Modern protein crystal structures are based nearly exclusively on X-ray data collected at cryogenic temperatures (generally 100 K). The cooling process is thought to introduce little bias in the ...functional interpretation of structural results, because cryogenic temperatures minimally perturb the overall protein backbone fold. In contrast, here we show that flash cooling biases previously hidden structural ensembles in protein crystals. By analyzing available data for 30 different proteins using new computational tools for electron-density sampling, model refinement, and molecular packing analysis, we found that crystal cryocooling remodels the conformational distributions of more than 35% of side chains and eliminates packing defects necessary for functional motions. In the signaling switch protein, H-Ras, an allosteric network consistent with fluctuations detected in solution by NMR was uncovered in the room-temperature, but not the cryogenic, electron-density maps. These results expose a bias in structural databases toward smaller, overpacked, and unrealistically unique models. Monitoring room-temperature conformational ensembles by X-ray crystallography can reveal motions crucial for catalysis, ligand binding, and allosteric regulation.
Many advances in the understanding of radiation damage to protein crystals, particularly at cryogenic temperatures, have been made in recent years, but with this comes an expanding literature, and, ...to the new breed of protein crystallographer who is not really interested in X‐ray physics or radiation chemistry but just wants to solve a biologically relevant structure, the technical nature and breadth of this literature can be daunting. The purpose of this paper is to serve as a rough guide to radiation damage issues, and to provide references to the more exacting and detailed work. No attempt has been made to report precise numbers (a factor of two is considered satisfactory), and, since there are aspects of radiation damage that are demonstrably unpredictable, the `worst case scenario' as well as the `average crystal' are discussed in terms of the practicalities of data collection.
Radiation damage limits the accuracy of macromolecular structures in X-ray crystallography. Cryogenic (cryo-) cooling reduces the global radiation damage rate and, therefore, became the method of ...choice over the past decades. The recent advent of serial crystallography, which spreads the absorbed energy over many crystals, thereby reducing damage, has rendered room temperature (RT) data collection more practical and also extendable to microcrystals, both enabling and requiring the study of specific and global radiation damage at RT. Here, we performed sequential serial raster-scanning crystallography using a microfocused synchrotron beam that allowed for the collection of two series of 40 and 90 full datasets at 2- and 1.9-Å resolution at a dose rate of 40.3 MGy/s on hen egg white lysozyme (HEWL) crystals at RT and cryotemperature, respectively. The diffraction intensity halved its initial value at average doses (D
1/2) of 0.57 and 15.3 MGy at RT and 100 K, respectively. Specific radiation damage at RT was observed at disulfide bonds but not at acidic residues, increasing and then apparently reversing, a peculiar behavior that can be modeled by accounting for differential diffraction intensity decay due to the nonuniform illumination by the X-ray beam. Specific damage to disulfide bonds is evident early on at RT and proceeds at a fivefold higher rate than global damage. The decay modeling suggests it is advisable not to exceed a dose of 0.38 MGy per dataset in static and time-resolved synchrotron crystallography experiments at RT. This rough yardstick might change for proteins other than HEWL and at resolutions other than 2 Å.
The structures of Aspergillus oryzae α-amylase were determined in a tetragonal crystal, having one molecule as asymmetric unit, and a monoclinic crystal with two molecules as asymmetric unit. Both ...crystal forms were obtained from trace contaminants of an old commercial lipase preparation. Structures were determined and refined to 1.65 Å and 1.43 Å resolution respectively. The latter crystal has a non-crystallographic (NCS) twofold axis within the asymmetric unit. Glycosylation at Asn197 is evident, and in the tetragonal crystal can be seen to include three, partially disordered sugar residues following the initial N-acetyl glucosamine (NAG). Superposition of the tetragonal crystal model on the α-amylases from Bacillus subtilis (PDB:1BAG), pig pancreas (PDB:3L2L), and barley (PDB:1AMY), show a high degree of coincidence, particularly for the (β/α)8-barrel domains, and especially within the active site. Using this structural agreement between amylases, we extrapolated the binding model of a six residue, limit dextrin found in pig pancreas α-amylase to the A. oryzae enzyme model, which predicts substrate interacting amino acid residues.
To increase the power of X-ray crystallography to determine not only the structures but also the motions of biomolecules, we developed methods to address two classic crystallographic problems: ...putting electron density maps on the absolute scale of e ⁻/Å ³ and calculating the noise at every point in the map. We find that noise varies with position and is often six to eight times lower than thresholds currently used in model building. Analyzing the rescaled electron density maps from 485 representative proteins revealed unmodeled conformations above the estimated noise for 45% of side chains and a previously hidden, low-occupancy inhibitor of HIV capsid protein. Comparing the electron density maps in the free and nucleotide-bound structures of three human protein kinases suggested that substrate binding perturbs distinct intrinsic allosteric networks that link the active site to surfaces that recognize regulatory proteins. These results illustrate general approaches to identify and analyze alternative conformations, low-occupancy small molecules, solvent distributions, communication pathways, and protein motions.
