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A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.

ABSTRACT: At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabricated sample holder from single crystalline silicon with micropores, which carries up to thousands of crystals and significantly reduces the background scattering level. For loading, the suspended microcrystals are pipetted onto the chip and excess mother liquor is subsequently soaked off through the micropores. Crystals larger than the pore size are retained and arrange themselves according to the micropore pattern. Using our chip we were able to collect 1.5 Å high resolution diffraction data from protein microcrystals with sizes of 4 micrometers and smaller.

SUBMITTER: Roedig P

PROVIDER: S-EPMC4448500 | biostudies-literature | 2015 May

REPOSITORIES: biostudies-literature

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A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.

Roedig P P Vartiainen I I Duman R R Panneerselvam S S Stübe N N Lorbeer O O Warmer M M Sutton G G Stuart D I DI Weckert E E David C C Wagner A A Meents A A

Scientific reports 20150529

At low emittance synchrotron sources it has become possible to perform structure determinations from the measurement of multiple microcrystals which were previously considered too small for diffraction experiments. Conventional mounting techniques do not fulfill the requirements of these new experiments. They significantly contribute to background scattering and it is difficult to locate the crystals, making them incompatible with automated serial crystallography. We have developed a micro-fabri ...[more]

PMID: 26022615

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Project description:Most of the sample holders currently used in macromolecular crystallography offer limited storage density and poor initial crystal-positioning precision upon mounting on a goniometer. This has now become a limiting factor at high-throughput beamlines, where data collection can be performed in a matter of seconds. Furthermore, this lack of precision limits the potential benefits emerging from automated harvesting systems that could provide crystal-position information which would further enhance alignment at beamlines. This situation provided the motivation for the development of a compact and precise sample holder with corresponding pucks, handling tools and robotic transfer protocols. The development process included four main phases: design, prototype manufacture, testing with a robotic sample changer and validation under real conditions on a beamline. Two sample-holder designs are proposed: NewPin and miniSPINE. They share the same robot gripper and allow the storage of 36 sample holders in uni-puck footprint-style pucks, which represents 252 samples in a dry-shipping dewar commonly used in the field. The pucks are identified with human- and machine-readable codes, as well as with radio-frequency identification (RFID) tags. NewPin offers a crystal-repositioning precision of up to 10?µm but requires a specific goniometer socket. The storage density could reach 64 samples using a special puck designed for fully robotic handling. miniSPINE is less precise but uses a goniometer mount compatible with the current SPINE standard. miniSPINE is proposed for the first implementation of the new standard, since it is easier to integrate at beamlines. An upgraded version of the SPINE sample holder with a corresponding puck named SPINEplus is also proposed in order to offer a homogenous and interoperable system. The project involved several European synchrotrons and industrial companies in the fields of consumables and sample-changer robotics. Manual handling of miniSPINE was tested at different institutes using evaluation kits, and pilot beamlines are being equipped with compatible robotics for large-scale evaluation. A companion paper describes a new sample changer FlexED8 (Papp et al., 2017, Acta Cryst., D73, 841-851).

Project description:In this paper we present the development and validation of the "Computational Reverse-Engineering Analysis for Scattering Experiments" (CREASE) method for analyzing scattering results from vesicle structures that are commonly found upon assembly of synthetic, biomimetic, or bioderived amphiphilic copolymers in solution. The two-step CREASE method takes the amphiphilic polymer chemistry and small-angle scattering intensity profile, I exp(q), as input and determines the vesicles' structural features on multiple length scales ranging from assembled vesicle wall's individual layer thicknesses to the monomer-level packing and distribution of polymer conformations. In the first step of CREASE, a genetic algorithm (GA) is used to determine the relevant vesicle dimensions from the input macromolecular solution information and I exp(q) by identifying the structure whose computed scattering profile best matches the input I exp(q). Then in the second step, the GA-determined dimensions are used for molecular reconstruction of the vesicle structure. To validate CREASE for vesicles, we test CREASE on input scattering intensity profiles generated mathematically (termed as in silico I exp(q) vs q) from a variety of vesicle sizes with known dimensions. We also test CREASE on in silico I exp(q) vs q generated from vesicles with dispersity in all relevant dimensions, resembling real experiments. After successful validation of CREASE, we compare the CREASE-determined dimensions against those obtained from the traditional approach of fitting the scattering intensity profile to relevant analytical model in SASVIEW package. We show that CREASE performs better than or as well as the core-multishell analytical model's fitting in SASVIEW in determining vesicle dimensions with dispersity. We also show that CREASE provides structural information beyond those possible from traditional scattering analysis using the core-multishell model, such as the distribution of solvophilic monomers between the vesicle wall's inner and outer layers in the vesicle wall and the chain-level packing within each vesicle layer.

Dataset Information

A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.

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A micro-patterned silicon chip as sample holder for macromolecular crystallography experiments with minimal background scattering.

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