2024-03-13RELRCSBRCSBRCSB2017-05-242017-06-142017-06-142024-03-13National Institutes of Health/National Institute of Biomedical Imaging and Bioengineering (NIH/NIBIB)DP2EB020402United StatesRabbit muscle aldolase using 200keVHerzik Jr MAWu MLander GCglycolytic enzyme, LYASEHerzik MAWu MLander GCAchieving better-than-3- angstrom resolution by single-particle cryo-EM at 200 keV.Nat. MethodsUS1410751078201728991891doi:10.1038/nmeth.44611548-71055vy5FULLOVERLAPRabbit Muscle AldolaseRabbit Muscle Aldolase01Rabbit Muscle Aldolase (reconstituted from lyophilized form)Oryctolagus cuniculus0.15Fructose-bisphosphate aldolase AOryctolagus cuniculusMuscle0.0392636724Escherichia coliLEVOPHSHPALTPEQKKELSDIAHRIVAPGKGILAADESTGSIAKRLQSIGTENTEENRRFYRQLLLTADDRVNPCIGGVILFH
ETLYQKADDGRPFPQVIKSKGGVVGIKVDKGVVPLAGTNGETTTQGLDGLSERCAQYKKDGADFAKWRCVLKIGEHTPSA
LAIMENANVLARYASICQQNGIVPIVEPEILPDGDHDLKRCQYVTEKVLAAVYKALSDHHIYLEGTLLKPNMVTPGHACT
QKYSHEEIAMATVTALRRTVPPAVTGVTFLSGGQSEEEASINLNAINKCPLLKPWALTFSYGRALQASALKAWGGKKENL
KAAQEEYVKRALANSLACQGKYTPSGQAGAAASESLFISNHAYP008834.1.2.13singleParticleparticle1.67.520.0C8H18N2O4SHEPES50.0NaClsodium chlorideQuantifoil, UltrAuFoil, R1.2/1.3GOLD300PLASMA CLEANINGOTHER0.00900000000000000115 WattsETHANE100277.15HOMEMADE PLUNGER3 uL of sample/grid was manually blotted for 4 seconds prior to immediate plunge-freezing in liquid nitrogen-cooled ethane.. Specimen was prepared from lyophilized rabbit muscle aldolase (Sigma Aldrich)FEI TALOS ARCTICAFLOOD BEAMBRIGHT FIELDFIELD EMISSION GUN20070.02.70.62.745000.0NITROGENGATAN K2 SUMMIT (4k x 4k)SUPER-RESOLUTION742076761-44181011.068.0Images collected using stage position navigation to target exposure.1Movies were collected in super-resolution mode and Fourier-binned by two prior to motion correction and dose weighting using MotionCor2 program.1009341Template-based particle picking was performed using templates generated from reference-free 2D classification of an initial set of automated particle picks.6ALDInitial model was low-passed filtered to 30 Angstrom resolutionD2BACK PROJECTION2.6FSC 0.143 CUT-OFFRELION2.083910PROJECTION MATCHINGRELION2.0PROJECTION MATCHINGRELION2.0OTHERStarting model was generated by stripping PDB entry 6ALD of all ligands and alternate conformations, then refining into the EM density using imposed symmetry while adjusting weighting/scoring according to estimated map resolution. The top 10 generated models (ranked based on quality metrics) were real-space refined using Phenix software.Maximum LikelihoodREAL35.0emd_8743_additional_1.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
128
128128
0
00128128128116.48116.48116.4890.090.090.0XYZ-0.070882630.1694407-0.00047059350.0138251720.910.910.91Unsharpened map of rabbit muscle aldolaseemd_8743_half_map_1.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
368
368368
-120
-120-120368368368334.88334.88334.8890.090.090.0XYZ-0.0228483530.061095220.000009329730.00159858950.910.910.91Rabbit muscle aldolase, odd half mapemd_8743_half_map_2.map.gz1IMAGE STORED AS FLOATING POINT NUMBER (4 BYTES)
368
368368
-120
-120-120368368368334.88334.88334.8890.090.090.0XYZ-0.0263472740.0614603270.000010850310.0016036710.910.910.91Rabbit muscle aldolase, even half map