High-resolution imaging enabled by 100-kW-peak-power parametric source at 5.7 THz.

Similar to x-ray imaging, THz imaging will require high power and high resolution to advance relevant applications. Previously demonstrated THz imaging usually experiences one or several difficulties in insufficient source power, poor spectral tunability, or limited resolution from a low-wavelength source. A short-wavelength radiation source in the 5-10 THz is relatively scarce. Although a shorter wavelength improves imaging resolution, widely used imaging sensors, such as microbolometers, Schottky diodes, and photoconductive antennas, are usually not sensitive to detect radiation with frequencies above 5 THz. The radiation power of a high-frequency source becomes a key factor to realize low-noise and high-resolution imaging by using an ordinary pyroelectric detector. Here, we report a successful development of a fully coherent, tunable, > 100-kW-peak-power parametric source at 5.7 THz. It is then used together with a low-cost pyroelectric detector for demonstrating high-resolution 5.7-THz imaging in comparison with 2-THz imaging. To take advantage of the wavelength tunability of the source, we also report spectrally resolved imaging between 5.55 and 5.87 THz to reveal the spectroscopic characteristics and spatial distribution of a test drug, Aprovel.

Structural insight into the hydrolase and synthase activities of an alkaline α-galactosidase from Arabidopsis from complexes with substrate/product.

The alkaline α-galactosidase AtAkαGal3 from Arabidopsis thaliana catalyzes the hydrolysis of α-D-galactose from galacto-oligosaccharides under alkaline conditions. A phylogenetic analysis based on sequence alignment classifies AtAkαGal3 as more closely related to the raffinose family of oligosaccharide (RFO) synthases than to the acidic α-galactosidases. Here, thin-layer chromatography is used to demonstrate that AtAkαGal3 exhibits a dual function and is capable of synthesizing stachyose using raffinose, instead of galactinol, as the galactose donor. Crystal structures of complexes of AtAkαGal3 and its D383A mutant with various substrates and products, including galactose, galactinol, raffinose, stachyose and sucrose, are reported as the first representative structures of an alkaline α-galactosidase. The structure of AtAkαGal3 comprises three domains: an N-terminal domain with 13 antiparallel ß-strands, a catalytic domain with an (α/ß)8-barrel fold and a C-terminal domain composed of ß-sheets that form two Greek-key motifs. The WW box of the N-terminal domain, which comprises the conserved residues FRSK75XW77W78 in the RFO synthases, contributes Trp77 and Trp78 to the +1 subsite to contribute to the substrate-binding ability together with the (α/ß)8 barrel of the catalytic domain. The C-terminal domain is presumably involved in structural stability. Structures of the D383A mutant in complex with various substrates and products, especially the natural substrate/product stachyose, reveal four complete subsites (-1 to +3) at the catalytic site. A functional loop (residues 329-352) that exists in the alkaline α-galactosidase AtAkαGal3 and possibly in RFO synthases, but not in acidic α-galactosidases, stabilizes the stachyose at the +2 and +3 subsites and extends the catalytic pocket for the transferase mechanism. Considering the similarities in amino-acid sequence, catalytic domain and activity between alkaline α-galactosidases and RFO synthases, the structure of AtAkαGal3 might also serve a model for the study of RFO synthases, structures of which are lacking.

Structures of honeybee-infecting Lake Sinai virus reveal domain functions and capsid assembly with dynamic motions.

Understanding the structural diversity of honeybee-infecting viruses is critical to maintain pollinator health and manage the spread of diseases in ecology and agriculture. We determine cryo-EM structures of T = 4 and T = 3 capsids of virus-like particles (VLPs) of Lake Sinai virus (LSV) 2 and delta-N48 LSV1, belonging to tetraviruses, at resolutions of 2.3-2.6 Å in various pH environments. Structural analysis shows that the LSV2 capsid protein (CP) structural features, particularly the protruding domain and C-arm, differ from those of other tetraviruses. The anchor loop on the central ß-barrel domain interacts with the neighboring subunit to stabilize homo-trimeric capsomeres during assembly. Delta-N48 LSV1 CP interacts with ssRNA via the rigid helix α1', α1'-α1 loop, ß-barrel domain, and C-arm. Cryo-EM reconstructions, combined with X-ray crystallographic and small-angle scattering analyses, indicate that pH affects capsid conformations by regulating reversible dynamic particle motions and sizes of LSV2 VLPs. C-arms exist in all LSV2 and delta-N48 LSV1 VLPs across varied pH conditions, indicating that autoproteolysis cleavage is not required for LSV maturation. The observed linear domino-scaffold structures of various lengths, made up of trapezoid-shape capsomeres, provide a basis for icosahedral T = 4 and T = 3 architecture assemblies. These findings advance understanding of honeybee-infecting viruses that can cause Colony Collapse Disorder.

