Table 2. Apparent air-water interface, particle, and ice behavior of the same samples in Table 1 using the descriptions in Figure 1.
Tilt-series were aligned and reconstructed using the same workflow and thus are oriented in the same direction. However, the direction relative to the sample application is not known. The bottom air-water interface corresponds to lower z-slice values, and the top to higher z-slice values as rendered in 3dmod from the IMOD package (Kremer et al., 1996). ‘A’ means that the air-water interface is apparently clean and cannot be visually differentiated between A1, A2 (primary structure), or A3. Percentages in parentheses are particle layer saturation estimates. Reported angles are the angles (absolute value) between the particle layer’s normal and the electron beam direction, measured using ‘Slicer’ in 3dmod. It is often difficult to distinguish between flat and curved ice at the air-water interfaces (e.g. Figure 2, ‘C1 or C2’ or ‘C2 or C3’) because most fields of view do not span entire holes. ‘‡’ indicates that the top layer of objects is the same layer as the bottom layer. ‘--’ indicates that these values were not measurable.
| Sample # | Sample name | Air-water interface, particle behavior, and layer/ice angle (bottom, center) |
Air-water interface, particle behavior, and layer/ice angle (bottom, edge) |
Ice behavior (bottom) | Air-water interface, particle behavior, and layer/ice angle (top, center) |
Air-water interface, particle behavior, and layer/ice angle (top, edge) |
Ice behavior (top) |
Notes |
|---|---|---|---|---|---|---|---|---|
| 1* | 32 kDa Kinase | A, B1 or B2 or B3 (50%), 8° | A, B1 or B2 or B3 (50%), 10° | C2 | A, B1 or B2 or B3‡ (50%), 8° | A, B1 or B2 or B3‡ (50%), 10° | C2 | Particles aggregate into clouds. |
| 2 | 32 kDa Kinase | A, B1 or B2 or B3 (50%), 4–8° |
-- | C1 or C2 | A, B1 or B2 or B3‡ (50%), 4–8° | -- | C1 or C2 | Gold beads are glow discharge contamination. |
| 3 | Insulin Receptor | A, B1 or B2 or B3 (100%), 3–5° | -- | C2 or C3 | A, B1 or B2 or B3‡ (100%), 3–5° | -- | C2 or C3 | Gold beads are glow discharge contamination. |
| 4*† | Hemagglutinin | A2, No particles, 3–7° | A, B3 (40%), 5° or A, B3 (40%), 3° |
C3 or C4 | A2‡, No particles, 3–7° or A, B3 (50%), 7° |
A, B3 (50%), 5–7° | C3 or C4 | Where very thin ice in the center of holes excludes particles, protein fragments remain. |
| 5* | HIV-1 Trimer Complex 1 | A2, B1, B3 (30%), 1–5° | -- | C1, C2, or C3 | A2, B1, B3 (30%), 1–5° | -- | C1, C2, or C3 | Trimer domains and/or unbound receptors are adsorbed to air-water interfaces. |
| 6* | HIV-1 Trimer Complex 1 | A2, B3 (80%), 6° | -- | C2 | A2, B3‡ (80%), 6° | -- | C2 | Trimer domains and/or unbound receptors are adsorbed to air-water interfaces. |
| 7* | HIV-1 Trimer Complex 2 | A, B2 or B3 (50%), 1° | A, B2 or B3 (50%), 3° | C1 or C2 | A, B2 or B3 (70%), 1° | A, B2 or B3 (70%), 3° | C1 or C2 | |
| 8 | 147 kDa Kinase | A, B2 or B3 (50%), 0° | -- | C2 or C3 | A, B2 or B3‡ (50%), 0° | -- | C2 or C3 | Gold beads are glow discharge contamination. |
| 9 | 150 kDa Protein | A, B2 or B3 (60%), 7–10° | A, B2 or B3 (60%), 8° | C2 or C3 | A, B2 or B3‡ (60%), 7° | A, B2 or B3 (40%), 9° | C2 or C3 | |
