The biological activity of macromolecules is accompanied by rapid structural changes. The photosensitivity of the carbon monoxide complex of myoglobin was used at the European Synchrotron Radiation Facility to obtain pulsed, Laue x-ray diffraction data with nanosecond time resolution during the process of heme and protein relaxation after carbon monoxide photodissociation and during rebinding. These time-resolved experiments reveal the structures of myoglobin photoproducts, provide a structural foundation to spectroscopic results and molecular dynamics calculations, and demonstrate that time-resolved macromolecular crystallography can elucidate the structural bases of biochemical mechanisms on the nanosecond time scale.
The structure of the catalytically active, reduced, form of the enzyme hydroxymethylbilane synthase (HMBS, Lys59Gln mutant) has been studied by Laue diffraction as the substrate, porphobilinogen (PBG), was fed to an immobilised crystal in a flow cell. Laue data at short time-scale time points (i.e. 1, 2, 4 and 8 min) were measured using several crystals and then averaged. Longer time-point data sets (i.e. 25 min ± 7 min and 2 h 23 min ± 9 min) were measured from individual crystals. All data sets benefited from using rapid exposures on ESRF ID09 (≈1 ms) and the fast duty cycle ESRF II CCD (readout time 8s). In one case, the substrate supply to a crystal in the flow cell was stopped at 4 h and monochromatic data collected at 12 h ± 30 min on ESRF BM14 (i.e. about 8 h after the substrate supply was stopped). Structural analysis of these data sets at all the time points was undertaken commencing with rigid-body refinement based upon the molecular model of the active, reduced, enzyme. The rigid-body refinement showed that rotational and translational movements of individual domains of the protein are less than 0.7° and 0.2 Å, respectively in the crystal with respect to the wild-type active form model. Moreover, difference Fourier maps at different time points versus the wild-type reduced form were calculated based upon the calculated phases from the wild-type reduced form model. These maps show that at 8 min, 25 min and 2 h, extended electron density appears in the active site region. This electron density is not visible in the 12 h case. Detailed structural refinement on the 2 h data, for which the extra electron density is most prominent, allowed an improved omit-type difference map to be calculated. This shows electron density in the active site adjacent and above the side-chain of Asp84, which plays a pivotal role throughout the catalytic reaction cycle. The density peak commences at the cofactor C2 ring (oxidised form) position (earlier proposed as a binding site for PBG). It then extends up towards Arg149, past Arg155 (residues whose mutation causes build up of ES1 and ES4 intermediate enzyme–substrate complexes, respectively) and out towards the open solvent channel of the crystal.
A time-resolved Laue X-ray diffraction technique has been used to explore protein relaxation and ligand migration at room temperature following photolysis of a single crystal of carbon monoxymyoglobin. The CO ligand is photodissociated by a 7.5 ns laser pulse, and the subsequent structural changes are probed by 150 ps or 1 μs X-ray pulses at 14 laser/X-ray delay times, ranging from 1 ns to 1.9 ms. Very fast heme and protein relaxation involving the E and F helices is evident from the data at a 1 ns time delay. The photodissociated CO molecules are detected at two locations: at a distal pocket docking site and at the Xe 1 binding site in the proximal pocket. The population by CO of the primary, distal site peaks at a 1 ns time delay and decays to half the peak value in 70 ns. The secondary, proximal docking site reaches its highest occupancy of 20% at ∼100 ns and has a half-life of ∼10 μs. At ∼100 ns, all CO molecules are accounted for within the protein: in one of these two docking sites or bound to the heme. Thereafter, the CO molecules migrate to the solvent from which they rebind to deoxymyoglobin in a bimolecular process with a second-order rate coefficient of 4.5 × 105 M-1 s-1. Our results also demonstrate that structural changes as small as 0.2 Å and populations of CO docking sites of 10% can be detected by time-resolved X-ray diffraction.
Catalase-1 (Cat1) of Neurospora crassa was crystallized by the hanging drop method and a crystal was diffracted at the Stanford Synchrotron Radiation Laboratory.X-ray reflection data were analyzed with denzo, xdisp and CCP4 programs.The crystal belongs to the space group C2, having unit cell parameters of a = 130.007, b = 182.242,c = 90.364Å, β = 133.413°and a dimer in the asymmetric unit.Cat1 structure was determined at 1.75 A resolution using the molecular replacement method and a Escherichia coli catalase (HPII) as starting model.The final R and Rfree values were 18.64 and 20.29, respectively.Cat1 is a large catalase with a C-terminal flavodoxin-like domain, similar in structure to the HPII.Heme in Cat1 was a mixture of protoheme IX (57%) and a heme with two hydroxyl groups in ring D, one of which formed a spirolactone with the propionyl group (43%).HPII also has a heme with a spirolactone.In HPII, the Tyr that coordinates the heme Fe(III) proximally makes a covalent bond with a neighbor His.Interestingly, the equivalent Tyr in Cat-1 makes a covalent bond with a Cys, instead.The corresponding bond being formed between Cys 356 sulfur and Tyr 379 Cb.