Cochlear Obliteration after Translabyrinthine Resection for Large Cerebellopontine Angle Tumor.

INTRODUCTION: The aim of this study was to better understand the onset time and factors associated with cochlear obliteration following translabyrinthine approach (TLA) surgery for large cerebellopontine angle tumors. METHODS: This retrospective cohort study included 117 patients with large cerebellopontine angle tumor (tumor diameter >2 cm) treated by TLA surgery from June 2011 to March 2019 in a single tertiary referral center. The Kaplan-Meier method with log-rank test was used to estimate cochlear patency survival and the association between survival and covariates, and the Cox proportional hazards regression analysis was used to identify possible factors associated with cochlear obliteration. RESULTS: Of the 117 patients included in our analysis, the median follow-up was 24.8 months. There were 30 (25.6%) patients in the cochlear obliteration group, and 87 (74.4%) in the patent cochlear group. Various degrees of cochlear obliteration was found in 25.6% patients in final MRI scan, comprised of 50% grade I, 30% grade II, and 20% grade III. Cochlear patency survival curves showed 94.0% at 3 months, 73.0% at 18 months, which plateaued after 20 months with a survival rate of 71.6%. In the multivariate Cox proportional hazards model, patients presented with postoperative hyperintense T1W cochlear signal had poorer cochlear patency survival compared to isointense T1W (HR = 4.15). Similarly, postoperative deteriorated facial function (HR = 4.52) and full IAC involvement of tumor (HR = 2.33) demonstrated a higher risks of cochlear obliteration after TLA surgery. CONCLUSION: The 2-year estimated cochlear patency rate was 71.6% in patients that received TLA. Cochlear obliteration can develop as early as 3 months post-surgery, with no new obliteration 20 months after the surgery and half of these patients got severe obliteration. Three factors associated with cochlear obliteration were identified including full IAC involvement of tumor, postoperative facial function deterioration, and postoperative hyperintense T1W cochlear signal.

Conformational Changes of α-Crystallin Proteins Induced by Heat Stress.

α-crystallin is a major structural protein in the eye lenses of vertebrates that is composed of two relative subunits, αA and αB crystallin, which function in maintaining lens transparency. As a member of the small heat-shock protein family (sHsp), α-crystallin exhibits chaperone-like activity to prevent the misfolding or aggregation of critical proteins in the lens, which is associated with cataract disease. In this study, high-purity αA and αB crystallin proteins were expressed from E. coli and purified by affinity and size-exclusion chromatography. The size-exclusion chromatography experiment showed that both αA and αB crystallins exhibited oligomeric complexes in solution. Here, we present the structural characteristics of α-crystallin proteins from low to high temperature by combining circular dichroism (CD) and small-angle X-ray scattering (SAXS). Not only the CD data, but also SAXS data show that α-crystallin proteins exhibit transition behavior on conformation with temperature increasing. Although their protein sequences are highly conserved, the analysis of their thermal stability showed different properties in αA and αB crystallin. In this study, taken together, the data discussed were provided to demonstrate more insights into the chaperone-like activity of α-crystallin proteins.

Octave-spanning supercontinuum generation from off-axis Raman oscillation in a monolithic KTP crystal.

We report generation of a visible and near-infrared supercontinuum from a high-gain, ultra-broadband, and mirrorless Raman oscillator in a monolithic KTP crystal. The plane transverse to the pump axis resonates and traps off-axis Stokes waves and their frequency-upconverted components bouncing between two crystal surfaces via total internal reflection. The Raman gain is maximized with the Stokes polarization perpendicular to the plane of reflections. When pumped by a Q-switched Nd:YAG laser, the monolithic oscillator generates quasi-mode-locked Stokes pulses with octave-spanning spectral groups across the visible and near-infrared spectra between 540 and 1800 nm.

Dark-line laser.