| 10* | Stick-like Protein 1 | A and A2, B4 and B5 (1%), 10° | -- | C2 | A2, B4 and B5 (50%), 10° | -- | C2 | |
| 11 | Stick-like Protein 2 (150 kDa) |
A2, B3 and B4 and B5 (70%), 7° | A2, B3 and B4 and B5 (70%), 7° | -- | A2, B3 and B4 and B5‡ (70%), 7° | A2, B3 and B4 and B5‡ (70%), 7° | -- | Determinations are not accurate due to over focusing and minimal tilt angles. |
| 12* | Stick-like Protein 2 | A2, B3 (80%), 0° | -- | C2 or C3 | A2, B3 (1%), 0° | -- | C2 or C3 | Note 1. Note 2. |
| 13* | Neural Receptor | A2, B3 (80%), 3–10° | -- | C2 or C3 | A2, No particles, 3–10° | -- | C2 or C3 | Note 1. Note 2. |
| 14* | Neural Receptor | -- | A2, No particles, 2–7° or A2, B3 (70%), 5° | C3 | -- | A2, B3 (70%), 7° or A2, No particles, 7° | C3 | Note 1. Note 2. Two tomograms have one orientation, one has the opposite. |
| 15 | 200 kDa Protein | A, B2 or B3 (60%), 2° | A, B2 or B3 (50%), 4° | C3 | No particles or A, B2 or B3‡ (60%), 2° | A, No particles, 11° | C3 | |
| 16 | Small, Popular Protein | A, B2 or B3 (90%), 6° | A, B2 or B3 (90%), 9° | C2 | A, B2 or B3‡ (90%), 6° | A, B2 or B3 (90%), 1° | C3 | |
| 17* | Glycoprotein with Bound Lipids (deglycosylated) | A, B3 (70%), 4° | A, B3 (80%), 10° | C3 | A, B3‡ (70%), 4° | A, B3 (80%), 11° | C3 | Lipid membrane dissociates from protein in center. |
| 18 | Glycoprotein with Bound Lipids (glycosylated) | A, B3 (50%), 10° | -- | C2 or C3 | A, B3 (60%), 4° | -- | C2 or C3 | |
| 19* | Lipo-protein | No particles or A, B2, 3° | A, B3, 11° | C3, C4 | No particles or A, B2‡, 5° | A, B3, 11° | C3, C4 | Particles are uniformly distributed in the ice. |
| 20* | GPCR | A, B2 or B3 (70%), 3° | A, B2 or B3 (60%), -- | C3 | A, B2 or B3‡ (70%), 3° | A, B2 or B3 (60%), -- | C3 | |
| 21*† | Rabbit Muscle Aldolase (1 mg/mL) | A, B2 or B3 (90%), 3–9° | A, B2 or B3 (80%), 6° | C3 | A, B2 or B3‡ (90%), 3–9° | A, B2 or B3 (80%), 10° | C3 | |
| 22*† | Rabbit Muscle Aldolase (6 mg/mL) | A, B1, B2 or B3 (90%), 5° | A, B1, B2 or B3 (90%), 5° | C2 or C3 | A, B1, B2 or B3 (90%), 5° | A, B1, B2 or B3 (90%), 5° | C2 or C3 | |
| 23 | Un-named Protein | A, B3 (40%), 0–3° | -- | C2 or C3 | A, B3‡ (40%), 0–3° | -- | C2 or C3 | |
| 24 | Un-named Protein | A, B3 (80%), 2° | A, B3 (60%), 4–6° | C3 | A, B3‡ (80%), 2° | A, B3 (60%), 4–9° | C3 | |
| 25* | Protein in Nanodisc (0.58 mg/mL) |
A, B2 (80%), 8–10° | A, B2 (80%), 8–10° | C2 or C3 | A, B2‡ (80%), 8–10° | A, B2 (80%), 8–10° | C2 or C3 | |
| 26 | IDE | A2, B2 or B3 and B4 and B5 (50%), 0° | A2, B1, B2 or B3 and B4 and B5 (50%), 5° | C3 | A2, B2 or B3 and B4 and B5‡ (50%), 0° | A2, B1, B2 or B3 and B4 and B5 (50%), 2° | C3 | Note 1. |
| 27* | IDE | A, B2 or B3 (95%), 0–4° | -- | C2 | A, B2 or B3 (95%), 0–4° | -- | C2 | |
| 28 | Small, Helical Protein | A, B2 or B3 (80%), 5° | A, B2 or B3 (70%), 3° | C3 | A, B2 or B3‡ (80%), 5° | A, B2 or B3 (70%), 7° | C3 | |
| 29 | 300 kDa Protein | A or A2, B2 or B3 (70%), 7° | A or A2, B2 or B3 (50%), 13° | C3 | A or A2, B2 or B3‡ (70%), 7° | A or A2, B2 or B3 (50%), 9° | C3 | |
| 30*† | GDH | A, B3 (70%), 10° | A, B1, B3 (50%), 1° | C2 | A, B3‡ (70%), 10° | A, B1, B3 (50%), 16° | C3 | Note 2. Some non-adsorbed particles stack between layers. |
