunit. While dramatic functional diversity exists among HIV-1 Env isolates-observable even in the context of monomeric gp120 proteins as differences in antigenicity and immunogenicity-we have little understanding of the structural features that distinguish Env isolates and lead to isolate-specific functional differences, as crystal structures of truncated gp120 "core" proteins from diverse isolates reveal a high level of structural conservation. Because gp120 proteins are used as prospective vaccine immunogens, it is critical to understand the structural factors that influence their reactivity with antibodies. Here, we studied four full-length, glycosylated gp120 monomers from diverse HIV-1 isolates by using small-angle X-ray scattering (SAXS) to probe the overall subunit morphology and hydrogen/deuterium-exchange with mass spectrometry (HDX-MS) to characterize the local structural order of each gp120. We observed that while the overall subunit architecture was similar among isolates by SAXS, dramatic isolate-specific differences in the conformational stability of gp120 were evident by HDX-MS. These differences persisted even with the CD4 receptor bound. Furthermore, surface plasmon resonance (SPR) and enzyme-linked immunosorbance assays (ELISAs) showed that disorder was associated with poorer recognition by antibodies targeting conserved conformational epitopes. These data provide additional insight into the structural determinants of gp120 antigenicity and suggest that conformational dynamics should be considered in the selection and design of optimized Env immunogens.

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Section: Methodsmentioning

confidence: 99%

“…SAXS measurements were conducted on beam line 4-2 at the Stanford Synchrotron Radiation Laboratory (69). In-line size exclusion with SAXS was used to generate high-quality data sets for each gp120 protein.…”

Section: Methodsmentioning

confidence: 99%

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Davenport

Guttman

Guo

et al. 2013

J Virol

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“…The X-ray wavelength was 1.38 Å (SSRL) or 1.03 Å (ALS). The sample-to-detector distance ranged from 1.6 -2.5 m. Circular averaging, intensity scaling and background subtraction, and radial averaging were done by using either MarParse (26) or Ogre. Samples were loaded into sample cells with mica windows and kept at 20 -22°C.…”

Section: Methodsmentioning

confidence: 99%

Molecular organization of Gram-negative peptidoglycan

Gan¹,

Chen²,

Jensen

2008

Proc. Natl. Acad. Sci. U.S.A.

249

238

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The stress-bearing component of the bacterial cell wall-a multigigadalton bag-like molecule called the sacculus-is synthesized from peptidoglycan. Whereas the chemical composition and the 3-dimensional structure of the peptidoglycan subunit (in at least one conformation) are known, the architecture of the assembled sacculus is not. Four decades' worth of biochemical and electron microscopy experiments have resulted in two leading 3-D peptidoglycan models: ''Layered'' and ''Scaffold'', in which the glycan strands are parallel and perpendicular to the cell surface, respectively. Here we resolved the basic architecture of purified, frozenhydrated sacculi through electron cryotomography. In the Gramnegative sacculus, a single layer of glycans lie parallel to the cell surface, roughly perpendicular to the long axis of the cell, encircling the cell in a disorganized hoop-like fashion.cell wall ͉ Cryo-EM ͉ sacculus ͉ tomography ͉ cell shape T he organization of peptidoglycan within the sacculus is a longstanding question in microbiology. Although the sacculus completely envelopes the cell, protecting it from osmotic and mechanical lysis, it is also porous, and thus sieves nutrients and small proteins. Sacculi are constantly remodeled, but they are robust enough that they can be purified from cells and still retain their original shape. Early electron microscope images of purified sacculi were difficult to interpret because the images were 2-D projections through heavy metal-stained sacculi (1, 2). Nevertheless the slender appearance of peptidoglycan in thinsectioned cells (3) and the gaps perpendicular to the polar axis produced by a peptidoglycan-specific endopeptidase (4) lead to the (Circumferential) Layered model (Fig. 1A ) in which the glycan strands lie in the plane of the cell wall and wrap around the cell. The alternative Scaffold model (5) (Fig. 1B) was proposed to fit biochemical data such as pore size and crosslinkage. Various arguments have been made in favor of (6) and against (7) the Scaffold model, but the debate intensified after a recent NMR structure of a synthetic peptidoglycan subunit dimer was interpreted to support the Scaffold model (8). To gain further insight into this issue, we imaged unstained sacculi in 3-D by electron cryotomography (9). Results and DiscussionElectron Cryotomography of Purified Sacculi. Sacculi isolated from C. crescentus strain CB15N and E. coli strains MG1655 and XL-10 were vitrified across holey carbon grids in thin (Ͻ 100 nm) ice. Whereas the gossamer sacculi were barely discernable in the low-dose, 2-D projection images, they were clearly visible in the 3-D tomograms and resembled flattened cells [ Fig. 2 and supporting information (SI) Movie S1 and Movie S2]. In stark contrast to intact cells, the sacculi were flexible and in some positions had radii of curvature less than 20 nm, highlighting how cell shape is not determined by the ''rigidity'' of the sacculus, but rather the cytosolic turgor (osmotic pressure) pushing the cytoplasmic membrane outward against the sacc...