A laser is meant to emit coherent radiation at a particular wavelength. Here, we demonstrate a laser that is prohibited to emit at a particular wavelength, called a dark line in the emission background of its gain spectrum. Specifically, we installed a 150 µm thick etalon mirror on an ytterbium-doped fiber laser. The laser suffers from 100% loss at the resonance of the etalon and generates a dark line in its emission spectrum. The interplay among the etalon resonance, homogenous gain broadening, and gain competition allows wavelength tuning and multiple-color emission from the laser.

Structural insights into the histidine-containing phospho-transfer protein and receiver domain of sensor histidine kinase suggest a complex model in the two-component regulatory system in Pseudomonas aeruginosa.

In Pseudomonas aeruginosa, an important opportunistic pathogen that causes numerous acute and chronic infections, the hybrid two-component system (TCS) regulates the swarming ability and biofilm formation with a multistep phospho-relay, and consists of hybrid-sensor histidine kinase (HK), histidine-containing phospho-transfer protein (Hpt) and response regulator (RR). In this work, two crystal structures of HptB and the receiver domain of HK PA1611 (PA1611REC) of P. aeruginosa have been determined in order to elucidate their interactions for the transfer of the phospho-ryl group. The structure of HptB folds into an elongated four-helix bundle - helices α2, α3, α4 and α5, covered by the short N-terminal helix α1. The imidazole side chain of the conserved active-site histidine residue His57, located near the middle of helix α3, protrudes from the bundle and is exposed to solvent. The structure of PA1611REC possesses a conventional (ß/α)5 topology with five-stranded parallel ß-sheets folded in the central region, surrounded by five α-helices. The divalent Mg2+ ion is located in the negatively charged active-site cleft and interacts with Asp522, Asp565 and Arg567. The HptB-PA1611REC complex is further modeled to analyze the binding surface and interactions between the two proteins. The model shows a shape complementarity between the convex surface of PA1611REC and the kidney-shaped HptB with fewer residues and a different network involved in interactions compared with other TCS complexes, such as SLN1-R1/YPD1 from Saccharomyces cerevisiae and AHK5RD/AHP1 from Arabidopsis thaliana. These structural results provide a better understanding of the TCS in P. aeruginosa and could potentially lead to the discovery of a new treatment for infection.

Determination and evaluation of secondary structure content derived from calcium-induced conformational changes in wild-type and mutant mnemiopsin 2 by synchrotron-based Fourier-transform infrared spectroscopy.

Mnemiopsin 2 from a luminous ctenophore with two functional EF-hand motifs is a calcium-regulated photoprotein that is responsible for emitting a bright blue bioluminescence upon reacting with coelenterazine and calcium ions in Mnemiopsis leidyi. Synchrotron radiation-based Fourier-transform infrared (SR-FTIR) spectroscopy was applied to analyze the distribution of secondary structures, the conformational changes resulting from calcium binding and the structural stabilities in wild-type mnemiopsin 2, as well as its mutant type that possesses three EF-hand motifs. The distribution of secondary structures of these proteins indicates that mutant apo-mnemiopsin 2 has a more stable secondary structure than the wild-type. Analyses of the SR-FTIR spectra revealed that the conformational changes at the secondary structures of both mnemiopsin 2 depend on the calcium concentrations, such that the most noticeable changes in structures of wild-type and mutant mnemiopsin 2 occur at optimum concentrations 6 and 2 mM of calcium chloride, respectively. The addition of calcium to both proteins increases the proportions of their secondary structures in the amide I and II regions. The major amide I bands in the IR spectra of both mnemiopsin­calcium complexes shift towards smaller wavenumbers, whereas their main amide II bands are identified at larger wavenumbers.

Characterization of Dimeric Interactions within Protrusion-Domain Interfaces of Parallel and X-Shaped Conformations of Macrobrachium rosenbergii Nodavirus: A Theoretical Study Using the DFT Method along with QTAIM and NBO Analyses.