A synthetic data set demonstrating a particularly challenging case of indexing ambiguity in the context of radiation damage was generated. This set shall serve as a standard benchmark and reference ...point for the ongoing development of new methods and new approaches to robust structure solution when single‐crystal methods are insufficient. Of the 100 short wedges of data, only the first 36 are currently necessary to solve the structure by `cheating', or using the correct reference structure as a guide. The total wall‐clock time and number of crystals required to solve the structure without cheating is proposed as a metric for the efficacy and efficiency of a given multi‐crystal automation pipeline.
Synthetic macromolecular crystallography diffraction‐image data were generated to demonstrate the challenges of combining data from multiple crystals with indexing ambiguity in the context of heavy radiation damage. The nature of the problems encountered using contemporary data‐processing programs is summarized.
In this work, classic intensity formulae were united with an empirical spot‐fading model in order to calculate the diameter of a spherical crystal that will scatter the required number of photons per ...spot at a desired resolution over the radiation‐damage‐limited lifetime. The influences of molecular weight, solvent content, Wilson B factor, X‐ray wavelength and attenuation on scattering power and dose were all included. Taking the net photon count in a spot as the only source of noise, a complete data set with a signal‐to‐noise ratio of 2 at 2 Å resolution was predicted to be attainable from a perfect lysozyme crystal sphere 1.2 µm in diameter and two different models of photoelectron escape reduced this to 0.5 or 0.34 µm. These represent 15‐fold to 700‐fold less scattering power than the smallest experimentally determined crystal size to date, but the gap was shown to be consistent with the background scattering level of the relevant experiment. These results suggest that reduction of background photons and diffraction spot size on the detector are the principal paths to improving crystallographic data quality beyond current limits.
Abstract
Light-driven oxidation of water to molecular oxygen is catalyzed by the oxygen-evolving complex (OEC) in Photosystem II (PS II). This multi-electron, multi-proton catalysis requires the ...transport of two water molecules to and four protons from the OEC. A high-resolution 1.89 Å structure obtained by averaging all the S states and refining the data of various time points during the S
2
to S
3
transition has provided better visualization of the potential pathways for substrate water insertion and proton release. Our results indicate that the O1 channel is the likely water intake pathway, and the Cl1 channel is the likely proton release pathway based on the structural rearrangements of water molecules and amino acid side chains along these channels. In particular in the Cl1 channel, we suggest that residue D1-E65 serves as a gate for proton transport by minimizing the back reaction. The results show that the water oxidation reaction at the OEC is well coordinated with the amino acid side chains and the H-bonding network over the entire length of the channels, which is essential in shuttling substrate waters and protons.
Oxidation states of individual metal atoms within a metalloprotein can be assigned by examining X‐ray absorption edges, which shift to higher energy for progressively more positive valence numbers. ...Indeed, X‐ray crystallography is well suited for such a measurement, owing to its ability to spatially resolve the scattering contributions of individual metal atoms that have distinct electronic environments contributing to protein function. However, as the magnitude of the shift is quite small, about +2 eV per valence state for iron, it has only been possible to measure the effect when performed with monochromated X‐ray sources at synchrotron facilities with energy resolutions in the range 2–3 × 10−4 (ΔE/E). This paper tests whether X‐ray free‐electron laser (XFEL) pulses, which have a broader bandpass (ΔE/E = 3 × 10−3) when used without a monochromator, might also be useful for such studies. The program nanoBragg is used to simulate serial femtosecond crystallography (SFX) diffraction images with sufficient granularity to model the XFEL spectrum, the crystal mosaicity and the wavelength‐dependent anomalous scattering factors contributed by two differently charged iron centers in the 110‐amino‐acid protein, ferredoxin. Bayesian methods are then used to deduce, from the simulated data, the most likely X‐ray absorption curves for each metal atom in the protein, which agree well with the curves chosen for the simulation. The data analysis relies critically on the ability to measure the incident spectrum for each pulse, and also on the nanoBragg simulator to predict the size, shape and intensity profile of Bragg spots based on an underlying physical model that includes the absorption curves, which are then modified to produce the best agreement with the simulated data. This inference methodology potentially enables the use of SFX diffraction for the study of metalloenzyme mechanisms and, in general, offers a more detailed approach to Bragg spot data reduction.
Electronic configurations at distinct metal centers within a metalloprotein may be characterized by inspecting the scattering factors at the X‐ray absorption edge. Such experiments may be feasible at XFEL sources, using Bayesian data analysis.
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