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“…Previous SAXS studies of water have mostly focused on the supercooled region and given contradictory results where both positive (10 -12) and zero enhancement (13,14) at low Q have been reported. With the development of third-generation synchrotron light sources the ability to perform SAXS has been greatly advanced and measurements can now be performed in a large Q-range with high accuracy and reproducibility (15). .…”

mentioning

confidence: 99%

The inhomogeneous structure of water at ambient conditions

Huang

Wikfeldt

Tokushima

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

562

610

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Small-angle X-ray scattering (SAXS) is used to demonstrate the presence of density fluctuations in ambient water on a physical length-scale of Ϸ1 nm; this is retained with decreasing temperature while the magnitude is enhanced. In contrast, the magnitude of fluctuations in a normal liquid, such as CCl4, exhibits no enhancement with decreasing temperature, as is also the case for water from molecular dynamics simulations under ambient conditions. Based on X-ray emission spectroscopy and X-ray Raman scattering data we propose that the density difference contrast in SAXS is due to fluctuations between tetrahedral-like and hydrogen-bond distorted structures related to, respectively, low and high density water. We combine our experimental observations to propose a model of water as a temperature-dependent, fluctuating equilibrium between the two types of local structures driven by incommensurate requirements for minimizing enthalpy (strong near-tetrahedral hydrogen-bonds) and maximizing entropy (nondirectional H-bonds and disorder). The present results provide experimental evidence that the extreme differences anticipated in the hydrogen-bonding environment in the deeply supercooled regime surprisingly remain in bulk water even at conditions ranging from ambient up to close to the boiling point.density fluctuations ͉ liquid-liquid hypothesis ͉ small angle X-ray scattering ͉ water structure ͉ X-ray spectroscopy L iquid water shows many anomalies in its thermodynamic properties such as compressibility, density variation and heat capacity (1-4). In the low-temperature regime, below the freezing point, these properties deviate strongly from normal and theories, related to a liquid-liquid phase transition between high and low density water, have been proposed to account for these anomalies (5). Although the anomalies are extreme in the supercooled region they are also present at ambient conditions where most of waters' physical, chemical and biological processes of importance occur. In contrast, water at ambient conditions has traditionally been considered as a homogeneous distribution of near-tetrahedral hydrogen-bonded (H-bonded) structures with thermal fluctuations increasing with temperature. This picture has been challenged by recent studies based on X-ray Raman (XRS) and conventional X-ray absorption spectroscopy (XAS) (6), and X-ray emission spectroscopy (XES) (7), suggesting two distinct local structures with tetrahedral as a minority and a highly hydrogen-bond (H-bond) distorted asymmetrical as the majority. In particular the proposed predominant asymmetrical structure has caused intense debate in the last years (see refs. 7 and 8 for detailed discussion).SAXS and small-angle neutron scattering (SANS) provide the most direct probes of density variations or fluctuations on large length scales in a liquid. Through an enhancement of the structure factor at low momentum transfer, Q, small deviations from the average electron density at different length scales can be reliably identified (9). Previous SAXS studies of w...

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Biological small-angle X-ray scattering facility at the Stanford Synchrotron Radiation Laboratory

Cited by 104 publications

References 25 publications

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Isolate-Specific Differences in the Conformational Dynamics and Antigenicity of HIV-1 gp120

Molecular organization of Gram-negative peptidoglycan

The inhomogeneous structure of water at ambient conditions

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