MAX IV is the first 'fourth-generation' synchrotron in the world [1] and it will start to produce the brightest light from this summer and onwards.As the first MX beamline at MAX IV, BioMAX will not only have highly brilliant X-ray beam but also high performance hardware [2,3], including the next generation hybrid pixel-array detector EIGER 16M, a newly developed fast sample changer ISARA and a high precision MD3 diffractometer.To take full advantage of these cutting edge technologies and to provide a more user friendly software interface particularly for the arising remote access, a new web-based version of a beamline and experiment control software MXCuBE3 has been being developed by MAX IV in strong collaboration with the ESRF.The software will support both single sample and in situ plate manipulation with modern acquisition methods, such as mesh scan and raster scan.Users can carry out their experiments through web browsers from any place and any operating system, without installation of any additional software.Following these high throughput experiments, a fast and accurate data processing pipeline becomes critical to meet the high data rate.While testing and comparing various existing processing pipelines that utilize XDS and Dials, we are also seeking new approaches that can handle the diffraction images.Finally, the experimental meta-data and processing results will be stored in our ISPyB server and users can access them via web browsers.
Laue diffraction patterns with an exposure time of ca 60 ps have been acquired at the European Synchrotron Radiation Facility (ESRF) on protein crystals by using the single-bunch mode of the storage ring. A 10 ns laser pulse initiating photodissociation was synchronized with the X-ray pulse. The potential for a quantitative detection of conformational changes in proteins on the nanosecond timescale with this technique is demonstrated using the example of carbonmonoxymyoglobin, from simulations and real data. The instrumental aspects of the experiment (highly intense X-ray beam, fast shutter system, Laue camera, detector, laser apparatus and synchronization technique) are emphasized.
The MAX IV laboratory operates two Macromolecular Crystallography (MX) beamlines, namely BioMAX and MicroMAX.BioMAX is a highly versatile beamline that became operational in 2017.It offers beam sizes ranging from 20 x 5 µm 2 FWHM (focused) to 100 x 100 µm 2 (defocused) with a maximum photon flux of 7 x 10 12 photons/s and energies between 6 and 24 keV.The experimental hutch contains an MD3 microdiffractometer, an IRELEC Isara sample changer, and a 16M Eiger detector.The beamline is easily operated with the MXCuBE data acquisition software and offers a wide range of experiments, including fully remote data collection at cryogenic temperatures, collection of small crystals with large unit cells, experimental phasing, and state-of-the-art synchrotron serial crystallography (SSX) setups.In addition, all collected data sets are immediately processed by multiple auto-processing pipelines, and the results and metadata are stored in the ISPyB/EXI laboratory information management system [1].BioMAX is able to collect almost 500 datasets per day and this exceptional throughput has prompted the development of the FragMAX platform for crystal-based fragment screening [2].The platform consists of three primary components: (i) a crystal preparation facility, (ii) automated diffraction data collection at the BioMAX beamline, and (iii) FragMAXapp, an intuitive web application for large-scale data processing [3].The crystal preparation facility is co-located with the Lund Protein Production Platform (LP3) and offers a comprehensive set of tools for protein crystallization, fragment libraries, pucks and pins, automated crystal soaking, and robot-assisted crystal mounting.BioMAX and FragMAX are accessible via the MAX IV user program, the MAX IV Industrial Relations Office (IRO), and the iNEXT Discovery program.Both are continuously evolving and will soon include in-situ data collection, remote data collection for room temperature experiments, new sample delivery instruments, and software tools for accelerated structure refinement and deposition.
The macromolecular crystallography beamline I911-3, part of the Cassiopeia/I911 suite of beamlines, is based on a superconducting wiggler at the MAX II ring of the MAX IV Laboratory in Lund, Sweden. The beamline is energy-tunable within a range between 6 and 18 keV. I911-3 opened for users in 2005. In 2010-2011 the experimental station was completely rebuilt and refurbished such that it has become a state-of-the-art experimental station with better possibilities for rapid throughput, crystal screening and work with smaller samples. This paper describes the complete I911-3 beamline and how it is embedded in the Cassiopeia suite of beamlines.
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