Rapid mixing Laue Experiments for Time-resolved Structural Biology of Copper Nitrite Reductase
Lead Research Organisation:
University of Essex
Department Name: Biological Sciences
Abstract
United States of America
Publications
Hough MA
(2021)
Serial synchrotron and XFEL crystallography for studies of metalloprotein catalysis.
in Current opinion in structural biology
Moreno-Chicano T
(2022)
Complementarity of neutron, XFEL and synchrotron crystallography for defining the structures of metalloenzymes at room temperature.
in IUCrJ
| Description | In the experiments funded by this award, two researchers travelled to the BioCARS beamline, Advanced Photon Source, USA for experimental work. The aim was to measure Laue diffraction data using a rapid mixing system to mix reagents with microcrystal suspensions as a way of conducting time-resolved X-ray crystallography. Key findings are that: 1. Laue structures of good quality may be determined from microcrystals We used microcrystals of Cu nitrite reductase mounted on a fixed target 'chip' support (Heidelberg Design) to test the diffraction of these crystals in the short-pulse Laue beam and determine initial structures of resting state enzyme to compare with time-resolved structures. Measurement of trial data allowed for optimisation of the X-ray beam (pulse length and multi-bunch structure). Good quality diffraction data were measured with a dataset being produced allowing a medium resolution crystal structure to be solved and refined. 2. Crystals survive multiple exposures to the Laue beam We tested the crystal lifetime in the X-ray beam and also induced structural changes to the Cu nitrite reductase enzyme by measuring repeated exposures from each microcrystal. This was an extensive experiment in which we were able to produce an X-ray dose series from the Laue images. Data analysis remains in progress. 3. Reaction initiation by mixing presents a significant challenge Extensive experiments were conducted using our protein microcrystals within a microfluidic rapid-mixing delivery system. Despite our best efforts, a combination of sample viscosity and heterogeneity in crystal sizes led to frequent blocking of the microfluidic system and did not allow for diffraction data to be measured. Further difficulties were encountered in recording data from fast moving crystals using the Laue beam. The results of our work have informed subsequent design developments in the microfluidic system. Structures determined under this award for the basis of a manuscript in preparation that compares serial Laue crystallography data with that obtained using other serial crystallography methods. |
| Exploitation Route | 1. Extension of chip based Laue method to additional protein/enzyme classes 2. Laue dose series could be used for many other proteins 3. Informing future developments in the design of the microfluidic mixing system |
| Sectors | Environment,Healthcare |
| Description | Experience gained in growing, handling and measuring from microcrystals fed directly into our current serial femtosecond crystallography programme. Experience in the use of a large scale facility in the USA provided excellent training and career development. |
| First Year Of Impact | 2017 |
| Sector | Other |
| Description | Breaking the Cage: Transformative Time-resolved Crystallography using Fixed Targets at Synchrotrons and XFELs |
| Amount | £446,146 (GBP) |
| Funding ID | BB/W001950/1 |
| Organisation | Biotechnology and Biological Sciences Research Council (BBSRC) |
| Sector | Public |
| Country | United Kingdom |
| Start | 01/2022 |
| End | 01/2025 |
| Title | LAUE-MSOX |
| Description | Chip-Laue MSOX method. We extended our method of measuring multiple structures from a single crystal with increasing dose to Laue radiation at the APS. Th protein target was Cu nitrite reductase |
| Type Of Material | Improvements to research infrastructure |
| Year Produced | 2017 |
| Provided To Others? | No |
| Impact | None yet |
| Description | Collaboration on rapid mixing and time-resolved structural biology of Cu nitrite reductase |
| Organisation | University of Hamburg |
| Department | Centre for Ultrafast Imaging |
| Country | Germany |
| Sector | Academic/University |
| PI Contribution | Discussions based on BBSRC award outcomes led to submission of a successful joint proposal to the Advanced Photon Source, USA for time-resolved Laue crystallography of AcNiR. We provide enzyme expertise, protein samples, microcrystals for experimental work together with analysis of resulting structures. |
| Collaborator Contribution | Novel microfluidic rapid mixing chips to mix CuNiR with substrates and reductants for time-resolved structure determination at ultra-low doses Collaboration with Dr Diana Monteiro and Professor Martin Trebbin |
| Impact | Successful beamtime award for 2 years to Advanced Photon Source, Chicago, USA |
| Start Year | 2016 |
| Description | Collaborative experiments in lauee |
| Organisation | Argonne National Laboratory |
| Country | United States |
| Sector | Public |
| PI Contribution | Participation in beamtime - Laue experiments at BioCARS facility, APS (two visits) |
| Collaborator Contribution | Training, experimental participation and data processing |
| Impact | Manuscript in preparation |
| Start Year | 2017 |