The protrusion domain (P-domain; MrNVPd) of Macrobrachium rosenbergii nodavirus (MrNV) exists in two conformations, parallel and X-shaped. We have performed a theoretical study to gain insight into the nature of the dimeric interactions involving the dimeric interfaces within parallel and X-shaped conformations of MrNVPd by applying the quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses in the framework of the density functional theory (DFT) approach. The results reveal that the dimer-dimer interfaces of MrNVPd have hydrogen bonds of common types. Leu255-Lys287, Tyr257-Lys287, Lys287-Ser253, Met294-Cys328, Asp295-Lys327, Ser298-Ser324, Ile326-Asp295, and Cys328-Met294 are the key residue pairs of the dimer-dimer interfaces to maintain the dimer-dimer structures of MrNVPd through charge-charge, charge-dipole, dipole-dipole, hydrophobic, and hydrogen bonding interactions. The strengths of these intermolecular dimer-dimer interactions in the parallel conformation are much greater than those in the X-shaped conformation. The parallel trimeric interface is held basically by electrostatic and hydrophobic interactions. The electrostatic interactions accompanying a strong hydrogen bond of Oγ1-Hγ1···Oγ1 in the Thr276 A-Thr276 D pair maintain the intermolecular interface of two X-shaped MrNVPd dimers.

The atomic structures of shrimp nodaviruses reveal new dimeric spike structures and particle polymorphism.

Shrimp nodaviruses, including Penaeus vannamei (PvNV) and Macrobrachium rosenbergii nodaviruses (MrNV), cause white-tail disease in shrimps, with high mortality. The viral capsid structure determines viral assembly and host specificity during infections. Here, we show cryo-EM structures of T = 3 and T = 1 PvNV-like particles (PvNV-LPs), crystal structures of the protrusion-domains (P-domains) of PvNV and MrNV, and the crystal structure of the ∆N-ARM-PvNV shell-domain (S-domain) in T = 1 subviral particles. The capsid protein of PvNV reveals five domains: the P-domain with a new jelly-roll structure forming cuboid-like spikes; the jelly-roll S-domain with two calcium ions; the linker between the S- and P-domains exhibiting new cross and parallel conformations; the N-arm interacting with nucleotides organized along icosahedral two-fold axes; and a disordered region comprising the basic N-terminal arginine-rich motif (N-ARM) interacting with RNA. The N-ARM controls T = 3 and T = 1 assemblies. Increasing the N/C-termini flexibility leads to particle polymorphism. Linker flexibility may influence the dimeric-spike arrangement.

Structural insights into the electron/proton transfer pathways in the quinol:fumarate reductase from Desulfovibrio gigas.

The membrane-embedded quinol:fumarate reductase (QFR) in anaerobic bacteria catalyzes the reduction of fumarate to succinate by quinol in the anaerobic respiratory chain. The electron/proton-transfer pathways in QFRs remain controversial. Here we report the crystal structure of QFR from the anaerobic sulphate-reducing bacterium Desulfovibrio gigas (D. gigas) at 3.6 Å resolution. The structure of the D. gigas QFR is a homo-dimer, each protomer comprising two hydrophilic subunits, A and B, and one transmembrane subunit C, together with six redox cofactors including two b-hemes. One menaquinone molecule is bound near heme bL in the hydrophobic subunit C. This location of the menaquinone-binding site differs from the menaquinol-binding cavity proposed previously for QFR from Wolinella succinogenes. The observed bound menaquinone might serve as an additional redox cofactor to mediate the proton-coupled electron transport across the membrane. Armed with these structural insights, we propose electron/proton-transfer pathways in the quinol reduction of fumarate to succinate in the D. gigas QFR.

Bacteriostatic Effect of a Calcined Waste Clamshell-Activated Plastic Film for Food Packaging.

The addition of calcined waste clamshells (CCS) into polyethylene (PE) plastic bags imparts antibacterial properties due to the presence of CaO. In this study, different proportions of calcined waste clamshells were added to PE to explore its bacteriostatic effects. The PE plastic bags with 9% and 11% of CCS exhibited antibacterial efficacy. Further, total aerobic viable count (TVC) values for raw fish fillet packaging in 9% and 11% CCS-PE plastic bags for five days were similar to the 0% CCS-PE plastic bag group after three days of incubation. In addition, the CCS-PE plastic bag demonstrated stability against solvents when examined using the metal migration test under heptane, ethanol, and acetic acid treatments. The results revealed that the CCS-PE bag retains its CaO bacteriostatic efficacy and that the addition of CCS powder to PE prolongs the shelf life of raw fish fillets, as well as mitigating safety concerns from metal leakage.

Domain swapping and SMYD1 interactions with the PWWP domain of human hepatoma-derived growth factor.