| 31*† | GDH | A, B3 (40%), -- | A, B1, B3 (40%), 10° | C3 | A, B3‡ (40%), -- | A, B1, B3 (40%), 2° | C2 | |
| 32*† | GDH (2.5 mg/mL)+0.001% DDM | A, B3 (40%), 4° | A, B1, B3 (40%), 7° | C2 | A, B3‡ (30%), 4° | A, B1, B3 (30%), 6° | C3 | Some non-adsorbed particles stack between layers. |
| 33*† | DnaB Helicase-helicase Loader | A, B2 or B3 (90%), 1° | A, B2 or B3 (90%), 4° | C3 | A, B2 or B3 (<5%), 1° | A, B2 or B3 (<5%), 1° | C2 | Gold flakes from Quantifoil are on the top. |
| 34*† | Apoferritin | A2, B2 or B3 (50%), 4–6° | -- | C2 or C3 | A2, B2 or B3‡ (50%), 4–6° | -- | C2 or C3 | Note 1. Note 2. |
| 35*† | Apoferritin | A2, B2 or B3 (60%), 4–12° | -- | C2 or C3 | A2, B2 or B3‡ (60%), 4–12° | -- | C2 or C3 | Note 1. Note 2. |
| 36*† | Apoferritin | A2, B3 (50%), 5° | A2, B1, B3 (50%), 10° | C3 | A2, B3‡ (70%), 5° | A2, B1, B3 (60%), 3° | C3 | Note 1. Note 2. |
| 37*† | Apoferritin (1.25 mg/mL) | A2, B2 or B3 (50%), 4–7° | A2, B1, B2 or B3 (50%), 6° | C3 | A2, B2 or B3‡ (40%), 4° | A2, B1, B2 or B3 (30%), 4° | C3 | Note 1. Note 2. |
| 38*† | Apoferritin (0.5 mg/mL) | A2, B2 or B3 (20%), 5° | -- | C2 or C3 | A2, B2 or B3‡ (20%), 1° | -- | C2 or C3 | Note 1. Note 2. |
| 39*† | Apoferritin with 0.5 mM TCEP | A, B2 or B3 (40%), -- or A, B2 or B3 (50%), 3° |
A, B1, B2 or B3 (40%), 5–9° | C3 | A, B2 or B3 (40%), -- or A, B2 or B3‡ (50%), 3° |
A, B1, B2 or B3 (40%), 2–8° | C3 | Note 1. Note 2. |
| 40 | Protein with Carbon Over Holes | Carbon, B1 (30%), B3 (60%), 5° | Carbon, B1 (30%), B3 (60%), 5–9° | C2 | A, B3 (5%), 5° | A, B3 (5%), 5° | C1 or C2 | Note 3. |
| 41 | Protein and DNA Strands with Carbon Over Holes | A, No particles, 2–3° | -- | C2 or C3 | Carbon, B1 (20%), B3 (60%), 2–3° | -- | C2 | Some non-adsorbed particles make contact with particle layer. Most non-adsorbed particles are attached to DNA strands. |
| 42*† | T20S Proteasome | A, B3 (80%), 3° | A, B1 (5%), B3 (80%), 14° |
C3 | A, B3‡ (80%), 3° | A, B1 (5%), B3 (20%), 3° |
C2 | Note 2. Note 3. |
| 43*† | T20S Proteasome | A, B3 (10%), 2–5° | A, B3 (10%), 2–5° | C2 | A, B1 (20%), B3 (90%), 5–7° |
A, B1 (20%), B3 (95%), 5–7° |
C3 | Note 3. |
| 44*† | T20S Proteasome | A, B1 (10%), B3 (80%), 11° | -- | C3 | A, B3 (2%), 11° | -- | C2 | Note 2. Note 3. |
| 45*† | Mtb 20S Proteasome | -- | A, B1, B2 or B3 (30%), 6° | C3 | -- | A, B1, B2 or B3 (30%), 11° | C3 | Heavy contamination. |
| 46 | Protein on Streptavidin | Streptavidin, B2 (10–30%), 0° or Streptavidin, No particles, 12° |
Streptavidin or A2, 2 (10–30%), 12° | C1, C2, or C3 | Streptavidin, B2 (10–30%), 0° or Streptavidin‡, No particles, 12° |
Streptavidin, 2 (10–30%), 13–14° | C1, C2, or C3 | Note 1. Some holes have a layer of streptavidin only on top, some have a layer on top and bottom. Particles are attached to streptavidin and sometimes the apposed air-water interface. |
*A video is included for sample.
†A dataset is deposited for sample.
Note 1: Apparent protein fragments/domains are adsorbed to the air-water interfaces.
Note 2: Partial particles exist.
Note 3: Non-adsorbed particles make contact with particle layer.