The human hepatoma-derived growth factor (HDGF), containing the chromatin-associated N-terminal PWWP domain capable of binding the SMYD1 promoter, participates in various cellular processes and is involved in human cancers. We report the first crystal structures of the human HDGF PWWP domain (residues 1-100) in a complex with SMYD1 of 10 bp at 2.84 Å resolution and its apo form at 3.3 Å, respectively. The structure of the apo PWWP domain comprises mainly four ß-strands and two α-helices. The PWWP domain undergoes domain swapping to dramatically transform its secondary structures, altering the overall conformation from monomeric globular folding into an extended dimeric structure upon DNA binding. The flexible loop2, as a hinge loop with the partially built structure in the apo PWWP domain, notably refolds into a visible and stable α-helix in the DNA complex. The swapped PWWP domain interacts with the minor grooves of the DNA through residues Lys19, Gly22, Arg79 and Lys80 in varied ways on loops 1 and 4 of the two chains, and the structure becomes more rigid than the apo form. These novel structural findings, together with physiological and activity assays of HDGF and the PWWP domain, provide new insights into the DNA-binding mechanism of HDGF during nucleosomal functions.

Discovery of high-gain stimulated polariton scattering near 4 THz from lithium niobate.

Lithium niobate is the most popular material for terahertz wave generation via stimulated polariton scattering (SPS), previously known to have a gain peak near 2 THz. Here we report the discovery of another phase-matched gain peak near 4 THz in lithium niobate, which greatly extends the useful gain spectrum of lithium niobate. Despite the relatively high 4 THz absorption in lithium niobate, the 4 THz SPS becomes dominant over the 2 THz one in an intensely pumped short lithium niobate crystal due to less diffraction-induced absorption and mode-area mismatch. We also demonstrate a signal-seeded OTPO that generates 1.4 nJ at 4.2 THz from lithium niobate with 17.5 mJ pump energy.

DFT, QTAIM, and NBO studies on the trimeric interactions in the protrusion domain of a piscine betanodavirus.

Crystal structure of the protrusion domain (P-domain) of the grouper nervous necrosis virus (GNNV) shows the presence of three-fold trimeric protrusions with two asymmetrical calcium cations along the non-crystallographic three-fold axis. The trimeric interaction natures of the interacting residues and the calcium cations with the neighboring residues within the trimeric interface have been studied by the quantum theory of atoms in molecules (QTAIM) and natural bond orbital (NBO) analyses in the framework of the density-functional theory (DFT) approach. The results revealed that residues Leu259, Val274, Trp280, and Gln322 of subunit A, Arg261, Asp275, Ala277, and Gln322 of subunit B, Leu259, Asp260, Arg261, Ala277, Val278, and Leu324 of subunit C are the main residues involved in the trimeric interactions. Charge-dipole, dipole-dipole, and hydrogen bonding interactions make the significant contributions to these trimeric interactions. Among different interacting residues within trimeric interface, residue pair Arg261 B-Leu259C forms the strongest hydrogen bond inside the interface between subunits B and C. It was also found that calcium cations interact with residues Asp273, Val274, and Asp275 of subunits A, B, and C through charge-charge and charge transfer interactions.

Terahertz parametric generation and amplification from potassium titanyl phosphate in comparison with lithium niobate and lithium tantalate.

We report superior terahertz parametric generation from potassium titanyl phosphate (KTP) over congruent-grown lithium niobate (CLN) and lithium tantalate (CLT) in terms of parametric gain and laser damage resistance. Under the same pump and crystal configurations, the signal emerged first from KTP, 5% Mg-doped CLN, CLN, and then finally from CLT. The signal growth rate in KTP was comparable to that in 5%-Mg-doped CLN, but the signal power from KTP reached a much higher value after all the other crystals were damaged by the pump laser. We further demonstrate seeded terahertz parametric amplification in an edge-cut KTP at 5.74 THz. The THz parametric amplifier (TPA) employs a 17-mm long KTP gain crystal, pumped by a passively Q-switched pump laser at 1064 nm and seeded by a continuous-wave diode laser tuned to the signal wavelength at 1086.2 nm. With 5.8-mJ energy in a 520-ps pump pulse and 100-mW seed signal power, we measured 5-W peak-power THz output from the KTP TPA with 22% pump depletion. In comparison, we measured no detectable THz output power from a similar edge-cut CLN TPA under the same pump power, detection scheme, and crystal configuration, when tuning the seed laser wavelength to 1072.2 nm and attempting to generate a radiation at 2.1 THz.

Efficient 750-nm LED-pumped Nd:YAG laser.

We report an Nd:YAG laser pumped by light emission diodes (LEDs) at 750 nm. With 1% output coupling from a linear cavity containing a 2-cm long Nd:YAG crystal, the laser generated 37.5 µJ pulse energy at 1064 nm with M2 = 1.1 when pumped by 2.73-mJ LED energy in a 1-ms pulse at a 10 Hz rate. The measured optical and slope efficiencies for this linear-cavity laser are 1.36, and 9%, respectively. With 1 and 5% output couplings from a Z-cavity containing the same laser crystal, the lasers generated 346 and 288 µJ pulse energy with an optical efficiency of 3.4 and 2.8% and slope efficiency of 6.6 and 14%, respectively, for the same 1-ms pump pulse repeating at a 10 Hz rate. At the highest output from the Z-cavity, the measured M2 for the beam is 3.6.

Regular screening of esophageal cancer for 248 newly diagnosed hypopharyngeal squamous cell carcinoma by unsedated transnasal esophagogastroduodenoscopy.

OBJECTIVES: Esophageal squamous cell carcinoma (ESCC) is common in hypopharyngeal squamous cell carcinoma (HSCC) patients. This prospective study is to reveal the prevalence of simultaneous ESCC in newly diagnosed HSCC patients by unsedated transnasal esophagogastroduodenoscopy (EGD) and to analyze the clinical predictors for simultaneous esophageal lesions and their survival. MATERIALS AND METHODS: 248 patients with newly diagnosed HSCC and without previous head and neck cancer between 2007 and 2014 were prospectively evaluated for HSCC and simultaneous esophageal lesions by unsedated transnasal EGD. The clinical factors for simultaneous esophageal lesions were evaluated. Survival analysis of the HSCC patients receiving complete treatment was done. RESULTS: The mean age was 58years. 170 HSCC (68.5%) were classified as T3-T4. The procedures were successfully performed (98.4%), except 4 huge tumors. 174 HSCC (85.7%, out of 203 tumors biopsied) were pathologically proved malignancy by this technique. Regarding esophageal lesions (45.5%), ESCC occurred in 36 patients (14.8%), dysplasia without ESCC occurred in 23 (9.4%) and Lugol voiding lesion without ESCC or dysplasia occurred in 52 (21.3%). Alcohol drinking (adjusted OR: 6.95, p<0.05) and N3 classification (adjusted OR: 2.41, p<0.05) of HSCC were the independent risk factors for the presence of esophageal lesions. The overall survival of the HSCC patients with ESCC was significantly lower than those without ESCC (p=0.013). CONCLUSIONS: Unsedated transnasal EGD is a promising technique for diagnosis of HSCC and simultaneous ESCC. Simultaneous esophageal lesions including ESCC (15%) are common in newly diagnosed HSCC patients, especially with alcohol drinking or N3 disease.

Direct phase selection of initial phases from single-wavelength anomalous dispersion (SAD) for the improvement of electron density and ab initio structure determination.

Optimization of the initial phasing has been a decisive factor in the success of the subsequent electron-density modification, model building and structure determination of biological macromolecules using the single-wavelength anomalous dispersion (SAD) method. Two possible phase solutions (φ1 and φ2) generated from two symmetric phase triangles in the Harker construction for the SAD method cause the well known phase ambiguity. A novel direct phase-selection method utilizing the θ(DS) list as a criterion to select optimized phases φ(am) from φ1 or φ2 of a subset of reflections with a high percentage of correct phases to replace the corresponding initial SAD phases φ(SAD) has been developed. Based on this work, reflections with an angle θ(DS) in the range 35-145° are selected for an optimized improvement, where θ(DS) is the angle between the initial phase φ(SAD) and a preliminary density-modification (DM) phase φ(DM)(NHL). The results show that utilizing the additional direct phase-selection step prior to simple solvent flattening without phase combination using existing DM programs, such as RESOLVE or DM from CCP4, significantly improves the final phases in terms of increased correlation coefficients of electron-density maps and diminished mean phase errors. With the improved phases and density maps from the direct phase-selection method, the completeness of residues of protein molecules built with main chains and side chains is enhanced for efficient structure determination.